JP2007526919A - HSV-2 type specific immunoassay using glycoprotein G2 peptide - Google Patents

Abstract

  The present invention relates to peptides derived from HSV-2 glycoprotein G2 and derived from HSV-2 type specific epitopes. The present invention provides a composition comprising a peptide for type-specific serological diagnosis of HSV-2 infection. Further provided may be methods of using these peptides for type-specific detection of HSV-2 antibodies and methods of distinguishing HSV-2 virus infection from HSV-1 virus infection and other herpes family virus infections.

Description

  The present invention provides a novel peptide sequence of glycoprotein G2 of HSV-2 that can be used in HSV-2 type specific diagnosis.

  [0001] Genital herpes caused by herpes simplex virus type 2 (HSV-2) infection is the most common sexually transmitted disease in humans. The prevalence of HSV-2 infection is over 20% among adults in the United States (Ashley and Wald, Clin. Microbiol. Rev. 12: 1-8, 1999). This disease is a major concern in public health because of its morbidity, frequency of recurrence, and life-threatening severity in the case of newborns infected with viruses after parturition infection.

  However, serological diagnosis of HSV-2 infection has been hampered by the fact that there is a strong cross-reactivity of HSV-2 antibody to herpes simplex virus type 1 (HSV-1). The two subtypes of HSV have important differences in epidemiology and natural history: HSV-1 usually causes lip disease, while HSV-2 most often causes genital herpes. For a general review of HSV epidemiology and diagnosis, see Brugha et al., Int. J. Epidemiol. 26: 698-709, 1997; Ashley and Wald.

  [0003] Various approaches have been developed in an attempt to identify HSV-2 specific antibodies. The most reliable method to date for type-specific detection of HSV-2 antibodies is an immunoblot assay, preferably a Western blot assay. A significant drawback of the method is that it is labor intensive and requires the investigator to have a certain level of skill in order to achieve a definite result. In the last decade or so, several HSV glycoproteins have been identified as viral proteins containing type-specific epitopes. Immunoassays have been developed based on these glycoproteins for type-specific determination of HSV-2 infection. For example, Lee et al., J. Clin. Microbiol. 22: 641-644 (1985); Eis-Hubinger et al., J. Clin. Microbiol. 37: 1242-1246 (1999); Groen et al., J. Clin. Microbiol. 36 845-847 (19989; Ashley et al., J. Clin. Microbiol. 36: 294-295 (1998); Hashido et al., J. Med. Virol. 53: 319-323 (1997); and U.S. Pat.No. 4,764,459. See

  [0004] As these methods exhibit varying sensitivity and specificity, there are general problems associated with these glycoprotein-based immunoassays for type-specific detection of HSV-2 antibodies. Full length glycoproteins are obtained from isolating natural viral proteins or recombinantly expressed proteins. These methods are expensive and susceptible to impurities and thus susceptible to cross-reactivity.

  [0005] The fact that peptides corresponding to partial sequences of certain viral proteins of HSV-2 can represent several HSV-2 type-specific epitopes, so that they are useful in HSV-2 type-specific detection. Research has shown. For example, Levi et al., Clin. Diagn. Lab. Immunol. 3: 265-269 (1996); Ackermann et al., J. Med. Virol. 55: 281-287 (1998); Marsden et al., J. Med. Virol. 56 79-84 (1998); Lijeqvist et al., J. Gen. Virol. 79: 1215-1224 (1998); Grabowska et al., J. Gen. Virol. 80: 1789-1798 (1999); U.S. Pat.No. 5,919,616 and See 5,965,357.

The present invention provides a novel peptide sequence of glycoprotein G2 of HSV-2 that can be used in HSV-2 type specific diagnosis.
[0006] All disclosure documents and patents cited herein are hereby incorporated by reference.
[0007] In one aspect, the invention includes a peptide that specifically binds to an HSV-2 antibody and minimally reacts to an HSV-1 specific antibody or antibody to other herpes family viruses. Relates to the composition. The peptide consists of 24-36 consecutive amino acids of SEQ ID NO: 1. In one preferred embodiment, the peptide has a sequence consisting of amino acids 5-32 of SEQ ID NO: 1. In a more preferred embodiment, the peptide has a sequence consisting of amino acids 9-32 of SEQ ID NO: 1. In the most preferred embodiment, the peptide has the amino acid sequence of SEQ ID NO: 1.

  [0008] In some embodiments, the peptide is dimerized. Such dimerization can be achieved via disulfide bonds. In another embodiment, the peptide is bound to a carrier. Examples of suitable carriers are carboxylated microspheres, preferably carboxylated latex or magnetic microspheres.

で示される。 [0009] In some embodiments, the peptide is linked to a carrier via a linker at the N-terminus or C-terminus of the peptide. On the other hand, in another embodiment, the peptide is linked to a carrier via a linker at an internal amino acid residue of the peptide. In some cases, the linker is a heterologous peptide or comprises a heterologous peptide. In other cases, the linker is or comprises a heterologous protein. In yet another case, both heterologous proteins and peptides are used. In some other embodiments, the entire linker is a heterologous peptide. In one preferred embodiment, the heterologous protein is bovine serum albumin (BSA), while in another preferred embodiment, the heterologous protein is keyhole limpet hemocyanin (KLH). In some embodiments, the heterologous peptide comprises 1 cysteine residue, 1 lysine residue, and at least 2 glycine residues. In some other embodiments, the linker comprises a branched amino acid polymer, preferably having the structure:

Indicated by

  [0010] In one preferred embodiment, the peptide has the amino acid sequence of SEQ ID NO: 1, the carrier is a carboxylated magnetic microsphere, and the linker is 4- (maleimidomethyl) -1-cyclohexanecarboxylic acid (SMCC) ) And a GGCK amino acid sequence, and the heterologous peptide is linked to the peptide at the C-terminus of the peptide. In another preferred embodiment, the linker comprises a heterologous protein BSA directly attached to a carrier, and SMCC is attached to the BSA and attached to the peptide via the heterologous peptide.

  [0011] In another preferred embodiment, the peptide has an amino acid sequence consisting of amino acids 5 to 32 of SEQ ID NO: 1, the carrier is a carboxylated magnetic microsphere, and the linker comprises the amino acid sequences of SMCC and GGGGCK. And having the heterologous peptide attached to the peptide at the C-terminus of the peptide. In another preferred embodiment, the linker further comprises a heterologous protein BSA directly attached to a carrier, and SMCC is attached to BSA and attached to the peptide via a heterologous peptide.

  [0012] In still another preferred embodiment, 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, and the linker has the amino acid sequences of SMCC and GGGGCK. And having the heterologous peptide attached to the peptide at the C-terminus of the peptide. In another preferred embodiment, the linker further comprises a heterologous protein BSA directly attached to the carrier, and the SMCC is attached to the BSA and attached to the peptide via the heterologous peptide.

  [0013] In still another preferred embodiment, 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, and the linker includes the amino acid sequences of SMCC and KCGGGGG. Having a heterologous peptide, and the heterologous peptide is linked to the peptide at the N-terminus of the peptide. In another preferred embodiment, the linker further comprises a heterologous protein BSA directly attached to the carrier, and the SMCC is attached to the BSA and attached to the peptide via the heterologous peptide.

で表される構造の末端Ｋ残基に直接結合されるＣＫという短いペプチドを含む。 [0014] In a further preferred embodiment, the peptide has an amino acid sequence consisting of amino acids 9-32 of SEQ ID NO: 1, the carrier is a carboxylated microsphere, and the linker is a branched chain having the following structure: Including amino acid polymers and further via the C residue:

A short peptide called CK that is directly linked to the terminal K residue of the structure represented by

  [0015] In a second aspect, the present invention relates to a type-specific diagnostic method for HSV-2 infection. The method for specific detection of HSV-2 antibody in a biological sample comprises two steps. The first step is to contact the biological sample with a composition consisting of 24-36 contiguous amino acids of SEQ ID NO: 1 and comprising a peptide bound to a carrier. The second step is to detect whether antigen-antibody binding has occurred between the peptide and the antibody component of the biological sample. In this step, detection of antigen-antibody binding indicates the presence of HSV-2 antibody in the biological sample. In some preferred embodiments, the second step is performed by flow cytometry. Preferably, the biological sample is whole blood, serum, plasma, cerebrospinal fluid, tissue from a swab device (tissue from a swab device), or seminal vesicle fluid.

