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WO1986001807A1 - Anticorps monoclonaux et leur utilisation - Google Patents

Anticorps monoclonaux et leur utilisation Download PDF

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Publication number
WO1986001807A1
WO1986001807A1 PCT/GB1985/000409 GB8500409W WO8601807A1 WO 1986001807 A1 WO1986001807 A1 WO 1986001807A1 GB 8500409 W GB8500409 W GB 8500409W WO 8601807 A1 WO8601807 A1 WO 8601807A1
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Prior art keywords
pseudomonas
monoclonal antibody
kit
antigen
labelled
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PCT/GB1985/000409
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English (en)
Inventor
Bruce William Wright
Peter John Cox
Alice Margaret Noyes
Danny Widdows
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Technology Licence Company Limited
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Publication date
Application filed by Technology Licence Company Limited filed Critical Technology Licence Company Limited
Publication of WO1986001807A1 publication Critical patent/WO1986001807A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1214—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Pseudomonadaceae (F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • MONOCLONAL ANTIBODIES AND THEIR USE This invention relates to monoclonal antibodies and their use.
  • Pseudomonas is a bacterium which is widely dispersed in nature and is a common environmental contaminant in hospitals, where it infects patients with altered defence against infection. These patients include burn patients, patients with cancer or underlying metabolic diseases and those who had have tubes or lines placed into various body orifices. The use of antibiotics for the treatment of other infection ofen leads to Pseudomonas infection. In burn and cancer centres, Pseudomonas may cause as many as 30% of all infections. The Pseudomonas species most frequently associated with human disease is Pseudomonas aeruginosa which is described in Zinsser Microbiology (17th ed.) 761-5.
  • this organism causes 10 to 20% of the nosocomial infections. It has replaced Staphylococcus aureus as the major pathogen of cystic fibrosis patients and is frequently isolated from individuals with neoplastic disease or severe burns. The mechanism by which Pseudomonas aeruginosa produces disease in man is not understood.
  • Pseudomonas divisions have been made among the Pseudomonas species.
  • the more commonly known Pseudomonas members include Pseudomonas cepacia, Pseudomonas fluorescens, Pseudomonas stutzeri, Pseudomonas maltophilia and Pseudomonas aeruginosa.
  • the serotyping of Pseudomonas is not uniform among medical scientists.
  • the system adopted herein is the Liu system, as defined by P.V.Liu, which sub-divides Pseudomonas aeruginosa into 18 serotypes.
  • Pseudomonas is known to produce exotoxins, many of which are poorly-defined substances with toxic potential, i.e. Lps, proteases and phospholipase and others being known as exotoxin A and exoenzymes.
  • Lps low-power substance
  • proteases and phospholipase and others being known as exotoxin A and exoenzymes.
  • the extreme specificity of antigen-antibody reactions has made it possible to recognise differences between strains of such bacterial species which are indistinguishable on the basis of other phenotypic criteria.
  • Pseudomonas is known to cause gram-negative sepsis which is a bloodstream infection. It is one of the major infectious disease problems encountered in modern medical centres. While it can be transient and self-limited, severe gram-negative sepsis constitutes a medical emergency.
  • Pseudomonas is also known to cause urinary tract infection. Gram-negative sepsis can occur during the course of a urinary tract infection, and is occasionally fatal. At the present time, the test for gram-negative sepsis involves processing blood and urine cultures and -other procedures on occasion. In addition to being expensive, blood culture tests are cumbersome. They require a day, and often several days, to return results. They require expert laboratory skills because of the complex nature of human blood which tends to interact non-specifically with many of the test reagents.
  • a microscopic examination is made, to determine the presence of micro-organisms as a preliminary screening.
  • the microscopic examination cannot distinguish among the gram-negative bacteria.
  • a second step is a urine culture to identify the organism isolated in the urine .sample. A delay in diagnosis and initiation of treatment can result in serious complications.
