WO2003018634A1 - Monoclonal antibody cb874 directed to an unknown antigen from umbilical cord blood - Google Patents

Abstract

A method is disclosed for producing monoclonal antibodies against antigens that are associated with the presence of a disease or condition. The method includes the steps of immunising a non-human animal with a sample of biological matter obtained from at least one patient afflicted with said disease or condition, from which sample have been removed antigens that are present in corresponding biological matter of a normal individual or of an individual who is not afflicted with the disease or condition by contacting the sample with antibodies, which have been raised against antigens present in the corresponding biological matter, to form antigen-antibody complexes, and separating the antigen-antibody complexes from the sample, immortalising antibody-producing cells of the animal to produce immortalised cell lines, producing antibodies from individual immortalised cell lines, and testing the antibodies for specific reactivity with another sample of the biological fluid to thereby identify a monoclonal antibody, which specifically detects the disease or condition. The invention further discloses monoclonal antibody-secreting hybridoma cells produced by the method of the invention, monoclonal antibodies produced therefrom as well as the use of these antibodies for detecting the presence of, or for treating and/or preventing, a disease or condition.

Description

Monoclonal antibody CB874 directed to an unknown antigen from umbilical cord blood

FIELD OF THE INVENTION

THIS INVENTION relates generally to a method of producing antigen-binding molecules. More particularly, the invention relates to a method of producing monoclonal antibodies that recognise antigens whose presence in a biological sample is indicative of the presence of a disease or condition. The method accommodates inter alia for antigens which are present in relatively small amounts or which are weakly immuiiogenic, or antigens of which only very small amounts are available or antigens which easily lose their in vivo conformation. The invention further relates to monoclonal antibody-secreting hybridoma cells produced by the method of the invention, to monoclonal antibodies produced therefrom, and to the use of these antibodies for detecting the presence of, or for treating and/or preventing, a disease or condition.

BACKGROUND OF THE INVENTION The seminal work of George Kδhler and Cesar Milstein (1975, Nature 256: 495-

497) described the first method for obtaining hybridomas in which an antibody-secreting immune cell, isolated from an immunised mouse, is fused with a myeloma cell, a type of B cell tumour. The resultant hybrid cells (i.e., hybridomas) can be maintained in vitro and continue to secrete antibodies that recognise a single determinant or epitope on an antigen. Thus, the development of monoclonal antibodies paved the way for the unlimited production and batch-to-batch consistency of homogeneous preparations of an antibody with a defined specificity.

Monoclonal antibodies have been used widely for diagnostic and therapeutic applications. However, the development of these applications is typically limited by the availability of antigens, which are indicative of, or which underlie, a particular disease or condition. The discovery of such antigens is generally a time consuming and laborious process. Further, if a desired antigen is present in limiting quantities, or is very unstable or weakly immunogenic, the production of monoclonal antibodies against it is typically hampered by the screening of an unfavourably large number of hybridoma clones. Many of the disadvantages of prior art monoclonal antibody-producing methods described above as well as many of the needs not met by them are addressed by the present invention which, as described more fully hereinafter, provides numerous advantages over the aforementioned methods.

SUMMARY OF THE INVENTION

Accordingly, in one aspect of the present invention, there is provided a method for preparing a monoclonal antibody which specifically detects a disease or condition, comprising: -

(a) contacting a sample of biological matter obtained from at least one patient afflicted with said disease or condition with a plurality of antibodies, which have been raised against antigens present in corresponding biological matter of a normal individual or of an individual who is not afflicted with the disease or condition, to form antigen- antibody complexes;

(b) separating the antigen-antibody complexes from the sample; (c) immunising a non-human animal with the sample from which the antigen- antibody complexes have been separated;

(d) immortalising antibody-producing cells of the animal to produce immortalised cell lines;

(e) producing antibodies from individual immortalised cell lines; and (f) testing said antibodies for specific reactivity with another sample of said biological matter obtained from said at least one patient to thereby identify a monoclonal antibody, which specifically detects said disease or condition.

In another aspect, the invention contemplates a method for preparing a monoclonal antibody which specifically detects a disease or condition, comprising: - (a) separating at least one major antigen from a sample of biological matter obtained from at least one patient afflicted with said disease or condition;

(b) contacting the sample from which said at least one major antigen has been separated with a plurality of antibodies, which have been raised against antigens present in corresponding biological matter of a normal individual or of an individual who is not afflicted with the disease or condition, to form antigen-antibody complexes;

(c) separating the antigen-antibody complexes from the sample; (d) immunising a non-human animal with the sample from which the antigen- antibody complexes have been separated;

(e) immortalising antibody-producing cells of the animal to produce immortalised cell lines; (f) producing antibodies from individual immortalised cell lines; and

(g) testing said antibodies for specific reactivity with another sample of said biological matter obtained from said at least one patient to thereby identify a monoclonal antibody, which specifically detects said disease or condition.

In a preferred embodiment, the at least one major antigen is separated from the biological matter using any one or more of affinity separation, gel filtration and ultraflltration.

In yet another aspect, the invention encompasses a method for preparing a monoclonal antibody which specifically detects a disease or condition, comprising: -

(a) contacting a sample of biological matter obtained from at least one patient afflicted with said disease or condition with a plurality of antibodies, which have been raised against antigens present in corresponding biological matter of a normal individual or of an individual who is not afflicted with the disease or condition, to form antigen- antibody complexes;

(b) separating the antigen-antibody complexes from the sample; (c) fractionating the sample from which the antigen-antibody complexes have been separated according to one or more physical or functional parameters to provide two or more antigen-containing fractions;

(d) immunising a non-human animal with an individual antigen-containing fraction;

(e) immortalising antibody-producing cells of the animal to produce immortalised cell lines;

(f) producing antibodies from individual immortalised cell lines; and

(g) testing said antibodies for specific reactivity with another sample of said biological matter obtained from said at least one patient to thereby identify a monoclonal antibody, which specifically detects said disease or condition. In still yet another aspect, the invention embraces a method for preparing a monoclonal antibody which specifically detects a disease or condition, comprising: - (a) fractionating the antigens present in a sample of a biological matter obtained from at least one patient afflicted with said disease or condition according to one or more physical or functional parameters selected from size, density, surface charge, biological activity or a combination of these to provide two or more antigen-containing fractions;

(b) contacting an individual antigen-containing fraction with a plurality of antibodies, which have been raised against antigens present in corresponding biological matter of a normal individual or of an individual who is not afflicted with the disease or condition, to form antigen-antibody complexes; (c) separating the antigen-antibody complexes from said antigen-containing fraction;

(d) immunising a non-human animal with said antigen-containing fraction from which said complexes have been separated;

(e) immortalising antibody-producing cells of the animal to produce immortalised cell lines; (f) producing antibodies from individual immortalised cell lines; and

(g) testing said antibodies for specific reactivity with another sample of said biological matter to thereby identify a monoclonal antibody, which specifically detects said disease or condition.

Another aspect of the invention contemplates a method for preparing a monoclonal antibody which specifically detects a disease or condition and which is not inrmuno-interactive with a known antigen associated with said disease or condition, comprising: -

(a) contacting a sample of biological matter obtained from at least one patient afflicted with said disease or condition with a plurality of antibodies, which have been raised against antigens present in corresponding biological matter of a normal individual or of an individual who is not afflicted with the disease or condition, to form antigen- antibody complexes;

(b) separating the antigen-antibody complexes from the sample;

(c) separating the known antigen from the sample; (d) immunising a non-human animal with the sample from which the antigen- antibody complexes and the known antigen have been separated; (e) immortalising antibody-producing cells of the animal to produce immortalised cell lines;

(f) producing antibodies from individual immortalised cell lines; and

(g) testing said antibodies for specific reactivity with another sample of said biological matter obtained from said at least one patient to thereby identify a monoclonal antibody, which specifically detects said disease or condition, but which is not immuno-interactive with said known antigen.

In yet a further aspect, the invention extends to a method for preparing a monoclonal antibody which detects a first disease or condition, comprising: - (a) contacting a sample of biological matter obtained from at least one patient afflicted with a second disease or condition that is related to said first disease or condition with a plurality of antibodies, which have been raised against antigens present in corresponding biological matter of a normal individual or of an individual who is not afflicted with the first disease or condition or with the second disease or condition, to form antigen-antibody complexes;

(b) separating the antigen-antibody complexes from the sample;

(c) immunising a non-human animal with the sample from which the antigen- antibody complexes have been separated;

(d) immortalising antibody-producing cells of the animal to produce immortalised cell lines;

(e) producing antibodies from individual immortalised cell lines; and

(f) testing said antibodies for specific reactivity with a sample of biological matter obtained from at least one patient afflicted with said first disease or condition to thereby identify a monoclonal antibody, which detects said first disease or condition. In still a further aspect, the invention extends to a method for preparing a monoclonal antibody which specifically detects a disease or condition, comprising: -

(a) immunising a non-human animal with a sample of biological matter obtained from at least one patient afflicted with said disease or condition, from which sample have been removed antigens that are present in corresponding biological matter of a normal individual or of an individual who is not afflicted with the disease or condition by contacting the sample with antibodies, which have been raised against antigens present in the corresponding biological matter, to form antigen-antibody complexes, and separating the antigen-antibody complexes from the sample;

(b) immortalising antibody-producing cells of the animal to produce immortalised cell lines; (c) producing antibodies from individual immortalised cell lines; and

(d) testing said antibodies for specific reactivity with another sample of said biological matter obtained from said at least one patient to thereby identify a monoclonal antibody, which specifically detects said disease or condition.

According to another aspect of the present invention, there is provided a method for preparing a monoclonal antibody which detects a first disease or condition, comprising: -

(a) immunising a non-human animal with a sample of biological matter obtained from at least one patient afflicted with a second disease or condition that is related to said first disease or condition, from which sample have been removed antigens that are present in corresponding biological matter of a normal individual or of an individual who is not afflicted with the first disease or condition or with the second disease or condition by contacting said sample with antibodies, which have been raised against antigens present in the corresponding biological matter, to form antigen-antibody complexes, and separating the antigen-antibody complexes from the sample; (b) immortalising antibody-producing cells of the animal to produce immortalised cell lines;

(c) producing antibodies from individual immortalised cell lines; and

(d) testing said antibodies for specific reactivity with a sample of biological matter obtained from at least one patient afflicted with said first disease or condition to identify a monoclonal antibody, which detects said first disease or condition.

In a further aspect, the invention contemplates an immortalised cell line, preferably a hybridoma cell line that secretes a monoclonal antibody, which specifically detects a disease or condition and which is obtained by a method as broadly described above. In another aspect, the invention provides a monoclonal antibody which specifically detects a disease or condition, and which is produced by the method as broadly described above. In yet another aspect, the invention contemplates a synthetic or recombinant antigen-binding molecule having specificity for the antigenic determinant or epitope recognised by the monoclonal antibody as broadly described above.

In a preferred embodiment, the synthetic or recombinant antigen-binding molecule is a humanised antibody, e.g. a chimeric or complementarity determining region (CDR)-grafted antibody.

In still yet another aspect, the invention provides an isolated antigen characterised by its selective binding to a monoclonal antibody as broadly described above.

The present invention further provides, in another aspect, a method of detecting the presence of an antigen in a biological sample, wherein said antigen is immuno- interactive with a monoclonal antibody as broadly described above, comprising: -

(a) contacting the biological sample with the monoclonal antibody or an analogue thereof ; and

(b) detecting the presence of a complex comprising the antigen and the monoclonal antibody, or analogue, in said contacted sample.

In a preferred embodiment, the monoclonal antibody analogue is a synthetic or recombinant antigen-binding molecule having specificity for the antigenic determinant or epitope recognised by the monoclonal antibody.

In yet another aspect, the invention contemplates a method of detecting the presence in a patient of a disease or condition associated with an antigen that is immuno- interactive with a monoclonal antibody as broadly described above, comprising: -

(c) contacting a biological sample obtained from the patient with the monoclonal antibody or analogue thereof; and

(d) detecting the presence of a complex comprising the antigen and the monoclonal antibody, or analogue, in said contacted sample.

In another aspect of the invention, there is provided a method of diagnosing a disease or condition associated with an aberrant concentration of an antigen in a biological sample of a patient, wherein said antigen is immuno-interactive with a monoclonal antibody as broadly described, comprising: - (a) contacting the biological sample with the monoclonal antibody or analogue thereof; (b) measuring the concentration of a complex comprising the antigen and the monoclonal antibody, or analogue, in said contacted sample; and

(c) relating said measured complex concentration to the concentration of antigen in said sample, wherein the presence of said aberrant concentration is indicative of said disease or condition.

In a further aspect, the invention provides a composition for the treatment or prevention of a disease or condition, said composition comprising a monoclonal antibody as broadly described above or an analogue thereof, which is immuno-interactive with an antigen associated with said disease or condition, together with a pharmaceutically acceptable carrier.

In yet another aspect of the invention, there is provided a method for treating or preventing a condition associated with the presence of an antigen in a biological sample obtained from an animal, comprising administering to said animal an effective amount of a composition as broadly described above. In yet another aspect of the invention, there is provided a method for treating or preventing a condition associated with an aberrant concentration of an antigen in a biological sample obtained from an animal, comprising administering to said animal an effective amount of a composition as broadly described above.

The invention also extends to the use of the monoclonal antibodies as broadly described above, or analogue thereof, in a kit for detecting and/or measuring a target antigen in a biological sample.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a photographic representation showing SDS-PAGE performed with a homogeneous 12.5% polyacrylamide gel. Lanes 2, 4, 6, 8 show samples of cord blood pools (n=4) processed by immunoaffinity chromatography and molecular weight separation. Lanes ,3, 5, 7 and 9 show the corresponding S2 fractions of the cord bloods post immunoaffinity and processed using a membrane with a 100-kDa cut-off. Molecular weight standards are shown in Lane 1.

