EP1725687A4 - SPECIFIC IMMUNOLOGICAL ASSAYS OF TYPE 2 VHS USING GLYCOPROTEIN G2 PEPTIDES - Google Patents

SPECIFIC IMMUNOLOGICAL ASSAYS OF TYPE 2 VHS USING GLYCOPROTEIN G2 PEPTIDES

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Publication number
EP1725687A4
EP1725687A4 EP04718011A EP04718011A EP1725687A4 EP 1725687 A4 EP1725687 A4 EP 1725687A4 EP 04718011 A EP04718011 A EP 04718011A EP 04718011 A EP04718011 A EP 04718011A EP 1725687 A4 EP1725687 A4 EP 1725687A4
Authority
EP
European Patent Office
Prior art keywords
peptide
heterologous
amino acid
carrier
linker
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.)
Ceased
Application number
EP04718011A
Other languages
German (de)
English (en)
French (fr)
Other versions
EP1725687A2 (en
Inventor
Peilin Chen
Peter Su
Hao Yu
Larry Blecka
Patrick F Coleman
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.)
Bio Rad Laboratories Inc
Original Assignee
Bio Rad Laboratories Inc
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Filing date
Publication date
Application filed by Bio Rad Laboratories Inc filed Critical Bio Rad Laboratories Inc
Publication of EP1725687A2 publication Critical patent/EP1725687A2/en
Publication of EP1725687A4 publication Critical patent/EP1725687A4/en
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16622—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/03—Herpetoviridae, e.g. pseudorabies virus
    • G01N2333/035—Herpes simplex virus I or II
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • HSV-2 herpes simplex virus type 2
  • HSV-1 herpes simplex virus type 1
  • HSV-1 usually causes oro labial disease
  • HSV-2 almost always leads to genital disease.
  • HSV-2 specific antibodies have been developed in an effort to identify HSV-2 specific antibodies.
  • the most reliable method for a type-specific detection of HSV-2 antibodies to date is an immunoblot assay, preferably a Western blot assay.
  • the significant drawback of this method is that the procedure is labor-intensive and requires the investigator to have a certain level of skill in order to achieve unequivocal results.
  • HSV glycoproteins have been identified as the viral proteins that contain type- specific epitopes.
  • hnmunoassays have been developed based on these glycoproteins for type- specific determination of HSV-2 infection. See, e.g., Lee et al., J. Clin. Microbiol.
  • glycoprotein-based immunoassays for HSV-2 antibody type-specific detection.
  • the full length glycoproteins are obtained through isolation of either naturally-occurring viral proteins or recombinantly expressed proteins. These procedures can be costly and susceptible to impurities and thus cross-reactivity.
  • peptides corresponding to partial sequences of certain viral proteins of HSV-2 may be useful in HSV-2 type-specific detection, as these peptides may represent some HSV-2 type-specific epitopes. See, e.g., Levi et al., Clin. Diagn. Lab. Immunol.
  • the present invention provides novel peptide sequences of HSV-2 glycoprotein G2 that can be used in HSV-2 type-specific diagnosis.
  • the present invention relates to a composition containing a peptide that binds specifically to HSV-2 antibodies and reacts minimally to HSV-1 specific antibodies or antibodies to any other herpes family viruses.
  • This peptide consists of 24 to 36 contiguous amino acids of SEQ ID NO:l.
  • the peptide has a sequence consisting of amino acids 5 to 32 of SEQ ID NO:l.
  • the peptide has a sequence consisting of amino acids 9 to 32 of SEQ ID NO:l.
  • the peptide has an amino acid sequence of SEQ ID NO:l.
  • the peptide is dimerized. Such dimerization may be achieved via a disulfide bond.
  • the peptide is linked to a carrier. Examples of suitable carriers are carboxylated microspheres, preferably carboxylated latex or magnetic microspheres.
  • the peptide is linked to a carrier via a linker at the N- terminus or the C-terminus of the peptide, whereas in other embodiments the peptide is linked to a carrier via a linker at an internal amino acid residue of the peptide.
  • the linker is or includes a heterologous peptide.
  • the linker is or includes a heterologous protein.
  • both a heterologous protein and a heterologous peptide are used.
  • the entire linker is a heterologous peptide.
  • the heterologous protein is bovine serum albumin (BSA), whereas in another preferred embodiment, the heterologous protein is Keyhole Limpet Hemocyanin (KLH).
  • the heterologous peptide includes one cysteine residue, one lysine residue, and at least two glycine residues.
  • the linker includes a branched amino acid polymer, whose structure is preferably that shown below:
  • the peptide has an amino acid sequence of SEQ ID NO: 1
  • the carrier is a carboxylated magnetic microsphere
  • the linker includes 4-
  • the linker further includes the heterologous protein BSA directly attached to the carrier, and SMCC is attached to BSA and, via the heterologous peptide, to the peptide.
  • the peptide has an amino acid sequence consisting of amino acids 5 to 32 of SEQ ID NO:l
  • the carrier is a carboxylated magnetic - . microsphere
  • the linker includes SMCC and a heterologous peptide with an amino acid sequence of GGGGCK, and the heterologous peptide is attached to the peptide at the C- terminus of the peptide.
  • the linker further includes the heterologous protein BSA directly attached to the carrier, and SMCC is attached to BSA and, via the heterologous peptide, to the peptide.
  • the peptide has an amino acid sequence consisting of amino acids 9 to 32 of SEQ ID NO:l
  • the carrier is a carboxylated magnetic microsphere
  • the linker includes SMCC and a heterologous peptide with an amino acid sequence of GGGGCK, and the heterologous peptide is attached to the peptide at the C- terminus of the peptide.
