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WO1999063349A1 - Methode de detection d'anticorps specifiques a des phases d'infection - Google Patents

Methode de detection d'anticorps specifiques a des phases d'infection Download PDF

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Publication number
WO1999063349A1
WO1999063349A1 PCT/EP1999/003841 EP9903841W WO9963349A1 WO 1999063349 A1 WO1999063349 A1 WO 1999063349A1 EP 9903841 W EP9903841 W EP 9903841W WO 9963349 A1 WO9963349 A1 WO 9963349A1
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WIPO (PCT)
Prior art keywords
infection
antibodies
antibody
compounds
phase
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Application number
PCT/EP1999/003841
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English (en)
Inventor
Fabrizio Bonelli
Luca Maria Barbero
Ugo Barbieri
Federico Corno
Antonio Soleti
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Diasorin International Inc.
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Filing date
Publication date
Application filed by Diasorin International Inc. filed Critical Diasorin International Inc.
Priority to CA002329687A priority Critical patent/CA2329687A1/fr
Priority to EP99929132A priority patent/EP1084411A1/fr
Priority to AU46050/99A priority patent/AU4605099A/en
Publication of WO1999063349A1 publication Critical patent/WO1999063349A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins

Definitions

  • the present invention relates to a method for the detection of antibodies and/or antibody isotypes specific for at least two different phases of an infection.
  • the method of the invention is characterized by the simultaneous incubation of a sample from a patient with at least two preferably antigenic compounds. These compounds comprise an epitope that is recognized by an antibody/antibody belonging to a particular isotype wherein said antibody is specifically occurring in one of said phases of said infection.
  • the method of the invention also requires that at least two epitopes are present in the incubation reaction that are specific for different phases of the infection and thus bind antibodies/antibody isotypes specific for said at least two different phases of said infection.
  • the invention also relates to a kit useful for carrying out the method of the invention.
  • EP-B1 0 328 588 describes a method of determining the amount of toxoplasmosis-associated IgM in a sample wherein a high IgM content is indicative of an acute infection of Toxoplasma.
  • US 4,877,726 discloses a method for distinguishing acute and chronic infection with Toxoplasma on the basis of the properties of a specific antibody.
  • the technical problem underlying the present invention was to provide an improved method for the convenient analysis of phases of an infection by a pathogen.
  • the solution to said technical problem is achieved by providing the embodiments characterized in the claims.
  • the present invention relates to a method for the detection of antibodies/antibody isotypes specific for at least two different phases of an infection comprising
  • each type of compound comprising a different epitope under conditions that allow binding of said antibodies to said epitopes, wherein each epitope is recognized by antibodies/antibodies belonging to a particular isotype specific for one of said phases of said infection, wherein further at least two epitopes are present in the incubation reaction recognized by antibodies/antibodies belonging to a particular isotype specific for at least two different phases of said infection, wherein further each type of compound/all types of compounds comprising epitopes recognized by antibodies/antibody isotypes indicative of the same phase of infection is/are affixed to the same type of support and wherein each type of support has properties that allow the physical distinction of said type of compounds from other types of support by physical means;
  • various phases of an infection can be identified in a convenient manner by incubating a sample from a patient known or suspected to suffer from infection by a pathogen with at least two compounds comprising different epitopes, wherein each epitope reacts with antibodies that are specifically found in a certain phase of the infection.
  • This will usually mean that the epitope is exposed to the immune system of this phase of the infection.
  • the present invention as outlined above envisages two different modes of determining the status or phase of an infection: either, two (or more) antibodies, e.g. of the ⁇ isotype (i.e.
  • IgM antibodies recognize two (or more) different epitopes wherein these two (or more) epitopes are characteristic of different phases of the infection; alternatively, two (or more) antibodies represent different isotypes and recognize different epitopes wherein said epitopes are again characteristic of different phases of the infection.
  • the antibodies specific for different phases of an infection alternatively belong to different isotypes.
  • binding by antibody is preferably specific, i.e. the antibodies do not cross-react with other epitopes. It is essential that antibodies reacting with one epitope used in the method of the invention do not cross-react with another epitope used in said method wherein said other epitope is indicative of a different phase of the infection.
  • fragments or derivatives of antibodies may also be employed as long as the physical distinction of said compounds (in step (b)) is not compromised.
  • each type of antibody would be considered indicative of a certain phase of infection.
  • the person skilled in the art will usually be in a position to identify the phase of infection, for example by evaluating the ratio of the different antibodies.
  • the reason for this type of finding may be, for example, that a certain type of antibody/antibody isotype is raised during one phase of infection, such as an early phase of infection, but may persist in the body during later phases of infection.
  • the actual phase of infection may be identified by way of determining the ratio of the amount of an antibody/antibody isotype indicative of a later phase of infection in relation to the amount of persisting antibodies.
  • the concomitant presence of two types of antibodies/antibody isotypes or the absence of one type of antibody/antibody isotype may be evaluated.
  • the method of the invention is also suitable for identifying the phase of an infection by detecting more than two of said antibodies/antibody isotypes.
  • the unique opportunity was provided to improve the reliability of the datation combining the results coming from two analytical outputs, selecting one marker positive in a shorter window period (typically 60 days), the other one or a prolonged one (up to 90 days).
  • the method of the invention further relies on the principle that the compounds comprising the various epitopes are affixed to different types of support that are distinguishable by their physical properties.
  • type of support may also, but not necessarily, mean that one type of support is disinguishable from another type of support by its chemical constitution.
  • the compound comprising the epitope may be affixed to said support by a variety of means known in the art. These means include chemical coupling, coating, attachment by van-der-Waals forces etc. It is important, however, that the epitope confirmation is not destroyed and that the epitope is accessible to binding by antibody.
  • Comprised by the method of the invention are also embodiments wherein different epitopes recognized by antibodies/antibody isotypes indicative of the same phase of infection are coupled to the same type of support. With these embodiments, a more accurate determination of the phase of infection may be achieved. Also, a stage of progress within a certain phase of infection/disease may be determined if the antibodies/epitopes are indicative of certain time points/time ranges within a certain phase of an infection.