  • the present invention provides novel monoclonal antibodies for use in accurately and rapidly diagnosing samples for the presence of Pseudomonas antigens and/or organisms.
  • the present invention comprises monoclonal antibodies specific for an antigen of Pseudomonas; in particular, the antigens or species of Pseudomonas cepacia, the antigens or species of
  • Pseudomonas fluorescens the antigens or species of Pseudomonas stutzeri, the antigens or species of Pseudomonas maltophilia, the antigens or species of Pseudomonas aeruginosa 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18, and the exotoxins (as described above) , e.g. exotoxin A, and exoenzymes, which may be produced by Pseudomonas aeruginosa, as well as a monoclonal antibody broadly cross-reactive with an antigen fo'r each species (or substantially all species) of the genus Pseudomonas.
  • the invention also comprises labelled monoclonal antibodies for use in diagnosing the presence of the Pseudomonas antigens, each comprising a monoclonal antibody against one of the above-mentioned antigens to Pseudomonas or to a particular species thereof and having linked thereto an appropriate label.
  • the label can be, for example, a radioactive isotope, enzyme, fluorescent compound, chemiluminescent compound, bioluminescent compound, ferromagnetic atom or particle.
  • the invention further comprises the process for diagnosing the presence of Pseudomonas antigens or organisms in a specimen, comprising contacting said specimen with the labelled monoclonal antibody in an appropriate immunoassay procedure.
  • the invention is also directed to a therapeutic composition
  • a therapeutic composition comprising a monoclonal antibody for an antigen of Pseudomonas and a carrier or diluent, as well as kits containing at least one labelled monoclonal antibody to an antigen of a Pseudomonas.
  • the monoclonal antibodies of the present invention are prepared by fusing spleen cells from a mammal which has been immunised against the particular Pseudomonas antigen, with an appropriate myeloma cell line, preferably NSO (uncloned), P3NS1-Ag4/1, or Sp2/0 Agl4. The resultant product is then cultured in a standard HAT (hypoxanthine, aminopterin and thymidine) medium. Screening tests for the specific monoclonal antibodies are employed utilising immunoassay techniques which will be described below.
  • the immunised spleen cells may be derived from any mammal, such as primates, humans, rodents (i.e. mice, rats and rabbits) , bovines, ovines and canines, but the present invention will be described in connection with mice.
  • the mouse is first immunised by injection of the particular Pseudomonas antigen chosen, e.g. for a period of approximately eleven weeks. When the. mouse shows sufficient antibody production against the antigen, as determined by conventional assay, it is given a booster injection of the appropriate Pseudomonas antigen, and then killed so that the immunised spleen may be removed.
  • the fusion can then be carried out utilising immunised spleen cells and an appropriate myeloma cell line.
  • the fused cells yielding an antibody which gives a positive response to the presence of the particular
  • Pseudomonas antigen are removed and cloned utilising any of the standard methods.
  • the monoclonal antibodies from the clones are then tested against standard antigens to determine their specificity for the particular Pseudomonas antigen.
  • the monoclonal antibody selected, which is specific for the particular Pseudomonas antigen or species, is then bound to an appropriate label.
  • Amounts of antibody sufficient for labelling and subsequent commercial production are produced by the known techniques, such as by batch or continuous tissue • culture or culture in vivo in mammals such as mice.
  • the monoclonal antibodies may be labelled with various labels, as exemplified above.
  • the present invention will be described with reference to the use of n enzyme-labelled monoclonal antibody. Examples of enzymes utilised as labels are alkaline phosphatase, glucose oxidase, galactosidase, peroxidase and urease.
  • Such linkage with enzymes can be accomplished by any known method, such as the Staphyl ⁇ coccal Protein A method, the glutaraldehyde method, the benzoquinone method, or the periodate method.