  [0016] In one preferred embodiment, the peptide has a sequence consisting of amino acids 5-32 of SEQ ID NO: 1. In a more preferred embodiment, the peptide has a sequence consisting of amino acids 9-32 of SEQ ID NO: 1. In the most preferred embodiment, the peptide has the amino acid sequence of SEQ ID NO: 1.

  [0017] In some embodiments, the peptide is dimerized. Such dimerization can be achieved via disulfide bonds. In some other embodiments, the peptide is bound to a carboxylated microsphere, a carboxylated latex, or a magnetic microsphere.

で表される。 [0018] In some embodiments, the peptide is linked to a carrier via a linker at the N-terminus or C-terminus of the peptide. On the other hand, in another embodiment, the peptide is bound to the carrier via a linker at an amino acid residue inside the peptide. In some embodiments, the linker comprises a heterologous peptide. In some other embodiments, the linker comprises a heterologous protein. In some other embodiments, the linker comprises a heterologous protein in addition to the heterologous peptide. In some other embodiments, the linker is a heterologous peptide. In one preferred embodiment, the heterologous protein is BSA, while in another preferred embodiment, the heterologous protein is KLH. In some embodiments, the heterologous peptide comprises 1 cysteine residue, 1 lysine residue, and at least 2 glycine residues. In some other embodiments, the linker comprises the following branched amino acid polymer, the structure of which is preferably:

It is represented by

  [0019] In one preferred embodiment, the peptide has the amino acid sequence of SEQ ID NO: 1, the carrier is a carboxylated magnetic microsphere, the linker comprises a heterologous peptide having the amino acid sequences of SMCC and GGCK, The heterologous peptide is bound to the peptide at the C-terminus of the peptide, and detection of antigen-antibody binding is performed by flow cytometry. In another preferred embodiment, the linker further comprises a heterologous protein BSA that is directly attached to the carrier, and the SMCC is attached to the BSA and to the peptide via the heterologous peptide.

  [0020] In another preferred embodiment, the peptide has an amino acid sequence consisting of amino acids 5 to 32 of SEQ ID NO: 1, the carrier is a carboxylated magnetic microsphere, and the linker includes the amino acid sequences of SMCC and GGGGCK. The heterologous peptide is bound to the peptide at the C-terminus of the peptide, and detection of antigen-antibody binding is accomplished by flow cytometry. In another preferred embodiment, the linker further comprises a heterologous protein BSA that is directly attached to the carrier, and the SMCC is attached to the BSA and attached to the peptide via the heterologous peptide.

  [0021] In still another preferred embodiment, 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, and the linker is an amino acid sequence of SMCC and GGGGCK. The heterologous peptide is bound to the peptide at the C-terminus of the peptide, and detection of antigen-antibody binding is achieved by flow cytometry. In another preferred embodiment, the linker further comprises a heterologous protein BSA directly attached to the carrier, and the SMCC is attached to the BSA and attached to the peptide via the heterologous peptide.

  [0022] In still another preferred embodiment, 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 is an amino acid sequence of SMCC, and KCGGGGG And the heterologous peptide is linked to the peptide at the N-terminus of the peptide. In another preferred embodiment, the linker further comprises a heterologous protein BSA directly attached to the carrier, and the SMCC is attached to the BSA and attached to the peptide via the heterologous protein.

で表される構造の末端Ｋ残基に直接結合されるＣＫという短いペプチドを含む。 [0023] In one further preferred embodiment, the peptide has an amino acid sequence consisting of amino acids 9-32 of SEQ ID NO: 1, the carrier is a carboxylated microsphere, and the linker has the following structure: Containing a branched amino acid polymer and further via a C residue:

A short peptide called CK that is directly linked to the terminal K residue of the structure represented by

I. Introduction
[0029] The present invention relates to a peptide consisting of a partial sequence of HSV-2 glycoprotein G2 and representing a HSV-2 type specific epitope. HSV-2 peptide consisting of 24-36 contiguous amino acids of SEQ ID NO: 1 binds to HSV-2 antibody with high specificity and sensitivity, while its cross-reactivity to HSV-1 antibody is minimal Was shown by this experiment. Thus, these peptides are useful for type-specific serological diagnosis of HSV-2 infection, particularly for distinguishing HSV-2 infection from HSV-1 infection.

  [0030] A composition comprising the peptide of the present invention is provided for type-specific serological diagnosis of HSV-2 infection. In a preferred embodiment, the peptides of the invention are conjugated to a carrier, such as a carboxylate, via a linker comprising a heterologous peptide and / or protein, such as bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH). Bonded to latex or magnetic microspheres. Other linkers, such as branched amino acid polymers, linear or molecular chain carbon linkers, heterocyclic carbon linkers (eg, SMCC), or polyether linkers can also be used in practicing the present invention. The HSV-2 peptides of the invention can be bound to the carrier at the N- or C-terminus of the peptide or via internal amino acid residues.

  [0031] Further provided are methods of using these peptides for type specific detection of HSV-2 antibodies. In a preferred embodiment, detection of HSV-2 specific antibodies is performed by flow cytometry.

II. Definition
[0032] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid mimetics and amino acid analogs that function in a manner similar to the naturally occurring amino acids. Natural amino acids are those encoded by the genetic code, as well as amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. The term “amino acid analog” refers to a compound having the same basic chemical structure as a natural amino acid, ie α, carbon bonded by hydrogen, carboxyl group, amino group, and R group, such as homoserine, norleucine, methionine sulfoxide, methionine. -Refers to methyl sulfonium. Such analogs have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. “Amino acid mimetics” refers to compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

  [0033] Amino acids may be represented herein by their commonly known three letter code or by the one letter code recommended by the IUPAC-IUB Biochemical Nomenclature Committee. .

  [0034] The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are the artificial chemical mimetics of the corresponding natural amino acid, and also applies to natural and non-natural amino acid polymers. As used herein, the term includes amino acid chains of any length, including full length proteins, where the amino acid residues are joined by covalent peptide bonds. As used in this application, HSV-2 peptide refers to a peptide having a sequence corresponding to a fragment of HSV-2 glycoprotein G2.

[0035] As used herein, "carrier" refers to natural materials such as glass and collagen, or synthetic materials such as acrylamide, cellulose, nitrocellulose, silicone rubber, polystyrene, polyethylene vinyl acetate, polypropylene. , Polymethacrylate, polyethylene, polysilicate, polyethylene oxide, polycarbonate, Teflon (registered trademark), fluorocarbon, nylon, polyanhydride, polyglycolic acid, polyacetic acid, polyorthoester, polypropyl fumarate, glycosaminoglycan, And an inert solid support such as a polyamino acid. Several functional groups originally present on the surface of the carrier, such as carboxylic acid (—COOH), free amine (—NH ₂ ), and sulfhydryl (—SH) groups, can be used for peptide bonds. In the absence of such groups originally available, the desired functional group, such as a carboxylic acid group, or a moiety known as a binding interaction partner (e.g., avidin capable of binding biotin) is present on such a solid support. Can be combined. Preferred carriers of the present invention are carboxylated latex or magnetic microspheres.

  [0036] As used herein, "linker" refers to any means for attaching a peptide of the invention to a carrier. The linkage can be located at either the N-terminus or C-terminus of the peptide. In some embodiments, linkage can be achieved through the side chain of an internal amino acid residue of the peptide, such as the second carboxyl group of aspartic acid that is not present at the N or C terminus of the peptide. The linker may comprise a peptide (eg, a short amino acid sequence such as GGGGCK or GGCK) or a protein (eg, BSA or KLH) that is heterologous to the peptide of the invention. In addition to the heterologous peptide, the linker may further comprise a heterologous protein, such as BSA or KLH. Non-polypeptide linkers, such as linear or branched carbon linkers, heterocyclic carbon linkers, or polyethylene linkers are suitable for the purpose of attaching the peptides of the invention to a carrier or as part of multiple component linkers. May be appropriate. One preferred non-polypeptide linker is SMCC. In addition, the linker may comprise a branched amino acid polymer capable of attaching more than one peptide of the invention to the carrier. Heterogeneous bifunctional and identical bifunctional linkers are suitable in the practice of the invention. The linker may comprise a covalent bond, such as a peptide bond or a disulfide bond, or a non-covalent bond, such as an ionic bond or a hydrophobic bond, when the peptide of the invention is directly attached to the carrier. Examples of non-covalent bonds are antigen-antibody or biotin-avidin interactions.