Figure 2 is a photographic representation showing a Western blot performed with a homogeneous 12.5% polyacrylamide gel, showing reactivity of MAb CB874 to one protein (CB marked) having an apparent molecular weight of about 58 kDa. The reactivity is directed to a single protein in each of the four S2 fractions prepared from the original cord blood pools (Lanes 3, 5, 7, 9). No reactivity was observed to four random maternal sera (post immunoaffinity) run on the same gel (Lanes 2, 4, 6, 8). Standards (faint) are also shown. Figure 3 is a graphical representation showing the results of an indirect 1-site enzyme immunoassay which measures the reactivity of MAb CB874 to 10 random untreated cord bloods (+ve samples) and 10 random blood sera (-ve samples) obtained from normal pregnant women.

DETAILED DESCRIPTION OF THE INVENTION

1. Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

By "affinity" is meant the strength of the interaction between an individual antigen binding site on an antigen-binding molecule and its corresponding site on the antigen.

By "antibody" is meant a protein of the immunoglobulin family that is capable of combining, interacting or otherwise associating with an antigen.

The term "antigen" is used herein in its broadest sense to refer to a substance that is capable of reacting in and/or inducing an immune response. Typically, but not necessarily, antigens are foreign to the host animal in which they produce immune reactions. By "antigen-binding molecule" is meant any molecule that has binding affinity for a target antigen. It will be understood that this term extends to immunoglobulins (e.g., polyclonal or monoclonal antibodies), immunoglobulin fragments and non- immunoglobulin derived protein frameworks that exhibit antigen-binding activity.

By "antigenic determinant" or "epitope" is meant that part of an antigenic molecule against which a particular immune response is directed. Typically, in an animal, antigens present several or even many antigenic determinants simultaneously.

By "associated with a disease or condition" is meant that the target antigen may be linked directly or indirectly with the disease or condition. For instance, the target antigen may be an epitope of an antigen that indirectly causes, contributes to, or is symptomatic or indicative of the disease or condition.

The term "biological matter", "biological sample" and the like as used herein refer to a sample of tissue or fluid that may be extracted, untreated, treated, diluted or concentrated from an animal. The biological matter may be selected from whole blood, serum, plasma, saliva, urine, sweat, ascitic fluid, peritoneal fluid, synovial fluid, amniotic fluid, cerebrospinal fluid, tissue biopsy, and the like.

By "carrier" is meant any substance of typically high molecular weight to which a non- or poorly immunogenic substance (e.g., a hapten) is naturally or artificially linked to enhance its immunogenicity.

As used herein, the term "CDR structural loops" refers to the three light chain and the three heavy chain regions in the variable portion of an antibody that bridge β strands on the binding portion of the molecule. These loops have characteristic canonical structures (Chothia et al, 1987, J Mol. Biol. 196: 901; Chothia et al, 1992, J. Mol Biol 227:799). Throughout this specification, unless the context requires otherwise, the words

"comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

The term "condition" is used herein to describe any condition of an organ or organ part. It may include a healthy condition or an unhealthy condition such as a disorder of the organ or organ part.

By "corresponding biological matter" is meant biological matter obtained from the same or related anatomical, physiological, pathological and/or symptomatic source as a reference biological matter. The term "diagnosis" is used herein in its broadest sense to include detection of an antigen that is immuno-interactive with an antigen-binding molecule produced in accordance with the present invention. Also included within its scope, is the analysis of diseases, disorders or conditions. Accordingly, the term "diagnosis" includes the use of monoclonal antibodies for research purposes as tools to detect and understand mechanisms associated with a disease or condition of interest. The term "effective amount" as used herein is an amount of an antigen-binding molecule such as a monoclonal antibody that will treat or prevent a particular disease or condition. The effective amount may be an amount that can inhibit an action of a target antigen for which the monoclonal antibody is interactive therewith. The effective amount will vary depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

By "framework region" is meant region of an immunoglobulin light or heavy chain variable region, which is interrupted by three hypervariable regions, also called complementarity determining regions (CDRs). The extent of the framework region and

CDRs have been precisely defined (see for example Kabat et al, "Sequences of Proteins of

Immunological Interest", U.S. Department of Health and Human Services, 1983). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. As used herein, a "human framework region" is a framework region that is substantially identical (about 85% or more, usually 90-95% or more) to the framework region of a naturally occurring human immunoglobulin. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs. The CDRs are primarily responsible for binding to an epitope of an antigen.

By "hapten" is meant a substance that can combine specifically with an antibody but that cannot or poorly induces an immune response unless bound to a carrier. A hapten typically comprises a single antigenic determinant or epitope.

As used herein, the term "heavy chain variable region" means a polypeptide which is from about 110 to 125 amino acid residues in length, the amino acid sequence of which corresponds to that of a heavy chain of a monoclonal antibody of the invention, starting from the amino-terminal (N-terminal) amino acid residue of the heavy chain. Likewise, the term "light chain variable region" means a polypeptide which is from about 95 to 130 amino acid residues in length, the amino acid sequence of which corresponds to that of a light chain of a monoclonal antibody of the invention, starting from the N- terminal amino acid residue of the light chain. Full-length immunoglobulin "light chains" (about 25 Kd or 214 amino acids) are encoded by a variable region gene at the NH₂- terminus (about 110 amino acids) and a K or λ constant region gene at the COOH— terminus. Full-length immunoglobulin "heavy chains" (about 50 Kd or 446 amino acids), are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes, e.g., gamma (encoding about 330 amino acids).

The term "immunogenicity" is used herein in its broadest sense to include the property of evoking an immune response within a patient. Immunogenicity typically depends partly upon the size of the substance in question, and partly upon how unlike host molecules it is. It is generally considered that highly conserved proteins tend to have rather low immunogenicity.

The term "immunoglobulin" is used herein to refer to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. The recognised immunoglobulin genes include the K, a, γ (IgG_l5 IgG₂, IgG₃, IgG₄), δ, e and μ constant region genes, as well as the myriad immunoglobulin variable region genes. One form of immunoglobulin constitutes the basic structural unit of an antibody. This form is a tetramer and consists of two identical pairs of immunoglobulin chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions are together responsible for binding to an antigen, and the constant regions are responsible for the antibody effector functions, hi addition to antibodies, immunoglobulins may exist in a variety of other forms including, for example, Fv, Fab, Fab' and (Fab')₂.

Reference herein to "immuno-interactive" includes reference to any interaction, reaction, or other form of association between molecules and in particular where one of the molecules is, or mimics, a component of the immune system.

By "isolated' is meant material that is substantially or essentially free from components that normally accompany it in its native state.

By "obtained from" is meant that a sample of biological matter that is isolated from, or derived from, a particular source of the host.

The term "patient" refers to patients of human or other animal origin and includes any individual it is desired to examine or treat using the antigen-binding molecules and methods of the invention. However, it will be understood that "patient" does not imply that symptoms are present. Suitable animals that fall within the scope of the invention include, but are not restricted to, primates, livestock animals (e.g., sheep, cows, horses, donkeys, pigs), laboratory test animals (e.g., rabbits, mice, rats, guinea pigs, hamsters), companion animals (e.g., cats, dogs) and captive wild animals (e.g., foxes, deer, dingoes), aves (e.g., chicken, geese, duck, emu, ostrich), reptile or fish. By "pharmaceutically acceptable carrier" is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in systemic administration.

The term "recombinant antigen-binding molecule" is used herein to denote an antigen-binding molecule produced using recombinant nucleic acid techniques.

The term "recombinant polynucleotide" as used herein refers to a polynucleotide formed in vitro by the manipulation of nucleic acid into a form not normally found in nature. For example, the recombinant polynucleotide may be in the form of an expression vector. Generally, such expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleotide sequence.

By "recombinant polypeptide" is meant a polypeptide made using recombinant techniques, i. e. , through the expression of a recombinant polynucleotide.

The term "related disease or condition" is used herein to refer to a disease or condition that is related anatomically, physiologically, pathologically and/or symptomatically to a reference disease or condition. For example, diseases or conditions may be related to one another by affecting similar anatomical locations (e.g., affecting the same organ or body part), affecting different organs or body parts with similar physiological function (e.g., the oesophagus, duodenum and colon which rely on peristalsis to move food from one end of the alimentary canal to the other), by having similar or overlapping pathologies (e.g., infection, tissue damage or rupture, apoptosis, necrosis) or by having similar or overlapping symptoms (i.e., allergic response, inflammation, lymphocytosis). Thus, for example, in accordance with the present invention, an antigen associated with ulcerated colitis may also be associated with perforation of the colon because these disease affect the same organ (i.e., colon).

By "target antigen" is meant an antigen that is associated with a disease or condition for which treatment or diagnosis is sought. The term "treating" is used herein in its broadest sense to include both therapeutic and prophylactic (i.e., preventative) treatment designed to ameliorate the disease or condition.

By "vector" is meant a nucleic acid molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage, or plant virus, into which a nucleic acid sequence may be inserted or cloned. A vector preferably contains one or more unique restriction sites and may be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible. Accordingly, the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. A vector system may comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants. Examples of such resistance genes are well known to those of skill in the art.

2. Method of producing monoclonal antibodies that specifically detect a disease or condition The present invention is directed, in one aspect, to a method of producing antibodies that specifically recognise a disease or condition of interest. The invention permits the manufacture of monoclonal antibodies and other antigen-binding molecules with desired specificities, including monoclonal antibodies that have eluded production using established protocols despite aggressive efforts in many laboratories. In this regard, the present invention is predicated in part on the determination that a purified target antigen which has been established to be indicative of, or to underlie, a particular disease or condition is not necessary to produce a monoclonal antibody that specifically detects that disease or condition. Instead, it has been found quite unexpectedly that such a monoclonal antibody can be obtained efficiently by separating a plurality of antigens, which are not associated with the disease or condition under test, from a sample of biological matter obtained from at least one patient afflicted with that disease or condition. The separation is effected by contacting the sample of biological matter with a plurality of antibodies, which have been raised against antigens present in corresponding biological matter of a normal individual or of an individual who is not afflicted with the disease or condition, to form antigen-antibody complexes and then separating the antigen-antibody complexes from the sample. The resulting sample is enriched for antigens which are not present in a normal individual or in an individual who is not afflicted with the disease or condition under test and which may, therefore, associate with that disease or condition. A non-human animal is then immunised with the sample and immortalised antibody- producing cells are derived from the immunised animal. Antibodies produced from these cells are screened for specific reactivity with another sample of the same biological matter from the patient(s) to identify one or more monoclonal antibodies, which are immuno- interactive with a disease or condition-associating target antigen and which are, therefore, capable of detecting said disease or condition.

In one embodiment, the plurality of antibodies is prepared by isolating the corresponding biological matter from a normal individual or from an individual who is not afflicted with the disease or condition. Preferably, the corresponding biological matter is isolated from at least five, more preferably from at least eight and even more preferably from at least ten such individuals and pooling the biological matter isolated therefrom. Generally, the larger the number of normal individuals or individuals who are not afflicted with the disease or condition from whom the biological matter is obtained the greater the probability will be that the pool of biological matter will contain 'normal' antigens which do not associate with the particular disease or condition of interest and which are common between individuals. In accordance with the present invention, such common antigens are advantageously removed from the sample of biological matter obtained from the patient(s) afflicted with said disease or condition to permit enrichment for the disease- or condition- associating antigens. In a preferred embodiment, substantially equivalent amounts of biological matter are pooled from each individual to produce the pool of biological matter. A non-human animal can then be immunised with the corresponding biological matter to obtain polyclonal antibodies or monoclonal antibodies according to standard protocols known to persons of skill in the art. Exemplary protocols for producing polyclonal antibodies are described for example in Coligan et al, "Current Protocols In Immunology", (John Wiley & Sons, Inc, 1991), and Ausubel et al, "Current Protocols in Molecular Biology", (John Wiley & Sons fric, 1994-1998, in particular Section III of Chapter 11). Monoclonal antibodies may be produced using the standard method as described, for example, by Kόhler and Milstein (1975, Nature 256, 495-497), or by more recent modifications thereof as described, for example, in Coligan et al, (1991, supra) by immortalising spleen or other antibody-producing cells derived from the immunised non- human animal. The plurality of antibodies may also be in the form of antibody fragments, including Fv, Fab, Fab' and F(ab')₂ immunoglobulin fragments. Synthetic antigen-binding molecules such as, but not limited to, stabilised Fv fragments are also contemplated for use in separating non-disease or non-condition associated antigens from the sample. The step of contacting the sample of biological matter with the antibodies and the separation of antigen-antibody complexes may be effected using any suitable immunoaffinity based method including, for example, immunochromatography and immunoprecipitation. A preferred method utilises solid phase adsorption in which the antibodies are attached to a suitable resin, the resin is contacted with the sample, and the sample is separated from the resin. Preferred resins include: Sepharose® (Pharmacia), Poros® resins (Roche Molecular Biochemicals, Indianapolis), Actigel Superflow™ resins (Sterogene Bioseparations Inc., Carlsbad Calif), and Dynabeads™ (Dynal Inc., Lake Success, N.Y.).

Suitably, the method further comprises separating from said sample at least one major antigen, which is present in said corresponding biological matter. In a preferred embodiment of this type, the at least one major antigen is separated from the sample before contacting the sample with said plurality of antibodies. The or each major antigen is suitably a major or dominant antigen that dominates an immune response by virtue of its size, abundance and/or immunogenicity relative to other antigens present in the same matter. For example, the major antigen(s) may be selected from albumin, globulins (e.g., immunoglobulins including, but not restricted to, IgG, IgA, IgM and IgE, 3S γ₁ -globulin, ι8₂-microglobulin, GC globulin, α₂-macroglobulin, haptoglogulin), lipoproteins (e.g., NLDL apoprotein A, B and C), transferrin, C-reactive protein, mucoprotein, cή-anti- trypsin, orosomucoid, /?₂-glycoprotein I, 0!₂ HS glycoprotein or hemopexin, or any combination of these. It will be understood by those of skill in the art that the presence or otherwise of such antigens will be dependent on the particular sample of biological matter employed. In an especially preferred embodiment of this type, the at least one major antigen is selected from any one or more of albumin, inrmunoglobulins, haptoglogulin, C- reactive protein and transferrin.