  • the linker further includes the heterologous protein BSA directly attached to the carrier, and SMCC is attached to BSA and, via the heterologous peptide, to the peptide.
  • the peptide has an amino acid sequence consisting of amino acids 9 to 32 of SEQ ID NO:l
  • the carrier is a carboxylated magnetic microsphere
  • the linker includes SMCC and a heterologous peptide with an amino acid sequence of KCGGGG
  • the heterologous peptide is attached to the peptide at the N- terminus of the peptide.
  • the linker further includes the heterologous protein BSA directly attached to the carrier, and SMCC is attached to BSA and, via the heterologous peptide, to the peptide.
  • the peptide has an amino acid sequence consisting of amino acids 9 to 32 of SEQ ID NO:l
  • the carrier is a carboxylated microsphere
  • the linker includes a branched amino acid polymer that has the following structure and further includes a short peptide of CK, which via the C residue is directly attached to the last K residue of the structure:
  • the present invention relates to a method for type-specific diagnosis of HSV-2 infection.
  • the method for specific detection of HSV-2 antibodies in a biological sample includes two steps.
  • the first step is contacting the biological sample with a composition that includes a peptide consisting of 24 to 36 contiguous amino acids of SEQ ID NO: 1, linked to a carrier.
  • the second step is detecting whether antigen-antibody binding has occurred between the peptide and an antibody component of the biological sample.
  • the detection of antigen-antibody binding indicates the presence of HSV-2 antibodies in the biological sample.
  • the second step is performed by flow cytometry.
  • the biological sample be whole blood, serum, plasma, cerebrospinal fluid, tissue from a swab device, or vesicle fluid.
  • the peptide has a sequence consisting of amino acids 5 to 32 of SEQ ID NO: 1. In a more preferred embodiment, the peptide has a sequence consisting of amino acids 9 to 32 of SEQ ID NO:l. In a most preferred embodiment, the peptide has an amino acid sequence of SEQ ID NO:l. [0017] In some embodiments, the peptide is dimerized. Such dimerization may be achieved via a disulfide bond. In some other embodiments, the peptide is linked to a carboxylated microsphere, preferably a carboxylated latex or magnetic microsphere.
  • the peptide is linked to the carrier via a linker at the N- terminus or the C-terminus of the peptide, whereas in other embodiments the peptide is linked to a carrier via a linker at an internal amino acid residue of the peptide.
  • the linker includes a heterologous peptide.
  • the linker includes a heterologous protein.
  • the linker includes a heterologous protein in addition to a heterologous peptide.
  • the linker is a heterologous peptide.
  • the heterologous protein is BSA, whereas in another preferred embodiment, the heterologous protein is KLH.
  • the heterologous peptide includes one cysteine residue, one lysine residue, and at least two glycine residues.
  • the linker includes a branched amino acid polymer, whose structure is preferably that shown below:
  • the peptide has an amino acid sequence of SEQ ID NO: 1
  • the carrier is a carboxylated magnetic microsphere
  • the linker includes SMCC and a heterologous peptide with an amino acid sequence of GGCK
  • the heterologous peptide is attached to the peptide at the C-terminus of the peptide
  • the detection of antigen-antibody binding is achieved by flow cytometry.
  • the linker further includes the heterologous protein BSA directly attached to the carrier, and SMCC is attached to BSA and, via the heterologous peptide, to the peptide.
  • the peptide has an amino acid sequence consisting of amino acids 5 to 32 of SEQ ID NO:l
  • the carrier is a carboxylated magnetic microsphere
  • the linker includes SMCC and a heterologous peptide with an amino acid sequence of GGGGCK
  • the heterologous peptide is attached to the peptide at the C-terminus of the peptide, and the detection of antigen-antibody binding is achieved by flow cytometry.
  • the linker further includes the heterologous protein BSA directly attached to the carrier, and SMCC is attached to BSA and, via the heterologous peptide, to the peptide.
  • the peptide has an amino acid sequence consisting of amino acids 9 to 32 of SEQ ID NO:l
  • the carrier is a carboxylated magnetic microsphere
  • the linker includes SMCC and a heterologous peptide with an amino acid sequence of GGGGCK
  • the heterologous peptide is attached to the peptide at the C-terminus of the peptide, and the detection of antigen-antibody binding is achieved by flow cytometry.
  • the linker further includes the heterologous protein BSA directly attached to the carrier, and SMCC is attached to BSA and, via the heterologous peptide, to the peptide.
  • the peptide has an amino acid sequence consisting of amino acids 9 to 32 of SEQ ID NO: 1
  • the carrier is a carboxylated magnetic microsphere
  • the linker includes SMCC and a heterologous peptide with an amino acid sequence of KCGGGG
  • the heterologous peptide is attached to the peptide at the N- terminus of the peptide.
  • the linker further includes the heterologous protein BSA directly attached to the carrier, and SMCC is attached to BSA and, via the heterologous peptide, to the peptide.
  • the peptide has an amino acid sequence consisting of amino acids 9 to 32 of SEQ ID NO:l
  • the carrier is a carboxylated microsphere
  • the linker includes a branched amino acid polymer that has the following structure and further includes a short peptide of CK, which via the C residue is directly attached to the last K residue of the structure :
  • Figure 1 shows SEQ ID NO: 1 , ⁇ PGSPAPPPPEHRGGPEEFEGAGDGEPPEDDDSATGL 36
  • Figure 2 shows the correlation between HSV-2 specific antibody detection using a commercial HSV-2 type-specific enzyme-linked immunoassay and using the method of the present invention where peptide 1-SMCC-BSA conjugate,
  • PSPAPPPPEHRGGPEEFEGAGDGEPPEDDDSATGLGGCK is used as an HSV-2 specific antigen.