  [0037] When used in the context of describing a peptide or protein as a linker or linker component, the term "heterologous" refers to a peptide of the invention that is linked to a carrier via a linker peptide or protein and a linker peptide. Means that they are not seen in the same relationship with each other. In other words, the linker peptide and / or linker protein and the peptide of the invention do not produce the originally discovered amino acid sequence when combined in certain embodiments of the invention. By way of example, the peptides of the present invention are derived from HSV-2 glycoprotein G2, and therefore the heterologous peptide or protein is derived from the sequence of HSV-2 glycoprotein G2 adjacent to the sequence from which the peptide of the present invention is derived. do not do.

  [0038] As used herein, "specific detection" refers to the detection of any antibody that binds to a peptide of the present invention, often in a heterogeneous collection of other antibodies and proteins, in the presence of an HSV-2 antibody. It means the fact of deciding. In particular, the presence of HSV-1 antibody will not result in a detectable amount of antibody bound to the peptide. Thus, the term “specific detection” specifically includes the use of the HSV-2 peptides of the present invention to distinguish HSV-2 infection from HSV-1 infection. Under the conditions of a specified immunoassay, a detectable signal refers to a signal that is at least twice the background signal. Thus, a specific antibody-polypeptide bond should produce a signal that is at least twice, preferably 10 times, and more preferably 100 times background.

  [0039] When used to describe a peptide of the invention, the term "dimerization" or "dimer" refers to the same peptide covalently bonded, such as via a disulfide bond, or non-covalently bonded, such as Refers to a complex formed by two molecules comprising the same peptide via a binding interaction between a known tag and a tag binding pair (eg biotin and avidin). Covalent or non-covalent bonds typically occur between the linker portions of two peptide-containing molecules.

  [0040] As used herein, the term "internal amino acid residue" refers to any amino acid residue that is not the first residue of either the N-terminus or C-terminus of the HSV-2 peptide of the invention. Point to.

  [0041] The term "biological sample" refers to tissue sections such as biopsy and autopsy anatomical samples, and frozen sections taken for histological purposes. Such samples can be whole blood, serum, plasma, cerebrospinal fluid, saliva, tissue, cultured cells such as primary cultures, explants, transformed cells, stool, urine, seminal vesicle fluid, mucus, and other body secretions, or It can include tissue that can be sampled with a cotton swab. Typically, the biological sample is obtained from a human who may have been infected with HSV-2.

[0042] The term "antibody" refers to an immunoglobulin family protein or a polypeptide comprising a fragment of an immunoglobulin capable of binding reversibly and specifically to the corresponding antigen by non-covalent bonds. A typical antibody structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one light chain (about 25 kD) and one heavy chain (about 50-70 kD). They are linked through disulfide bonds. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as kappa or lambda. Heavy chains are classified as γ, μ, α, δ, or ε, 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 100-110 or more amino acids that primarily contributes to antigen recognition. The terms variable light chain (V _L ) and variable heavy chain (V _H ) refer to the light chain and heavy chain variable regions, respectively.

[0043] The term "complementarity determining domain" or "CDR" refers to the hypervariable regions of V _L and V _H. The CDR is the target protein binding site of an antibody chain with specificity for the target protein. human V _L or three CDR in V _H (CDRs 1 to 3, sequentially named from the N-terminus) is present, .CDR constitutes about 15-20% of the variable region are structurally complementary to the epitope of the target protein and, and directly contribute to binding specificity. stretch remaining V _L or V _H, the so-called framework regions, exhibit only low variability for amino acid sequence (Kuby, Immunology, fourth Edition, Chapter IV, WH Freeman & Co., New York, 2000).

  [0044] The location of CDRs and framework regions is determined by various well-known definitions in the art, such as Kabat, Chothia, International ImMunoGeneTics (IMGT) database, and AbM (eg, Johnson et al., Nucleic Acids Res., 29: 205-206 (2001); Chothia and Lesk, J. MoI. Biol, 196: 901-917 (1987); Chothia et al., Nature, 342: 877-883 (1989); Chothia et al., J. MoI. Biol., 227: 799-817 (1992); Al-Lazikani et al., J. Mol. Biol, 273: 927-748 (1997)). Definitions of antigen binding sites are also described below: Ruiz et al., Nucleic Acids Res., 28: 219-221 (2000); and Lefranc, MP, Nucleic Acids Res., 29: 207-209 (2001); MacCallum et al., J. MoI. Biol, 262: 732-745 (1996); and Martin et al., Proc. Natl. Acad. Sci. USA, 86: 9268-9272 (1989); Martin et al., Methods Enzymol, 203: 121. -153 (1991); and Rees et al., In Sternberg MJ.E. (ed), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996).

The term [0045] "Antibody light chain" and "antibody heavy chain" refers to a V _L or V _H, respectively. V _L is encoded by gene fragments V (variable part) and J (junction), and V _H is encoded by V, D (different part) and J. Each V _L or V _H comprises CDR, and the framework regions.

[0046] Antibodies exist as intact immunoglobulins or as many well-characterized fragments produced by cleavage with various peptidases. Thus, for example, pepsin cleaves the antibody below the disulfide bond in the hinge region, resulting in a dimer of F ( _ab ) ' ₂ , _Fab '. F _ab ′ itself is a light chain linked to V _H -C _H 1 by a disulfide. F ( _ab ) ' ₂ is reduced under mild conditions, breaking the disulfide bond in the hinge region, thereby converting the F ( _ab )' ₂ dimer into a Fab 'monomer. Fab ′ monomers are essentially Fabs that have part of the hinge region (Paul, Fundamental Immunology 3rd edition, 1993). While various antibody fragments have been defined in terms of cleaving intact antibodies, it will be appreciated by those skilled in the art that such fragments can be synthesized chemically or by using recombinant DNA methodology. Thus, the term antibody as used herein refers to antibody fragments produced by modification of whole antibodies, or antibody fragments newly synthesized using recombinant DNA methodology (eg, single chain FV Or antibody fragments identified using a phage display library (see, eg, McCafferty et al., Nature, 348: 552-554 (1990)).

  [0047] For preparation of monoclonal or polyclonal antibodies, any technique known in the art can be used (eg, Kohler & Milstein, Nature, 256: 495-497 (1975); Kozbor et al., Immunology Today, 4 : 72 (1983); see Cole et al., Monoclonal Antibodies and Cancer Therapy, pp. 77-96. Alan R. Liss, Inc. 1985). Techniques for producing single chain antibodies (US Pat. No. 4,946,778) can be applied to produce antibodies to the polypeptides of the invention. Also, transgenic mice, or other organisms, such as other mammals, can be used to express humanized antibodies. Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments, or scFV fragments that specifically bind to a selected antigen (see, eg, McCafferty et al .; Marks et al., Biotechnology, 10 : 779-783 (1992)).

  [0048] As used in this application, "HSV-2 antibody" refers to an antibody that specifically reacts with an HSV-2 antigen, but does not react with antigens of other origins, particularly HSV-1.

III. Peptide synthesis
A. Synthesis of peptides by chemical methods
[0049] The peptides of the present invention may be chemically synthesized using conventional peptide synthesis or other protocols well known in the art.

  [0050] Peptides are described in 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 (ed. ), Academic Press, NY, vol. 2, pp.3-284 (1980); and Stewart et al., Solid Phase Peptide Synthesis Second Edition, Pierce Chem. Co., Rockford, 111. (1984). Can be synthesized by solid phase peptide synthesis methods using methods similar to. During synthesis, N-α-protected amino acids with protected side chains were sequentially added to the extended polypeptide chain linked by the C-terminus and to the solid support, ie polystyrene beads. Peptides are synthesized by attaching the amino group of an N-α-deprotected amino acid to the α-carboxy group of the N-α-protected amino acid. The N-α-protected amino acid was activated by reacting it with a reagent such as dicyclohexylcarbodiimide. Binding of the free amino group to the activated carboxyl group results in the formation of a peptide bond. The most commonly used N-α-protecting groups include Boc which is acid labile and Fmoc which is base labile.

  [0051] Suitable materials for use as a solid support are well known to those skilled in the art and include, but are not limited to, the following: halomethyl resins, such as chloromethyl resins, or bromomethyl resins; Resins; phenol resins, such as 4- (α- [2,4-dimethoxyphenyl] -Fmoc-aminomethyl) phenoxy resin; tert-alkyloxycarbonyl-hydrazide resins and the like. Such resins are commercially available and their production methods are known by those skilled in the art.