  • the method of the invention can be combined with established prior art analytical techniques, as demonstrated, for example, in the appended examples to confirm or further analyze the results.
  • said antibodies recognizing said different epitopes are IgM antibodies.
  • IgM antibodies i.e. IgM antibodies with different binding specificities
  • the same type of antibody may first occur in a certain (first) phase of an infection and persist during the second phase of infection.
  • IgM antibodies recognizing different epitopes may first occur in the same (first) phase, but not persist in the later phase.
  • occurrence of the first and the second antibody in this scenario would be indicative of the early phase, whereas detection of only one (e.g. the first) antibody would be indicative of the later phase of the infection.
  • isotypes such as IgG and IgA, as well.
  • said antibodies belonging to a particular isotype are IgM, IgG or IgA antibodies.
  • IgM or IgA detection would be indicative of an earlier phase of infection
  • IgG antibodies would be indicative of a later phase of infection.
  • said at least two different phases of an infection include the acute phase, the post-acute phase, the chronic phase, the remission phase of an infection or a phase past infection.
  • the above indicated phases of an infection are well accepted in the medical field, but may, with respect to their distinction from earlier or later phases, in various diseases not be clear-cut for every type of infection. In such cases, the respective phase is to be interpreted in accordance with what the person skilled in the art understands by the respective term.
  • phase past infection is intended to mean a phase where infectious pathogen is no longer detectable in the organism and pathogenic effects are no longer detectable.
  • said infection is an infection with a virus, a bacterium or a protozoon.
  • said virus is human Cytomegalovirus, Hepatitis C virus, Hepatitis A virus, Hepatitis B virus, Epstein-Barr virus, HIV or Herpes simplex virus.
  • said bacterium is Borrelia burgdorferi, Treponema pallidum or Helicobacter pylori.
  • said protozoon is Toxoplasma gondii or Trypanosoma cruzi.
  • said sample from a patient is blood, serum or is derived therefrom. It is well-known in the art that blood and serum may be treated prior to analysis and/or that certain fractions thereof may be used for analysis. The term "is derived therefrom" is intended to include these treated body fluids or fractions thereof.
  • the compound may be a carrier of a natural or a non-natural origin.
  • the invention in another preferred embodiment relates to a method wherein said compound is an antigen.
  • said antigen is a natural or a recombinant antigen.
  • said antigen is treated with detergents, like SDS, Triton ® -X (Triton ® X-100) or other detergents known to the person skilled in the art prior to step (a).
  • detergents like SDS, Triton ® -X (Triton ® X-100) or other detergents known to the person skilled in the art prior to step (a).
  • Further detergents known to the skilled artisan comprise Tween ® 20, Tween ® 40, Tween ® 80, CHAPS and CTAB.
  • said antigen is a (poly)peptide or DNA.
  • (poly)peptide in accordance with the present invention, refers to either a peptide or a polypeptide.
  • the term "(poly)peptide” also includes the term "protein”.
  • the (poly)peptide, in accordance with this invention, may comprise naturally occurring peptides or proteins, as well as synthetic or recombinantly produced peptides/proteins.
  • the (poly)peptide may encompass amino acid chains of any length, wherein the amino acid residues are linked by covalent peptide bonds.
  • a (poly)peptide may comprise different (poly)peptide species.
  • a (poly)peptide species is defined by its chemical composition and modifications of said peptide(s)/polypeptide(s) by, inter alia, glycosylations, acetylations, phosphorylations, lipidations or by amino acid exchanges.
  • the term (poly)peptide species is therefore defined as the smallest unit of protein classification, defined by its chemical structure.
  • polypeptide comprises, in accordance with this invention, antigenic fragments of said (poly)peptide(s) and/or fusion proteins. Additionally, the term refers also to a mixture of (poly)peptides, like the mixture in lysates.
  • DNA refers to polynucleotides and/or nucleic acid molecules and refers to coding as well as to non-coding sequences.
  • DNA comprises also any feasible derivative of a nucleic acid and peptide nucleic acids (PNAs) containing DNA analogs with amide backbone linkage (Nielson, Science 254 (1991 ), 1497-1500).
  • PNAs peptide nucleic acids
  • said antigen or (poly)peptide is CM2 fusion protein (HCMV) (Vornhagen et al., J. of Virological Methods 60: 73-80 (1996) and Vornhagen et al., DE 4 435 789 C1 (1995)), p52 (HCMV) (Vornhagen et al., J.
  • Such supports may comprise, inter alia, plates, stripes, wells, microchips or containers.
  • Suitable materials for such supports or materials for further coating of said supports include, but are not limited to, glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified celluloses, like nitrocellulose, polyacrylamide, agaroses, magnetide and metals, like colloidal gold.
  • said support is a bead.
  • Said beads preferably have a range of diameter between 1.5 and 2 ⁇ m such as 1.5, 1.6, 1.7, 1.8, 1.9 and 2.0 ⁇ m.
  • Compounds comprising an epitope recognized by an antibody specific for an early phase of an infection may thus, for example, be coupled to beads having a diameter of 1.5 ⁇ m whereas compounds comprising an epitope recognized by an antibody specific for a later phase of an infection may be coupled to beads having a diameter of 1.9 ⁇ m.
  • said bead is a latex bead, a colloid metal particle, such as a gold particle, or any combination thereof.
  • said property that allows the physical distinction of said types of compounds is mass, size, refractive index, a magnetic property, electric property or a combination thereof.
  • the physical distinction of said compounds is effected by measuring a change in said property caused by the agglutination of said compounds.
  • the agglutination of said compounds is effected by their crosslinking by antibody binding of the epitopes.
  • a smaller or larger degree of agglutination will occur.
  • IgM is usually in a pentameric structure whereas IgA or IgG are dimeric. IgM will thus normally give rise to larger agglutination structures.
  • said agglutination is an agglutination of two or three compounds.
  • said measurement is the measurement of light scattering, magnetic field variation or electric field variation.