  • Figure 3 shows the correlation between HSV-2 specific antibody detection using a commercial HSV-2 type-specific enzyme-linked immunoassay and using the method of the present invention where peptide 2-SMCC-BSA conjugate,
  • FIG. 4 shows the correlation between HSV-2 specific antibody detection using a commercial HSV-2 type-specific enzyme-linked immunoassay and using the method of the present invention where BSA-SMCC-peptide 5 conjugate, BSA-SMCC- (KCGGGGPEHRGGPEEFEGAGDGEPPEDDDS), is used as an HSV-2 specific antigen.
  • Figure 5 shows the correlation between HSV-2 specific antibody detection using a commercial HSV-2 type-specific enzyme-linked immunoassay and using the method of the present invention where peptide 5, KCGGGGPEHRGGPEEFEGAGDGEPPEDDDS, is used as an HSV-2 specific antigen.
  • the present invention relates to peptides that consist of partial sequences of HSV-2 glycoprotein G2 and represent HSV-2 type-specific epitopes.
  • HSV-2 peptides consisting of 24 to 36 contiguous amino acids of SEQ ID NO:l bind HSV-2 antibodies with high specificity and sensitivity, while their cross-reactivity to HSV-1 antibodies is minimal.
  • these peptides are useful for type-specific sero logical diagnosis of HSV-2 infection, particularly for differentiation of HSV-2 infection from HSV-1 infection.
  • compositions comprising peptides of the present invention are also provided for type-specific sero logical diagnosis of HSV-2 infection.
  • the peptides of the present invention are linked to a carrier, such as a carboxylated latex or magnetic microsphere, via a linker that includes a heterologous peptide and/or protein, such as bovine serum albumin (BSA) and Keyhole Limpet Hemocyanin (KLH).
  • BSA bovine serum albumin
  • KLH Keyhole Limpet Hemocyanin
  • Other linkers such as branched amino acid polymers, straight or branched-chain carbon linkers, heterocyclic carbon linkers (e.g., SMCC), or polyether linkers, may also be used in practicing the present invention.
  • An HSV-2 peptide of this invention may be conjugated to a carrier at the N- or C-terminus of the peptide, or via an internal amino acid residue.
  • HSV-2 antibodies are provided.
  • the detection of HSV-2 specific antibodies is performed by flow cytometry.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ - carboxyglutamate, and O-phosphoserine.
  • amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g. , norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
  • polypeptide refers to a polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
  • the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.
  • An HSV-2 peptide as used in the present application refers to a peptide that has a sequence corresponding to a segment of the HSV-2 glycoprotein G2.
  • a “carrier” as used herein refers to an inert solid support of natural material, such as glass and collagen, or synthetic material, such as acrylamide, cellulose, nitrocellulose, silicone rubber, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polysilicates, polyethylene oxide, polycarbonates, teflon, fluorocarbons, nylon, polyanhydrides, polyglycolic acid, polylactic acid, polyorthoesters, polypropylfumarate, glycosaminoglycans, and polyamino acids.
  • natural material such as glass and collagen
  • synthetic material such as acrylamide, cellulose, nitrocellulose, silicone rubber, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polysilicates, polyethylene oxide, polycarbonates, teflon, fluorocarbons, nylon, polyanhydrides, polyglycolic acid, polylactic
  • a carrier of the present invention is a carboxylated latex or magnetic microsphere.
  • a "linker" as used herein refers to any means of connecting a peptide of the present invention to a carrier.
  • the linkage may be located at either the N-terminus or the C- terminus of the peptide. In some embodiments, the linkage can be effectuated through a side group of an internal amino acid residue of the peptide, such as a second carboxyl group of an aspartate residue that is not located at the N- or C-terminus of the peptide.
  • a linker may comprise a peptide (e.g., the short amino acid sequence of GGGGCK or GGCK) or a protein (e.g., BSA or KLH) that is heterologous to the peptide of the present invention.
  • a linker may further comprise a heterologous protein, such as BSA or • KLH.
  • Non-polypeptide linkers such as straight or branched-chain carbon linkers, heterocyclic carbon linkers, or polyether linkers, may also be suitable for the purpose of connecting a peptide of the invention to a carrier, or as a part of a multiple-component linker.
  • One preferred non-polypeptide linker is SMCC.
  • a linker may comprise a branched amino acid polymer that is capable of attaching more than one peptide of the invention to a carrier. Both heterobifunctional and monobifunctional linkers are suitable in the practice of the present invention.
  • a linker may also comprise a covalent bond, such as a peptide bond or a disulfide bond, or a noncovalent bond, such as an ionic or hydrophobic bond, in the case where a peptide of the present invention is attached directly to a carrier.
  • a non-covalent bond is the interaction between antigen-antibody or biotin-avidin.
  • the linker peptide and/or linker protein and the peptide of the invention do not produce an amino acid sequence that is found in nature.
  • the peptides of the present invention are derived from HSV-2 glycoprotein G2, a heterologous peptide or protein is therefore not derived from any sequence of HSV-2 glycoprotein G2 that is contiguous to the sequence from which the peptides of the present invention are derived.
  • Specific detection refers to the fact that detection of any antibody bound to the peptides of the present invention is determinative of the presence of HSV-2 antibody, often in a heterogeneous population of other antibodies and proteins. In particular, the presence of HSV-1 antibodies will not result in a detectable amount of antibody bound to the peptides.
  • the term “specific detection” particularly encompasses the use of HSV-2 peptides of the present invention to differentiate HSV-2 infection from HSV-1 infection. Under designated immunoassay conditions, a detectable signal is designated as one that is at least twice the background signal. Thus, specific antibody-peptide binding should yield a signal at least two times, preferably more than 10 times, and more preferably more than 100 times the background.