The major or dominant antigen(s) can be separated from the biological matter by any suitable means. For example, the separation may take advantage of any one or more of the antigen's surface charge properties, size, density, biological activity and its affinity for another entity (e.g., another protein or chemical compound to which it binds or otherwise associates). Thus, for example, separation of the antigen from the biological matter may be achieved by ultracentrifugation, ion-exchange chromatography (e.g., anion exchange chromatography, cation exchange chromatography), electrophoresis (e.g., polyacrylamide gel electrophoresis, isoelectric focussing), size separation (e.g., gel filtration, ultrafiltration), affinity-mediated separation (e.g., immunoaffinity separation including, but not limited to, magnetic bead separation such as Dynabead™ separation, immunochromatography, immunoprecipitation) or combination of these. Choice of the separation technique(s) employed may depend on the biological activity or physical properties that may be known for a particular antigen, or group of antigen, to be separated. For example, when the antigen to be separated is immunoglobulin, methods usually used to purify immunoglobulins from serum or plasma such as, for example, precipitation with ammonium sulphate, fractionation with caprylic acid, ion exchange chromatography or by binding and elution from immobilized protein G or protein A may be utilised. Alternatively, when the antigen to be separated is albumin, albumin-binding supports such as Affi-Gel Blue™ (Biorad) may be employed for separation.

Optionally, the method further comprises separating from said sample at least one known antigen associated with said disease or condition. This step is employed advantageously to discover novel antigens that associate with the disease or condition of interest. This separation may take advantage of the known antigen's surface charge properties, size, density, biological activity, its affinity for another entity (e.g., another protein or chemical compound to which it binds or otherwise associates) or combination of these. However, separation is preferably effected using immunoaffinity based separation such as by contacting the sample with an antigen-binding molecule that is irnmuno- interactive with the known antigen to produce a complex comprising the known antigen and the antigen-binding molecule and separating the complex from the sample. Preferably, the separation of the antigen from the biological matter preserves conformational epitopes present on the antigen surface and, thus, suitably avoids techniques that cause denaturation of the antigen. Persons of skill in the art will recognise the importance of maintaining or mimicking as close as possible physiological conditions peculiar to the antigen (e.g., the biological tissue or fluid from which they are obtained) to ensure that the antigenic determinants or active site/s on the antigen, which are exposed to the animal, are structurally identical to that of the native antigen. This would ensure the raising of appropriate antibodies in the immunised animal that would recognise the native antigen. In a preferred embodiment of this type, the antigen is separated from the biological matter using any one or more of affinity separation, gel filtration and ultrafiltration. hi another embodiment, the method further comprises fractionating the biological matter according to one or more physical or functional parameters to provide two or more antigen-containing fractions. The antigens in the sample can be fractionated before contacting a resulting antigen-containing fraction with the plurality of antibodies. However, the antigens are preferably fractionated after separating the antigen-antibody complexes from the sample. The physical or functional parameters are suitably selected from size, density, surface charge, biological activity or combination of these. Accordingly, such fractionation may be performed, for example, using any of the separation techniques mentioned above. In a preferred embodiment of this type, fractionation is carried out using size separation including, but not limited to, ultrafiltration and gel filtration. In another preferred embodiment of this type, the method further includes conjugating carriers to antigens of antigen-containing fractions that have native molecular weights of less than 5,000, preferably less than 10,000, more preferably less than 20,000 and even more preferably less than 30,000 to enhance their immunogenicity. Useful carriers are well known in the art and include for example: keyhole limpet haemocyanin (KLH); thyroglobulin; albumins such as human serum albumin; toxins, toxoids or any mutant cross-reactive material (CRM) of the toxin from tetanus, diphtheria, pertussis, Pseudomonas, E. coli, Staphylococcus, and Streptococcus; polyamino acids such as poly(lysine:glutamic acid); influenza; Rotavirus NP6, Parvovirus NP1 and NP2; hepatitis B virus core protein; hepatitis B virus recombinant vaccine and the like. Alternatively, a fragment or epitope of a carrier protein or other immunogenic protein may be used. For example, the carriers may comprise a T cell epitope of a bacterial toxin, toxoid or CRM as for example described in U.S. Patent No 5,785,973.

In another embodiment, the method comprises isolating biological matter from at least five patients, more preferably from at least eight patients, even more preferably from at least ten patients afflicted with the disease or condition and pooling the biological matters isolated therefrom. Generally, the larger the number of patients afflicted with the disease condition from whom the biological matter is obtained the greater the probability would be that the pool of biological matter would contain one or more common antigens associating with the particular disease or condition of interest. Preferably, roughly equivalent amounts of biological matter are pooled from each patient to produce the pool of biological matter. In a preferred embodiment of this type, equivalent volumes of biological fluid are pooled from each patient to produce the pool of biological matter. Preferably, the volume of the pool for immunisation is at least at least 10 mL, more preferably at least 15 mL and still more preferably at least 20 mL. By way of example, the biological matter may comprise any one or more of whole blood, serum, plasma, saliva, urine, sweat, ascitic fluid, peritoneal fluid, synovial fluid, amniotic fluid, cerebrospinal fluid, and the like.

Immunisation and subsequent production of monoclonal antibodies can be carried out using standard protocols as for example described by Kδhler and Milstein (1975, supra; 1976, Eur. J. Immunol. 6(7): 511-519) or by more recent modifications thereof as described, for example, in Coligan et al. (Current Protocols in Immunology, John Wiley & Sons, Inc, 1991-1997) and in Toyama et al, ("Monoclonal Antibody, Experiment Manual", published by Kodansha Scientific, 1987). Essentially, an animal is immunised with an antigen-containing biological fluid or fraction thereof by standard methods to produce antibody-producing cells, particularly antibody-producing somatic cells (e.g., B lymphocytes). These cells can then be removed from the immunised animal for immortalisation. Immortalisation of antibody-producing cells may be carried out using methods, which are well-known in the art. For example, the immortalisation may be achieved by the transformation method using Epstein-Barr virus (EBN) (Kozbor et al, 1986, Methods in

Enzymology 121: 140). In a preferred embodiment, antibody-producing cells are immortalised using the cell fusion method (described by Coligan et al. supra), which is widely employed for the production of monoclonal antibodies. In this method, somatic antibody-producing cells with the potential to produce antibodies, particularly B cells, are fused with a myeloma cell line. These somatic cells may be derived from the lymph nodes, spleens and peripheral blood of primed animals, preferably rodent animals such as mice and rats, hi the exemplary embodiment of this invention mice spleen cells are used. It would be possible, however, to use rat, rabbit, sheep or goat cells, or cells from other animal species instead.

Specialised myeloma cell lines have been developed from lymphocytic tumours for use in hybridoma-producing fusion procedures (Kδhler and Milstein, 1976, supra; Shuhnan et al, 1978, Nature 276: 269-270; Nolk et al, 1982, J Virol. 42(1): 220-227). These cell lines have been developed for at least three reasons. The first is to facilitate the selection of fused hybridomas from unfused and similarly indefinitely self-propagating myeloma cells. Usually, this is accomplished by using myelomas with enzyme deficiencies that render them incapable of growing in certain selective media that support the growth of hybridomas. The second reason arises from the inherent ability of lymphocytic tumour cells to produce their own antibodies. To eliminate the production of tumour cell antibodies by the hybridomas, myeloma cell lines incapable of producing endogenous light or heavy immunoglobulin chains are used. A third reason for selection of these cell lines is for their suitability and efficiency for fusion. Many myeloma cell lines may be used for the production of fused cell hybrids, including, e.g., P3X63-Ag8, P3X63-AG8.653, P3/ΝSl-Ag4-l (NS-1), Sp2/0-Agl4 and S194/5.XXO.Bu.l. The P3X63-Ag8 and NS-1 cell lines have been described by Kohler and Milstein (1976, supra). Shuhnan et al. (1978, supra) developed the Sp2/0-Agl4 myeloma line. The S194/5.XXO.Bu.l line was reported by Trowbridge (1978, J Exp. Med. 148(1): 313-323).

Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually involve mixing somatic cells with myeloma cells in a 10:1 proportion (although the proportion may vary from about 20:1 to about 1:1), respectively, in the presence of an agent or agents (chemical, viral or electrical) that promotes the fusion of cell membranes. Fusion methods have been described by Kδbler and Milstein, supra, Gefter et al. (1977, Somatic Cell Genet. 3: 231-236), and Nolk et al (1982, supra). The fusion-promoting agents used by those investigators were Sendai virus and polyethylene glycol (PEG).

Because fusion procedures produce viable hybrids at very low frequency (e.g., when spleens are used as a source of somatic cells, only one hybrid is obtained for roughly every lxlO⁵ spleen cells), it is preferable to have a means of selecting the fused cell hybrids from the remaining unfused cells, particularly the unfused myeloma cells. A means of detecting the desired antibody-producing hybridomas among other resulting fused cell hybrids is also necessary. Generally, the selection of fused cell hybrids is accomplished by culturing the cells in media that support the growth of hybridomas but prevent the growth of the unfused myeloma cells, which normally would go on dividing indefinitely. The- somatic cells used in the fusion do not maintain long-term viability in in vitro culture and hence do not pose a problem. In the example of the present invention, myeloma cells lacking hypoxanthine phosphoribosyl transferase (HPRT-negative) were used. Selection against these cells is made in hypoxanthine/aminopterin/thymidine (HAT) medium, a medium in which the fused cell hybrids survive due to the HPRT-positive genotype of the spleen cells. The use of myeloma cells with different genetic deficiencies (drug sensitivities, etc.) that can be selected against in media supporting the growth of genotypically competent hybrids is also possible.

Several weeks are required to selectively culture the fused cell hybrids. Early in this time period, it is necessary to identify those hybrids which produce the desired antibody, so that they may subsequently be cloned and propagated. Generally, around 10% of the hybrids obtained produce the desired antibody, although a range of from about 1 to about 30%) is not uncommon. The detection of antibody-producing hybrids can be achieved by any one of several standard assay methods, including enzyme-linked immunoassay and radioimmunoassay techniques as for example described in Monoclonal Antibodies and Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (ed), pp. 376-384, Plenum Press, New York, 1980. Once the desired fused cell hybrids have been selected and cloned into individual antibody-producing cell lines, each cell line may be propagated in either of two standard ways. A suspension of the hybridoma cells can be injected into a histocompatible animal.

The injected animal will then develop tumours that secrete the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can be tapped to provide monoclonal antibodies in high concentration.

Alternatively, the individual cell lines may be propagated in vitro in laboratory culture vessels. The culture medium containing high concentrations of a single specific monoclonal antibody can be harvested by decantation, filtration or centrifugation, and subsequently purified.

The cell lines are tested for their specificity to detect the disease or condition of interest by any suitable immunodetection means as for example described in Section 4 infra. For example, cell lines can be aliquoted into a number of wells and incubated and the supernatant from each well is analysed by enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibody technique, or the like. The cell line(s) producing a monoclonal antibody capable of recognising the disease or condition and which is immuno-interactive specifically with biological fluids from patients with that disease or condition but which does not recognise biological fluids from normal patients or from individuals who are not afflicted with that disease or condition, are isolated , and then, directly cultured in vitro or injected into a histocompatible animal to form tumours and to produce, collect and purify the required antibodies.

Thus, the present invention further provides monoclonal antibodies which specifically detect a disease or condition and which are produced by the method as broadly described above as well as hybridomas from which such monoclonal antibodies can be produced.

3. Monoclonal antibody analogues

The invention also contemplates the use and generation of fragments of monoclonal antibodies produced by the method of the invention including, for example, Fv, Fab, Fab' and F(ab')₂ fragments. Such fragments may be prepared by standard methods as for example described by Coligan et al (supra). The present invention also contemplates synthetic or recombinant antigen-binding molecules with the same or similar specificity as the monoclonal antibodies of the invention. Antigen binding molecules of this type may comprise a synthetic stabilised Fv fragment. Exemplary fragments of this type include single chain Fv fragments (sFv, frequently termed scFv) in which a peptide linker is used to bridge the N terminus or C terminus of a V_# domain with the C terminus or N-terminus, respectively, of a V∑ domain. ScFv lack all constant parts of whole antibodies and are not able to activate complement. Suitable peptide linkers for joining the Y_H and Nz, domains are those which allow the V# and N domains to fold into a single polypeptide chain having an antigen binding site with a three dimensional structure similar to that of the antigen binding site of a whole antibody from which the Fv fragment is derived. Linkers having the desired properties may be obtained by the method disclosed in U.S. Patent No 4,946,778. However, in some cases a linker is absent. ScFvs may be prepared, for example, in accordance with methods outlined in Krebber et al (Krebber et al. 1997, J. Immunol. Methods; 201(1): 35-55). Alternatively, they may be prepared by methods described in U.S. Patent No 5,091,513, European Patent No 239,400 or the articles by Winter and Milstein (1991, Nature 349:293) and Plϋckthun et al (1996, n. Antibody engineering: A practical approach. 203-252).

Alternatively, the synthetic stabilised Fv fragment comprises a disulphide stabilised Fv (dsFv) in which cysteine residues are introduced into the N_# and Vι domains such that in the fully folded Fv molecule the two residues will form a disulphide bond therebetween. Suitable methods of producing dsFv are described for example by Glockshuber et al (1990, Biochem. 29: 1363-1367) Reiter et al. (1994, J. Biol. Chem. 269: 18327-18331), Reiter et al. (1994, Biochem. 33: 5451-5459), Reiter et al. (1994. Cancer Res. 54: 2714-2718), and Webber et al. (1995, Mol. Immunol 32: 249-258). Also contemplated as synthetic or recombinant antigen-binding molecules are single variable region domains (termed dAbs) as for example disclosed by Ward et al. (1989, Nature 341: 544-546), Hamers-Casterman et al. (1993, Nature. 363: 446-448) and Davies & Riechmann (1994, FEBSLett. 339: 285-290).