  [0052] Briefly, a C-terminal N-α-protected amino acid is first bound to a solid support. Next, the N-α-protecting group is removed. The deprotected α-amino group is then coupled to the activated α-carboxyl group of the N-α-protected amino acid. This process is repeated until the desired peptide is synthesized. The resulting peptide is then cleaved from the insoluble polymer support and the amino acid side chains are deprotected. Longer peptides can be obtained by condensing protected peptide fragments. Details about suitable chemical reactions, resins, protecting groups, protected amino acids, and reagents are well known in the art and are not described in detail herein (Atherton et al., Solid Phase Peptide Synthesis: A Practical (See Approach, IRL Press (1989), and Bodanszky, Peptide Chemistry, A Practical Textbook, Second Edition, Springer-Verlag (1993)).

B. Production of peptides by recombinant methods
[0053] As will be appreciated by those skilled in the art, the peptides of the present invention can also be made by recombinant methods. Although it is often preferred to chemically synthesize peptides according to the methods described above, in some cases there may be some advantage to obtaining peptides by recombinant methods. For example, when the HSV-2 peptide of the invention is conjugated to a heterologous peptide and / or heterologous protein, the nucleic acid sequence encoding the HSV-2 peptide is introduced into a suitable expression vector and subsequently the heterologous peptide and And / or fused in-frame with the coding sequence of the protein. As a result, when transfected or transformed into an appropriate host cell line, a fusion polypeptide of HSV-2 peptide and heterologous peptide / protein can be produced and purified. A wide variety of expression vectors and host cells well known to those skilled in the art can be used for this purpose.

C. Peptide purification
[0054] The purification of synthetic peptides uses various methods of chromatography, such as reverse phase HPLC, gel permeation, ion exchange, size exclusion, affinity, partitioning, or countercurrent partitioning. Achieved. The selection of appropriate matrices and buffers is well known in the art.

  [0055] Purifying recombinantly produced peptides until they are substantially pure includes standard techniques, including selective precipitation with substances such as ammonium sulfate, column chromatography, immunopurification, and other methods. Can be achieved using. For example, Scopes, Protein Purification: Principles and Practice (1982); U.S. Pat.No. 4,673,641; Ausubel et al., Current Protocols in Molecular Biology (1994); and Sambrook and Russell, Molecular Cloning: A Laboratory Manual 3rd edition (2001). See In particular, when the recombinant polypeptide comprises an HSV-2 peptide fused to a heterologous protein with known adhesive properties, it is relatively easy and relatively high by passing an appropriate binding partner through an immobilized column. It can be purified with purity.

D. Confirmation of peptide sequence
[0056] The amino acid sequence of peptides prepared for HSV-2 specific detection can be confirmed by a number of established methods. For example, conventional methods of Edman degradation can be used to determine the amino acid sequence of a peptide. Several variations of sequencing methods based on Edman degradation, such as methods based on microsequencing and mass spectrometry, are frequently used for this purpose.

E. Peptide modification
[0057] The peptides of the present invention can be modified to achieve more desirable properties. Chemically modified peptides and peptide mimetics that are resistant to proteolytic degradation or have improved solubility or binding ability are well known.

  [0058] The modified amino acid or chemical derivative of the peptide used for HSV-2 type specific detection usually includes an additional chemical moiety of the modified amino acid that is not part of glycoprotein G2. Covalent modifications of the peptide are within the scope of the present invention. Such modifications can be introduced into the peptide by reacting the target amino acid residue of the peptide with an organic derivative agent capable of reacting with selected side or terminal residues. The following examples for chemical derivatives are provided for purposes of illustration and not for purposes of limitation.

  [0059] The design of peptide mimetics that are resistant to degradation by proteolytic enzymes is known to those skilled in the art. See, for example, Sawyer, Structure-Based Drug Design, P. Verapandia, N. Y. (1997); US Pat. Nos. 5,552,534 and 5,550,251. Both peptide backbone and side chain modifications can be used in designing secondary structures to mimic. Possible modifications include substitutions for D-amino acids, N-α-Me-amino acids, Cα-Me-amino acids, and dehydroamino acids. To date, various secondary structure mimetics have been designed and incorporated into peptides or peptide mimetics.

  [0060] Other modifications include substitution of a natural amino acid with an unnatural hydroxylated amino acid, substitution of a carboxy group of an acidic amino acid with a nitrile derivative, substitution of a hydroxyl group of a basic amino acid with an alkyl group, or methionine Of methionine sulfoxide. Further, the amino acids of the peptide for type-specific detection of HSV-2 can be replaced by amino acids that are the same amino acid but have the opposite chirality. That is, a natural L-amino acid may be substituted by its D configuration.

  [0061] The peptides of the present invention may be modified to increase their ability to specifically bind to HSV-2 antibodies. For example, a linker that may include heterologous peptides, heterologous proteins, and / or virtually non-polypeptide chemicals to facilitate the immobilization of the claimed peptide to a carrier, as well as the peptide to the HSV-2 antibody. And can be introduced at either end of the peptides of the invention. As with other examples, the peptides of the invention can be dimerized to achieve high levels of specific binding to HSV-2 antibodies. Various methods are used for dimerization, and the most commonly used method is through a disulfide bond. Since natural cysteine residues are not present in the peptide, cysteine residues can be included as part of the linker. Methods for adding linkers to the peptides of the invention will be described in detail in the next section.

IV. Immobilization of peptide to carrier
[0062] The peptide disclosed in the present invention for type-specific detection of HSV-2 antibody is preferably immobilized on a solid support or carrier. The carrier is often a synthetic polymeric material, but may be a natural material. Examples of carrier materials are acrylamide, cellulose, nitrocellulose, glass, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polysilicate, polyethylene oxide, polycarbonate, Teflon, fluorocarbon, nylon, silicone Rubber, collagen, polyanhydride, polyglycolic acid, polyacetic acid, polyorthoester, polypropyl fumarate, glycosaminoglycan, and polyamino acid. Carriers include thin films or membranes, beads, bottles, dishes, fibers, woven fibers, shaped polymers, particles, and microparticles such as microspheres. Preferred forms of the support are plates and beads. The most preferred form of beads is magnetic beads or latex beads.

A. Binding to linker
[0063] The peptides of the present invention can be attached to the carrier via various linkers. The linker can be attached to the N or C terminus of the peptide. In the case of an indirect bond between the peptide and the carrier, the linker may consist of a second peptide that is heterologous to the peptide of the invention, or in addition to the heterologous peptide and further heterologous to the peptide of the invention. It further includes proteins that are sex. Branched amino acid polymers are sometimes used as linkers and utilize multiple functional groups on amino acid residues, such as two amine groups on lysine residues. There are other suitable linkers well known to those skilled in the art, including, but not limited to, linear or molecular chain carbon linkers, heterocyclic carbon linkers (eg SMCC), or polyether linkers. These linkers can be used in addition to or in place of heterologous peptides and / or proteins.

  [0064] In the case of a direct bond, the linker can be a covalent bond (eg, a disulfide bond) or a non-covalent bond (eg, an ionic bond) between the peptide and the carrier.

1. Indirect coupling
[0065] When the peptides of the present invention are obtained via synthetic methods, heterologous peptides that serve as linkers may be included during peptide synthesis. For example, a linker consisting of one cysteine residue, one lysine residue, and at least two glycine residues can be included as part of the peptide sequence synthesized at the N- or C-terminus. Other linkers such as linear or branched carbon linkers, heterocyclic carbon linkers, or polyethylene linkers may be used to link the HSV-2 peptides of the invention in combination with heterologous peptides and / or proteins or alone. May be. In addition to heterologous peptides in the linker, when heterologous proteins such as BSA and KLH are used, the heterologous proteins can be linked to the peptide by chemical methods after peptide synthesis and purification. Here, a heterologous peptide has already been added to the peptide during synthesis. Such linkages are linked to the amino acid residues of the peptide through the α-carbon amino and the carboxyl group of the terminal amino acid residue, for example by linking the peptide and a heterologous protein through a peptide bond, or through a side chain group, for example through a disulfide bond. This can be achieved by linking a heterologous protein. Linkers such as heterologous peptides and / or proteins can be linked to the HSV-2 peptides of the present invention through functional groups of internal amino acid residues of the HSV-2 peptide (eg, the second carboxyl group of the aspartic acid residue).

  [0066] When the peptide of the present invention is obtained via a recombinant method, the nucleotide sequence encoding the heterologous peptide or protein is included during the subcloning method, and the recombinant polypeptide thus obtained is ligated. Already have a suitable linker.