  • the term "dimerized” or “dimer” when used to describe a peptide of the present invention refers to the complex formed by two molecules comprising the same peptide via a covalent bond, such as a disulfide bond, or a noncovalent bond, such as via the binding interaction between a known tag and tag-binder pair (e.g., biotin and avidin).
  • a covalent bond such as a disulfide bond
  • a noncovalent bond such as via the binding interaction between a known tag and tag-binder pair (e.g., biotin and avidin).
  • the covalent bond or noncovalent bond typically occurs between the linker portions of the two peptide-containing molecules.
  • an internal amino acid residue refers to any amino acid residue that is not the first residue from either the N-terminus or the C-terminus of an HSV-2 peptide of the present invention.
  • biological sample refers to sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histological purposes. Such samples may include whole blood, serum, plasma, cerebrospinal fluid, sputum, tissue, cultured cells, e.g., primary cultures, explants, transformed cells, stool, urine, vesicle fluid, mucus, and other bodily secretion, or tissue that could be sampled with a swab device.
  • a biological sample is typically obtained from a human who may have been infected with HSV-2.
  • antibody denotes a protein of the immunoglobulin family or a polypeptide including fragments of an immunoglobulin that is capable of noncovalently, reversibly, and in a specific manner binding a corresponding antigen.
  • An illustrative antibody structural unit includes a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy” chain (about 50-70 kD), connected through one or more disulfide bonds.
  • the recognized immunoglobulin genes include the K, ⁇ , a, ⁇ , ⁇ , e, and ⁇ constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Light chains are classified as either K or ⁇ Heavy chains are classified as y, ⁇ , a, ⁇ , or e, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively.
  • the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the terms variable light chain (VL) and variable heavy chain (VH) refer to these regions of light and heavy chains respectively.
  • CDRs complementarity-determining domains
  • the CDR is the target protein-binding site of the antibody chain that harbors specificity for that target protein.
  • CDR1- 3 There are three CDRs (CDR1- 3, numbered sequentially from the N-terminus) in each human VL or V H , constituting about 15-20% of the variable domains.
  • the CDRs are structurally complementary to the epitope of the target protein and are thus directly responsible for the binding specificity.
  • the remaining stretches of the V L or VH, the so-called framework regions exhibit less variation in amino acid sequence (Kuby, Immunology, 4th ed., Chapter 4, W.H.
  • the positions of the CDRs and framework regions are determined using various well known definitions in the art, e.g., Kabat, Chothia, International ImMunoGeneTics database ( GT), and AbM (see, e.g., Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol, 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J. Mol. Biol, 227:799-817 (1992); Al-Lazikani et al., J.Mol.Biol.
  • antibody light chain and “antibody heavy chain” denote the V L or
  • VH VH, respectively.
  • the V is encoded by the gene segments V (variable) and J (junctional), and the VH is encoded by V, D (diversity), and J.
  • V L or V H includes the CDRs as well as the framework regions.
  • Antibodies exist as intact immunoglobulins or as a number of well- characterized fragments produced by digestion with various peptidases.
  • pepsin digests an antibody below the disulfide linkages in the hinge region to produce F( ab )' 2 , a dimer of F a b' which itself is a light chain joined to V H -CH1 by a disulfide bond.
  • the F( aD )' 2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F( at> )' 2 dimer into an F a b' monomer.
  • the F ab ' monomer is essentially F ab with part of the hinge region (Paul, Fundamental Immunology 3d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain F v ) or those identified using phage display libraries (see, e.g., McCafferty et al., Nature, 348:552-554 (1990))
  • any technique known in the art can be used (see, e.g., Kohler & Milstein, Nature, 256:495-497 (1975); Kozbor et al., Immunology Today, 4:72 (1983); Cole et al., Monoclonal Antibodies and Cancer Therapy, pp. 77-96. Alan R. Liss, Inc. 1985).
  • Techniques for the production of single chain antibodies can be adapted to produce antibodies to polypeptides of this invention.
  • transgenic mice, or other organisms such as other mammals may be used to express humanized antibodies.
  • phage display technology can be used to identify antibodies, and heteromeric F a fragments, or scFv fragments that specifically bind to selected antigens (see, e.g., McCafferty et al., supra; Marks et al., Biotechnology, 10:779- 783, (1992)).
  • HSV-2 antibody refers to an antibody that is specifically reactive to HSV-2 antigens but not to antigens of any other source, particularly HSV-1.
  • the peptides of the present invention may be synthesized chemically using conventional peptide synthesis or other protocols well known in the art.
  • Peptides may be synthesized by solid-phase peptide synthesis methods using procedures similar to those described by Merrifield et al., J. Am. Chem. Soc, 85:2149-2156 (1963); Barany and Merrifield, Solid-Phase Peptide Synthesis, in The Peptides: Analysis, Synthesis, Biology Gross and Meienhofer (eds.), Academic Press, N.Y., vol. 2, pp. 3-284 (1980); and Stewart et al., Solid Phase Peptide Synthesis 2nd ed., Pierce Chem. Co., Rockford, 111. (1984).
  • N- ⁇ -protected amino acids having protected side chains are added stepwise to a growing polypeptide chain linked by its C-terminal and to a solid support, i.e., polystyrene beads.
  • the peptides are synthesized by linking an amino group of an N- ⁇ -deprotected amino acid to an ⁇ -carboxy group of an N- ⁇ -protected amino acid that has been activated by reacting it with a reagent such as dicyclohexylcarbodiimide. The attachment of a free amino group to the activated carboxyl leads to peptide bond formation.
  • the most commonly used N- ⁇ -protecting groups include Boc, which is acid labile, and Fmoc, which is base labile.