  • EIA enzyme-linked immunosorbent assay
  • Fluorescent-immunoassay is based on the labelling of antigen or antibody with fluorescent probes. A non-labelled antigen and a specific antibody are combined with identical fluorescently-labelled antigen. Both labelled and unlabelled antigen compete for antibody binding sites. The amount of labelled antigen bound to the antibody is dependent upon, and therefore a measurement of, the concentration of non-labelled antigen. Examples of this particular type of fluorescent-immunoassay include heterogeneous systems such as Enzyme-Linked Fluorescent Immunoassay, or homogeneous systems such as the Substrate-Labelled
  • Fluorescent Immunoassay The most suitable fluorescent probe, and the one most widely used, is fluorescein. While fluorescein can be subject to considerable interference from scattering, sensitivity can be increased by the use of a fluorometer optimised for the probe utilised in the particular assay, and in which the effect of scattering can be minimised.
  • Fluorescence polarisation In fluorescence polarisation, a labelled sample is excited with polarised light and the degree of polarisation of the emitted light is measured. As the antigen binds to the antibody, its rotation slows down and the degree of polarisation increases. Fluorescence polarisation is simple, quick and precise. However, at the present time, its sensitivity is limited to the micromole per litre range and upper nanomole per litre range with respect to antigens in biological samples.
  • Luminescence is the emission of light by an atom or molecule as an electron is transferred to the ground state from a higher energy state.
  • the free energy of a chemical reaction provides the energy required to produce an intermediate reaction or product in an electronically-excited state. Subsequent decay back to the ground state is accompanied by emission of light.
  • Bioluminescence is the name given to a special form of chemiluminescence found in biological systems, in which a catalytic protein or enzyme, such as luciferase, increases the efficiency of the luminescent reaction. The best known chemiluminescent substance is luminol.
  • a further aspect of the present invention is a therapeutic composition
  • a therapeutic composition comprising one or more of the monoclonal antibodies to the particular Pseudomonas antigen or species, as well as a pharmacologically- acceptable carrier or diluent.
  • Such compositions can be used to treat humans and/or animals afflicted with some form of Pseudomonas infection and they are used in amounts effective to cure; the amount may vary widely, depending upon the individual " being treated and the severity of the infection.
  • One or more of the monoclonal antibodies can be assembled into a diagnostic kit for use in diagnosing for the presence of an antigen, antigens or species of Pseudomonas in various specimens.
  • kits could be used in pathology laboratories for the rapid detection of gram-negative bacteria in urine, or on an out-patient basis.
  • conjugated or labelled monoclonal antibodies for antigens and/or species of Pseudomonas and other gram-negative bacteria can be utilised in a kit to identify such antigens and organisms in blood samples taken from patients for the diagnosis of possible Pseudomonas or other gram-negative sepsis.
  • the monoclonal test is an advance over existing procedures in that it is more accurate than existing tests; it gives "same day” results, ' provides convenience to the patient and improves therapy as a result of early, accurate diagnosis; and it reduces labour costs and laboratory time required for administration of the tests.
  • the kit may be sold individually or included as a component in a comprehensive line of compatible immunoassay reagents sold to reference laboratories to detect the species and serotypes of Pseudomonas.
  • One preferred embodiment of the present invention is a diagnostic kit comprising at least one labelled monoclonal antibody against a particular Pseudomonas antigen or species, as well as any appropriate stains, counterstains or reagents. Further embodiments include kits containing at least one control sample of a Pseudomonas antigen and/or a cross-reactive labelled monoclonal antibody which would detect the presence of any of the given particular Pseudomonas organisms in a particular sample. Monoclonal diagnostics which detect the presence of Pseudomonas antigens can also be used in periodic testing of water sources, food supplies and food processing operations.
  • the present invention describes the use of the labelled monoclonal antibodies to determine the presence of a standard antigen
  • the invention can have many applications in diagnosing the presence of antigens by determining whether specimens, such as urine, blood, stool, water and milk, contain the particular Pseudomonas antigen. More particularly, the invention could be utilised as a public health and safety diagnostic aid, whereby specimens such as water or food could be tested for possible contamination.
  • the monoclonal antibodies of the present invention were prepared generally according to the method of Kohler and Milstein, supra.