  [0067] Those skilled in the art will recognize that a variety of other linkers with appropriate functional groups, such as carbon linkers, or polyether linkers, will be useful for practicing the present invention. . These linkers can be linked to the constituent amino acids of the peptide through side chain groups of amino acids (eg, via a disulfide bond to cysteine). The linker can be linked to the α-carbon amino group or carboxyl group of the terminal amino acid of the peptide.

2. Direct binding
[0068] The peptide of the present invention can be directly immobilized on an insoluble carrier. Strategies for conjugating peptides to carriers (the peptides of such strategies are usually various functional groups such as carboxylic acid (—COOH), free amine (—NH ₂ ), or sulfhydryl (—SH) groups on the carrier). (Including functional groups that can be used to react with the appropriate functional groups to provide conjugation) is similar to several approaches for attaching peptide or protein linkers to carriers, details of which are described in the next section. Has been.

B. Binding to carrier
1. Covalent binding
[0069] Peptides for HSV-2 type specific detection may be bound to the carrier via a covalent bond with or without a linker. Often the carrier has several functional groups such as amines, carboxylic acids, and sulfhydryl groups. Here, these functional groups and the functional group of the peptide or linker can easily react and create a covalent bond that connects the peptide and the carrier. The covalent bond linking the peptide of the present invention and the carrier can exist between the carrier and the terminal amino acid residue of the peptide, or can exist between the carrier and the internal amino acid residue of the peptide. For this purpose, if there are no functional groups originally present on the carrier, the carrier is derivatized prior to conjugation to expose or attach additional reactive functional groups. Derivatization can involve attaching any of a number of molecules commercially available from Pierce Chemical Company, Rockford, Illinois.

2. Non-covalent bond
[0070] Alternatively, the peptide can be bound to the carrier via a known interaction between the tag and the tag binding agent. One partner of the binding interaction, such as a tag, is bound to the peptide as a linker, while the other partner, such as a tag binding agent, can be bound to a carrier. Many tags and tag binding substances can be used based on the molecular interactions described in the literature. For example, if the tag has a natural binding agent, such as biotin, biotin can be used in conjunction with a suitable tag binding agent (avidin, streptavidin, neutravidin, etc.). Receptor-ligand interactions are also suitable as tag and tag binding agent pairs. Cell membrane receptor agonist and antagonist (eg cell receptor-ligand) interaction, eg transferrin, c-kit, viral receptor ligand, cytokine receptor, chemokine receptor, interleukin receptor, cadherin family, integrin family For the selectin family and the like, see, for example, Pigott & Power, The Adhesion Molecule Facts Book I (1993). Similarly, the effects of various small ligands including toxins and toxins, viral epitopes, hormones (eg opiates, steroids, etc.), intracellular receptors (eg steroids, thyroid hormones, retinoids and vitamin D; peptides) Mediating receptors), drugs, lectins, sugars, nucleic acids (linear and cyclic polymer forms), oligosaccharides, proteins, phospholipids, antibodies, etc. can all interact with various cellular receptors. In addition, some synthetic polymers such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, and polyacetates can similarly form suitable tags or tag binding materials. .

  [0071] A linker comprising a tag can be attached to the peptide via a number of methods as described above. On the other hand, the tag binding substance can be immobilized on a solid support (ie, carrier) using any of a variety of currently available methods. The solid support is generally derivatized or functionalized by exposing all or part of the support to a chemical reagent that immobilizes the chemical group on the surface, and the chemical group is then one of the tag binding substances. React with the department. For example, groups suitable for attachment to long chain moieties will include amine, hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes and hydroxyalkylsilanes can be used to functionalize various surfaces, such as glass surfaces. The construction of such solid phase biopolymer arrays is well documented in the literature. For example, Merrifield, J. Am. Chem. Soc. 85: 2149-2154 (1963) (eg, describing solid phase synthesis of peptides); Geysen et al., J. Immun. Meth. 102: 259-274 (1987) (Describes the synthesis of solid phase components on pins); Frank & Doting, Tetrahedron 44: 6031-6040 (1988) (describes the synthesis of various peptide sequences on cellulose discs); Fodor et al., Science, 251: 767-777 (1991); Sheldon et al., Clinical Chemistry 39 (4): 718-719 (1993); and Kozal et al., Nature Medicine 2 (7): 753759 (1996) (all these are solid carriers (Describes an array of immobilized biopolymers). Non-chemical approaches for immobilizing the tag binding substance to the carrier include other common methods such as heating, cross-linking by UV radiation, and the like.

V. Assay for detection of type-specific HSV-2 antibody
A. Detection of HSV-2 antibody using the peptide of the present invention
[0072] In order for the peptides of the present invention to be useful for HSV-2 type specific detection, the peptides of the present invention must be capable of binding to HSV-2 antibodies with specificity. Many well-known immunological binding assays can be performed to test for specific binding. See, for example, U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168. For a general review of immunoassays, see Asai, Methods in Cell Biology, Volume 37: Antibodies in Cell Biology, Academic Press, Inc. NY (1993).

  [0073] Typically, the peptides of the invention can be immobilized and used as so-called "supplementing agents" for HSV-2 antibodies. Samples that contain HSV-2 antibodies, but are known not to contain HSV1 antibodies, may be used in binding assays to screen for peptides that have specificity and can bind to HSV-2 antibodies. Appropriate binding conditions are known in the art, and general instructions for performing such binding assays can be found in many chemical literatures. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988). When an antibody-peptide complex is formed, a labeling agent is used to indicate the presence of such complex. In this case, there are several ways of using labeling agents for this purpose. For example, the labeling agent can be a secondary antibody that can recognize the antibody peptide complex and has a label. Alternatively, the secondary antibody may not have its own label, but may instead be bound by a labeled tertiary antibody specific for the antibody of the species from which the secondary antibody was derived. The secondary antibody may be modified with a detectable moiety, such as biotin. Here, a tertiary labeled molecule (for example, streptavidin having a label) can specifically bind to the biotin. In addition, other proteins that can specifically bind to the immunoglobulin constant region, such as protein A or protein G, can also be used as labeling agents. These proteins are normal components of streptococcal cell walls and exhibit strong non-immunogenic reactivity to immunoglobulin constant regions from various species. See generally Kronval et al., J. Immunol, 111: 1401-1406 (1973); and Akerstrom et al., J. Immunol, 135: 2589-2592 (1985).

  [0074] Throughout the assay, 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 24 hours. Incubation times will vary depending on the assay format, the particular peptide, solution volume, concentration, etc. The assay is often performed at room temperature, but the assay can be performed in a temperature range such as about 10 ° C to about 40 ° C.

[0075] Different labeling methods can be used to detect antibody-peptide complexes. Labeling moieties are, for example, fluorescent molecules (e.g., fluorescein, rhodamine, Texas red, and phycoerythrin) or enzyme molecules (e.g., horseradish peroxidase, alkaline phosphatase, and β-galactosidase conjugated to a secondary or tertiary antibody ) And allows detection based on chemical reaction products catalyzed by fluorescence or enzyme. Radiolabels containing various isotopes such as ³ H, ¹²⁵ I, ³⁵ S, ¹⁴ C, or ³² P are bound to the appropriate molecule, and detection of the antibody-peptide complex is optional Can be performed by any suitable method such as autoradiography. See, for example, Tijssen, “Practice and Theory of Enzyme Immunoassays,” Laboratory Techniques in Biochemistry and Molecular Biology, edited by Burdon and van Knippenberg, Elsevier (1985), pp. 9-20. Label introduction, labeling method, and label detection are described by Polak and Van Noorden, Introduction to Immunocytochemistry, Second Edition, Springer Verlag, NY (1997); and Haugland, Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc. It can be found in the published handbook and catalog combination (1996).

