EP0527200A1 - Procede servant a determiner des anticorps specifiques au type du virus de l'herpes simple des types 1 et 2 - Google Patents

Procede servant a determiner des anticorps specifiques au type du virus de l'herpes simple des types 1 et 2

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Publication number
EP0527200A1
EP0527200A1 EP19910909576 EP91909576A EP0527200A1 EP 0527200 A1 EP0527200 A1 EP 0527200A1 EP 19910909576 EP19910909576 EP 19910909576 EP 91909576 A EP91909576 A EP 91909576A EP 0527200 A1 EP0527200 A1 EP 0527200A1
Authority
EP
European Patent Office
Prior art keywords
hsv
antibodies
labeled
antibody
monoclonal antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19910909576
Other languages
German (de)
English (en)
Inventor
Mark Norman Bobrow
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
EIDP Inc
Original Assignee
EI Du Pont de Nemours and Co
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by EI Du Pont de Nemours and Co filed Critical EI Du Pont de Nemours and Co
Publication of EP0527200A1 publication Critical patent/EP0527200A1/fr
Withdrawn legal-status Critical Current

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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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56994—Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus

Definitions

  • This invention concerns a method of serotyping antibodies to herpes simplex virus types 1 and 2, and, in particular, to a method of serotyping which employs monoclonal antibodies to HSV-1 gG and HSV-2 gG which are not susceptible to heterologous blocking.
  • herpes simplex virus HSV
  • HSV herpes simplex virus
  • types 1 and 2 which crossreact.
  • One of the major problems in seroepidemiologic studies of HSV infection is the differentiation of antibodies in human serum directed against HSV-1 and HSV-2.
  • researchers have attempted to overcome these problems using purified HSV glycoproteins and absorption to remove cross reacting antibodies in an effort to develop a rapid method of distinguishing HSV-1 and HSV-2 antibodies.
  • Such methods have important clinical and epide iological significance because it permits diagnosis of persons with asymptomatic HSV infection, identification of candidates for antiviral therapy, as well as identification of pregnant women who may transmit a potentially fatal HSV infection to the newborn.
  • the absorbance index defined as the ratio of the absorbance generated by a serum sample absorbed with a heterologous virus- infected cell extract versus the absorbance generated by a serum sample absorbed with an uninfected cell extract, was used to determine the presence or absence of antibodies to HSV-1 and HSV-2.
  • This invention concerns a method of serotyping antibodies to HSV 2 or 1 or in a sample suspected to contain HSV antibodies which comprises: a) reacting the sample and at least one monoclonal antibody specific for HSV-2 or HSV-1 glycoprotein G with an antigen wherein said monoclonal antibody is not susceptible to heterologous blocking; and b) determining whether antibody binding has occurred.
  • the serotyping method of the invention can be done in a variety of formats such as sequential or simultaneous.
  • this invention concerns kits for serotyping antibodies to HSV-1 and HSV-2.
  • BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph depicting the absorbance values for an assay used to determine anti-HSV-1 antibodies.
  • Figure 2 is a graph depicting the absorbance values for an assay to determine HSV-2 antibodies.
  • HSV-2 glycoprotein G refers to the HSV-2 glycoprotein as described by Roizman et al., Virology 133 242-247, 1984; Marsden et al., J. Virology 50. 547-554, 1984; Lee et al., J. Clin. Microbiol. 22., 641-644, 1985; Balachandran et al., J. Virology $£, 825-832, 1985; and Su et al., J. Virology£., 3668-3674, 1988.
  • HSV-1 glycoprotein G refers to the HSV-1 glycoprotein as described by Ackermann et al., Virology 150. 207-220, 1986; Richman et al., J. Virology 5L, 647-655, 1986; and Sullivan et al., J. Gen. Virol. £&, 2587-2598, 1987.
  • the assay of the present invention is an extraordinarily simple and specific method to serotype antibodies to HSV-2 or HSV-1 which overcomes the cross- reactivity problems which have plagued researchers in differentiating these antibodies.
  • HSV-1 antibodies are serotyped by using at least one monoclonal antibody specific for HSV-1 glycoprotein G which is not susceptible to heterologous blocking.
  • HSV-2 antibodies are serotyped by using at least one monoclonal antibody specific for HSV-2 glycoprotein G which is not susceptible to heterologous blocking. These monoclonal antibodies can be unlabeled or labeled as is discussed below.
  • HSV antigen, types 1 and 2 is immobilized on a suitable support.
  • a sample usually serum or plasma
  • HSV antibodies which, if present, will bind to the antigen, and, thus, block binding of the monoclonal antibody.
  • a labeled monoclonal antibody is incubated with the mixture.
  • Antibodies to HSV-1 are detected by using a monoclonal antibody specific for HSV-1 glycoprotein G .