  • API Analytical Profile Index (ref. Ayerst Laboratories)
  • DMEM Dulbeccos Modified Eagles Medium
  • FCS Foetal Calf Serum
  • % T refers to vaccine concentrations measured in a 1 cm light path
  • CFA Complete Freunds Adjuvant
  • Pseudomonas fluorescens antigen was obtained from the National Collection of Type Cultures (NCTC accession No. 10038) and tested by standard biochemical methods of microbial identification to confirm its identity (using API profiles) .
  • the Pseudomonas fluorescens was -removed from the lyophile> grown on blood agar, and tested by API to confirm its identity and purity.
  • the bacteria were transferred for growth on to TSB and harvested for use as a source of antigen. The organisms were boiled and washed in formed saline by repeated centrifugation, and they were then resuspended in 1% formol saline.
  • mice were injected with the prepared antigen. They were given one intraperitoneal injection per week for three weeks (0.05 ml 80% T vaccine), followed by 8 iv • injections after intervals of 1, 4, 1, .4 x 2 and 3 weeks. The mice were bled approximately six days after the last injection and the serum tested for antibodies by assay. The conventional assay used for this serum titer testing was the enzyme-linked immunosorbent assay system. When the mice showed antibody production after this regimen, generally a positive titer of at least 10,000, a mouse was selected as a fusion donor and given a booster injection (0.05 ml 80% T vaccine) intraperitoneally, three days prior to splenectomy.
  • 0.05 ml 80% T vaccine 0.05 ml 80% T vaccine
  • Spleen cells from the immune mice were harvested three days after boosting, by conventional techniques. First, the donor mouse selected was killed and surface-sterilised by immersion in 70% ethyl alcohol.
  • the spleen was then removed and immersed in approximately 2.5 ml DMEM to which had been added 3% FCS.
  • the spleen was then gently homogenised in a LUX homogenising tube until all cells had been released from the membrane, and the cells were washed in 5 ml 3% FCS-DMEM.
  • the cellular debris was then allowed to settle and the spleen cell suspension placed in a 10 ml centrifuge tube.
  • the debris was then rewashed in 5 ml 3% FCS-DMEM.
  • 50 ml suspension were then made in 3% FCS-DMEM.
  • the myeloma cell line used was NSO (uncloned) , obtained from the.MRC Laboratory of Molecular Biology in Cambridge, England.
  • the myeloma cells were in the log growth phase, and rapidly dividing. Each cell line was washed using, as tissue culture medium, DMEM containing 3% FCS.
  • the spleen cells were then spun down at the same time that a relevant volume of myeloma cells were spun down (room temperature for 7 minutes at 600 g) , and each resultant pellet was then separately resuspended in 10 ml 3% FCS-DMEM.
  • 0.1 ml of the suspension was diluted to 1 ml and a haemacytometer with phase microscope was used.
  • 0.1 ml of the suspension was diluted to 1 ml with Methyl Violet-citric acid solution, and a haemacytometer and light microscope were used to count the stained nuclei of the cells.
  • 10 ml serum-free tissue culture medium DMEM were then slowly added, followed by up to 50 ml of such culture medium, centrifugation and removal of all the supernatant, and resuspension of the cell pellet in 10 ml of DMEM containing 18% by weight FCS.
  • 10 ⁇ l of the mixture were placed in each of 480 wells of standard multiwell tissue culture plates. Each well contains 1.0 ml of the standard HAT medium
  • the wells were kept undisturbed and cultured at 37°C in 9% CO- air at approximately 100% humidity.
  • the wells were analysed for growth, utilising the conventional inverted microscope procedure, after about 5 to 10 days.
  • screening tests for the specific monoclonal antibody were made utilising the conventional enzyme immunoassay screening method described below.