B. Flow cytometry
[0076] Flow cytometry is one of the preferred methods for detecting the presence of HSV-2 type specific antibodies, wherein the peptides of the invention are bound to appropriate particles and HSV-2 Specific binding of the antibody is detected, for example, through the binding of a fluorescently labeled third molecule. Flow cytometry methods and devices for flow cytometry are known in the art and can be used in the practice of the present invention. A common flow cytometry is to pass a suspension of microparticles as a flow across a laser beam and detect the fluorescence emission from each particle with a photomultiplier tube. Detailed descriptions of apparatus and methods for flow cytometry are given in the literature. Examples of literature are: McHugh, "Flow Microsphere Immunoassay for the Quantitative and Simultaneous Detection of Multiple Soluble Analytes," Methods in Cell Biology 42, Part B (Academic Press, 1994); McHugh et al., "Microsphere-Based Fluorescence Immunoassays Using Flow Cytometry Instrumentation, "Clinical Flow Cytometry, Bauer, KD et al. (Baltimore, Maryland, USA: Williams and Williams, 1993), pp. 535-544; Lindmo et al.," Immunometric Assay Using Mixtures of Two Particle Types of Different Affinity , "J. Immunol. Meth. 126: 183-189 (1990); McHugh," Flow Cytometry and the Application of Microsphere-Based Fluorescence Immunoassays, "Immunochemica 5: 116 (1991); Horan et al.," Fluid Phase Particle Fluorescence Analysis : Rheumatoid Factor Specificity Evaluated by Laser Flow Cytophotometry, "Immunoassays in the Clinical Laboratory, 185-189 (Liss 1979); Wilson et al.," A New Microsphere-Based Immunofluorescence Assay Using Flow Cytometry, "J. Immunol. Meth. 107: 225 -230 (1988); Fulwyler et al., "Flow Micr osphere Immunoassay for the Quantitative and Simultaneous Detection of Multiple Soluble Analytes, "Meth. Cell Biol. 33: 613-629 (1990); Coulter Electronics Inc., British Patent No. 1,561,042 (published February 13, 1980); and Steinkamp Et al., Review of Scientific Instruments 44 (9): 1301-1310 (1973).

  [0077] The particles used in practicing the present invention are preferably microscopically sized and formed from a polymeric material. Polymers that are useful as microparticles are chemically inert compared to the components of biological samples and compared to assay reagents other than binding member films that are immobilized on the surface of the microparticles. Suitable particulate material has minimal autofluorescence, is solid and is insoluble in the sample and any buffer, solvent, carrier, diluent, or suspension used in the assay, And it would be possible to fix the appropriate coating substance, preferably via a covalent bond. Examples of suitable polymers are polyesters, polyethers, polyolefins, polyalkylene oxides, polyamides, polyurethanes, polysaccharides, celluloses, and polyisoprenes. Crosslinking is useful to give structural integrity and rigidity to many polymers. The particle size range varies and the particle size range is not critical to the invention. In many cases, the microparticles will range in diameter from about 0.5 μm to about 100 μm, and preferably from about 0.3 μm to about 40 μm.

[0078] In order to enhance the particle recovery and wash steps of the assay, the particles preferably comprise a magnetically reactive material, ie any material that is responsive to a magnetic field. Separation of the solid and liquid phases after the incubation or washing step can be accomplished by applying a magnetic field to the reaction vessel in which the suspension is subsequently incubated, causing the particles to adhere to the vessel walls, thereby decanting or aspirating the liquid. This can be achieved by allowing removal by Magnetically responsive materials of interest of the present invention include paramagnetic materials, ferromagnetic materials, ferrimagnetic materials, and metamagnetic materials. Paramagnetic materials are preferred. Examples of this are iron, nickel and cobalt, and metal oxides such as Fe ₃ O ₄ , BaFe ₁₂ O ₁₉ , CoO, NiO, Mn ₂ O ₃ , Cr ₂ O ₃ and CoMnP.

  [0079] The magnetically responsive material is dispersed through the polymer and applied as a coating on the polymer surface or as one of two or more coatings on the surface, or the material is incorporated into the particles. It can be incorporated or fixed in any other manner of fixation. The amount of magnetically reactive material in the particles is not critical and can vary over a wide range. The amount can affect the density of the microparticles, but both the amount and the particle size can cause the microparticles in suspension to achieve maximum contact between the liquid and the solid phase and to perform flow cytometry. It can affect the ease of maintenance. An excess of magnetically reactive material in the microparticles can produce a level of autofluorescence that is high enough to interfere with the assay results. Therefore, it is preferred that the concentration of the magnetically responsive material should be low enough to minimize the autofluorescence emitted from the material. In view of these, the magnetically reactive material in the particles according to the present invention ranges from about 0.05% to about 75% of the total weight of the particles. A more preferred weight percent range is from about 1% to about 50%, a further preferred weight percent range is from about 2% to about 25%, and an even more preferred weight range is from about 2% to about 8%.

[0080] Coating the particle surface with a suitable assay reagent may be accomplished by electrostatic attraction, specific affinity interactions, hydrophobic interactions, or covalent bonds. A covalent bond is preferred. The polymer can be derivatized with a functional group that covalently binds the assay reagent by conventional methods, in particular by using monomers containing the functional group. Such monomers serve as a single monomer or as a comonomer. 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). ). Monomers useful for introducing carboxylic acid groups into the polyolefin are, for example, acrylic acid and methacrylic acid.

  [0081] A linker is an epitope present on the particle surface that can be used as a means to increase the density of epitopes that can be recognized by an antibody and to reduce steric hindrance. This increases the range and sensitivity of the assay. When it is necessary to immobilize certain coating materials of the present invention, linkers can be used as a method of adding specific types of reactive groups to the solid surface. Examples of suitable and useful functional groups are polylysine, polyaspartic acid, polyglutamic acid, and polyarginine.

  [0082] In general, care should be taken to avoid the use of particles that exhibit strong autofluorescence. Particles formed from a variety of starting monomers by conventional suspension polymerization techniques are generally suitable. This is because it exhibits only a low level of autofluorescence at most. Conversely, particles that have been modified to increase their porosity and thus increase their surface area, ie particles referred to in the literature as “macroporous”, are undesirable. Because they tend to show high autofluorescence. It is further considered that autofluorescence increases with increasing size and with increasing proportion of divinylbenzene monomer.

  [0083] The label used in the labeled binding member may be any label capable of emitting a detectable signal. A preferred such label is a fluorophore. Every fluorophore is reported in the literature and is thus known to those skilled in the art, and many are readily available from commercial sources in the biotechnology industry. References to fluorophores can be found in Cardullo et al., Proc. Natl. Acad. ScL USA 85: 8790-8794 (1988); Dexter, DL, J. of Chemical Physics 21: 836-850 (1953); Hochstrasser et al., Biophysical Chemistry. 45: 133-141 (1992); Selvin, P., Methods in Enzymology 246: 300-334 (1995); Steinberg, I. Ann. Rev. Biochem., 40: 83- 114 (1971); Stryer, L. Ann. Rev. Biochem., 47: 819-846 (1978); Wang et al., Tetrahedron Letters 31: 6493-6496 (1990); Wang et al., Anal Chem. 67: 1197-1203 (1995).

[0084] The following is an example of a fluorophore.
4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid acridine isothiocyanic acid acridine 5- (2'-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS)
4-amino-N- [3-vinylsulfonyl) phenyl] naphthalimide-3,5-disulfonate N- (4-anilino-1-naphthyl) maleimide anthranilamido BODIPY
Brilliant yellow,
Coumarin 7-Amino-4-methylcoumarin (AMC, Coumarin 120)
7-amino-4-trifluoromethylcoumarin (coumarin 151)
Cyanine dye Cyanosine 4 ', 6-diamidino-2-phenylindole (DAPI)
5 ', 5 "-Dibromopyrogallol-Sulfonaphthalene (Bromopyrogallol Red)
7-diethylamino-3- (4'-isothiocyanatophenyl) -4-methylcoumarin diethylenetriamine pentaacetate 4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid 4,4'-di Isothiocyanatostilbene-2,2′-disulfonic acid 5- [dimethylamino] naphthalene-1-sulfonyl chloride (DNS, dansyl chloride)
4- (4'-Dimethylaminophenylazo) benzoic acid (DABCYL)
4-Dimethylaminophenylazophenyl-4 ′ isothiocyanate (DABITC)
Eosin isothiocyanate eosin erythrosine B
Erythrosine isothiocyanate ethidium 5-carboxyfluorescein (FAM)
5- (4,6-Dichlorotriazin-2-yl) aminofluorescein (DTAF)
2 ′, 7′dimethoxy-4′5′dichloro-6-carboxyfluorescein (JOE)
Fluorescein Fluorescein isothiocyanate Fluoresamine IR144
IR1446
Malachite green isothiocyanate 4-methylumbelliferone ortho-cresolphthalein nitrotyrosine pararosaniline phenol red phycobilin protein (B-phycoerythrin, R-phycoerythrin, etc.)
o-phthaldialdehyde pyrene pyrene butyrate succinimidyl 1-pyrene butyrate quantum dot reactive red (Cibacron (registered trademark) brilliant red 3B-A)
6-carboxy-X-rhodamine (ROX)
6-carboxyrhodamine (R6G)
Lisaamine Rhodamine B sulfonyl chloride Rhodamine (Rhod)
Rhodamine B
Rhodamine 123
Rhodamine X isothiocyanate sulforhodamine B
Sulforhodamine 101
Sulforhodamine 101 sulfonyl chloride derivative (Texas Red)
N, N, N ′, N′-tetramethyl-6-carboxyrhodamine (TAMRA)
Tetramethyl rhodamine Tetramethyl rhodamine isothioanate (TRITC)
Riboflavin rosolic acid
Lanthanide chelate derivatives

  [0085] Attaching any of these fluorophores to the binding molecule to form an assay reagent for use in practicing the present invention can be performed on the appropriate functional group on the fluorophore and on the binding member. This is accomplished by conventional covalent bonding with appropriate functional groups. The reaction of recognizing such groups and forming bonds will be readily apparent to those skilled in the art.