Alternatively, the synthetic or recombinant antigen-binding molecule may comprise a "minibody". In this regard, minibodies are small versions of whole antibodies, which encode in a single chain the essential elements of a whole antibody. Suitably, the minibody is comprised of the N_# and Nz, domains of a native antibody fused to the hinge region and CH3 domain of the immunoglobulin molecule as, for example, disclosed in U.S. Patent No 5,837,821.

In an alternate embodiment, the synthetic or recombinant antigen binding molecule may comprise non-immunoglobulin derived, protein frameworks. For example, reference may be made to (Ku & Schultz, 1995, Proc. Natl Acad. Sci. USA, 92: 652-6556) which discloses a four-helix bundle protein cytochrome b562 having two loops randomised to create complementarity determining regions (CDRs), which have been selected for antigen binding.

The synthetic or recombinant antigen-binding molecule may be multivalent (i.e., having more than one antigen binding site). Such multivalent molecules may be specific for one or more antigens. Multivalent molecules of this type may be prepared by dimerisation of two antibody fragments through a cysteinyl-containing peptide as, for example disclosed by (Adams et al, 1993, Cancer Res. 53: 4026-4034; Cumber et al, 1992, J Immunol. 149: 120-126). Alternatively, dimerisation may be facilitated by fusion of the antibody fragments to amphiphilic helices that naturally dimerise (Pack P. Plϋnckthun, 1992, Biochem. 31: 1579-1584), or by use of domains (such as leucine zippers jun and fos) that preferentially heterodimerise (Kostelny et al. 1992, J Immunol. 148: 1547-1553).

In an alternate embodiment, the multivalent molecule may comprise a multivalent single chain antibody (multi-scFv) comprising at least two scFvs linked together by a peptide linker. In this regard, non-covalently or covalently linked scFv dimers termed "diabodies" may be used. Multi-scFvs may be bispecific or greater depending on the number of scFvs employed having different antigen-binding specificities. Multi-scFvs may be prepared for example by methods disclosed in U.S. Patent No. 5,892,020. The invention also encompasses chimeric antibodies having the same or similar specificity as the monoclonal antibodies prepared according to the invention. Chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin variable and constant region genes belonging to different species. Thus, in accordance with the present invention, once a hybridoma producing the desired monoclonal antibody is obtained, techniques can be used to produce interspecific monoclonal antibodies wherein the binding region of one species is combined with a non-binding region of the antibody of another species (Liu et al, 1987, Proc. Natl Acad. Sci. USA 84:3439-3443). For example, the CDRs from a non-human (e.g., a rodent) monoclonal antibody can be grafted onto a human antibody, thereby "humanising" the rodent antibody (EPO Publication No. 0239400; Jones et al, 1986, Nature 321: 522-525; Nerhoeyen et al, 1988, Science 239: 1534-1536; Riechmann et al, 1988, Nature 332: 323-327). More particularly, the CDRs can be grafted onto a human antibody variable region with or without human constant regions. The non-human antibody providing the CDRs is typically referred to as the "donor" and the human antibody providing the framework is typically referred to as the "acceptor". Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, preferably about 95% or more identical. Hence, all parts of a humanised antibody, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. Thus, a "humanised antibody" is an antibody comprising a humanised light chain and a humanised heavy chain immunoglobulin. For example, a humanised antibody would not encompass a typical chimeric antibody as defined above because, for example, the entire variable region of a chimeric antibody may be non-human. A donor antibody is said to be "humanised", by the process of "humanisation", because the resultant humanised antibody is expected to bind to the same antigen as the donor antibody that provides the CDRs. It will be understood that the humanised antibodies may have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. Exemplary conservative substitutions may be made according to TABLE A:

TABLE A

Exemplary methods which may be employed to produce humanised antibodies according to the invention are described for example by Riechmann et al. (supra), Winter (EPO Publication No. 0239400), Chou et al. (U.S. Patent No 6,056,957), Queen et al. (U.S. Patent No 6,180,370) and Morgan et al. (U.S. Patent No. 6,180,377).

Thus, in a preferred embodiment, the invention contemplates a humanised antibody molecule having specificity for the epitope recognised by a monoclonal antibody prepared according to the present invention, wherein at least one of the complementarity determining regions (CDRs) of the variable domain is derived from the said monoclonal antibody and the remaining immunoglobulin-derived parts of the humanised antibody molecule are derived from a human immunoglobulin or an analogue thereof, said humanised antibody molecule.

4. Isolation and characterisation of antigens

The invention also resides in methods for isolating an antigen characterised by its selective immuno-interaction with a monoclonal antibody according to the present invention. Any suitable method which takes advantage of the immuno-interaction between the antigen and the monoclonal antibody is contemplated. In one embodiment, for example, the antigen may be isolated using immunoaffinity techniques as for example described by Coligan et al, (supra, in particular Chapter 9.5) and Ausubel et al. (^'Current Protocols in Molecular Biology", John Wiley & Sons Inc, 1994-1998, in particular Chapter 10.11), by immunoblotting, as for example described by Ausubel et al. (supra, in Chapter 10.8), or by immunoprecipitation, as for example described by Ausubel et al. (supra, in Chapter 10.16). For example, the antigen may be isolated by immunoblotting techniques including, for example, Western blotting in which proteinaceous material present in a biological fluid sample is suitably separated by electrophoretic techniques including sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE), which may be optionally native or denaturing SDS-PAGE. The separated proteins may then be transferred to a solid support sheet such as nitro-cellulose, nylon or other sheets. The sheet is probed with the a monoclonal antibody which specifically detects the disease or condition of interest, or with another antigen-binding molecule having the same or similar specificity as the monoclonal antibody. The region(s) of the sheet which are immuno- interactive with the monoclonal antibody or other antigen-binding molecule are then excised to isolate the corresponding antigen. The antigen so isolated may then be subjected to physical analytical methods including N- and/or C-terminal amino acid sequence analysis to obtain sufficient sequence information, for example, to clone a genetic sequence encoding the antigen using conventional recombinant DNA techniques

In an alternate embodiment, the monoclonal antibody or other antigen-binding molecule may be used for affinity purification, preferably immuno-affinity purification of antigen. For example, the monoclonal antibody or other antigen-binding molecule can be coupled to suitable supports including, but not limited to, CNBr-activated Sepharose 4B

(Pharmacia) Affi-gel (Bio-RAD) or other affinity chromatography supports able to bind proteins. Immobilized supports of this type can then be applied to the fractionation and purification of specific antigen from a corresponding biological fluid by affinity chromatography. After binding of antigen to the immobilized antigen-binding molecule, unbound macromolecular species can be washed away from the solid support with, e.g. buffers containing 1.5 M NaCl. Subsequently the antigen can be eluted from the affinity column with, e.g. low or high pH buffer or buffers containing chaotropic ions, e.g. 0.5- 3.0 M sodium thiocyanate.

The application of the monoclonal antibody or other antigen-binding molecule to affinity chromatography enables sufficient quantities of specific antigens to be rapidly isolated from the biological fluid for biochemical characterisation, amino acid sequencing and cloning studies. Having identified the antigen(s) molecular biology or chemical techniques, e.g. cloning techniques may be used to produce unlimited amounts of this antigen.

In an alternate embodiment, the monoclonal antibody or other antigen-binding molecule may be used to isolate the antigen by probing a cDNA expression library as for example described by Ausubel et al. (supra).

5. Diagnostic supports

The invention also envisions a diagnostic device which utilises the antigen- binding molecules of the invention, or antigens that are immuno-interactive therewith, for diagnosing the presence or absence of a disease or condition. Accordingly, the invention contemplates a diagnostic insoluble support to which the disease or conditionΛspecific antigen binding molecule or antigen is bound.

An antigen-binding molecule of the invention, or an antigens that is immuno- interactive therewith, may be bound to an insoluble support by conventional processes. Procedures for binding of antigen-binding molecules to insoluble supports are described, for example, in U.S. Pat. Nos. 3,551,555, 3,553,310, 4,048,298 and RE-29,474. Binding of antibodies to polystyrene by adsorption has been described, for example, in U.S. Pat. Nos. 3,646^346 and 4,092,408. Binding of protein-containing antigens to a variety of insoluble supports has been described, for example, in U.S. Pat. No. 3,720,760. A variety of materials may be used as the insoluble support, the primary consideration being the binding characteristics of the antigen-binding molecule or the antigen to the surface, the absence of interference with the antigen-binding molecule and antigen conjugating reaction or with other reactions which may be employed to determine the presence and extent of the conjugating reaction. Organic and inorganic polymers, both natural and synthetic, can be used as the insoluble support. Examples of suitable polymers include polyethylene, polypropylene, polybutylene, poly(4-methylbutlyene), butyl rubber, silastic polymers, polyesters, polyamides, cellulose and cellulose derivatives (such as cellulose acetate, nitrocellulose and the like), acrylates, methacrylates, vinyl polymers (such as polyvinyl acetate, polyvinyl chloride, polyvinylidene chloride, polyvinyl fluoride, and the like), acrylates, methacrylates, vinyl polymers (such as polyvinyl acetate, polyvinyl chloride, polyvinylidene chloride, polyvinyl fluoride, and the like), polystyrene and styrene graft copolymers, rayon, nylon, polyvinylbutyrate, polyformaldehyde, etc. Other materials which can be used as the insoluble support can the latexes of the above polymers, silica gel, silicon wafers, glass, paper, insoluble protein, metals, metalloids, metal oxides, magnetic materials, semi-conductive materials, cermets and the like. In addition are included substances which form gels, such as proteins such as gelatins, lipopolysaccharides, silicates, agarose, polyacrylamides or polymers which form several aqueous phases such as dextrans, polyalkylene glycols (alkylene with 2 to 3 carbon atoms) or surfactants, e.g. amphophilic compounds such as phosphohpids, long chain (12-24 carbon atoms) alkyl ammonium salts and the like.

One diagnostic support comprises polystyrene, styrene copolymers, or polyolefins such as polyethylene or polypropylene, and acrylate and methacrylate polymers and copolymers. The disease or condition-specific antigen-binding molecule or antigen can be bound to the insoluble support by adsorption, ionic bonding, van der Waals adsorption, electrostatic bonding, or other non-covalent bonding, or it can be bound to the insoluble support by covalent bonding. A particularly advantageous support for this procedure comprises a microtiter plate having a plurality of wells. The well surface or plastic cup inserts therein can constitute the antigen or antigen-binding molecule support. If the determination will require the use of fluorometric measurements, the microtiter plate or the well inserts are advantageously opaque to light so that excitation light applied to a well does not reach or influence contents of the surrounding wells. Procedures for non-covalent bonding are described, for example, in U.S. Pat. No.

4,528,267. Procedures for covalently bonding antibodies and antigens to insoluble supports are described by Ichiro Chibata (Immobilized Enzymes, Halsted Press: New York, 1978) and A. Cuatrecasa, (1970, J Biol. Chem. 245: 3059). The surface can be coated with a protein and coupled with the antigen-binding molecule or antigen using procedures described, for example, in U.S. Pat. No. 4,210,418 using glutaraldehyde as a coupling agent. In a still further procedure, the well can be coated with a layer having free isocyanate groups such as a polyether isocyanate, and application of the antigen-binding molecule or antigen in aqueous solution thereto effects the requisite bonding. In another procedure, the antibody or antigen can be coupled to a hydroxylated material by means of cyanogen bromide as described, for example, in U.S. Pat. No. 3,720,760.

6. Detection of specific disease or conditions

The present invention also features a method for detecting in a biological sample an antigen present in, and indicative of the presence of, a particular disease or condition which comprises contacting the biological sample with the monoclonal antibody prepared for example according to Section 2, or with an analogue thereof prepared for example according to Section 3, which is immuno-interactive with the antigen and detecting the presence of a conjugate or complex comprising the antigen and the monoclonal antibody, or analogue, in the contacted sample. The invention also extends to a method of diagnosing a disease or condition in a patient, which disease or condition is associated with an aberrant concentration of an antigen that is immuno-interactive with a monoclonal antibody of the invention. The method comprises contacting a biological sample obtained from the patient with a monoclonal antibody or analogue thereof, measuring the concentration of a complex comprising the antigen and the monoclonal antibody, or analogue, in said contacted sample; and relating the measured complex concentration to the concentration of antigen in said sample, wherein the presence of said aberrant concentration is indicative of said disease or condition.

Any suitable technique for determining formation of the complex may be used. For example, an antigen-binding molecule according to the invention, having a reporter molecule associated therewith may be utilised in immunoassays. Such immunoassays include, but are not limited to, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs) and immunochromatographic techniques (ICTs), Western blotting which are well known those of skill in the art. For example, reference may be made to Coligan et al. (supra) which discloses a variety of immunoassays that may be used in accordance with the present invention. Immunoassays may include competitive assays as understood in the art or as for example described infra. It will be understood that the present invention encompasses qualitative and quantitative immunoassays.

Suitable immunoassay techniques are described for example in U.S. Patent Nos.

4,016,043, 4, 424,279 and 4,018,653. These include both single-site and two-site assays of the non-competitive types, as well as the traditional competitive binding assays. These assays also include direct binding of a labelled antigen-binding molecule to a target antigen.

Two site assays are particularly favoured for use in the present invention. A number of variations of these assays exist all of which are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabelled antigen-binding molecule such as an unlabelled antibody is immobilised on a solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen complex, another antigen-binding molecule, suitably a second antibody specific to the antigen, labelled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labelled antibody. Any unreacted material is washed away and the presence of the antigen is determined by observation of a signal produced by the reporter molecule. The results may be either qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of antigen. Variations on the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including minor variations as will be readily apparent. In accordance with the present invention, the sample is one that might contain an antigen including blood, serum, plasma, saliva, urine, sweat, ascitic fluid, peritoneal fluid, synovial fluid, amniotic fluid, cerebrospinal fluid, and the like. In the typical forward assay, a first antibody having specificity for the antigen or antigenic parts thereof is either covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs or microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well known in the art and generally consist of cross-linking, covalently binding or physically adsorbing, the polymer-antibody complex to the solid support, which is then washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient and under suitable conditions to allow binding of any antigen present to the antibody. Following the incubation period, the antigen- antibody complex is washed and dried and incubated with a second antibody specific for a portion of the antigen. The second antibody has generally a reporter molecule associated therewith that is used to indicate the binding of the second antibody to the antigen. The amount of labelled antibody that binds, as determined by the associated reporter molecule, is proportional to the amount of antigen bound to the immobilised first antibody.