  • the monoclonal antibodies from the clones were screened by the standard techniques for binding to
  • Pseudomonas fluorescens NCTC 10038 prepared as in the immunisation, and for specificity in a test battery of Pseudomonas fluorescens species and related genera bearing different antigens. Specifically, a grid of microtitre plates containing a representative selective of Pseudomonas organisms was prepared, boiled, and utilised as a template to define the specificity of the parent group. The EIA immunoassay noted above may be used. The monoclonals had the appropriate specificity (to NCTC 10038) , and were negative to other Pseudomonas, IS. coli, Shigella, Salmonella, Proteus, Providencia and Serratia.
  • mice were primed with pristane, for at least
  • the -_ precipitate was dissolved in a minimum volume of cold phosphate/EDTA buffer (20 mM " sodium phosphate, 10 mM EDTA, pH 7.5, + 0.02% sodium azide) .
  • the solution was dialysed versus 2x1000 ml of the same buffer, at +4°C.
  • the dialysed, redissolved precipitate was centrifuged at 30,000 g for 10 minutes and applied to a 10 ml column of DEAE-cellulose, previously equilibrated in phosphate/EDTA buffer.
  • the monoclonal antibody was eluted with phosphate/EDTA buffer.
  • Monoclonal antibody was dialysed with alkaline phosphatase (Sigma Type VII-T) against 2 x 1000 ml- of PBS pH 7.4, at +4°C. After dialysis, the volume was made up to 2.5 ml with PBS and 25 ⁇ l of a 20% solution of glutaraldehyde in PBS was added. The conjugation mixture was left at room temperature for 1.5 hours.
  • the enzyme immunoassay method was used for testing. This method comprises coating the wells of a standard polyvinyl chloride (PVC) microtitre tray with the antigen, followed by addition of monoclonal antibody enzyme conjugate, and finally addition of the enzyme substrate, para-nitrophenyl phosphate.
  • PVC polyvinyl chloride
  • the monoclonal antibodies were found to be specific for the antigen of Pseudomonas fluorescens.
  • The'monoclonal antibody was tested and shown to be of the Classes IgG- .
  • the particular epitopic site to which the antibody attaches to the antigen can also be determined.
  • the same enzyme immunoassay method can also be used to determine whether diagnostic specimens such as urine, blood, stool, water or milk contain the antigen. In such cases, the antibody can first be bound to the plate. Examples 2 to 14
  • Example 2 The procedure of Example 1 was followed in each of 13 cases, with differences outlined below, to prepare monoclonal antibodies and conjugates for various antigens of the genus Pseudomonas.
  • the antigen was Pseudomonas stutzeri, NCTC 10475; in Example 3, Pseudomonas maltophilia, NCTC 10257; in Example 4, Pseudomonas cepacia, NCTC 10743; and in Examples 5 to 13, Pseudomonas aerogenes serotypes 1 (NCTC 11440) , 3 (NCTC 11442) , 4 (NCTC 11443) , 5 (NCTC 11444) , 5d (Public Health Laboratory Service Type 5d) , 6 (NCTC 11446) , 8 (NCTC 11447) , 9 (NCTC 11448) and 11 (PA103) , respectively.
  • Example 14 used the same antigen as Example 6 (NCTC 11442) .
  • Example 4 blood agar was the growth medium, and a distilled water sonicate was taken.
  • the growth medium was DMEM, and the organisms were boiled and washed in saline before being suspended in phenol saline.
  • Example 13 after growth in TSB, the supernatant was taken and the protein fraction separated.
  • Example 3 The sequence for Example 3 was ip-2-ip-2-ip-l- iv-4-iv-l-iv-2-iv-4-iv-2-iv-2-iv.
  • the sequence for Example 4 was im (in CFA)-16-iv(in formol saline).
  • Example 5 the sequence was ip-2-ip-2-ip-l-iv-4-iv-2-iv-2-iv-2-iv-2-iv-20-iv.
  • Examples 6 and 14 the sequence was ip-2-ip-2-ip-l-iv-4-iv-l-iv-2-iv-2-iv-2-iv-2-iv-2-iv.