  • Materials suitable for use as the solid support include, but are not limited to, the following: halomethyl resins, such as chloromethyl resin or bromomethyl resin; hydroxymethyl resins; phenol resins, such as 4-(c-- [2,4-dimethoxyphenyl]-Fmoc-aminomethyl)phenoxy resin; tert-alkyloxycarbonyl- hydrazidated resins, and the like.
  • halomethyl resins such as chloromethyl resin or bromomethyl resin
  • hydroxymethyl resins such as phenol resins, such as 4-(c-- [2,4-dimethoxyphenyl]-Fmoc-aminomethyl)phenoxy resin
  • tert-alkyloxycarbonyl- hydrazidated resins and the like.
  • the C-terminal N- ⁇ -protected amino acid is first attached to the solid support.
  • the N- ⁇ -protecting group is then removed.
  • the deprotected c--amino group is coupled to the activated ⁇ -carboxylate group of the next N- ⁇ -protected amino acid.
  • the process is repeated until the desired peptide is synthesized.
  • the resulting peptides are then cleaved from the insoluble polymer support and the amino acid side chains deprotected. Longer peptides can be derived by condensation of protected peptide fragments.
  • the peptides of the present invention can also be generated by recombinant means. Although it is often prefened to have the peptides synthesized chemically, according to the methods described above, there may be some advantages to obtain the peptides recombinantly in certain cases.
  • an HSV-2 peptide of the present invention when an HSV-2 peptide of the present invention is to be conjugated with a heterologous peptide and/or a heterologous protein, the nucleic acid sequence encoding the HSV-2 peptide can be introduced into a suitable expression vector, and subsequently fused in-frame with the coding sequence(s) of the heterologous peptide and/or protein, so that upon transfection or transformation of an appropriate host cell line, the fusion polypeptide of the HSV-2 peptide and the heterologous peptide/protein can be produced and purified.
  • a large variety of expression vectors and host cells well known to those skilled in the art can be used for this purpose.
  • a recombinant polypeptide comprises an HSV-2 peptide fused to a heterologous protein with known molecular adhesion properties
  • it can be purified with relative ease and to a relatively high purity by passing through a column to which a proper binding partner is immobilized.
  • the amino acid sequence of a peptide prepared for HSV-2 type-specific detection can be confirmed by a number of well established methods.
  • the conventional method of Edman degradation can be used to determine the amino acid sequence of a peptide.
  • sequencing methods based on Edman degradation including microsequencing, and methods based on mass spectrometry are also frequently used for this purpose.
  • the peptides of the present invention can be modified to achieve more desirable properties.
  • the design of chemically modified peptides and peptide mimics which are resistant to degradation by proteolytic enzymes or have improved solubility or binding ability is well known.
  • Modified amino acids or chemical derivatives of the peptides used for HSV-2 type-specific detection may contain additional chemical moieties of modified amino acids not normally a part of the glycoprotein G2. Covalent modifications of the peptide are within the scope of the present invention. Such modifications may be introduced into a peptide by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. The following examples of chemical derivatives are provided by way of illustration and not by way of limitation. [0059] The design of peptide mimics which are resistant to degradation by proteolytic enzymes is known to those skilled in the art. See e.g., Sawyer, Structure-Based Drug Design, P.
  • modifications include substitution of a natural amino acid with an unnatural hydroxylated amino acid, substitution of the carboxy groups in acidic amino acids with nitrile derivatives, substitution of the hydroxyl groups in basic amino acids with alkyl groups, or substitution of methionine with methionine sulfoxide.
  • an amino acid of a peptide for type-specific detection of HSV-2 can be replaced by the same amino acid but of the opposite chirality, i.e., a naturally-occurring L-amino acid may be replaced by its D- configuration.
  • the peptides of the present invention can also be modified to enhance their ability to specifically bind to HSV-2 antibodies.
  • a linker which may comprise a heterologous peptide, a heterologous protein, and/or chemicals of non-polypeptide in nature, may be introduced to either terminus of a peptide of the invention in order to facilitate the immobilization of the claimed peptide to a canier, as well as to better present the peptide to HSV-2 antibodies.
  • the peptides of the invention may be dimerized to achieve higher level of specific binding to HSV-2 antibodies. A variety of means can be used for dimerization, and the most commonly used method is through a disulfide bond. Since there is no naturally occurring cysteine residue in the peptides, a cysteine residue can be included as a part of the linker. The process of adding a linker to a peptide of the present invention will be discussed in detail in the next section.
  • HSV-2 antibodies are preferably immobilized to a solid support, or a carrier.
  • a carrier is often a synthetic polymeric material, but may also be naturally-occurring. Examples of carrier material are acrylamide, cellulose, nitrocellulose, glass, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polysilicates, polyethylene oxide, polycarbonates, teflon, fluorocarbons, nylon, silicon rubber, collagen, polyanhydrides, polyglycolic acid, polylactic acid, polyorthoesters, polypropylfumarate, glycosaminoglycans, and polyamino acids.
  • a carrier may be in one of the many useful forms including thin films or membranes, beads, bottles, dishes, fibers, woven fibers, shaped polymers, particles, and microparticles such as microspheres.
  • Prefened forms of supports are plates and beads. The most prefened form of beads is magnetic beads or latex beads.
  • a peptide of the present invention can be attached to a carrier via various linkers.
  • a linker can be attached to the N- or C-terminus of a peptide.
  • a linker may consist of a second peptide that is heterologous to the peptide of the invention, or may, in addition to a heterologous peptide, further comprise a protein heterologous to the peptide of the invention.
  • Branched amino acid polymers can also be used as linkers, taking advantage of multiple functional groups on amino acid residues such as the two amine groups on a lysine residue.