An alternative method involves immobilising the antigen in the biological sample and then exposing the immobilised antigen to specific antibody that may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound antigen may be detectable by direct labelling with the antibody. Alternatively, a second labelled antibody, specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.

From the foregoing, it will be appreciated that the reporter molecule associated with the antigen-binding molecule may include the following:-

(a) direct attachment of the reporter molecule to the antigen-binding molecule;

(b) indirect attachment of the reporter molecule to the antigen-binding molecule; i.e., attachment of the reporter molecule to another assay reagent which subsequently binds to the antigen-binding molecule; and (c) attachment to a subsequent reaction product of the antigen-binding molecule. The reporter molecule may be selected from a group including a chromogen, a catalyst, an enzyme, a fluorochrome, a chemiluminescent molecule, a paramagnetic ion, a lanthanide ion such as Europium (Eu³⁴), a radioisotope and a direct visual label.

In the case of a direct visual label, use may be made of a colloidal metallic or non- metallic particle, a dye particle, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like.

A large number of enzymes suitable for use as reporter molecules is disclosed in United States Patent Specifications U.S. 4,366,241, U.S. 4,843,000, and U.S. 4,849,338. Suitable enzymes useful in the present invention include alkaline phosphatase, horseradish peroxidase, luciferase, β-galactosidase, glucose oxidase, lysozyme, malate dehydrogenase and the like. The enzymes may be used alone or in combination with a second enzyme that is in solution.

Suitable fluorochromes include, but are not limited to, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), R-Phycoerythrin (RPE), and Texas Red. Other exemplary fluorochromes include those discussed by Dower et al. (International Publication WO 93/06121). Reference also may be made to the fluorochromes described in U.S. Patents 5,573,909 (Singer et al), 5,326,692 (Brinkley et al). Alternatively, reference may be made to the fluorochromes described in U.S. Patent Nos. 5,227,487, 5,274,113, 5,405,975, 5,433,896, 5,442,045, 5,451,663, 5,453,517, 5,459,276, 5,516,864, 5,648,270 and 5,723,218.

In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognised, however, a wide variety of different conjugation techniques exist which are readily available to the skilled artisan. The substrates to be used with the specific enzymes are generally chosen for the production of, upon hydrolysis by the corresponding enzyme, a detectable colour change. Examples of suitable enzymes include those described supra. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labelled antibody is added to the first antibody-antigen complex, allowed to bind, and then the excess reagent washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of antigen which was present in the sample.

Alternately, fluorescent compounds, such as fluorescein, rhodamine and the lanthanide, europium (EU), may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope. The fluorescent-labelled antibody is allowed to bind to the first antibody-antigen complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to light of an appropriate wavelength. The fluorescence observed indicates the presence of the antigen of interest. Immunofluorometric assays (IFMA) are well established in the art and are particularly useful for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules may also be employed.

7. Compositions

The invention also extends to a composition for treating or preventing a disease or condition associated with an antigen, comprising a monoclonal antibody that is immuno- interactive with the antigen, which is prepared, for example, according to Section 2 or with an analogue thereof that is suitably prepared according to Section 3 ^therapeutic agents"), together with a pharmaceutically acceptable carrier.

The invention also encompasses a composition for use in eliciting an immune response in a mammal which response includes production of elements that specifically bind an antigen associated with a particular disease or condition, comprising an antigen that is immuno-interactive with a monoclonal antibody as broadly described above ("immunogenic agents"), together with a pharmaceutically acceptable carrier. Optionally, said composition further comprises an adjuvant.

Depending on the specific conditions being treated, therapeutic agents may be formulated and administered systemically or locally. Techniques for formulation and administration may be found in "Remington's Pharmaceutical Sciences," Mack Publishing

Co., Easton, Pa., latest edition. Suitable routes may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. For injection, the therapeutic agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. Intra-muscular and subcutaneous injection is appropriate, for example, for administration of immunogenic compositions, vaccines and DNA vaccines. The agents can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the compounds of the invention to be formulated in dosage forms such as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. These carriers may be selected from sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulphate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and pyrogen-free water.

Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. The dose of agent administered to a patient should be sufficient to effect a beneficial response in the patient over time such as a reduction in the symptoms associated with the disease or condition. Alternatively, the dose of immunogenic agent administered to a mammal should be sufficient to elicit an immune response that includes the production of elements that specifically bind to the antigen recognised by the monoclonal antibody of the invention. The quantity of the agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof. In this regard, precise amounts of the agent(s) for administration will depend on the judgement of the practitioner. In determining the effective amount of the therapeutic agent to be administered in the treatment or prophylaxis of the disease or condition, the physician may evaluate tissue or circulating fluid levels of an antigen that is immuno-interactive with a monoclonal antibody of the invention, and progression of the disease or condition or the production of antibodies that are immuno-interactive with the antigen associated with the disease or condition. In any event, those of skill in the art may readily determine suitable dosages of the therapeutic agents of the invention.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipopbilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilisers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as., for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PNP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more therapeutic agents as described above with the carrier which constitutes one or more necessary ingredients. In general, the pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilising processes.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterise different combinations of active compound doses.

Pharmaceutical which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticiser, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and or lubricants such as talc or magnesium stearate and, optionally, stabilisers, hi soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilisers may be added. Dosage forms of the therapeutic agents of the invention may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release of an agent of the invention may be effected by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose, hi addition, controlled release may be effected by using other polymer matrices, liposomes and/or microspheres.

Therapeutic agents of the invention may be provided as salts with pharmaceutically compatible counterions. Pharmaceutically compatible salts may be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms.

For any agent used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture (e.g., the concentration of a test agent, which achieves a half-maximal inhibition in the activity of the corresponding antigen). Such information can be used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of such therapeutic agents can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit large therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilised. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See for example Fingl et al, 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 pi).

Dosage amount and interval may be adjusted individually to provide plasma levels of the active agent which are sufficient to maintain a reduction in antigen levels or a reduction or inhibition of antigen activity or effects. Usual patient dosages for systemic administration range from 1-2000 mg/day, commonly from 1-250 mg/day, and typically from 10-150 mg/day. Stated in terms of patient body weight, usual dosages range from 0.02-25 mg/kg/day, commonly from 0.02-3 mg/kg/day, typically from 0.2-1.5 mg/kg/day.

Alternately, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a tissue often in a depot or sustained release formulation. Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with tissue-specific antibody. The liposomes will be targeted to and taken up selectively by the tissue.

In any event, those of skill in the art may readily determine suitable dosages of the immunogenic and therapeutic agents of the invention. Such dosages maybe in the order of nanograms to milligrams of the immunogenic agents of the invention.

An immunogenic agent according to the invention can be mixed, conjugated or fused with other antigens, including B or T cell epitopes of other antigens. In addition, it can be conjugated to a carrier as described below.

When an haptenic peptide is used (i.e., a peptide which reacts with cognate antibodies, but cannot itself elicit an immune response), it can be conjugated with an immunogenic carrier. Useful carriers are well known in the art and include for example those mentioned in Section 2.

The immunogenic compositions may include an adjuvant as is well known in the art. Suitable adjuvants include, but are not limited to: surface active substances such as hexadecylamine, octadecylamine, octadecyl amino acid esters, lysolecithin, dimethyldioctadecylammonium bromide, N, N-dicoctadecyl-N', N'bis(2-hydroxyethyl- propanediamine), methoxyhexadecylglycerol, and pluronic polyols; polyamines such as pyran, dextransulfate, poly IC carbopol; peptides such as muramyl dipeptide and derivatives, dimethylglycine, tuftsin; oil emulsions; and mineral gels such as aluminum phosphate, aluminum hydroxide or alum; lymphokines, and QuilA.

8. Detection kits

The present invention also provides kits for the detection of an antigen present in, and indicative of the presence of, a particular disease or condition. These will contain one or more of the monoclonal antibody, analogue thereof or antigen that is immuno- interactive therewith described above depending upon the nature of the test method employed. The kits may also optionally include appropriate reagents for detection of labels, positive and negative controls, washing solutions, dilution buffers and the like.

In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non- limiting examples.

EXAMPLES

EXAMPLE 1

Antigen Preparation

Typically, a pool of biological fluid is prepared from equal volumes of sample fluid (blood plasma, blood serum, urine, amniotic fluid, cerebrospinal fluid etc.) obtained from different patients afflicted with a disease or condition of interest. The entire pool is pretreated with Affi-Gel Blue Gel (AGB, Biorad) to remove as much of the endogenous albumin as possible. The AGB is prepared by washing with PBS in a 50-nιL conical bottom culture tube and centrifuged at 2000 rpm for 10 min. The supernatant is removed and the same volume of fresh buffer is added to the gel, mixed (vortexed), and again centrifuged as above. This procedure is repeated 5 times. AGB is added to the antigen pool in a 1:1 [v/v] ratio and mixed end-over-end for four hours. The solution is centrifuged at 2000 rpm for 20 min and the supernatant collected into a fresh 50 mL sterile culture tube. Protein G-Agarose (or Sepharose 4B): 100 μL (also pre-washed in PBS with five PBS buffer changes) is then added to the pool and allowed to incubate for 4-6 hrs at R.T. then 12 hrs at 4° C. After centrifugation at 2000 rpm for 30 min, the supernatant is collected (50 mL sterile tube) for further processing. It is preferred that the foregoing procedure is completed within two (2) days.

The next stage in the preparation of the antigen involves the use of an immunoaffinity chromatography column to which is coupled a population of antibodies that recognise 'normal antigens'. These antibodies are conveniently prepared by immunising mice with a corresponding pool of biological fluid obtained from normal patients or from patient who are not afflicted with the specified disease or condition, using the protocol outlined in Example 4. The immunochromatography column can be prepared according to the general protocol described in Example 2.

The partially processed antigen pool, which is suspected to contain disease- or condition-associated antigen, is then loaded onto the column equilibrated with 0.05 M Tris glycine, 0.05M NaCl buffer pH 7.0 and the elution profile of the protein monitored at

254 nm. The unbound fractions (1 mL) are collected; and once the base line is re- established, the bound fraction is eluted with 0.1 M sodium acetate pH 3.5 and 2 mL fractions collected. Once the baseline is re-established again, the column is equilibrated in running buffer and the next pool is chromatographed as before. The fractions are examined using homogeneous (10-15%) or gradient (4-20%) SDS-PAGE and protein levels determined using a suitable protein assay (BioRad). Protein fractions are pooled and processed in accordance with the protocol described below.

The next stage in the preparation of the antigen involves molecular weight (MW) separation using centrifugal microconcentrators with two distinct MW cutoffs. The protocol is as follows:

• Microconcentrator (Filtron™, Amicon) devices with a molecular weight cutoff of (a) 300 kDa and (b) 100 kDa are rinsed with 70% ethanol and washed with

PBS by centrifugation at 2500 rpm for 10 min.

• 3.5 mL of unbound antigen fraction resulting from the immunochromatography step is added to the 300 kDa cutoff microconcentrator and centrifuged for 90 min at 2500 rpm. The procedure is repeated until one-half of the filtrate (1.7- 1.8) mL has passed through into the filtrate receiver. PBS is added to the sample reservoir (containing the original pool), mixed, and centrifuged again for 90 min at 2500 rpm. The microconcentrator is again centrifuged for 45 min at 2500 rpm and the supernatant containing the protein fraction with a MW of > 300 kDa is transferred to a sterile 12 mL conical-bottomed tube (designated supernatant "SI").

• The filtrate from the first centrifugation stage is then transferred to the second microconcentrator with a 100 kDa cutoff membrane. The microconcentrator is centrifuged for 90 min at 2500 rpm, PBS is added to the sample reservoir (made up to the starting volume of 3.5mL) and again centrifuged for 120 min at 2500 rpm.

• Both the supernatant ("S2") and filtrate ("FI") are collected following centrifugation with a 100 kDa membrane into sterile 12 mL conical-bottomed tubes. • Protein estimations are performed on all three collected fractions (ie SI, S2, FI). 150-200 μg of each fraction is mixed with adjuvant for the immunisation schedule.

The processed antigen is preferably used within 1-2 days of the immunisation schedule. The remainder of the pool is stored suitably at -20° C in aliquots containing sufficient antigen (i.e., 150-200 μg) for subsequent booster injections.

EXAMPLE 2

Immunoaffinity chromatosraphy column preparation

Immunoaffinity chromatography is performed by coupling an antibody pool onto an Affi-gel hydrazide gel matrix as per the manufacturers instructions (BioRad). In brief,

12 mL of an antibody pool (1.7 mg/niL) purified by protein A chromatography is dialysed against three changes of 0.1 M NaHCO₃, 0.15M NaCl/ Acetic acid pH 5.5 (coupling buffer) for 24 hrs at 4° C. Sodium periodate (25 mg/1.2 mL distilled water) is added to the dialysed antibody in a ratio of 1 :9 and mixed end-over-end in the dark at room temperature (R.T.) for 4 hrs and again dialysed overnight in coupling buffer. Following this, the activated antibody is added to the Affi-gel Hydrazide gel (2 mL) which is pre-washed in coupling buffer and the antibody:gel slurry allowed to mix end-over-end at R.T. for a further 12 hrs. The slurry is removed, packed into a glass column (1.5cm x 10cm

[BioRad]) and washed with 20 column volumes of 0.02 M Tris/HCl, 0.5 M NaCl pH 7.4 at a rate of 0.8 mL/min.