  • Example 7 the sequence was ip-l-ip-l-ip-l-iv-4-iv-2-iv- 2-iv-2-iv-2-iv-4-iv.
  • Example 9 the sequence was im (in CFA)-3-ip-2-iv.
  • the sequence for Example 10 was ip-2-ip-2-ip-l-iv-4-iv-2-iv-20-iv.
  • the sequence for Example 11 was ip-2-ip-2-ip-l-iv-4-iv-2-iv-2-iv-2-iv-2-iv-20-ip-l-ip.
  • the sequence for Example 12 was ip-2-ip-
  • the limiting dilution method was used instead or (in Examples 3, 6 and 14) in addition to the agar method.
  • dilutions of cell suspensions in 18% FCS-DMEM + Balb/c mouse macrophages were made to achieve one cell/well and one-half cell/well in a 96-well microtitre plate. The plates were incubated for 7-14 days at 37 C, 97% RH, 7-9% C0 2 until semi-confluent. The superhatants were assayed for specific antibody by the standard enzyme immunoabsorbent assay.
  • Example 10 In Examples 10, 11, 13 (in addition to the procedure given in Example 1) and 14, the antibody production step was conducted by growing cells of the monoclonal antibody-cell line in batch tissue culture. DMEM-10% FCS was used to support growth in mid-log phase, to 1 litre volume, and the culture was then allowed to overgrow to allow maximum antibody production. The culture was then centrifuged at 1200 g for approximately 10 minutes, the cells discarded and the antibody-rich supernatant collected.
  • DMEM-10% FCS was used to support growth in mid-log phase, to 1 litre volume, and the culture was then allowed to overgrow to allow maximum antibody production. The culture was then centrifuged at 1200 g for approximately 10 minutes, the cells discarded and the antibody-rich supernatant collected.
  • the antibody purification step for Examples 2, 3, 6, 7 and 14 involved the Protein A-Sepharose method. Ascites fluid was filtered through glass wool and centrifuged at 30,000 g for 10 minutes. The ascites was then diluted with twice its own volume of cold phosphate buffer (0.1M sodium phosphate, pH 8.2). The diluted ascites was applied to a 2 ml column of Protein A-Sepharose, previously equilibrated with phosphate buffer. The column was washed with 40 ml of phosphate buffer. The monoclonal antibody was eluted with citrate buffer (0.1M sodium citrate, pH 3.5) into sufficient IM TRIS buffer, pH 9.0 to raise the pH immediately to about 7.5. The eluate was dialysed in PBS, pH 7.4, at 4 C and stored at -20 C.
  • citrate buffer 0.1M sodium citrate, pH 3.5
  • the suspension was stirred for a further 30 minutes, and then the precipitate was harvested by centrifugation at 10,000 g for 10 minutes.
  • the precipitate was dissolved in a minimum volume of cold phosphate/EDTA buffer (20 mM sodium phosphate, 10 mM EDTA pH 7.5 + 0.02% sodium azide) . " The dialysed, redissolved precipitate was centrifuged at 30,000 g for 10 minutes and applied to a 10 ml column of DEAE-cellulose, previously equilibrated in phosphate/EDTA buffer. The monoclonal antibody was eluted with phosphate/EDTA buffer.
  • Example 13 antibody purification was conducted by the following method:
  • TRIS buffered supernatant was applied at a flow rate of 1 ml/min to a 1 ml column of Protein A-Sepharose, previously equilibrated with 0.1M TRIS buffer, pH 8.2. The column was then washed with 40 ml of 0.1M TRIS buffer.
  • the monoclonal antibody was eluted with citrate buffer (0.1M sodium citrate, pH 3.5) into sufficient IM TRIS buffer, pH 9.0, to raise the pH immediately to about 7.5.
  • the eluate was dialysed in PBS, pH 7.4, at 4 C, and stored at -20 C.