  • Antibodies to HSV-2 are detected by using a monoclonal antibody specific for HSV-2 glycoprotein G.
  • the mixture is washed again and antibody binding is determined by measuring the amount of reporter bound to the solid support.
  • the sample need not be limited to serum or plasma. Any biological fluid suspected to contain HSV antibodies can be used as the sample.
  • the antibody can be labeled with a reporter or with a member of an immune or non-immune specific binding pair. When the latter is used, detection is effected by reacting the complex with the other member of the binding pair which is labeled.
  • Immunospecific binding pairs suitable for practicing the invention can be of the immune or non- immune type.
  • immune specific binding pairs include antigen-antibody systems or hapten-anti-hapten systems.
  • the antibody member of the specific binding pair can be produced by customary methods familiar to those skilled in the art. Such methods involve immunizing an animal with the antigen member of the specific binding pair. If the antigen member of the specific binding pair is not immunogenic, i.e., a hapten, it can be covalently coupled to a carrier protein to render it immunogenic.
  • the antibody member whether polyclonal, monoclonal, or an immunoreactive fragment thereof can be produced by conventional techniques well known to those skilled in the art.
  • the terms immunoreactive antibody fragment or immunoreactive fragment mean fragments which contain the binding region of the antibody.
  • Such fragments can be Fab-type fragments which are defined as fragments devoid of the Fc portion, e.g.. Fab, Fab' and F(ab') 2 fragments, or may be so-called "half-molecule" fragments obtained by reductive cleavage of the disulfide bonds connecting the heavy chain components of the intact antibody.
  • Non-immune binding pairs include systems wherein the two components share a natural affinity for each other but are not antibodies.
  • Exemplary non-immune binding pairs are biotin-avidin or biotin-streptavidin, folic acid-folate binding protein, complementary probe nucleic acids, etc.
  • Biotin can be covalently coupled to monoclonal antibodies by utilizing commercially available active derivatives. Some of these are biotin-N-hydroxysuccinimide which binds to amine groups on proteins; biotin hydrazide which binds to carbohydrate moieties, aldehydes and carboxyl groups via a carbodiimide coupling; and biotin maleimide and iodoacetyl biotin which bind to sulfhydryl groups.
  • Fluorescein can be coupled to protein amine groups using fluorescein isothiocyanate. Dinitrophenyl groups can be coupled to protein amine groups using 2,4-dinitrobenzene sulfonate or 2,4-dinitrofluoro- benzene.
  • Other standard methods of conjugate may be employed to couple monoclonal antibodies to a member of a specific binding pair including dialdehyde, carbodiimide coupling, homofunctional crosslinking, and heterobifunctional crosslinking.
  • Carbodiimide coupling is an effective method of coupling carboxyl groups on one substance to amine groups on another.
  • Carbodiimide coupling is facilitated by using the commercially available reagent, l-ethyl-3-(dimethylaminopropyl)- carbodiimide (EDAC) .
  • Homobifunctional crosslinkers including the bifunctional i idoesters and bifunctional N-hydroxy- succinimide esters, are commercially available and are employed for coupling amine groups on one substance to amine groups on another.
  • Heterobifunctional crosslinkers are reagents which possess different functional groups. The most common commercially available heterobifunctional crosslinkers have an amine reactive N-hydroxysuccinimide ester as one functional group, and a sulfhydryl reactive group as the second functional group.
  • the most common sulfhydryl reactive groups are maleimides, pyridyl disulfides and active halogens.
  • One of the functional groups may be a photoactive aryl nitrene, which upon irradiation reacts with a variety of groups.
  • a reporter can be linked directly or indirectly, covalently or non-covalently to the monoclonal antibodies or to a member of a specific binding pair. Reporters can be radioactive isotopes, enzymes, fluorogenic, chemiluminescent or electrochemical materials. Two commonly used radioisotopes are 125 I and 3 H.
  • Standard radioactive isotopic labeling procedures include the chloramine T, lactoperoxidase and Bolton- Hunter methods for 125 I and reductive methylation for 3 H.
  • Enzymes are also used as reporters for immunoassays. These include, but are not limited to, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, glucose oxidase, luciferase, ⁇ -lactamase, urease and lysozyme. Labeling with enzymes is facilitated by using dialdehyde, carbodiimide coupling, homobifunctional crosslinkers and heterobifunctional crosslinkers as described above for labeling monoclonal antibodies with members of specific binding pairs.
  • the labeling method chosen depends on the functional groups available on the enzyme and the material to be labeled, and the tolerance of both to the conjugation conditions.