  • linkers well known to those of skill in the art, including but not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers (e.g., SMCC), or polyether linkers. These linkers can be used in addition to or in place of a heterologous peptide and/or protein.
  • a linker in the case of direct linkage, can be a covalent bond (e.g., a disulfide bond) or a noncovalent bond (e.g., an ionic bond) between the peptide and the carrier.
  • a covalent bond e.g., a disulfide bond
  • a noncovalent bond e.g., an ionic bond
  • heterologous peptides serving as linkers may be included at the time of peptide synthesis.
  • a linker consisting of one cysteine residue, one lysine residue, and at least two glycine residues can be included as a part of the peptide sequence to be synthesized, either at the N- or C-terminus.
  • Other linkers such as straight or branched-chain carbon linkers, heterocyclic carbon linkers, or polyether linkers may also be used to join an HSV-2 peptide of the present invention alone or in combination with a heterologous peptide and/or protein.
  • a heterologous protein such as BSA and KLH
  • a heterologous peptide may be joined to a peptide, to which a heterologous peptide is already added during synthesis, by chemical means following the synthesis and purification of the peptide.
  • conjugation may be accomplished by joining the peptide and the heterologous protein through the ⁇ -carbon amino and carboxyl groups of the terminal amino acid residues, such as through a peptide bond; or by joining the amino acid residues of the peptide and the heterologous protein through their side groups, such as through a disulfide bond.
  • Linkers such as heterologous peptides and/or proteins can also be joined to the HSV-2 peptides of the present invention through a functional group of an internal amino acid residue (e.g. , a second carboxyl group of an aspartate residue) of the HSV-2 peptides.
  • linkers with appropriate functional groups such as carbon linkers or polyether linkers may also be useful to practice the present invention. These linkers may be joined to a peptide's constituent amino acids through their side groups (for example, through a disulfide linkage to cysteine). The linkers may also be joined to the ⁇ -carbon amino or carboxyl groups of the peptide's terminal amino acids.
  • the peptides of the present invention can be immobilized to a insoluble carrier directly.
  • the strategies of attaching a peptide which usually contains a variety of functional groups such as carboxylic acid ( — COOH), free amine ( — NH 2 ), or sulfhydryl ( — SH) groups that are available for reaction with a suitable functional group on the canier to result in a linkage, are similar to some approaches of attaching a peptide or protein linker to a carrier, the detailed discussion of which is provided in the next section.
  • the peptides for HSV-2 type-specific detection, with or without a linker, may be attached to a carrier via a covalent bond.
  • a carrier has some functional groups, such as amine, carboxylic acid, and sulfhydryl groups, with which the functional groups of a peptide or a linker may easily react and establish a covalent bond that conjugates the peptide and the carrier.
  • a covalent bond joining a peptide of the present invention and a carrier can exist between the carrier and a terminal amino acid residue of the peptide, or between the carrier and an internal amino acid residue of the peptide.
  • the carrier may be derivatized to expose or to attach additional reactive functional groups prior to conjugation.
  • the derivatization may involve attachment of any of a number of molecules such as those available from Pierce Chemical Company, Rockford, Illinois.
  • a peptide can be linked to a carrier via the known interaction of a tag and a tag-binder.
  • One of the partners of this binding interaction e.g., a tag
  • the other partner e.g., a tag binder
  • tags and tag binders can be used, based upon known molecular interactions well described in the literature.
  • a tag has a natural binder, e.g., biotin
  • tag binders avidin, streptavidin, neutravidin, etc.
  • Receptor-ligand interactions are also appropriate as tag and tag-binder pairs.
  • agonists and antagonists of cell membrane receptors e.g., cell receptor- ligand interactions such as transferrin, c-kit, viral receptor ligands, cytokine receptors, chemokine receptors, interleukin receptors, the cadherein family, the integrin family, the selectin family, and the like; see, e.g., Pigott & Power, The Adhesion Molecule Facts Book I (1993).
  • toxins and venoms can all interact with various cell receptors.
  • hormones e.g., opiates, steroids, etc.
  • intracellular receptors e.g. which mediate the effects of various small ligands, including steroids, thyroid hormone, retinoids and vitamin D; peptides
  • lectins e.g. which mediate the effects of various small ligands, including steroids, thyroid hormone, retinoids and vitamin D; peptides
  • drugs lectins
  • sugars e.g., nucleic acids (both linear and cyclic polymer configurations), oligosaccharides, proteins, phospholipids and antibodies
  • nucleic acids both linear and cyclic polymer configurations
  • oligosaccharides oligosaccharides
  • proteins e.g.,
  • some synthetic polymers such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, and polyacetates can form an appropriate tag or tag binder as well.
  • a linker containing a tag can be attached to a peptide via a number of ways as described above.
  • a tag binder can be fixed to a solid substrate (i.e., a carrier) using any of a variety of methods cunently available.
  • Solid substrates are commonly derivatized or functionalized by exposing all or a portion of the substrate to a chemical reagent which fixes a chemical group to the surface, and the chemical group is in turn reactive with a portion of the tag binder.
  • groups which are suitable for attachment to a longer chain portion would include amines, hydroxyl, thiol, and carboxyl groups.
  • Aminoalkylsilanes and hydroxyalkylsilanes can be used to functionahze a variety of surfaces, such as glass surfaces.
  • the construction of such solid phase biopolymer anays is well described in the literature. See, e.g., Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963) (describing solid phase synthesis of, e.g., peptides); Geysen et al., J. Immun. Meth.
  • peptides of the present invention In order for peptides of the present invention to be useful for HSV-2 type- specific detection, they must first be able to bind HSV-2 antibodies with specificity. To test such specific binding, a number of well known immunological binding assays can be performed. See, e.g., U.S. Patent Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168. For a general review of immunoassay methods, see also Asai, Methods in Cell Biology, Volume 37: Antibodies in Cell Biology, Academic Press, Inc. NY (1993).