  • the antibody conjugation step for Examples 2 and 10 was conducted using benzoquinone, as follows: 24 mg alkaline phosphatase (Sigma Type VII-T) were dialysed against 2 x 500 ml of 0.25 M sodium phosphate buffer, pH 6.0, at +4 C. 18 mg p-benzoquinone were dissolved in 0.6 ml warm AR ethanol, and added to the dialysed alkaline phosphatase. The benzoquinone/alkaline phosphatase mixture was left in the dark at room temperature for 1 hour.
  • IM lysine was then added to give a final concentration of 0.1M. After 2 hours in the dark at room temperature, the conjugate was dialysed against 2 x 1000 ml PBS + 0.02% sodium azide at +4 C. An equal volume of glycerol was added. The conjugate was sterile-filtered through a 0.22 ⁇ m membrane filter into a sterile amber vial, and stored at +4 C.
  • Example 14 The monoclonal of Example 14 was cross-reactive with Pseudomonas aeruginosa and negative to other organisms.
  • Example 15 The Sub-classes IgG2a (Examples 2, 7, 10 and 13) , IgG3 (Examples 3, 4, 6 and 11), IgM (Examples 5 and 8), igG2b (Examples 9 and 14) and IgGl (Example 12) were found.
  • Example 15 The Sub-classes IgG2a (Examples 2, 7, 10 and 13) , IgG3 (Examples 3, 4, 6 and 11), IgM (Examples 5 and 8), igG2b (Examples 9 and 14) and IgGl (Example 12) were found.
  • Example 15 The Sub-classes IgG2a (Examples 2, 7, 10 and 13) , IgG3 (Examples 3, 4, 6 and 11), IgM (Examples 5 and 8), igG2b (Examples 9 and 14) and IgGl (Example 12) were found.
  • Example 15
  • Example 1 The procedure of Example 1 was repeated, to give a monoclonal antibody broadly cross-reactive with each species of the genus Pseudomonas.
  • Tests using the present invention are superior to existing tests, based on the following advantages: (i) greater accuracy; (ii) same day results, within an hour or two; (iii) reduction in amount of skilled labour required to administer laboratory procedures, resulting in reduced labour'costs; (iv) reduction in laboratory time and space used in connection with tests, resulting in reduced overhead expenses; and (v) improved therapy based upon early, precise diagnosis.

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Abstract

Des anticorps monoclonaux pour le genre Pseudomonas, les anticorps marqués, compositions et kits les contenant, et leur utilisation pour le diagnostic d'antigènes et le traitement.
PCT/GB1985/000409 1984-09-07 1985-09-09 Anticorps monoclonaux et leur utilisation WO1986001807A1 (fr)

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GB848422649A GB8422649D0 (en) 1984-09-07 1984-09-07 Monoclonal antibodies
GB8422649 1984-09-07

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EP0176365A2 (fr) * 1984-09-26 1986-04-02 Sumitomo Chemical Company, Limited Anticorps monoclonal humain et sa préparation
EP0229107A1 (fr) * 1985-06-06 1987-07-22 Genetic Systems Corporation ANTICORPS MONOCLONAUX HUMAINS DE PROTECTION CONTRE L'EXOTOXINE A PRODUITE PAR $i(PSEUDOMONAS AERUGINOSA)
EP0305960A2 (fr) * 1987-09-03 1989-03-08 Takeda Chemical Industries, Ltd. Une cellule transformée pour la production d'un anticorps monoclonal humain, hybridome, leur production et utilisation
WO1990013660A3 (fr) * 1989-05-09 1990-12-13 Cetus Corp Anticorps monoclonaux humains de determinants sero-specifiques de bacteries gram-negatives
AT399885B (de) * 1986-07-03 1995-08-25 Genetic Systems Corp Verfahren zur herstellung von zellinien, die monoklonale antikörper produzieren