  • WO92/21024 With respect to the embodiment where measurement is effected by light scattering, the technology developed in WO92/21024 and WO 94/15193, which are herewith incorporated by reference, is preferably employed.
  • the methodology as disclosed in WO 92/21024 relies on the use of a high resolution optical sheath flow cell, a single detector for measurement of pulse signals from unidirectional low angle forward light scatter from said differently-sized, differently-coated beads and their aggregated multimers, and a flow particle analyzer apparatus.
  • Said method involves the mixing of samples with said coated monomeric particles and an incubation period of said samples with the coated monomeric particles, to allow agglutination reactions to occur.
  • Said agglutination is measured in said flow particle analyzer, whereas monomeric, dimeric or n-meric particles pass in a sheath- type flow all through a finely focussed optical beam produced by a semiconductor laser. As each type of particle passes through the beam, a unique light scatter signal is produced, which is detected by a photodiode. These pulses are classified by amplitude into a histogram for electronic data analysis.
  • the method of the present invention relates to a method wherein in step (a) the sample from a patient is further incubated with an anti-human immunoglobulin antibody.
  • said anti-human immunoglobulin antibody is an anti-lgM antibody.
  • Said anti-human immunoglobulin antibody may be added prior, during or after contacting the sample from a patient with said at least two types of compounds comprising a different epitope.
  • the addition of said anti-human immunoglobulin antibody may improve the sensitivity for the detection of antibodies/antibody isotypes, as documented in the appended examples.
  • Said anti-human immunoglobulin antibody may be a monoclonal or polyclonal antibody.
  • Said anti-human immunoglobulin antibody comprises also synthetic antibodies, as well as fragments of antibodies, such as F(ab') 2 or scFv fragments (e.g. scFv fragments expressed as "phagobodies” (Felici et al., Biotechnol. Annu. Rev. 1 (1995), 149-183).
  • Such anti-human immunoglobulin antibodies or fragments thereof can be obtained by using methods which are described, e.g. in Harlow and Lane, (1988), -Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory.
  • fivalian/coupling/coating of said compound to said support is stabilized by surfactant treatment.
  • surfactant treatment An example of such a surfactant treatment is provided by appended Example 4.
  • the invention also relates to a kit comprising different types of supports of distinguishable physical properties wherein to each type of support a different compound comprising an epitope is covered, wherein further each epitope is recognized by an antibody/antibody belonging to a particular isotype and each antibody/antibody belonging to a particular isotype is specific for a phase of a disease and wherein said different compounds and epitopes, respectively, are recognized by antibodies/antibodies belonging to a particular isotype specific for at least two different phases of said infection.
  • kit of the invention is particularly useful in carrying out the method of the invention that has been described in detail herein above.
  • said supports are beads.
  • said beads are latex beads, colloid metal particles, preferrably gold particles, or combinations thereof.
  • said compound is CM2 fusion protein (HCMV), p52 (HCMV) or a viral particle from HCMV, gp36, gp33, gp27, gp30 or gp24 (hepatitis B virus), a Toxoplasma gondii lysate, optionally proteinase K-treated or deglycosylated, or 41 kDa protein or 31/34 kDa protein from Borrelia burgdorferi.
  • HCMV CM2 fusion protein
  • HCMV p52
  • a viral particle from HCMV gp36, gp33, gp27, gp30 or gp24
  • Toxoplasma gondii lysate optionally proteinase K-treated or deglycosylated
  • 41 kDa protein or 31/34 kDa protein from Borrelia burgdorferi is optionally proteinase K-treated or deglycosylated
  • said kit further comprises an anti-human immunoglobulin antibody.
  • said anti-human immunoglobulin is an anti-lgM antibody. It is also preferred in accordance with the present invention that the same type of support is coated with different epitopes or different compounds wherein said different epitopes or compounds are recognized by antibodies/antibody isotypes indicative of the same phase of infection.
  • the kit of the invention may comprise a carrier means being compartmentalized to receive in close confinement one or more container means such as vials, tubes and the like, each of the container means comprising one of the separate elements to be used in the method.
  • one of the containers may comprise, inter alia, the anti-human immunoglobulin antibody in lyophilized form or in solution.
  • the carrier means may also contain a plurality of containers each of which comprises, inter alia, different, predetermined amounts of the compound and/or beads useful in the method of the invention. These latter containers can then be used to prepare a standard curve into which can be interpolated the results obtained from the sample containing the unknown amount or unknown type of the detectable antibodies and/or antibody isotypes.
  • Figure 1 Seroconversion profiles evaluated with the Copalis I system, employing CM2 and p52 protein. Used beads are coated with CM2 protein or p52 protein. The Y axis depicts a signal in CTR, whereas on the X axis the days of the infection are blotted. The cut-off lines are printed for the two different beads (see legend).
  • FIG. 2 Seroconversion profiles evaluated with the Copalis I system, employing CM2 and VP. Used beads are coated with CM2 protein or viral particle (VP). The Y-axis depicts the signal in CTR, whereas on the X axis the days of infection are blotted. The cut-off lines are printed for the two beads (see legend).
  • Figure 3 Evaluation of patient sera, positive for anti-Toxoplasma gondii IgG and negative for anti-Toxoplasma gondii IgM.
  • Top, left Distribution frequency of sera which are negative for anti- Toxoplasma gondii-lgM and -IgG, as evaluated with the Copalis I system.
  • the used beads are coated with the Toxoplasma gondii lysate which was treated with proteinase K.
  • FIG. 4 Top: Distribution of sera which are positive for anti-Toxoplasma gondii IgM as evaluated with the Copalis I system (left) and the reference system (right) (Toxo Vidas IgM, # 30202 (TXM)). Bottom: Comparative results obtained with sera positive for anti- Toxoplasma gondii IgM as evaluated with Copalis I system and a reference system (Toxo Vidas IgM, # 30202 (TXM)).
  • Figure 5 Seroconversion profiles evaluated with the Copalis I system employing CM2, p52 and VP. Used beads are coated with CM2 protein, p52 protein or viral particle (VP).