EXAMPLE 3

Cell lines, growth media and antibiotics: general considerations

Mouse Cell Lines

The majority of mouse plasmacytoma cell lines have been obtained from the mineral oil induced tumour MOP-21. Early 8-azaguanine resistant derivatives still produced the λ or K chains (P3-X63/Ag8) or K chain (NSl/l.Ag.4.1). Hybridomas produced from either of these lines resulted in mixed specificities of the resultant antibodies, since both the light and heavy chains would assemble in relative proportions. In order to select for the growth of hybrid cells from the mixture of cells containing spleen cells, myeloma cells and myeloma-spleen cell hybrids (hybridomas), myeloma cell lines with drug resistant genetic markers have been established. Usually the 8-azaguanine/6-thioguanine-resistant [loss of hypoxanthine guanine phosphoribosyl transferase (HGPRT)] cell lines are used for this selection. Nariant mouse myeloma cells which lack the HGPRT enzyme cannot utilise exogenous hypoxanthine to synthesise purines via an alternate biosynthetic pathway. Therefore, these cells die when cultivated in the presence of aminopterin which blocks the endogenous synthesis of both purines and pyrimidines. The myeloma-spleen cell hybrids contain the spleen cell's HGPRT enzyme and the myeloma's property of "immortality." These cells only can survive in the presence of aminopterin in the selective hypoxanthine aminopterin thymidine (HAT) medium. The ΝS1 cell line is preferably employed since the hybridomas produced using this line have been very stable.

Media

In general, either RPMI (Roswell Park Memorial Hospital) or DMEM

(Dulbecco's Modified Eagles Medium) are used as the basic medium. There are few differences between these media, DMEM having pyruvate and a higher concentration of bicarbonate. Glutamine is required by cells at a relatively high concentration bit is unstable. For this reason, glutamine is stored as a stock solution at -20° C and is added to the medium immediately prior to use. Pyruvate (1 mM), glycine (up to 100 mM) and 2- mercaptoethanol (50 μM) are optional additives since none of these have been shown to be necessary for hybridoma production.

Antibiotics It is customary to add antibiotics to media to reduce the chances of microbial contamination, though other types need only be added in the event of infections such as those listed below:

1. Penicillin (100 i.u.cm^"3) and Streptomycin (100 μg cm^'3).

2. Gentamycin (50 μg cm^"3). 3. Fungizone [Amphoteracin B] (2.5 μg cm^" - toxic (only if necessary-antifungal)

4. Ciprofloxacin - use against Mycoplasma sp.

Buffering of medium is maintained by a bicarbonate/CO₂ system. Foetal calf serum [FCS](10-25 % v/v) must also be added to the medium. Parent cell lines and established clones may be maintained in 10% FCS medium but 25% FCS is used immediately after the fusion until the cells have been cloned three times and then expanded into 75 cm² flasks. FCS should be heated to 56° C for 30 min in order to destroy heat labile complement components.

Previous workers have claimed that hypoxanthine-aminopterin-thymidine (HAT) should be added 24 hours after the fusion, in order to allow hybridoma cells time to synchronise the cell cycle and express the HGPRT genes. This particular theory has been contested by others. Aminopterin has a very high affinity for dihydrofolate reductase and, therefore, a long maintenance period in HT medium is required before returning to normal media. The present inventor includes HT in the media on the day of the fusion to reduce HT media supplementation. Once the hybridomas have been sub-cloned three times by limited dilution and then expanded into 25 cm² flasks HT is reduced in 25% increments with each sub-culture.

EXAMPLE 4

Immunisation: general protocol Antigenic preparations may be injected at a dose of 150-200 μg of protein mixed with adjuvant per mouse. The adjuvant employed is preferably mineral oil with inactivated Mycobacterium sp. (MDP) i.e., Freund's Complete Adjuvant (FCA). The antigen/FCA is mixed in equal volumes and emulsification is achieved by vortexing the mixture and mixing immediately prior to injection using a 1-mL syringe and 21-G needle. Six- to ten- week old female BALB/c mice are injected subcutaneously/intradermally at 4-5 points with antigen/adjuvant. Twenty eight to thirty- five days after the initial injection, each animal is given an intraperitoneal boost of antigen, at a similar dose, prepared with Freund's Incomplete Adjuvant (FIA). 2-4 weeks after the second inoculation, the animals are bled from the retro-bulbar sinus and the serum assayed for antibody titre (ELISA) using appropriate controls. Serum is obtained from both immunised and non-immunised mice, the latter to be used as a negative control in the original screening procedure. Upon collection, the sera are transferred to Eppendorf™ tubes and allowed to clot for 2 hrs at room temperature and centrifuged at 10000 rpm for 10 min. Sera are removed and stored at 4° C until required for analysis by an enzyme immunoassay described below. The positive controls consist of the original protein fraction used in the immunisation schedule whose preparation is described above. Those animals responding well to the antigen are then boosted with the antigen at a 5x higher dose (in sterile saline), intravenously, four (4) days prior to the fusion.

EXAMPLE 5

Screening Assay for Immune Mouse Sera

In the indirect one-site enzyme immunoassay procedure, SI, S2, FI fractions are thawed at 37° C in a water bath. These fractions are applied neat and diluted in PBS (1/10, 1/50) and 100 μL of each sample apphed to each well of a single column across a 96 well microtitre plate (NUNC Immunoplate: maxisorp) and allowed to incubate for 1.5 hrs at room temperature, (approx. 25° C) for 1 hr. Plates are washed three times with PBS(low salt (LS)) (16 g/L NaCl; 1.15 g/L Na₂HPO₄.2H₂O; 0.2 g/L KH₂PO₄; 0.2 g/L KC1) and blocked with 0.2 mL/well PBS(LS) containing 1.0 % Ovalbumin for 2 hr at room temperature. Plates are washed 3 times with PBS(LS), and 0.1 mL of mouse sera supernatant is added to each well as follows; sera from mice immunised with the different protein fractions and non-immune mice were diluted 1/10, 1/50 and 1/100 with PBS. Fifty microlitres of each dilution added to three wells containing the corresponding fraction used in the immunisation schedule; non-immune sera (blanks) was similarly applied to all fractions and their respective dilutions. For example, fraction SI is applied neat and diluted 1/10 and /50 to wells A1-A3 on the microtitre plate, this process is repeated down the plate for the same sample. Mouse sera obtained from animals immunised with fraction SI are diluted in PBS and apphed as follows: Mouse sera at 1/10 is apphed to wells A1-A3, at a dilution of 1/50 it is apphed to wells B1-B3, at 1/100 it is applied to C1-C3, blank sera (non-immune mouse sera) is apphed to wells D1-D3. This procedure is repeated for every mouse being tested for each subsequent fraction]. NOTE: If there is a limited supply of the negative or non-immune mouse sera, then it should be tested at least one against all three fractions. After incubation at room temperature for 2 hr, plates are washed three times with PBS (LS) buffer containing 0.2 % (v/v) Tween-20 and 50 μL /well of peroxidase conjugated goat anti-mouse IgG diluted in PBS(LS)/Tween was added and incubated for a further hour at room temperature. Following a further four washes, 100 μL per well of ABTS substrate solution (5 mM citric acid; 5 mM tri-sodium citrate; 0.4 mM azinobis [3-ethylbenzthiazoline sulfonic acid] diammonium salt activated with 0.003 % (v/v) hydrogen peroxide) was added and the reaction allowed to proceed for 30 min. The reaction is stopped with 0.05 mL/well of 3.9 % oxalic acid and the absorbance measured in a plate reader (Medical Devices) at 405 nm.

EXAMPLE 6

Fusion Prior to the fusion, the NS-1 cells are growing well in 10% FCS medium. The cells are split to 20-25% confluency two (2) days before the fusion. One day before the fusion, the NS-1 cells are fed with fresh media containing 20% FCS to ensure that all cells are in the log phase of growth when harvested for fusion. Typically, NS-1 cells which had been split three or more days prior to fusion had produced considerably lower numbers of hybridomas.

One or more mice are sacrificed by CO₂ asphyxiation and the spleen is removed and placed into 50 mL sterile culture tube containing Hank's Balanced Salt Solution (without calcium & magnesium) [HBSS] prior to transporting the spleens to the laboratory and laminar flow cabinet. Under sterile conditions the spleens are removed from the HBSS media and placed in a 60-mm petri dish containing 5 mL RPMI culture medium. The spleen is perfused with medium by injecting it with a 21-guage needle at five sites, thereby forcing medium into the spleen to release the cells. The cells are transferred to a sterile 50-rnL conical-bottom culture tube, centrifuged (1200 rpm for 10 min) and the supernatant removed. The cells are resuspended in 10-mL culture medium and counted on a Coulter Counter [Coulter]. NS1 cells grown in RPMI/ 20 % FCS are similarly centrifuged and resuspended in 10-mL RPMI and counted.

A 50 % polyethylene glycol (PEG) blend is prepared by mixing equal volumes of PEG 1500 [Boehringer-Mannheim] and PEG 4000 [BDH] prepared in DMEM. The PEG mixture is dissolved at 50° C, adjusted to pH 7.8-8.0 with 5 M NaOH and filtered through a 0.22 μm filter. NOTE: This solution should be used within seven days of the fusion protocol. This solution is kept at 37° C on the day of the fusion protocol.

Splenic lymphocytes are mixed with NS1 myeloma cells at a ratio of 8:1. The cell suspension containing the splenocytes and NS-1 cells is mixed and four equal volumes of the cell suspension distributed between four sterile 50-mL conical-bottom culture tube. These tubes are centrifuged for 10 min at 1200 rpm one at a time just prior to their use in the fusion protocol and the supernatant is removed. The culture tube is gently tapped to break the pellet ensuring dispersion of the cells and minimisation of cell distribution throughout the tube. The cells are now ready for the fusion protocol outlined below.

Fusion Protocol (stepwise method)

• Add 1.2 mL of the PEG (50%) solution drop-wise using a sterile graduated 2 mL pipette over a 1 min period while continuously mixing the tube by hand.

• Continue stirring for 2 min.

• Allow tube to stand for a further 2 min. • Add 0.5 mL warm (37° C) RPMI/HT dropwise to the cells over a 1 min period while gently mixing the tube by hand.

• Add a further 0.5 mL RPMI/HT dropwise over a 1-min period while gently mixing.

• Add further 1 mL RPMI/HT dropwise over a 1-min period while continuously stirring.

• Add further 1 mL RPMI/HT dropwise over a 1-min period while continuously stirring.

• Add 5 mL RPMI/HT dropwise over a 2-min period while continuously stirring..

• Add 5 mL RPMJ7HT dropwise over a 2-min period while continuously stirring. • Add 10 mL RPMI/HT dropwise over a 5-min period while continuously stirring.

• Add 10 mL RPMI/HT dropwise over a 5-min period while continuously stirring.

• Add final 10 mL RPMI/HT media over a 5-min period while continuously stirring. • Centrifuge me tube at 1200 rpm for 10 min.

• Remove supernatant and add 40 mL RPMI/HT media containing 25% FCS; at this stage the cells are transferred to a 75 cm² culture flask and placed into a 37° C humidified 5% CO environment for four hours (minimum 4 hrs, can be left for 8 hrs with no adverse effect).

• Transfer cells to a sterile 210 mL culture vessel containing 480 mL RPMI containing 1% HAT/antibiotics and plated out into 24 x 1 mL tissue culture plates at 0.5 mL per well. To each well also add 0.5 mL spleen feeder cells (see below). Hybrids were grown in a selection medium containing HAT (10 mM hypoxanthine; 0.04 mM aminopterin; 1.6 mM thymidine), 2 x 10⁵ feeder cells/ mL, 25 % FCS, 100 U/mL penicillin, 100 U/mL streptomycin, 0.25 μg/mL Fungizone in RPMI.

• Plates are placed into a 37° C humidified 5% CO₂ environment and left for seven days without disturbing the cultures or opening the incubator doors during the first week.

• NOTE: It is essential to plate the plasmacytoma cell line (NS-1) in spare wells, with the HAT medium, in order to ensure that selection has occurred.

EXAMPLE 7

Feeding The Cultures One week following the fusion protocol, 1 mL fresh RPMI media containing HAT

(10 mM hypoxanthine; 0.04 mM aminopterin; 1.6 mM thymidine), 25 % FCS, 0.25 μg/mL Fungizone, 100 U/mL penicillin and 100 U/mL streptomycin is added to each well and the culture plates returned to the incubator (37° C humidified 5% CO₂ environment) environment for a further seven days. After two weeks of growth in HAT selective media, 1 mL of the supernatant from each culture well is removed and replenished with fresh media but containing HT (10 mM hypoxanthine, 1.6 mM thymidine).

During the third and forth weeks post-fusion large numbers of colonies are evident in the culture wells; in most instances there are large numbers of colonies evident in the same well. At this stage, it is important to have the feeder cells prepared at a density of 2xl0⁵ /mL of media (RPMI media containing HT (10 mM hypoxanthine, 1.6 mM thymidine, 25 % FCS, 100 U/mL penicillin, 100 U/mL streptomycin, 0.25 μg/mL Fungizone) and distributed into new culture plates at 1 mL per well. Using a 5-50 μL pipette (set to 20 μL), individual colonies are removed from the original culture wells. Cells from these colomes are collected from the centre of the colony in the first instance and transferred to a well in a new culture plate containing feeder cells. For larger colonies where multiple cell removals are required, the cells removed are placed into new culture wells preferably on the same plate. Culture plates with the original hybridoma colomes are maintained and any new colonies appearing 4-6 weeks post fusion are transferred to new 24-well plates with feeder cells.

All colonies are allowed to grow from the day of initial transfer for a period of 14 days in which time the colonies are supplemented with fresh media (RPMI media containing HT (10 mM hypoxanthine,1.6 mM thymidine), 25 % FCS, 100 U/mL penicillin, 100 U/mL streptomycin, 0.25 μg/mL Fungizone) on day 4 (no removal of media), day 8 (1 mL removed and fresh media to a similar volume added to each well), and day 14 (1 mL removed for first screening and replenished with fresh media at 1 mL per well).