  • the labeling method used in the present invention may be one of, but not limited to, any conventional methods currently employed including those described by Engvall and Pearlmann, Immunochemistry 8, 871 (1971), Avrameas and Ternynck, Immunochemistry 8, 1175 (1971), Ishikawa et al., J. Immunoassay 4(3):209- 327 (1983) and Jablonski, Anal. Biochem. 148:199 (1985). Labeling may be accomplished by indirect methods such as using spacers or other members of specific binding pairs.
  • biotinylated antibodies with unlabeled streptavidin and biotinylated enzyme, with the unlabeled streptavidin and biotinylated enzyme being added either sequentially or simultaneously. Detection of enzyme activity may be facilitated by measuring chromogenic, fluorogenic, che iluminescent or electrochemical changes by commonly known methods.
  • Reporters can be fluorogenic or chemiluminescent in nature. In addition, reporters can be detectable by electrochemical means. Some methods of labeling with these reporters are described above.
  • the enzyme, horseradish peroxidase is used as the reporter.
  • the monoclonal antibody need not be labeled. If it is unlabeled, it can be detected using a labeled anti-species immunoglobulin reagent.
  • the combination should be specific for either HSV-1 gG or HSV-2 gG.
  • Another embodiment involves the simultaneous, rather than sequential, addition of sample and monoclonal antibody to a support containing immobilized antigen. This is illustrated in the examples described below.
  • at least one monoclonal antibody can be immobilized on a support. It can then be reacted with labeled antigen and sample or the labeled antigen and sample can be preincubated and then reacted with the support containing immobilized monoclonal antibody.
  • Suitable supports include synthetic polymer supports, such as polystyrene, polypropylene, substituted polystyrene, e.g., aminated or carboxylated polystyrene; polyacrylamides; polyamides; polyvinyl- chloride, etc.; glass beads; agarose; nitrocellulose, etc. 10
  • kits for serotyping antibodies to HSV-1 or HSV-2 or both HSV-1 and HSV-2 comprises (a) a support containing immobilized HSV antigen, (b) a quantity of at least one monoclonal antibody specific for HSV-1, HSV-2 or a combination thereof wherein said antibody is not susceptible to heterologous blocking and further wherein said antibody is either (i) unlabeled, (ii) labeled with a reporter, or (iii) labeled with a first member of a specific binding pair, and (c) a quantity of labeled anti-antibody when the monoclonal antibody is unlabeled or a quantity of a labeled second member of the binding pair when the monoclonal antibody is labeled with the first member of the specific binding pair.
  • Monoclonal Antibodies were prepared according to the methods of L. Pereira et al., as described in Infection and Immunity, Vol. 29, No. 2, pp. 724-732 (Aug. 1980) and Oi, et al., as described in Immunoglobulin Producing Hybrid Cell Lines, pp. 351-371, in B. Mishell and S. Schiigi (ed.). Selected Methods in Cellular Immunology, W. H. Freeman Co., San Francisco. Monoclonal antibodies were purified from ascites fluid by salt fractionation with 40% saturated ammonium sulfate and gel filtration on Sephacryl S-300 (Pharmacia, Inc., Piscataway, NJ) . Purified monoclonal antibodies were labeled with horseradish peroxidase (HRP) according to the SMCC method described by Ishikawa 11
  • HR horseradish peroxidase
  • HSV-2 antigen Scripps Laboratories, San Diego, CA
  • PBS phosphate buffered saline
  • TMB tetramethylbenzidine
  • the monoclonal antibodies were diluted into the serum specimens, added to the wells, and incubated for 1 hr at 37°C. Following washing with PBST, TMB was added and absorbance determined as described for the sequential format.
  • Polystyrene microplate wells were coated with a combination of HSV-1 and HSV-2 antigens (Scripps Laboratories, San Diego, CA) as described in Example 1 above.
  • HRP labeled monoclonal antibody 55315 (anti-HSV-1 gG) diluted in 1% BSA-PBST, was added for 1 hr at 37°C. Following washing with PBST, TMB was added, and the absorbance determined as described in Example 1. 13
  • Figure 1 is a graph showing the absorbance values for the twenty anti-HSV-1 and twenty anti-HSV-2 antibody containing sera used in an assay to determine anti-HSV-1 antibodies.
  • the sera containing anti-HSV-1 antibodies blocked the binding of the anti-HSV-1 gG monoclonal antibody. No such blocking was observed with the sera containing anti-HSV-2 antibodies.
  • Figure 2 is a graph showing the absorbance values obtained for an assay to determine anti-HSV-2 antibodies.
  • the sera containing anti-HSV-2 antibodies blocked the binding of the anti-HSV-2 gG monoclonal antibody. No such blocking was observed with the sera containing anti-HSV-1 antibodies.
  • Polystyrene microplate wells were coated with a combination of HSV-1 and HSV-2 antigens as described in Example 1 above.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Virology (AREA)
  • Immunology (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
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Abstract