  • Latex beads of a diameter size of 1.8 ⁇ m were coated with CM2 fusion protein, latex beads of a diameter of 1.7 ⁇ m were coated with the p52 protein and latex beads with a diameter of 1.6 ⁇ m were coated with viral particle (VP).
  • the coated latex beads were used for simultaneous measurement of IgM or IgG in a sheath flow optical Flow Particle Analyzer (Hansen (WO92/21204)).
  • a coating buffer comprising 1.59 g/l Na 2 C0 3 , 2.93 g/l NaHC0 at pH 9.6 and an overcoating buffer (OB), comprising 7.51 g/l glycine, 10 g/l BSA fraction V protease free, 50 g/l sucrose and 2 g/l NaN 3 were used.
  • CB coating buffer
  • OB overcoating buffer
  • CM2 In order to coat CM2 (from Biotest AG, Germany; Vornhagen et al., J. of Virological Methods 60:73-80 (1996), and DE 4 435 789 C1 ) to the 1.8 ⁇ m latex beads, a 0.5% solution of latex beads in coating buffer was prepared. After 3 washes of the beads in coating buffer comprising centrifugation steps for 20 minutes at 4,000 rpm, the beads were resuspended in CB to a final concentration of 1%. The recombinant CM2 protein was prepared in coating buffer at a final concentration of 20 ⁇ g/ml. Drop by drop, the recombinant protein solution was combined with the 1.8 ⁇ m latex beads.
  • Hydrophobic coating of the latex beads with CM2 protein was carried out during a 1 hour incubation at room temperature on a drum roller. Coated latex beads were washed in OB by centrifugation for 20 minutes at 4,000 rpm. The coated 1.8 ⁇ m latex beads were resuspended in OB to a concentration of 1%. Post-coating in OB was carried out for 1 hour at room temperature on a drum roller. After centrifugation for 20 minutes at 4,000 rpm, the beads were resuspended in OB to a final concentration of 0.5% (w/v) and stored at 4°C until further use.
  • the p52 gene was isolated by PCR using the following primers:
  • the cellular paste was processed as follows: the p52 pellet obtained from a 125 mL culture was resuspended in 5 mL of 50 mM Tris pH 8, adding 50 ⁇ l benzonase (Benzonuclease from Merch, Germany) and 50 ⁇ l MgCI 2 0.1 M. After the incubation at 37°C for 3 hours, the sample was spun at 10°C for 30 min 11 ,000 rpm in a JA20 rotor. The resulting pellet was resuspended in 3 mL of 50 mM Tris, 8 M urea, 10 mM beta- OH pH 9.4 (buffer A), dissolved and sonicated 5 times for 30 sec.
  • buffer A buffer A
  • the obtained lysate was shaken for 1 hour at RT, then heated at 70°C for 10 min and spun at 4°C for 30 min 11 ,000 rpm.
  • the 8 M urea supernatant was gel-filtrated with the buffer A, and the pooled protein was dialyzed overnight at RT against 1.5 I of buffer 50 mM Tris, 6 M urea, 500 mM NaCI pH 8.8.
  • the protein has been purified through chelating chromatography (# 17040901 PHARMACIA, Sweden), exploiting the His tail property.
  • a 100 ⁇ g/ml p52 protein solution in coating buffer was prepared, SDS to a final concentration of 0.1% was added to the recombinant protein solution and incubated at room temperature for 30 minutes. After this incubation the 1.7 ⁇ m latex beads were combined, drop by drop, with the recombinant p52 protein solution. Hydrophobic coating of the latex beads with p52 was carried out by incubation for 2.5 hours at room temperature on a drum roller. The beads were washed twice in OB and post-coating was carried out for 1 hour at room temperature in OB.
  • the p52-coated 1.7 ⁇ m latex beads were resuspended in OB to a final concentration of 1 % (w/v) and stored at 4°C until further use.
  • Coating of 1.6 ⁇ m latex beads with viral particle (VP) was carried out by washing 1.6 ⁇ m latex beads three times in coating buffer. After the last washing step a 2% solution of 1.6 ⁇ m latex beads in OB was prepared. To 1 ⁇ g of viral particle (VP from AB USA) 80 ⁇ l 1 % SDS solution was added and incubated for 30 minutes at room temperature. The viral particle solution was prepared at a concentration of 160 ⁇ g/ml in coating buffer. Same volumes of viral particle solution and 1.6 ⁇ m latex beads solution were combined drop by drop and incubated for 1 hour at room temperature on a drum roller.
  • the latex beads were resuspended in OB and post-coating was carried out for 1 hour at room temperature on a drum roller. After further centrifugation steps at 4,000 rpm for 20 minutes, the beads were resuspended in OB to a final concentration of 1 % (w/v) and stored at 4°C until further use.
  • the dried latex-bead mixture was resuspended in 180 ⁇ l reaction buffer (comprising 0. 5 M KBr, 0.1 % BSA, 0.15% PEG-8000, 0.002% zwitterionic detergent, 1 mM EDTA and and 0.1 M Glycine at pH 9.0) and 20 ⁇ l of test sample was added. After an incubation for 10 minutes at RT, the sample was measured under Copalis I standard, procedures (CopalisTM System, Procedure Manual 1997, Sienna Biotech).
  • the measurement in Copalis I is based on a monitoring of light scattered from single particles or particle aggregates the instrument belongs to the class of Flow Particle Analyzes (FPA) (Hansen (WO92/21024)).
  • FPA Flow Particle Analyzes
  • Latex beads and n-meric microparticles can be discriminated in size by the scattered photon energy. If coating on the latex beads caused an agglutination of the microparticles in the presence of IgG or IgM in the sample, the latex beads changed their size distribution. The number of monomeric latex beads was depleted in a sample, while the number of n-meric particles was increased. The comparison of the number of monomeric latex beads when exposed to a non-reactive sample and the same monomer number when exposed to a reactive sample is compared.
  • CTR 100 x (negative control )/(sample under assay)
  • the CTR monitors the tested sample reactivity with respect to a non-reactive reference specimen/sample.