  [0086] Similarly, methods and devices for applying and removing a magnetic field as part of an automated assay are known to those skilled in the art and have been reported in the literature. Examples of references include Forrest et al., U.S. Pat.No. 4,141,687 (Technicon Instruments Corporation, February 27, 1979); Ithakissios, U.S. Pat.No. 4,115,534 (Minnesota Mining and Manufacturing Company, September 19, 1978); Vlieger, AM, et al., Analytical Biochemistry 205: 1-7 (1992); Dudley, Journal of Clinical Immunoassay 14: 77-82 (1991); and Smart, Journal of Clinical Immunoassay 15: 246-251 (1992). All references cited in this paragraph and in the previous paragraph are incorporated herein.

C. Non-reactivity of peptides of the invention to HSV-1
[0087] Another equally important aspect of the essential features of peptides used for HSV-2 type specific detection is the detectable specificity in the test format used for HSV-2 antibody detection. It should not bind to HSV-1 antibody. Once peptides were shown to react specifically with HSV-2 antibodies, they were further tested for potential cross-reactivity to antibodies against HSV-1 antibodies and other viruses such as the herpes family. . To test for non-reactivity to the HSV-1 antibody, the peptide was immobilized and the peptide was “captured in an immunoassay substantially identical to the immunoassay described in the last section. Use as However, a sample confirmed to contain HSV-1 antibody (excluding HSV-2 antibody) is used for the binding assay.

  [0088] The following examples are provided for purposes of illustration, and not for purposes of limitation.

I. Peptide synthesis
[0089] Peptides listed in Table 1 were synthesized as monomers using the N-α-protecting group Boc or Fmoc.

II. Dimerization
[0090] The optional steps of peptide dimerization were performed as follows: the monomeric peptide was dissolved in deionized water or 0.1 M sodium bicarbonate and stirred overnight at 4 ° C. The resulting oxidized dimerized peptide was purified by preparative HPLC using a C18 column.

III. Preparation of Monomer Peptide-BSA Conjugate
[0091] In a solution of 0.1 mM borate buffer (pH 8.0) (10 mg / ml) containing bovine serum albumin (BSA), 154 μl of SMCC, dimethyl sulfoxide (DMSO) containing N-hydroxysuccinimide (10 mg / ml) ml) was added. The mixture was incubated at room temperature for 2 hours, applied to a Sephadex G-25 column (30 ml) pre-equilibrated with 10 mM phosphate saline (PBS, pH 7.4) and eluted with PBS. The first peak was controlled and the protein concentration was estimated by absorbance at 280 nm.

  [0092] Monomer peptide (1 mg) was mixed with 1 mg modified BSA and stored overnight at 4 ° C. The peptide-BSA complex was purified via size exclusion chromatography.

IV. Preparation of dimerized peptide-BSA complex
[0093] The dimerized peptide-BSA complex can be prepared as described below.

1. SMCC-peptide dimer
[0094] 50 μl of DMSO solution (10 mg / ml) containing SMCC is added to 1 ml of 100 mM phosphate (pH 8.0) solution containing peptide dimer. After 2 hours of incubation, the modified peptide is purified by HPLC chromatography.

2. 2-Mercaptoacetyl-BSA
[0095] 50 μl of DMSO containing N-succinimidyl S-acetylthioacetate (SATA) (10 mg / ml) is added to 1 ml of 100 mM phosphate (pH 8.0) containing BSA (10 mg / ml). After 2 hours of incubation, 100 μl 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 subpurged onto a Sephadex G-25 column. The modified BSA is eluted from the column with 10 mM PBS.

3. Peptide dimer-BSA complex
[0096] 4 mg modified BSA is mixed with 4 mg modified peptide at 2-8 ° C. and the mixture is incubated overnight. The complex peptide is purified by size exclusion chromatography.

V. Preparation of magnetic beads coated with peptide or peptide-BSA
[0097] To 6 mg magnetic beads washed twice with 25 mM · 2- [N-morpholino] ethanesulfonic acid (MES) (pH 6.1), 588 μl of deionized water, 0.5 M · MES (pH 6.1) 80 μl of deionized water containing 100 mg / ml N-hydroxysuccinimide (NHS), 92 μl deionized water, and 50 mg / ml 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC). 40 μl of deionized water was added and the mixture was vortexed for 30 minutes at room temperature. The activated beads were washed twice with 25 mM MES (pH 6.1). Next, 100 μl of 0.5 M MES (pH 6.1), 720 μl of deionized water, and 180 μl of peptide (1 mg / ml) or peptide-BSA complex (1 mg / ml) were added and the mixture vortexed . The mixture was further incubated on the rotor for 2 hours at room temperature and centrifuged at 7000 rpm for 2 minutes. After discarding the supernatant, the beads were added PBS containing 1 ml of 1% BSA and 20 mM Tris base, mixed on a rotator for 2 hours at room temperature, and then centrifuged to remove the supernatant. . The beads were washed twice with 10 mM PBS containing 1% BSA and stored in 1 ml 1% BSA. Scheme 1 shows a method for attaching HSV-2 via a heterologous peptide already attached to SMCC at the C-terminus. This is then coupled to the microsphere via BSA:

VI. Flow cytometric immunoassay (FCIA)
[0098] Peptides or peptide-BSA complexes, such as those in Table 1, are bound to predetermined magnetic beads (all peptides or peptide-BSA complexes are bound to magnetic beads and the fluorescence of the magnetic beads Indicates specific dye amount ranges), and mixed together with similar bead numbers for each range of magnetic beads. The mixed beads were diluted in PBS containing 1% BSA, 0.1% Tween 20, and diluted to 1000 copies of each range of magnetic beads per ml.

  [0099] To a microtiter tube, 100 μl of sample diluent, 5 μl of patient sample, and 100 μl of bead mixture were added and incubated at 37 ° C. on a shaker for 20 minutes. After magnetic separation, the beads were washed twice with wash buffer (10 mM PBS with 0.1% Tween 20). 50 μl of anti-human IgG (Fc specific) -B-phycoerythrin complex was added. After incubation at 37 ° C. for 10 minutes on the shaker, the beads were washed again twice with wash buffer and suspended in 50 μl of wash buffer. The beads were counted and analyzed on a Luminex 100 instrument. The amount of antibody (IgG) bound to the magnetic beads was measured with anti-human IgG conjugated to phycoerythrin.

VII. Comparison with commercially available assay systems
[0100] According to a study using 137 clinically defined patient samples, an assay system using the peptide-BSA complex of the present invention has become a commercially available HSV-2 type IGG assay system based on gG-2. It was shown to be superior and have 100% consistency in the confirmation test of the Western blot assay (Table 2).

VIII. Non-reactivity to HSV-1 IgG
[0101] The HSV-2 specific immunoassay system of the present invention was tested for lack of cross-reactivity to antibodies against other viruses of the herpes family, particularly HSV-1. Table 3 shows that HSV-1 IgG has no cross-reactivity as confirmed by two commercially available HSV-2 type specific assay systems and Western blot assay.