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CHEMICAL ABSTRACTS, Volume 100, No. 25, 18 June 1984, Columbus, Ohio (US) D.R. GALLOWAY et al.: "Production and Characterization of Monoclonal Antibodies to Exotoxin A from Pseudomonas Aeruginosa", see page 422, column 1, Abstract No. 207654v & Infect. Immun. 1984, 262-267 (Eng) *
CHEMICAL ABSTRACTS, Volume 100, No. 5, 30 January 1984, Columbus, Ohio (US) L.M. MUTHARIA et al.: "Surface Localization of Pseudomonas Aeruginosa Outer Membrane Porin Protein F by using Monoclonal Antibodies", see page 333, column 2, Abstract No. 33069r & Infect. Immun., 1983, 1027-1033 (Eng) *
CHEMICAL ABSTRACTS, Volume 101, No. 25, 17 December 1984, Columbus, Ohio (US) S. SAWADA et al.: "Protection against Infection with Pseudomonas Aeruginosa by Passive Transfer of Monoclonal Antibodies to Lipopolysaccharides and Outer Membrane Proteins", see page 588, column 1, Abstract No. 228262b *
CHEMICAL ABSTRACTS, Volume 102, No. 13, 1 April 1985, Columbus, Ohio, (US) K. MURAKAMI et al.: "Monoclonal Antibodies against Species-Specific Cephalosporinase of Pseudomonas Aeruginosa", see page 517, column 1, Abstract No. 111126q, & Eur. J. Biochem. 1985, 693-697 (Eng) *
CHEMICAL ABSTRACTS, Volume 102, No. 19, 13 May 1985, Columbus, Ohio, (US) R.T. IRVIN et al.: "Immunochemical Examination of the Pseudomonas Aeruginosa Glycocalyx: A Monoclonal Antibody which Recognizes L- Guluronic Acid Residues of Alginic Acid", see page 473, column 2, Abstract No. 165021v & Can. J. Microbiol., 1985, 268-275 (Eng) *
CHEMICAL ABSTRACTS, Volume 103, No. 9, 2 September 1985, Columbus, Ohio, (US) R.E.W. HANCOCK et al.: "Immunotherapeutic Potential of Monoclonal Antibodies against Pseudomonas Aeruginosa Protein F", see page 493, column 1, Abstract No. 69460n & Eur. J. Clin. Microbiol., 1985, 224-227 (Eng) *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0176365A2 (fr) * 1984-09-26 1986-04-02 Sumitomo Chemical Company, Limited Anticorps monoclonal humain et sa préparation
EP0176365A3 (en) * 1984-09-26 1987-05-20 Sumitomo Chemical Company, Limited Human monoclonal antibody and its preparation
EP0229107A1 (fr) * 1985-06-06 1987-07-22 Genetic Systems Corporation ANTICORPS MONOCLONAUX HUMAINS DE PROTECTION CONTRE L'EXOTOXINE A PRODUITE PAR $i(PSEUDOMONAS AERUGINOSA)
EP0229107A4 (fr) * 1985-06-06 1987-11-09 Genetic Systems Corp ANTICORPS MONOCLONAUX HUMAINS DE PROTECTION CONTRE L'EXOTOXINE A PRODUITE PAR -i(PSEUDOMONAS AERUGINOSA).
AT399885B (de) * 1986-07-03 1995-08-25 Genetic Systems Corp Verfahren zur herstellung von zellinien, die monoklonale antikörper produzieren
EP0305960A2 (fr) * 1987-09-03 1989-03-08 Takeda Chemical Industries, Ltd. Une cellule transformée pour la production d'un anticorps monoclonal humain, hybridome, leur production et utilisation
EP0305960A3 (fr) * 1987-09-03 1989-06-28 Takeda Chemical Industries, Ltd. Une cellule transformée pour la production d'un anticorps monoclonal humain, hybridome, leur production et utilisation
WO1990013660A3 (fr) * 1989-05-09 1990-12-13 Cetus Corp Anticorps monoclonaux humains de determinants sero-specifiques de bacteries gram-negatives

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GB8422649D0 (en) 1984-10-10
JPS62500174A (ja) 1987-01-22
EP0192727A1 (fr) 1986-09-03

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