  • CTR indicates the coated microparticle tendency to aggregate in the presence of the tested sample, i.e. the number of reacted monomers.
  • CM2 coated latex beads were supposed to react with IgM during acute and postacute phase of an HCMV infection
  • p52 coated latex beads were supposed to react with IgM of the acute phase of an HCMV infection
  • VP coated latex beads were supposed to have a high sensitivity for IgG after an HCMV infection. Accordingly, the specificity and sensitivity of the different coated latex bead was tested. These relevant data are summarized in Tables 1 to 5. Specificity and sensitivity of the HCMV-antigen coated latex beads was tested, using anti-CMV IgG and anti-CMV IgM negative sera.
  • Table 2 shows the results for 25 Test-sera which were anti-CMV IgM-negative and anti-CMV IgG-positive. Only one serum (# 23208) showed reactivity with CM2- and p52-coated beads (probably due to anti-latex antibodies in the serum), whereas all tested samples showed reactivity with VP-coated latex beads, which documented the high sensitivity of VP-coated beads for anti-HCMV IgG.
  • Tables 3, 4 and 5 document the high sensitivity of CM2- and p52-coated latex beads for anti-CMV IgM antibodies. For the documented results, different anti-CMV IgM positive sera of low (Table 3), medium (Table 4) or high (Table 5) titers have been used.
  • Table 6 shows for each patient the test-results which were obtained at different days post-infection, using the CM2-, p52- and VP-coated latex beads in Copalis I (CopalisTM System, Procedure Manual 1997, Sienna Biotech).
  • CM2-coated beads demonstrate that this fusion protein was able to detect all anti-HCMV IgM antibodies up to the end of the post-acute phase (6 to 7 months post infection).
  • Results obained with p52-coated beads show (see e.g. seroconversion # 20675) that p52 was very specific for the acute phase of the HCMV infection: the signal generated by the p52-coated latex beads increased during the month of infection and decreased during the second month post infection. This decrease of p52-signal can be correlated to the end of the acute phase of HCMV infection.
  • Results obtained with VP-coated latex beads demonstrated the appearance of anti- HCMV IgG antibodies in the tested sera during the progression of HCMV infection.
  • multianalyte immunoassays for immunoglobulins of different subtypes can be carried out simultaneously, using specific antigen coated microparticles of different sizes, each of the microparticles being reactive towards a different antibody class in a biological fluid.
  • Hepatitis B differentiation of acute from remission infection phase
  • preS2-HBsAg The recombinant preS2-HBsAg protein (Sammata and Youn, Vaccine 7: 69-76 (1989); ay subtype), the so-called middle-protein, was expressed in human kidney cells 293. (ATCC, Maryland, USA) using a BK vector (Gallina et al., J. of General Virology 73:139-148 (1992)).
  • PreS2-HBsAg is a Hepatitis B surface protein (Heermann, J. Virol. 52: 396-402 (1984)), using 3 different in-frame start codons (Gallina et al., J. of General Virology 73:139-148 (1992)).
  • Initiation at the 3 rd AUG codon generates the major protein (HBsAg, p24 and gp27 in its glycosylated form); initiation at the 2 nd AUG codon generates the preS2-HBsAg protein (middle-protein p30, and its mono- and diglycosylated forms, gp33 and gp36 respectively) and initiation at the 1 st AUG codon adds a further 108 to 119 aa extension (preS1 region) to the N-term of the middle protein generating the so-called large-protein (p39).
  • preS1 region extension
  • the cell culture supernatant was neutralized with a solution containing 0.15 M NaCI, 0.01 M NaH 2 P0 4 , 1 mM MgCI 2 at pH 7.8.
  • Endonuclease (445 U/ ⁇ L, SIGMA) and 0.05 mM PMSF, 1 ⁇ g/mL Aprotinin, 1 ⁇ g/mL Leupeptin (anti-protease mixture) were added.
  • a first precipitation was carried out by slowly adding PEG (polyethylene glycol) to a final concentration of 6%. After an incubation for 30 min at 37°C on a shaker, the solution was discarded and a second precipitation with PEG (final concentration of 14%) was carried out.
  • the solution was centrifuged at 10,000 rpm for 25 min at room temperature.
  • the resulting pellet was resuspended in 1/34 initial volume in PBS/0.05% NaN 3 at pH 7.4 and supplemented with the anti-protease mixture.
  • 100 mL of the sample was dialyzed for 1 hour at room temperature with SpectraPor MWCO 50000 against 0.05 M citrate buffer at pH 2.4. After a centrifugation at 10,000 rpm for 15 min, the dialyzed samples were neutralized with 0.03 M NaHC0 3 .
  • the pellet was again dialyzed with SpectraPor MWCO 50000 against PBS, 0.05% NaN 3 at pH 7.4 and anti-protease mixture.
  • the solution was filtered (porosity of filter: 0.2 ⁇ M) and dispensed in aliquots until further use.
  • the gp36 polypeptide was the deglycosylated form of the preS2- HBsAg protein (Asn-123 within the preS2 region and Asn-320 within the S region), gp33 was the monoglycosylated form of the preS2 region (Asn-123), gp27 was the glycosylated form of the S region (Asn-320), p24 represented the non-glycosylated form of the S region (Meisel et al., Intervirology 37: 330-339 (1994)).
  • preS2-HBsAg protein was deglycosylated: Na 2 HP0 buffer pH 7.7 and a solution containing 2% SDS and 1 M 2-mercaptoethanol were added to the protein sample. After heating of the sample (140 ⁇ l) at 100°C for 5 minutes and immediately cooling with ice for 5 minutes, 12 ⁇ l Nonidet P-40 (99%) and 5 ⁇ l PNGaseF enzyme (BioRad, 2.5 Ul/ml) were added at 37°C for 2.5 hours. The obtained p30 protein was the non-glycosylated form of preS2-HBsAg protein.