EXAMPLE 8

Screening Assay (Indirect 1-Site Enzyme Immunoassay) In the indirect one-site enzyme immunoassay procedure, 100 μL of both positive and negative control pools (tissue homogenates, blood or other fluids obtained from patients (n=6- 10) clinically known to be positive and negative for the particular disease/condition are pre- treated with Affi-Gel blue, two separate pools for each control are prepared) are apphed in columns to a 96-well microtitre plates [NUNC nmunoplate: maxisorp] and incubated at room temperature (R.T. (approx. 25° C)) for 1 hr. Plates are washed three times with PBS(low salt (LS)) (16 g/L NaCl; 1.15 g/L Na₂HPO₄.2H₂O; 0.2 g/L KH₂PO₄; 0.2 g/L KC1) and blocked with 0.2 mL/well PBS(LS)/ 1.0 % Ovalbumin for 2 hr at room temperature. Plates are washed 3 times with PBS(LS), and 0.15 mL of supernatant is added to each well; blanks were similarly prepared but using 0.20 mL/well of culture supernatant prepared from NS-1 cells grown in identical media those clones being screened. After incubation at room temperature for 2 hr, plates are washed three times with PBS (LS) buffer containing 0.2 % (v/v) Tween-20 and 0.05 mL/well of peroxidase conjugated goat anti-mouse IgG diluted in PBS(LS)/Tween was added and incubated for a further hour at room temperature. Following a further four washes, 0.10 mL/well of ABTS substrate solution ( 5 mM citric acid; 5 mM tri- sodium citrate; 0.4 mM azinobis [3-ethylbenzthiazoline sulfonic acid] diammonium salt activated with 0.003 % (v/v) hydrogen peroxide) was added and the reaction allowed to proceed for 30 min. The reaction is stopped with 0.05 mL/well of 3.9 % oxalic acid and the absorbance measured in a plate reader (Medical Devices) at 405 nm.

Supernatants which display an absorbance reading 4-fold greater than the blank and negative controls are further tested 5-7 days using the same assay format but with new pools of both the negative and positive control prepared with different samples. Monoclonal antibodies at this stage that demonstrate specific reactivity to the positive control pools are cloned by limited dilution.

EXAMPLE 9

Cloning By Limited Dilution (Low Density Cloning)

Feeder cells are prepared as described above and plated out at 1 mL/well into 24- well culture plates one day prior to cloning. Hybridomas are cloned once the colony has reached at least 30 % confluency as determined by visual inspection, h practice, aspiration of 25 μL of cells will provide approximately 1000 cells which are to be used for cloning. The cells are suspended in 5 mL RPMI/HT/20% FCS/antibiotics at 25-50 cells per mL and plated out in 50 μL aliquots into new plates containing feeder cells across the culture plate. These cells are incubated for a period of 2-3 weeks until visible colonies appear. The supernatants are again removed and tested by the 1-site EIA using three different pools of both positive and negative controls. Typically, each hybridoma is cloned three times in order to obtain a stable line before freezing primary and secondary stocks of the hybridomas. Furthermore, it has been found that cloning at an average cell concentration of 1.5 cells/well works consistently. The first cloning need not produce a single antibody cell/well and instead may only be necessary to remove the cell from other non-secreting cells also present in the precloning well. Clones demonstrating highest reactivity are again sub-cloned as described above and tested by means of an EIA against three pools of both positive and negative controls. Those demonstrating strong reactivity to the positive controls are expanded into 25-cm² flasks without feeder cells. Cells in the flasks are sub-cultured (1/6) every 3-4 days at which time the concentration of HT in the medium is decreased by a factor of 25%. Cells are examined for growth at each stage to determine whether they are viable and observe the effects of deceased HT concentrations in the media. On the fourth passage, cultures are expanded into 75-cm flasks. Three to four days from this expansion, cells are frozen and stored in liquid nitrogen and the supernatants retained for further evaluation by EIA and western blotting to examine specificity. In those instances where there is considerable growth observed in the cultures particularly at the lower dilutions, these cultures are focused upon for further sub-cloning and expansion to flasks. Once the cultures have demonstrated continual growth in the 75-cm² flasks, the level of FCS is reduced by 25 % with each sub-culture (1/6) to a final 10% (v/v) in media (RPMIføntibiotics/glutamine). It should be noted that the inclusion of glutamine is only required when omitted from the original media source acquired from the manufacturer.

EXAMPLE 10

Feeder (Spleen) Cell Preparation

Typically, BALB/c mice (mice to be of the same genetic strain as the mouse used in the fusion), 8-12 weeks of age are employed for splenocyte preparation. Once the mouse is sacrificed, it is submerged into 70% ethanol for a minimum period of five minutes. Once removed from alcohol, the spleen is removed, washed gently with 70 % ethanol and transferred to a sterile 50 mL conical tube containing RPMI (R.T.) and transferred to the laminar flow hood. The spleen is placed into a 6 cm petri dish containing 5 mL RPMI. A small incision is made with a scalpel at one end of the spleen, and a 5 mL syringe filled with RPMI using a 21-G needle is inserted in the opposite end of the spleen and pressure applied gently to push the splenocytes out of the spleen. Splenocytes are collected into a fresh 50-mL culture tube and fresh media applied to the petri dish. The spleen is teased apart using a scalpel and forceps; the cells are released into fresh RPMI (5 mL) collected and transferred to the original splenocyte pool. This procedure is repeated until such as stage when the RPMI media is clear and no further cells are released from the tissue (visual interpretation). The splenocytes are centrifuged for 10 min at 1200 rpm, fresh RPMI media containing 25% FCS/antibiotics/glutamine/HAT is then added to the cell pellet which is resuspended to provide a cell density of 4xl0⁵/mL. Five hundred microlitres of this cell suspension is added to each well of a 24-well culture plate.

EXAMPLE 11

Purification and characterisation of monoclonal antibody pool

Purification of Monoclonal Antibody Pool

Antibody rich culture media harvested from each cultured hybridoma was pooled; centrifuged at 2500 rpm for 10 min and the aqueous fraction containing the antibody was collected, and stored at -70° C until required. To purify the monoclonal antibody, a 10-mL Protein A agarose (BioRad) column was equilibrated with 0.025 M glycine, 0.5M K₂HPO₄ buffer pH 9.0. 5 L of culture media was applied to the column per each chromatography run and the unbound material washed through with at least 20 column volumes of the same buffer until the base-line was re-established. The immunoglobulin fraction was eluted using 0.1M glycine pH 3.0 and 1.5 mL fractions collected. The purification and recovery of immunoglobulin was monitored by an indirect one-site enzyme immunoassay and by SDS- PAGE.

Recovery of Immunoglobulin

The recovery of immunoglobulin was measured by an indirect one-site ELISA. Aliquots of the immunoglobulin fractions were serially diluted in PBS and 0.1 mL/well was added to a microtitre plate plates [NUNC] and incubated at room temperature for 2 hr. The plate was washed three times with PBS and blocked with the same buffer containing 1 % OA for 2 hr at room temperature. The plate was washed three times with PBS/Tween and 50 μL/well of peroxidase conjugated goat anti-mouse IgG diluted 1:1,000 in PBS(LS)/Tween was added and incubated for a further hour at room temperature. Following a further four washes, 100 μL/well of ABTS substrate solution was added and the reaction allowed to proceed for 30 min. The reaction was stopped with 3.9 % oxalic acid and the absorbance measured at 405 nm. The purification process was monitored by SDS-PAGE under denaturing conditions using 10-12.5% homogeneous polyacrylamide gels which were silver stained upon completion of electrophoresis

Characterisation of Immunoglobulin The immunoglobulin isotype was determined from cell culture supernatant using a commercial mouse antibody isotyping kit [SIGMA].

EXAMPLE 12

Isolation of an anti-foetal-specific protein monoclonal antibody

Antigen preparation Pools from random unmatched cord blood sera were initially processed in order to identify a protein uniquely present in the amniotic fluid specimens. Four antigen pools were prepared from equal volumes of cord blood samples obtained from different patients (n=5 per pool). Each pool (20 mL) was pretreated with Affi-Gel Blue Gel (AGB, Biorad) to remove endogenous albumin and albumin-separated cord blood was subjected to immunochromatography as described in Example 1. The column employed for immunochromatography comprised antibodies raised against sera from pregnant subjects (n=10). The pH of each fraction was adjusted using 10 M Tris within the range of 7-7.5, as monitored by pH paper. The fractions were then examined using homogeneous (10-15%) or gradient (4-20%) SDS-PAGE and protein levels determined using a protein assay (BioRad). Unbound protein fractions enriched for foetal-specific antigens were pooled and processed by molecular weight (MW) separation using centrifugal microconcentrators with two distinct MW cutoffs to provide fractions SI, S2 and FI as described in Example 1. Protein estimations were performed on all three collected fractions (i.e. SI, S2, FI); each fraction being composed of a pool obtained from the four individual cord blood pools processed as described above. Fraction SI had very low protein levels and was not used in the immunisation schedule. 150-200 μg of fraction S2 and FI was mixed with adjuvant for the immunisation schedule. The remainder of the pool was stored at -20° C in aliquots containing sufficient antigen (i.e. 150-200 μg) for subsequent booster injections. SDS-PAGE of the four cord blood pools following the various stages of purification are shown in Figure 1. It is clear that the immunoaffinity chromatography and gel permeation procedure had removed a large proportion of contaminating proteins. Cord blood post irnmunoaffinity chromatography shows a number of prominent bands of varying molecular weight. A larger amount of the dominant protein bands are seen in the cord blood fractions following the molecular weight separation using the microconcentrators, particularly in the S2 fraction (Figure 1).

Monoclonal antibody production and screening immunoassay

For primary immunisations of six BALB/c mice, 0.15-0.2 mg of each fraction (S2 or FI) was emulsified in MPL (1:10 v/v [SIGMA]) and injected subcutaneously into each mouse. Booster immunisations using 0.15-0.2 mg of the respective cord blood fraction prepared with MPL (1:10 v/v [SIGMA]) were given by the same routes. The initial boost was given at four weeks post-immunisation with subsequent boosts prescribed at 7-10 day intervals for six weeks. Antibody levels were monitored by collecting eye bleeds four weeks post primary immunisation and then weekly until 10 weeks and testing the sera by an indirect one-site enzyme immunoassay.

In the indirect one-site enzyme immunoassay procedure, 100 μL of each pooled cord blood fraction (0.2 mg/mL) in Tris-HCl buffer pH 7.2 were added to each well of 96- well microtitre plates [NUNC Immunoplate: maxisorp] and incubated at R.T. (approx. 25° C) for 1 hr. Plates were washed three times with PBS(low salt (LS)VTween (16 g/L NaCl [BDH]; 1.15 g/L Na₂HPO₄.2H₂O [BDH]; 0.2 g/L KH₂PO₄ [BDH]; 0.2 g/L KC1 [BDH] containing 0.2% (v/v) Tween-20 [SIGMA]) and blocked with 0.1 mL/well PBS(LS) containing 0.5% ovalbumin for 2 hr at room temperature. Plates were washed 3 times with PBS(LS)/Tween, and 0.10 mL of mouse sera was then added to each well; blanks were similarly prepared but using 0.10 mL/well of sera collected from non- immunised BALB/c mice. After incubation at room temperature for 1 hr, plates were washed three times with wash buffer and 0.05 mL/well of peroxidase conjugated goat anti- mouse IgG [Dako] diluted 1:1,000 in PBS(LS)/Tween was added and incubated for a further hour at R.T. Following a further four washes, 0.10 mL/well of ABTS substrate solution (5 mM citric acid [BDH]; 5 mM tri-sodium citrate [BDH]; 0.4 mM azinobis [3- ethylbenzthiazoline sulfonic acid] diammonium salt [SIGMA] activated with 0.003 % (v/v) hydrogen peroxide) was added and the reaction allowed to proceed for 30 min. The reaction was stopped with 0.05 mL/well of 3.9 % oxalic acid [SIGMA] and the absorbance was measured in a plate reader (Molecular Devices) at 405 nm.

One mouse demonstrated a 12-fold increase over the blank absorbance and was chosen for further treatment and given a final subcutaneous boost of the pooled cord blood S2 fraction. Four days later, the mouse was sacrificed by CO₂ asphyxiation and the spleen was removed under sterile conditions and placed in a 60 mm petri dish in RPMI-1640 culture medium. The spleen was perfused with medium by injecting it with a 26-guage needle at five sites, thereby forcing medium into the spleen to release the cells. The cells were transferred to a sterile centrifuge tube, centrifuged (250 x g for 10 min) and the supernatant removed. The cells were resuspended in 10 mL culture medium and counted on the Coulter Counter [Coulter], lxl 0⁷ NS1 cells grown in RPMI-1640/ 10 % FCS were similarly centrifuged and resuspended in 10 mL RPMI-1640 and counted. Splenic lymphocytes were fused with NS1 myeloma cells at a ratio of 8:1 using 50 % PEG 1500/4000 in RPMI-1640. Hybrids were grown in a selection medium containing HAT (10 mM hypoxanthine; 0.04 mM aminopterin; 1.6 mM tliymidine) [GIBCO] 2 x 10⁵ feeder cells/ mL, 20 % FCS [TRACE], 100 U/mL penicillin, 50 U/mL streptomycin and 50 U/mL gentamycin in RPMI-1640 (Goding, 1986). The feeder cells were obtained from non- immunized BALB/c mice. 1023 hybrids were obtained. 46 clones secreting antibodies against components in the S2 fraction were detected by indirect one-site enzyme immunoassay as described above. These hybrids was cloned by limited dilution and further expanded in culture prior to them being cryopreserved. One clone (designated CB874) was further expanded using a 75-cm³ flask and grown in RPMI-1640 supplemented with 20% FCS, 2 mM L-Glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin at 37° C in a humid atmosphere of 5% CO in air for about 40 days with IgG rich media replenished with fresh media every 5-6 days. EXAMPLE 13

Identification and isolation of the foetal protein recognised by monoclonal antibody CB874

SDS-PAGE/Western blot SDS-polyacrylamide gel electrophoresis was performed on 10 and 12.5 % homogeneous polyacrylamide gels. Electrophoresis was performed on the Mini Protean II system [BioRad] for 60 min at 180 N. Western blotting was performed according to the method of Towbin (1984, J Immunol Methods 72(2): 313-340) with minor modifications; i.e. the use of 0.3 M Tris pH 10.4 containing 10% methanol as the anode buffer; 25 mM Tris, 192 mM glycine, pH 9.4 containing 20 % (v/v) methanol as the cathode buffer. Electrophoretic transfer was performed at 120 mA for 90 min using a semi-dry electrophoretic blotting system [Biometra; Fast Blot B33]. Calibration curves for molecular weight estimation were obtained from pre-stained standards [BioRad] similarly transferred to the membrane. Blots were soaked in blocking solution (5% [w/v] milk powder) overnight at R.T. and then washed in 3 x 10 min consecutive washes in TBS (20 mM Tris, 500 mM ΝaCl, pH 7.5). The CB874 MAb was diluted in this same wash buffer (1/100), was added to the blot and left overnight at 4° C. Blots were washed in 4 x 10 min consecutive washes in the same buffer supplemented with 0.02% Tween-20; at which time goat anti-mouse ALP conjugate (BioRad) diluted in the same buffer was added to the blot and allowed to incubate for a further four hours. Blots were rinsed as before and_. incubated with phosphatase substrate (BCIP/ΝBT [ICΝ]) solution for 10-30 min. The reactions were terminated by placing the blot into distilled water.