  • peptides of the present invention can be immobilized and used as a so-called "capture agent" for HSV-2 antibodies.
  • Samples that are known to contain HSV-2 antibodies but not HSV-1 antibodies may be used in binding assays to screen for peptides that can bind HSV-2 antibodies with specificity.
  • the proper binding conditions are well known in the art and general instructions on performing such binding assays may be found in many scientific publications. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988).
  • a labeling agent is used to indicate the presence of such complex. In the present case, there are several ways of using a labeling agent for this purpose.
  • the labeling agent may be a second antibody that can recognize an antibody-peptide complex and bears a label.
  • the second antibody may itself lack a label, but can in turn be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived.
  • the second antibody may also be modified with a detectable moiety, such as biotin, to which a third labeled molecule can bind with specificity, such as streptavidin with a label.
  • other proteins capable of specifically binding immnunoglobulin constant regions such as protein A or protein G, can also be used as labeling agents.
  • incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. The incubation time will vary, depending upon the assay format, particular peptides, volume of solution, concentrations, and the like.
  • the assays are frequently carried out at ambient temperature, although they can be conducted over a range of temperatures, such as from about 10°C to about 40°C.
  • a labeling moiety can be, e.g., a fluorescent molecule (such as fluorescein, rhodamine, Texas Red, and phycoerythrin) or an enzyme molecule (such as horseradish peroxidase, alkaline phosphatase, and ⁇ -galactosidase) attached to a second or a third antibody, allowing detection based on fluorescence emission or a product of a chemical reaction catalyzed by the enzyme.
  • a fluorescent molecule such as fluorescein, rhodamine, Texas Red, and phycoerythrin
  • an enzyme molecule such as horseradish peroxidase, alkaline phosphatase, and ⁇ -galactosidase
  • Radioactive labels involving various isotopes can also be attached to appropriate molecules, and detection of antibody- peptide complex can thus be made by any suitable methods that registers radioactivity, such as autoradiography. See, e.g., Tijssen, "Practice and Theory of Enzyme Immunoass ays, " Laboratory Techniques in Biochemistry and Molecular Biology, Burdon and van Knippenberg Eds., Elsevier (1985), pp. 9-20.
  • Flow cytometry is one of the prefened methods for detecting the presence of HSV-2 type-specific antibodies, where the peptides of the present invention are conjugated to suitable particles and specific binding of HSV-2 antibodies is detected through the binding of a third molecule labeled with, e.g., fluorescence.
  • Methods of and instrumentation for flow cytometry are known in the art, and can be used in the practice of the present invention.
  • Flow cytometry in general resides in the passage of a suspension of the microparticles as a stream past a laser beam and the detection of fluorescent emission from each particle by a photo multiplier tube. Detailed descriptions of instrumentation and methods for flow cytometry are found in the literature.
  • the particles used in the practice of this invention are preferably microscopic in size and formed of a polymeric material.
  • Polymers that will be useful as microparticles are those that are chemically inert relative to the components of the biological sample and to the assay reagents other than the binding member coatings that are affixed to the microparticle surface.
  • Suitable microparticle materials will also have minimal autofluorescence, will be solid and insoluble in the sample and in any buffers, solvents, carriers, diluents, or suspending agents used in the assay, and will be capable of affixing to the appropriate coating material, preferably through covalent bonding.
  • the particles preferably contain a magnetically responsive material, i.e., any material that responds to a magnetic field.
  • Magnetically responsive materials of interest in this invention include paramagnetic materials, fenomagnetic materials, ferrimagnetic materials, and metamagnetic materials. Paramagnetic materials are prefened. Examples are iron, nickel, and cobalt, as well as metal oxides such as Fe 3 O 4 , BaFe ⁇ 2 O ⁇ 9 , CoO, NiO, Mn 2 O 3 , Cr 2 O 3 , and CoMnP.
  • the magnetically responsive material can be dispersed throughout the polymer, applied as a coating on the polymer surface or as one of two or more coatings on the surface, or incorporated or affixed in any other manner that secures the material in to the particle.
  • the quantity of magnetically responsive material in the particle is not critical and can vary over a wide range. The quantity can affect the density of the microparticle, however, and both the quantity and the particle size can affect the ease of maintaining the microparticle in suspension for purposes of achieving maximal contact between the liquid and solid phase and for facilitating flow cytometry. An excessive quantity of magnetically responsive material in the microparticles may produce autofluorescence at a level high enough to interfere with the assay results.
  • the concentration of magnetically responsive material be low enough to minimize any autofluorescence emanating from the material.
  • the magnetically responsive material in a particle in accordance with this invention preferably ranges from about 0.05% to about 75% by weight of the particle as a whole.
  • a more prefened weight percent range is from about 1% to about 50%, a still more prefened weight percent range is from about 2% to about 25%, and an even more prefened weight percent range is from about 2% to about 8%.
  • Coating of the particle surface with the appropriate assay reagent can be achieved by electrostatic attraction, specific affinity interaction, hydrophobic interaction, or covalent bonding. Covalent bonding is prefened.
  • the polymer can be derivatized with functional groups for covalent attachment of the assay reagent by conventional means, notably by the use of monomers that contain the functional groups, such monomers serving either as the sole monomer or as a co-monomer. Examples of suitable functional groups are amine groups ( — NH 2 ), ammonium groups ( — NH 3 + or — NR 3 + ), hydroxyl groups ( — OH), carboxylic acid groups ( — COOH), and isocyanate groups ( — NCO).
  • Useful monomers for introducing carboxylic acid groups into polyolefins, for example, are acrylic acid and methacrylic acid.