Cette invention se rapporte à un procédé qui sert à effectuer le typage sérologique d'anticorps du virus de l'herpès simple (HSV) des types 2 ou 1 ou se trouvant dans un échantillon suspecté de contenir des anticorps anti-HSV et qui consiste: (a) à faire réagir l'échantillon ou au moins un anticorps monoclonal spécifique à la glycoprotéine G du virus HSV-2 ou HSV-1 avec un antigène dans lequel cet anticorps monoclonal n'est pas exposé à un blocage hétérologue; et (b) à déterminer si une liaison anticorpale se produit. Ce procédé de typage sérologique peut être utilisé selon une variété de formats, par exemple séquentiel ou simultané. Dans un autre mode de réalisation, cette invention décrit des kits servant à effectuer le typage sérologique d'anticorps des virus HSV-1 et HSV-2.
EP19910909576 1990-05-04 1991-04-30 Procede servant a determiner des anticorps specifiques au type du virus de l'herpes simple des types 1 et 2 Withdrawn EP0527200A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US51932990A 1990-05-04 1990-05-04
US519329 1990-05-04

Publications (1)

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EP0527200A1 true EP0527200A1 (fr) 1993-02-17

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EP19910909576 Withdrawn EP0527200A1 (fr) 1990-05-04 1991-04-30 Procede servant a determiner des anticorps specifiques au type du virus de l'herpes simple des types 1 et 2

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EP (1) EP0527200A1 (fr)
CA (1) CA2081888A1 (fr)
IE (1) IE911468A1 (fr)
WO (1) WO1991017443A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5965354A (en) * 1995-07-28 1999-10-12 Chiron Corporation Herpes simplex virus diagnostics
GB9615533D0 (en) * 1996-07-24 1996-09-04 Peptide Therapeutics Ltd Diagnostic test for herpes simplex virus type-2

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9117443A1 *

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IE911468A1 (en) 1991-11-06
WO1991017443A1 (fr) 1991-11-14
CA2081888A1 (fr) 1991-11-05

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