  • samples 1 , 2, 3, 4 The two aliquots (untreated and deglycosylated) were divided into four parts (samples 1 , 2, 3, 4), separated on SDS-PAGE (polyacrylamide gel 15%) and transferred onto a PVDF membrane (IMMOBILON P, Millipore) in order to perform Western blot analysis. Transferred samples 1 and 2 were incubated with a 1 :50 dilution of PHM 907-09 serum, a seroconversion marker for HBV infection (provided by Boston Biomedica Inc.) in dry low-fat milk in PBS pH 7.4. Samples 3 and 4 were incubated with a serum representative of the remission phase (from AALTO, Ireland) diluted 1 :50 in dry skimmed milk in PBS pH 7.4.
  • Samples 2 and 4 were incubated with a 1 :350 dilution of anti-human IgG goat antibodies (Atlantic Antibodies, Maine, USA), labeled with HRP. After an incubation period of 1 hour at 37 °C on a shaker, the membranes were washed and a revelation procedure was carried out, using 9-ethyl-amino-carbazol (AEC, Sigma).
  • AEC 9-ethyl-amino-carbazol
  • PHM907-09 serum (DNA HBV positive, HBsAg positive and HBeAg positive), being diagnostic for an acute HBV infection phase, reacted very strongly with the gp36 and gp33 antigens, showing immunoreactivity of the patient towards the glycosylated forms of preS2 region.
  • serum representative of the remission phase (anti-HBs positive, anti-HBc positive, anti-HBe positive) reacted mainly with gp27 and p24, showing high immunoreactivity towards the major protein (S region).
  • gp36, gp33 and p30 can be used as specific antigens for the detection of an acute phase of an HBV infection, whereas gp27 and p24 can be employed as antigens for the remission phase of an HBV infection.
  • Example 3
  • Toxoplasma gondii isotype antibody differentiation and preparation of isotype specific antigen-beads
  • gondii were obtained from Gaslini Hospital Genua, Italy and the lysate was prepared according to conventional procedures) in NP40 (concentration 5.11 mg/ml, lot 076, Pharmacia, USA) was added to the latex bead pellet, to a final antigen concentration of 1.5 mg/ml. After the addition of 200 ⁇ l Glycine Buffered Saline (GBS: 0.1 M glycine, 0.17 M NaCI, 9.15 mM NaN 3 at pH 9.2) and 180 ⁇ l deionized H 2 0, the beads were incubated with the Toxoplasma gondii lysate for 30 minutes at room temperature.
  • GSS Glycine Buffered Saline
  • the beads were washed twice in GBS (diluted 1 :15 with deionized H 2 0) and once in GBS containing 10g/l BSA (bovine serum albumine). Pelleted beads were resuspended in 1 ml GBS-BSA buffer and sonicated for 15 sec at 150 W.
  • GBS diluted 1 :15 with deionized H 2 0
  • BSA bovine serum albumine
  • TRIS-buffer 0.1 M TRIS, 20 mM CaCI 2 at pH 7.5. After the last wash, beads were resuspended in 1 ml TRIS-buffer containing 200 ⁇ m/ml proteinase K. After a 2- hour incubation at 37°C on an orbital shaker, the proteinase K reaction was blocked by addition of 10 ⁇ l 0.1 M PMSF (Sigma, USA) and further incubation for 30 minutes at room temperature.
  • the Tg/proteinase K-coated beads were tested using anti-Tg IgM and anti-Tg IgG negative control sera (doubly negative control sera). Furthermore, the beads were tested on sera, which were anti-Tg IgM negative and anti-Tg IgG positive. The results of the tests are shown in Figure 3.
  • the enzymatic deglycosylation kit enzymatically cleaves N-linked and sialic acid substituted Gal (b1-3) GalNAc (a1 ) O-linked oligosaccharides from glycoproteins.
  • the enzymatic deglycosylation was less harsh than chemical methods and provided deglycosylated glycoproteins that were suitable for further protein and carbohydrate analysis. Furthermore, the role of the glycosylation on protein bioactivity and antibody binding could be determined. Deglycosylation-reaction was carried out according to manufacturers' recommendation.
  • the deglycolsylated protein mixture was coated onto polystyrene particles of appropriate size, ranging from 1.6 through 1.9 ⁇ m.
  • a surfactant was added in order to stabilize the coating and a post-coating buffer (Overcoating buffer as described in example 1 ) was employed to reduce non-specific reactions.
  • the coated beads were treated with the deglycosylating mixture at 37°C for 2.5 hours and stored at 4°C until further use. After drying of the beads, the beads were resuspended in reaction buffer and mixed with test samples (as described in example 1 ). After an incubation for 10 min at room temperature, the sample was measured under Copalis I standard procedures (CopalisTM System, Procedure Manual 1997, Sienna Biotech). The findings from these experiments confirmed the results obtained by the Western blot analysis: anti-Tg IgM reactivity was reduced, whereas anti-Tg IgG reactivity was maintained.
  • the chemical disruption was performed by treating the main Toxoplasma g. protein with solutions of Nal0 4 at different concentrations (ranging from 0.01 to 40 mM). Reactions were carried out at room temperature for 30 minutes and at different pHs. The reactions were blocked by ethylenglycol treatment acid treatment. Residual aldehydic groups were blocked by the reduction with NaCNBH 3 in the presence of glycine or ethanolamine 0.1 M at basic pH. Efficiency of the reactions was confirmed by running samples (before and after the deglycosylation) on SDS-PAGE gels and Western blots. With these techniques, it was observed that the anti-Tg IgM reactivity was disrupted, whereas the anti-Tg IgG reactivity was completely maintained.
  • Toxoplasma gondii lysate-coated polystyrene beads were also treated with the periodate.
  • agglutination assays were performed with the Nal0 treated beads, the results as described for the Western blot assays could be confirmed.
  • a differentiation between a new and old Borrelia burgdorferi infection is based on the finding that no reactivity against the 31/34 kDa proteins can be detected during early phases of infection.
  • the three Borrelia burgdorferi proteins are easily purified by separation of the outer membrane fraction (31 kDa and 34 kDa antigens) of Borrelia burgdorferi from the periplasmatic flageliae (41 kDa protein), and further electrophoretic separation or electroelution.