  • Linkers can be used as a means of increasing the density of antibody- recognizable epitopes on the particle surface and decreasing steric hindrance. This will increase the range and sensitivity of the assay. Linkers can also be used as a means of adding specific types of reactive groups to the solid phase surface if needed to secure the particular coating materials of this invention. Examples of suitable useful functional groups are polylysine, polyaspartic acid, polyglutamic acid, and polyarginine.
  • the labels used in the labeled binding members may be any label that is capable of emitting detectable signal. Prefened such labels are fluorophores. A vast anay of fluorophores are reported in the literature and thus known to those skilled in the art, and many are readily available from commercial suppliers to the biotechnology industry. Literature sources for fluorophores include Cardullo et al, Proc. Natl. Acad. Sci. USA 85:
  • fluorophores 4-acetamido-4 ' -isothiocyanatostilbene-2,2 ' disulfonic acid acridine acridine isothiocyanate 5-(2'-aminoethyl)aminonaphthalene-l-sulfonic acid (EDANS) 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate N-(4-anilino- 1 -naphthyl)maleimide anthranilamide
  • peptides are shown to react to HSV-2 antibodies specifically, they will be further tested for any possible cross-reactivity to HSV-1 antibodies and antibodies against other viruses such as the herpes family.
  • the peptides are immobilized and used as "capture agents" in immunoassays essentially identical to those described in last section, except that samples confirmed to contain HSV-1 antibodies (but not
  • HSV-2 antibodies are used for the binding assays.
  • Peptides listed in Table 1 were synthesized as monomers using the N- ⁇ - protecting group Boc or Fmoc.
  • Table 1 gG-2 Peptides Synthesized Name Sequence 1 PGSPAPPP i PEHRGG PEEFEGAGDG EPPE ⁇ DDDS ATGL GG CK 2 APPP PEHRGG PEEFEGAGDG EPPEDDDS GGGG CK 3 -' PEHRGG PEEFEGAGDG EPPEDDDS GGGG CK 4 PEEFEGAGDG EPPEDDDS GGGG CK 5 KC GGGG PEHRGG PEEFEGAGDG EPPEDDDS 6 ' RGRAG RRRYAHPSVR GGGG CK 7 > WRGRAG RRRYAHPSVR Y GGGG CK 8 : RGRAG RRRYAHPSVR YVCLPPER : D GGGG CK 9 : !
  • RRRYAHPSVR YVCLPPER D GGGG CK 10 KC GGGG ; , WRGRAG RRRYAHPSVR Y 11 KC GGGG
  • peptide dimerization is performed as follows: a monomer peptide was dissolved in either deionized water or 0.1 M sodium bicarbonate and stined at 4°C overnight. The resulting oxidized, dimeric peptide was purified by preparative HPLC using a C 18 column.
  • the monomer peptide (1 mg) was mixed with 1 mg of the modified BSA and stored at 4 °C overnight.
  • the peptide-BSA conjugate was purified through size exclusion chromatography.
  • a dimer peptide-BSA conjugate can be prepared in accordance with the following description. 1. SMCC-Peptide Dimer
  • SATA N-succinimidyl S-acetylthioacetate
  • BSA lOmg/ml
  • 100 mM phosphate pH 8.0
  • 100 ⁇ l of 200 mM N-ethyl maleimide containing 10 mM EDTA is added.
  • the reaction mixture is incubated at room temperature for 1 hour and then applied to a Sephadex G-25 column.
  • the modified BSA is eluted from the column with 10 mM PBS.
  • the activated beads were washed twice with 25 mM MES (pH6.1). Subsequently, 100 ⁇ l of 0.5 M MES (pH 6.1), 720 ⁇ l of deionized water, and 180 ⁇ l of Peptide (1 mg/ml) or Peptide-BSA conjugate (1 mg/ml) were added and the mixture vortexed. The mixture was further incubated at room temperature on a rotator for 2 hours and centrifuged at 7000 rpm for 2 min. One ml of 1% BSA in PBS containing 20 mM Tris Base was added after discarding the supernatant, mixed for 2 hour at room temperature on a rotator and then centrifuged to remove the supernatant.
  • the peptides or peptide-BSA conjugates were coupled to predefined magnetic beads (every peptide or peptide-BSA conjugate was coupled to magnetic beads whose fluorescence is characteristic of a particular region of dye content) and were mixed together at similar bead number for each region of magnetic beads.
  • the mixed beads were diluted with 1% BSA in PBS with 0.1% Tween 20 to 1000 copies of each region magnetic beads per ml.
  • the beads were washed with wash buffer (10 mM PBS with 0.1% Tween 20) twice. 50 ⁇ l of antihuman IgG (Fc specific) -B-phycoerythrin conjugate was added. Following a 10-minute incubation on a shaker at 37 °C, the beads were washed again with the wash buffer twice and resuspended in 50 ⁇ l of the wash buffer. The beads were counted and analyzed on Luminex 100 instrument. The amount of antibodies (IgG) bound to the magnetic beads was determined with antihuman IgG conjugated to phycoerythrin.
  • wash buffer 10 mM PBS with 0.1% Tween 20
  • 50 ⁇ l of antihuman IgG (Fc specific) -B-phycoerythrin conjugate was added. Following a 10-minute incubation on a shaker at 37 °C, the beads were washed again with the wash buffer twice and resuspended in 50 ⁇ l of the wash buffer. The beads were counte
  • HSV-2 type-specific immunoassay systems of the present invention are tested for lack of cross-reactivity to antibodies against other viruses of the herpes family, particularly HSV-1.
  • Table 3 demonstrates the lack of cross-reactivity to HSV-1 IgG as confirmed by two commercially available HSV-2 type-specific assay systems and Western Blot assays.

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