Figure 2 shows the results of a Western blot analysis following SDS-PAGE electrophoresis in 10 % homogeneous gel in the presence of SDS//3-mercaptoethanol. Only one band of reactivity is seen and it is identical in two of the original cord blood pools; one newly prepared cord and in the purified protein used in the immunisation schedule (lane 2).

This band migrates with an apparent sub-unit molecular weight of 58 + 4 kD (Figure 2 ).

Isoelectric Focusing

Isoelectric focusing was performed on the BioRad mini-IEF system. Gels were prepared from 1% agarose (w/v), 3% sucrose and containing 2% ampholytes of different ranges (BioRad). Samples consisting of cord blood protein pools (S2) and IEF standards [BioRad] were applied to the gel on applicator strips and allowed to adsorb onto the gel for five minutes. The gel was stained for protein by initially fixing for 30 min using 35 % methanol, 13 %> tricarboxylic acid (ICN), 3.5 % sulfosalicylic acid [ICN], then suspended into 95%o ethanol for a further 20 min, and finally drying the gel under hot air. The dried gel was placed into a solution of 30% methanol, 10% acetic acid containing Coomassie blue R-250 (SIGMA[0.2% w/v]) for up to 20 min. The gel was then destained using 30 % methanol, 10 % acetic acid until the background was clear. Using this procedure, only one major band of reactivity against purified protein was evident which corresponds to an isoelectric point of 6.9 +0.1.

Gel Permeation Chromatography

The native molecular weight of the protein was again determined by testing the reactivity of fractions eluted from a pre-calibrated Sephacryl S-300 column using a 1-site EIA (section 2.4). Briefly, a column (2.5 cm x 100 cm) was packed with Sephacryl™ S- 300 and equilibrated at 1.0 mL/min with 50 mM Tris-HCl (pH 7.4) for 12 hr. Following column equilibration (10 column volumes), gel filtration standards [BioRad] were run and a standard curve constructed. Cord blood S2 fractions were spiked with 1 % Dextran Blue- 2000 ([Pharmacia]), added to the column and 2 mL fractions collected. Fractions were screened by a 1-site EIA (infra) and the apparent native molecular weight was interpolated from the standard curve as being 115+ 10 kD. No other fractions were found to show reactivity.

EIA protocol

The one-site ELISA procedure described in Example 8 was used to evaluate the specificity of the monoclonal antibody CB874 to cord blood. Before use, the plates were washed three times in PBS(LS). To perform the assay, samples of cord blood and maternal blood obtained from patients of varying gestational ages (18-40 weeks) were applied (0.1 mL/well) in duplicate and incubated at room temperature for 2 hr. Blanks containing PBS(LS) (0.1 mL/well) instead of sample, were also included. After a further three washes in PBS(LS)/Tween, 50 μL of the purified CB874 MAb diluted in PBS(LS)/Tween or 100 μL of the antibody rich cell culture supernatant was added to each well and incubated at room temperature for 2 hrs. After four washes with PBS(LS)/Tween, 50 μL goat anti- mouse-HRPO conjugate was added to each well and plated incubated for a further one hour. After four washes with PBS(LS)/Tween, 0.1 mL of ABTS solution was allowed to react with the enzyme for up to 2 hr. The reaction was stopped with 3.9 % oxalic acid (50 μL per well). Optical densities of samples (OD sample) were measured at a wavelength of 405 nm.

The results presented in Figure 3 show that there is strong reactivity to all of the cord blood samples tested and no reactivity to any of the maternal sera tested.

The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application

Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appendant claims.

Claims

WHAT IS CLAIMED IS:

1. A method for preparing a monoclonal antibody which specifically detects a disease or condition, comprising: -

(a) contacting a sample of biological matter obtained from at least one patient afflicted with said disease or condition with a plurality of antibodies, which have been raised against antigens present in corresponding biological matter of a normal individual or of an individual who is not afflicted with the disease or condition, to form antigen- antibody complexes;

(b) separating the antigen-antibody complexes from the sample; (c) immunising a non-human animal with the sample from which the antigen- antibody complexes have been separated;

(d) immortalising antibody-producing cells of the animal to produce immortalised cell lines;

(e) producing antibodies from individual immortalised cell lines; and (f) testing said antibodies for specific reactivity with another sample of said biological matter obtained from said at least one patient to thereby identify a monoclonal antibody, which specifically detects said disease or condition.

2. A method for preparing a monoclonal antibody which specifically detects a disease or condition, comprising: - (a) separating at least one major antigen from a sample of biological matter obtained from at least one patient afflicted with said disease or condition;

(b) contacting the sample from which said at least one major antigen has been separated with a plurality of antibodies, which have been raised against antigens present in corresponding biological matter of a normal individual or of an individual who is not afflicted with the disease or condition, to form antigen-antibody complexes;

(c) separating the antigen-antibody complexes from the sample;

(d) immunising a non-human animal with the sample from which the antigen- antibody complexes have been separated;

(e) immortalising antibody-producing cells of the animal to produce immortalised cell lines; (f) producing antibodies from individual immortalised cell lines; and

(g) testing said antibodies for specific reactivity with another sample of said biological matter obtained from said at least one patient to thereby identify a monoclonal antibody, which specifically detects said disease or condition.

3. A method for preparing a monoclonal antibody which specifically detects a disease or condition, comprising: -

(a) contacting a sample of biological matter obtained from at least one patient afflicted with said disease or condition with a plurality of antibodies, which have been raised against antigens present in corresponding biological matter of a normal individual or of an individual who is not afflicted with the disease or condition, to form antigen- antibody complexes;

(b) separating the antigen-antibody complexes from the sample;

(c) fractionating the sample from which the antigen-antibody complexes have been separated according to one or more physical or functional parameters to provide two or more antigen-containing fractions;

(d) immunising a non-human animal with an individual antigen-containing fraction;

(e) immortalising antibody-producing cells of the animal to produce immortalised cell lines;

(f) producing antibodies from individual immortalised cell lines; and (g) testing said antibodies for specific reactivity with another sample of said biological matter obtained from said at least one patient to thereby identify a monoclonal antibody, which specifically detects said disease or condition.

4. A method for preparing a monoclonal antibody which specifically detects a disease or condition, comprising: - (a) fractionating the antigens present in a sample of a biological matter obtained from at least one patient afflicted with said disease or condition according to one or more physical or functional parameters selected from size, density, surface charge, biological activity or combination of these to provide two or more antigen-containing fractions; (b) contacting an individual antigen-containing fraction with a plurality of antibodies, which have been raised against antigens present in corresponding biological matter of a normal individual or of an individual who is not afflicted with the disease or condition, to form antigen-antibody complexes;

(c) separating the antigen-antibody complexes from said antigen-containing fraction;

(d) immunising a non-human animal with said antigen-containing fraction from which said complexes have been separated;

(e) immortalising antibody-producing cells of the animal to produce immortalised cell lines;

(f) producing antibodies from individual immortalised cell lines; and

(g) testing said antibodies for specific reactivity with another sample of said biological matter to thereby identify a monoclonal antibody, which specifically detects said disease or condition.

5. A method for preparing a monoclonal antibody which specifically detects a disease or condition and which is not immuno-interactive with a known antigen associated with said disease or condition, comprising: - (a) contacting a sample of biological matter obtained from at least one patient afflicted with said disease or condition with a plurality of antibodies, which have been raised against antigens present in corresponding biological matter of a normal individual or of an individual who is not afflicted with the disease or condition, to form antigen- antibody complexes; (b) separating the antigen-antibody complexes from the sample;

(c) separating the known antigen from the sample

(d) immunising a non-human animal with the sample from which the antigen- antibody complexes and the known antigen have been separated;

(e) immortalising antibody-producing cells of the animal to produce immortalised cell lines;

(f) producing antibodies from individual immortalised cell lines; and

(g) testing said antibodies for specific reactivity with another sample of said biological matter obtained from said at least one patient to thereby identify a monoclonal antibody, which specifically detects said disease or condition, but which is not immuno-interactive with said known antigen.

6. A method for preparing a monoclonal antibody which detects a first disease or condition, comprising: -

(a) contacting a sample of biological matter obtained from at least one patient afflicted with a second disease or condition that is related to said first disease or condition with a plurality of antibodies, which have been raised against antigens present in corresponding biological matter of a normal individual or of an individual who is not afflicted with the first disease or condition or with the second disease or condition, to form antigen-antibody complexes;

(b) separating the antigen-antibody complexes from the sample; (c) immunising a non-human animal with the sample from which the antigen- antibody complexes have been separated;

(d) immortalising antibody-producing cells of the animal to produce immortalised cell lines;

(e) producing antibodies from individual immortalised cell lines; and (f) testing said antibodies for specific reactivity with a sample of biological matter obtained from at least one patient afflicted with said first disease or condition to thereby identify a monoclonal antibody, which detects said first disease or condition.

7. The method of any one of claims 2, wherein said at least one major antigen is selected from albumin, globulins, lipoproteins, transferrin, C-reactive protein, mucoprotein, αranti- trypsin, orosomucoid, fo-glycoprotein I, a_ HS glycoprotein, or hemopexin.

8. The method of claim 7, wherein said at least one major antigen is selected from albumin, immunoglobulins, haptoglogulin, C-reactive protein or transferrin.

9. The method of claim 3 or claim 4, wherein the antigens are fractionated by size separation and/or affinity-mediated separation techniques.

10. The method of claim 3 or claim 4, further comprising conjugating carriers to antigens of antigen-containing fractions that have native molecular weights of less than 30,000 to enhance their immunogenicity.

11. An immortalised cell line which secretes a monoclonal antibody that specifically detects a disease or condition and which is obtained by the method of any one or more of claims 1 to 10.

12. A monoclonal antibody which specifically detects a disease or condition, and which is produced by the method of any one or more of claims 1 to 10.

13. A synthetic or recombinant antigen-binding molecule having specificity for the antigenic determinant or epitope recognised by the monoclonal antibody of claim 12.

14. The synthetic or recombinant antigen-binding molecule of claim 13, which is a humanised antibody.

15. A diagnostic insoluble support to which the monoclonal antibody of claim 12, or analogue thereof is bound, or to which an antigen that is immuno-interactive with said monoclonal antibody is bound.

16. A method of detecting the presence of an antigen in a biological sample, wherein said antigen is immuno-interactive with the monoclonal antibody of claim 12, comprising: -

(a) contacting the biological sample with a monoclonal antibody or an analogue thereof ; and

(b) detecting the presence of a complex comprising the antigen and the monoclonal antibody, or analogue, in said contacted sample.

17. A method of detecting the presence in a patient of a disease or condition associated with an antigen that is immuno-interactive with the monoclonal antibody of claim 12, comprising: -

(a) contacting a biological sample obtained from the patient with the monoclonal antibody or analogue thereof; and

(b) detecting the presence of a complex comprising the antigen and the monoclonal antibody, or analogue, in said contacted sample.

18. A method of diagnosing a disease or condition associated with an aberrant concentration of an antigen in a biological sample of a patient, wherein said antigen is immuno-interactive with the monoclonal antibody of claim 12, comprising: -

(a) contacting the biological sample with the monoclonal antibody or analogue thereof;

(b) measuring the concentration of a complex comprising the antigen and the monoclonal antibody, or analogue, in said contacted sample; and (c) relating said measured complex concentration to the concentration of antigen in said sample, wherein the presence of said aberrant concentration is indicative of said disease or condition.

19. A composition for the treatment or prevention of a disease or condition, said composition comprising the monoclonal antibody of claim 12, or an analogue thereof, which is immuno-interactive with an antigen associated with said disease or condition, together with a pharmaceutically acceptable carrier.

20. A composition for the treatment or prevention of a disease or condition, said composition comprising an antigen which is associated with the disease or condition and which is immuno-interactive with the monoclonal antibody of claim 12, together optionally with a suitable adjuvant.

21. A method for treating or preventing a condition associated with the presence of an antigen in a biological sample obtained from an animal, comprising administering to said animal an effective amount of a composition comprising the monoclonal antibody of claim 12, or an analogue thereof, which is immuno-interactive with an antigen associated with said disease or condition, together with a pharmaceutically acceptable carrier.

22. A method for treating or preventing a condition associated with an aberrant concentration of an antigen in an animal, comprising administering to said animal an effective amount of a composition comprising the monoclonal antibody of claim 12, or an analogue thereof, which is immuno-interactive with an antigen associated with said disease or condition, together with a pharmaceutically acceptable carrier.

23. A method for treating or preventing a condition associated with an antigen in an animal which antigen is immuno-interactive with the monoclonal antibody of claim 12, comprising administering to said animal an effective amount of a composition comprising the antigen, together optionally with a suitable adjuvant.

24. A kit for detecting and/or measuring in a biological sample a target antigen associated with a disease or condition, comprising the monoclonal antibody of claim 12, or an analogue thereof, which is immuno-interactive with the target antigen .