  • outer envelope (OE) components of Borrelia burgdorferi Borrelia burgdorferi cultures were centrifuged (7000 g for 20 min at 20°C). The resulting pellet was washed and resuspended in saline. SDS (0.03%) was added at RT for 15 min and the suspension was centrifuged (25000 g for 90 min at 4°C). The supernatant containing the OE fraction was filtered and dialyzed to remove,
  • the pellet fraction resulting from the SDS treatment was resuspended in saline and blended in a Waring blender. The suspension was centrifuged (26000 g for 6 min at
  • PF periplasmic flageliae fraction
  • a preparative SDS-PAGE gel was loaded either with OE or PF fraction and run overnight at 30 mA.
  • the protein position was identified by Coomassie blue staining and subsequent isopropanol/acetic acid destaining.
  • the desired proteins were identified by comparison of the resulting gel with an analytical SDS-PAGE.
  • the gel portion containing the proteins was then electroeluted for 12 hours at 10 V in a BioRad Transblot. In order to remove SDS, a dialysis step followed.
  • the purified proteins were coated onto polystyrene microparticles of appropriate size, ranging from 1.6 through 1.9 ⁇ m, under either acidic, basic or neutral conditions, using either sodium carbonate (pH 9.6), glycine (pH 4.0) or HEPES (pH 7.0) as buffer systems.
  • a surfactant SDS or Tween 20 at concentrations ranging from 0.01 to 0.05%) was added in order to stabilize the coating.
  • a lysate of a Borrelia burgdorferi culture was coated onto the polystyrene particles.
  • the coated latex beads are then blocked by a neutral BSA-sucrose solution (1 % BSA, 5% sucrose), BSA concentration around 1% and the sucrose.
  • the results of the agglutination immunoassay gives the following reactivity patterns: anti 41 kDa anti 31/34 kDa anti lysate reactivity interpretation reactivity reactivity neg neg neg not exposed patient pos neg neg or slightly pos new infection pos pos pos old infection, living spirochetes neg pos pos or slighlty pos old infection
  • the CMV Multiplex Reagent Kit is an homogeneous test for the simultaneous determination of different isotypes of anti-CMV antibodies developed by DiaSorin s.r.l. on the instrument Copalis I (see Example 1 ).
  • the CMV Multiplex assay is a test able to differentiate between acute infection stages and immune status to Cytomegalovirus, using coupled particle light scattering.
  • Acute IgM antibodies are detected by the CM2 and the p52 antigens, however, an antibody response to the CM2 antigen but not to the p52 antigen can only be detected during the convalescent stage of an acute infection. Furthermore, antibodies to the VP appear during acute infection and persist. Single reactivity to VP is, in contrast, characteristic of a prior infection and therefore indicative of an immune status.
  • Antibody detection (as carried out by the Copalis I Immunoassay System) is here documented qualitatively for the IgM analytes and semi-quantitatively for the IgG analytes.
  • Past CMV infection 150 expected IgG positive samples
  • ETI-CYTOK-G and ETI-CYTOK-M reverse see Example 1 : available from DiaSorin #2860 and #3238 and the test developed by Revello M.G. et al., J._ Virol. Methods 35 (1991 ), 315-329). These tests were employed according to the manufacturers instructions.
  • a sample was considered positive when a Cl of 1 or higher was obtained; a "grey zone" of ⁇ 5%, ⁇ 15% and ⁇ 20% across the threshold was set for VP, CM2 and p52, respectively.
  • said grey zone (“equivocal") is set between negative and positive results.
  • the equivocal zone setting is marker dependent: for example, in this CMV assay, a grey zone of ⁇ 5%, ⁇ 15%, ⁇ 20% on the Copalis Index is set for the considered markers (VP, CM2 and p52, respectively).
  • the Copalis test detected anti-lgM antibodies two blood withdrawals earlier than the comparison assay.
  • the clinical significance of this marker is different from a typical IgM marker and the additional advantage of its presence is the ability for differentiation of the early phase of an acute CMV infection: the majority (90.9%; i.e. 40 out of 44 samples) of samples (having an infection in the range of 60-90 days) showed positive results.
  • test efficacy of Copalis CMV Multiplex can be considered as 96.4% (348 samples out of the 361 tested were correctly classified by the test).
  • the assay is able to simultaneously distinguish between the acute and convalescent stages of CMV infection.
  • the first version consists of cups prepared according to the procedure described in Example 1 , i.e. beads were coated with the three different antigen p52, CM2 and viral particle.
  • patient 1 (# VT-872562) VP (viral particle) was positive for all the withdrawals of blood in both cases, showing a higher COPALIS index (Cl) without the anti-lgM antibody; p52 was positive in 2 out of 3 bleedings in both cases (+/- anti- lgM), CM2 became positive in the 2 nd withdrawal with the anti-lgM antibody and positive only at the 3 rd without the anti-lgM antibody.
  • CM2 was positive only in 3 out of 6 withdrawals without the anti-lgM antibody, whilst it was positive in all the withdrawals with the anti-lgM antibody.
  • p52 coated bead was in the grey zone without the anti-lgM antibody and was above the cut-off with the anti-lgM antibody in the first withdrawal only; interestingly the VP was positive in all the bleedings without the anti-lgM antibody and became positive only in 3 out of 6 with the addition of the anti-lgM antibody. This means that the addition of the anti-lgM antibody mediates a better sensitivity for IgM detection, but gives also a better specificity.
  • IgM are the main agglutinating factors of the p52 and CM2 particles and IgG of the VP particle. Furthermore, the CMV Multiplex COPALIS assay is able to detect the appearance of CMV infection not only with immunocompetent subjects but also with immunocompromised subjects such as transplanted patients. Table 16: Sera from transplantation patients Tested without anti-lgM

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Abstract

La présente invention concerne une méthode de détection d'anticorps et/ou d'isotypes d'anticorps spécifiques à au moins deux phases différentes d'une infection. La méthode selon l'invention se caractérise par l'incubation simultanée d'un échantillon provenant d'un patient avec deux composés de préférence antigènes. Ces composés comprennent un épitope qui est reconnu par un anticorps/anticorps appartenant à un isotype particulier, cet anticorps intervenant spécifiquement dans une des phases de l'infection. La méthode selon l'invention nécessite également la présence dans la réaction d'incubation d'au moins deux épitopes spécifiques aux différentes phases de l'infection qui lient ainsi des isotypes anticorps/anticorps spécifiques à au moins deux phases différentes de l'infection. L'invention concerne également une trousse s'utilisant pour réaliser la méthode selon l'invention.
PCT/EP1999/003841 1998-06-02 1999-06-02 Methode de detection d'anticorps specifiques a des phases d'infection WO1999063349A1 (fr)

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CA002329687A CA2329687A1 (fr) 1998-06-02 1999-06-02 Methode de detection d'anticorps specifiques a des phases d'infection
EP99929132A EP1084411A1 (fr) 1998-06-02 1999-06-02 Methode de detection d'anticorps specifiques a des phases d'infection
AU46050/99A AU4605099A (en) 1998-06-02 1999-06-02 Method for detecting infection-phase-specific antibodies

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IT98TO000473A ITTO980473A1 (it) 1998-06-02 1998-06-02 Nuovo procedimento per la rivelazione di anticorpi e/o isotipi di anticorpi specifici per almeno due differenti fasi di un'infezione

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102539756A (zh) * 2011-12-23 2012-07-04 王滔 测定胃黏膜样品中幽门螺杆菌的免疫¾®球方法
CN102565391A (zh) * 2011-12-23 2012-07-11 王滔 胃黏膜样品中幽门螺杆菌的免疫测定法
US9523685B2 (en) 2007-06-08 2016-12-20 Bio-Rad Innovations Multiplex method for detecting an infection
CN107942068A (zh) * 2017-11-15 2018-04-20 浙江夸克生物科技有限公司 β2‑微球蛋白测定试剂盒
WO2021030935A1 (fr) * 2019-08-16 2021-02-25 深圳迈瑞生物医疗电子股份有限公司 Nouveau mode de dosage immunologique dans son ensemble pour déterminer un anticorps total

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GB2026691A (en) * 1978-07-28 1980-02-06 Abbott Lab Determination of class-specific antibody
WO1989001154A1 (fr) * 1987-07-28 1989-02-09 International Institute Of Cellular & Molecular Pa DETERMINATION D'ANTICORPS IgM
US4877726A (en) * 1988-03-02 1989-10-31 Research Institute Of Palo Alto Medical Foundation Method for the detection of acute-phase toxoplasma infection
EP0366092A2 (fr) * 1988-10-25 1990-05-02 Roche Diagnostics GmbH Procédé pour déterminer des anticorps antigène- et classes-spécifiques et réactif pour ce procédé
WO1992015707A1 (fr) * 1991-02-27 1992-09-17 New York University Titrage immunologique pour anticorps contre l'adn oxyde et utilisations
WO1992021024A1 (fr) * 1991-05-20 1992-11-26 Sienna Biotech, Inc. Procedes d'analyses multiples simultanees
DE4435789C1 (de) * 1994-10-06 1995-12-21 Biotest Ag Peptide und Fusionsproteine umfassend eine aus dem Leserahmen UL 57 des Cytomegalievirus stammende Aminosäuresequenz und diese enthaltende diagnostische Testkits
WO1996004300A1 (fr) * 1994-07-29 1996-02-15 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Procedes et compositions servant a effectuer un diagnostic differentiel d'infection aigue et chronique par le virus de l'hepatite c

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2026691A (en) * 1978-07-28 1980-02-06 Abbott Lab Determination of class-specific antibody
WO1989001154A1 (fr) * 1987-07-28 1989-02-09 International Institute Of Cellular & Molecular Pa DETERMINATION D'ANTICORPS IgM
US4877726A (en) * 1988-03-02 1989-10-31 Research Institute Of Palo Alto Medical Foundation Method for the detection of acute-phase toxoplasma infection
EP0366092A2 (fr) * 1988-10-25 1990-05-02 Roche Diagnostics GmbH Procédé pour déterminer des anticorps antigène- et classes-spécifiques et réactif pour ce procédé
WO1992015707A1 (fr) * 1991-02-27 1992-09-17 New York University Titrage immunologique pour anticorps contre l'adn oxyde et utilisations
WO1992021024A1 (fr) * 1991-05-20 1992-11-26 Sienna Biotech, Inc. Procedes d'analyses multiples simultanees
WO1996004300A1 (fr) * 1994-07-29 1996-02-15 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Procedes et compositions servant a effectuer un diagnostic differentiel d'infection aigue et chronique par le virus de l'hepatite c
DE4435789C1 (de) * 1994-10-06 1995-12-21 Biotest Ag Peptide und Fusionsproteine umfassend eine aus dem Leserahmen UL 57 des Cytomegalievirus stammende Aminosäuresequenz und diese enthaltende diagnostische Testkits

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9523685B2 (en) 2007-06-08 2016-12-20 Bio-Rad Innovations Multiplex method for detecting an infection
EP2160605B1 (fr) * 2007-06-08 2017-11-01 Bio-Rad Innovations Méthode multiplex de détection d'une infection
CN102539756A (zh) * 2011-12-23 2012-07-04 王滔 测定胃黏膜样品中幽门螺杆菌的免疫¾®球方法
CN102565391A (zh) * 2011-12-23 2012-07-11 王滔 胃黏膜样品中幽门螺杆菌的免疫测定法
CN107942068A (zh) * 2017-11-15 2018-04-20 浙江夸克生物科技有限公司 β2‑微球蛋白测定试剂盒
WO2021030935A1 (fr) * 2019-08-16 2021-02-25 深圳迈瑞生物医疗电子股份有限公司 Nouveau mode de dosage immunologique dans son ensemble pour déterminer un anticorps total

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