を示す。
図２は、市販のＨＳＶ-２型特異的酵素結合免疫学的検定を用いたＨＳＶ-２特異的抗体の検出と、ペプチド１-ＳＭＣＣ-ＢＳＡ複合体：

をＨＳＶ-２特異的抗原として用いる本発明の方法を使用したＨＳＶ-２特異的抗体の検出との間の相関を示す。
図３は、市販のＨＳＶ-２型特異的酵素結合免疫学的検定を用いたＨＳＶ-２特異的抗体の検出と、ペプチド２-ＳＭＣＣ-ＢＳＡ複合体：

をＨＳＶ-２特異的抗原として用いる本発明の方法を使用したＨＳＶ-２特異的抗体の検出との間の相関を示す。
図４は、市販のＨＳＶ-２型特異的酵素結合免疫学的検定を用いたＨＳＶ-２特異的抗体検出と、ＢＳＡ-ＳＭＣＣ-ペプチド５複合：

をＨＳＶ-２特異的抗原として用いる本発明の方法を使用したＨＳＶ-２特異的抗体の検出との間の相関を示す。
図５は、市販のＨＳＶ-２型特異的酵素結合免疫学的検定を用いたＨＳＶ-２特異的抗体検出と、ペプチド５：

をＨＳＶ-２特異的抗原として用いる本発明の方法を使用したＨＳＶ-２特異的抗体の検出との間の相関を示す。 FIG. 1 shows SEQ ID NO: 1:

Indicates.
FIG. 2 shows the detection of HSV-2 specific antibodies using a commercially available HSV-2 specific enzyme-linked immunoassay and the peptide 1-SMCC-BSA complex:

Figure 2 shows the correlation between detection of HSV-2 specific antibodies using the method of the invention using as the HSV-2 specific antigen.
FIG. 3 shows the detection of HSV-2 specific antibodies using a commercially available HSV-2 type specific enzyme-linked immunoassay and peptide 2-SMCC-BSA conjugate:

Figure 2 shows the correlation between detection of HSV-2 specific antibodies using the method of the invention using as the HSV-2 specific antigen.
FIG. 4 shows HSV-2 specific antibody detection using a commercially available HSV-2 specific enzyme-linked immunoassay and BSA-SMCC-peptide 5 conjugate:

Figure 2 shows the correlation between detection of HSV-2 specific antibodies using the method of the invention using as the HSV-2 specific antigen.
FIG. 5 shows HSV-2 specific antibody detection using a commercially available HSV-2 specific enzyme-linked immunoassay and peptide 5:

Figure 2 shows the correlation between detection of HSV-2 specific antibodies using the method of the invention using as the HSV-2 specific antigen.

Claims (59)

  A composition comprising an HSV-2 peptide consisting of 24-36 contiguous amino acids of SEQ ID NO: 1.   The composition according to claim 1, wherein the peptide has a sequence consisting of amino acids 5 to 32 of SEQ ID NO: 1.   The composition according to claim 1, wherein the peptide has a sequence consisting of amino acids 9 to 32 of SEQ ID NO: 1.   The composition of claim 1, wherein the peptide has the sequence of SEQ ID NO: 1.   The composition of claim 1, wherein the peptide is dimerized.   6. The composition of claim 5, wherein the peptide is dimerized via a disulfide bond.   The composition of claim 1, wherein the peptide is bound to a carrier.   8. The composition of claim 7, wherein the carrier is a carboxylated microsphere.   9. A composition according to claim 8, wherein the carrier is a carboxylated latex or magnetic microsphere.   The composition according to claim 7, wherein the peptide is bound to a carrier via a linker at the N-terminus or C-terminus of the peptide.   8. The composition of claim 7, wherein the peptide is bound to a carrier via a linker at an internal amino acid residue of the peptide.   The composition of claim 10, wherein the linker comprises a heterologous peptide.   The composition of claim 10, wherein the linker consists of a heterologous protein.   13. The composition of claim 12, wherein the linker further comprises a heterologous protein.   15. The composition of claim 14, wherein the heterologous protein is bovine serum albumin (BSA).   The composition according to claim 14, wherein the heterologous protein is keyhole limpet hemocyanin.   The composition of claim 12, wherein the linker consists of a heterologous peptide.   13. The composition of claim 12, wherein the heterologous peptide comprises one cysteine residue, one lysine residue, and at least two glycine residues.   12. The composition of claim 10, wherein the linker comprises a branched amino acid polymer. Said branched amino acid polymer is:

The composition of Claim 19 containing the structure represented by these. (1) the peptide has the amino acid sequence of SEQ ID NO: 1;
(2) the carrier is a carboxylated magnetic microsphere;
(3) the linker comprises 4- (maleimidomethyl) -1-cyclohexanecarboxylic acid (SMCC) and a heterologous peptide, the heterologous peptide having the amino acid sequence of GGCK; and
(4) The composition of claim 12, wherein the heterologous peptide binds to the peptide at the C-terminus of the peptide.   23. The composition of claim 21, wherein the linker further comprises a heterologous protein BSA that is directly attached to the carrier, wherein SMCC is attached to BSA and to the peptide via a heterologous peptide. (1) The peptide has an amino acid sequence consisting of amino acids 5 to 32 of SEQ ID NO: 1;
(2) the carrier is a carboxylated magnetic microsphere;
(3) the linker comprises SMCC and a heterologous peptide, the heterologous peptide having the amino acid sequence of GGGGCK; and
(4) the heterologous peptide binds to the peptide at the C-terminus of the peptide;
The composition according to claim 12.   24. The composition of claim 23, wherein the linker further comprises a heterologous protein BSA that is directly attached to the carrier, wherein SMCC is attached to the BSA and to the peptide via the heterologous peptide. . (1) The peptide has an amino acid sequence consisting of amino acids 9 to 32 of SEQ ID NO: 1;
(2) the carrier is a carboxylated magnetic microsphere;
(3) the linker comprises SMCC and a heterologous peptide, the heterologous peptide having the amino acid sequence of GGGGCK; and
(4) the heterologous peptide is bound to the peptide at the C-terminus of the peptide;
The composition according to claim 12.   26. The composition of claim 25, wherein the linker further comprises a heterologous protein BSA that is directly attached to the carrier, wherein SMCC is attached to the BSA and to the peptide via the heterologous peptide. . (1) The peptide has an amino acid sequence consisting of amino acids 9 to 32 of SEQ ID NO: 1;
(2) the carrier is a carboxylated magnetic microsphere;
(3) the linker comprises SMCC and a heterologous peptide, the heterologous peptide having an amino acid sequence of KCGGGGG; and
(4) the heterologous peptide is bound to the peptide at the N-terminus of the peptide;
The composition according to claim 12.   28. The composition of claim 27, wherein the linker comprises a heterologous protein BSA that is directly attached to the carrier, wherein SMCC is attached to BSA and to the peptide via the heterologous peptide. (1) The peptide has an amino acid sequence consisting of amino acids 9 to 32 of SEQ ID NO: 1;
(2) the carrier is a carboxylated microsphere; and
(3) The branched amino acid polymer further comprises a short peptide called CK, wherein the short peptide is directly linked to the terminal K residue of the branched amino acid polymer via its C residue. ,
21. The composition of claim 20. A method for the specific detection of HSV-2 antibody in a biological sample comprising the following steps:
(a) contacting a biological sample with a composition comprising 24 to 36 contiguous amino acids of SEQ ID NO: 1 comprising the peptide bound to a carrier;
(b) detecting whether antigen-antibody binding occurs between the peptide and the antibody component of the biological sample, wherein detection of the antigen-antibody binding is performed in the HSV in the biological sample. -2 indicates the presence of antibody,
The detection method comprising:   32. The method of claim 30, wherein step (b) is performed by flow cytometry.   32. The method of claim 30, wherein the biological sample is whole blood, serum, plasma, cerebrospinal fluid, seminal vesicle fluid, or mucus.   32. The method of claim 30, wherein the peptide has a sequence consisting of amino acids 5 to 32 of SEQ ID NO: 1.   32. The method of claim 30, wherein the peptide has a sequence consisting of amino acids 9-32 of SEQ ID NO: 1.   32. The method of claim 30, wherein the peptide has the sequence of SEQ ID NO: 1.   32. The method of claim 30, wherein the carrier is a carboxylated microsphere.   37. The method of claim 36, wherein the carrier is a carboxylated latex or magnetic microsphere.   32. The method of claim 30, wherein the peptide is dimerized.   40. The method of claim 38, wherein the peptide is dimerized via a disulfide bond.   31. The method of claim 30, wherein the peptide is attached to the carrier via a linker at the N-terminus or C-terminus of the peptide.   32. The method of claim 30, wherein the peptide is attached to the carrier via a linker at an internal amino acid residue of the peptide.   41. The method of claim 40, wherein the linker comprises a heterologous peptide.   41. The method of claim 40, wherein the linker consists of a heterologous protein.   43. The method of claim 42, wherein the linker further comprises a heterologous protein.   45. The method of claim 44, wherein the heterologous protein is BSA.   45. The method of claim 44, wherein the heterologous protein is KLH.   43. The method of claim 42, wherein the linker consists of a heterologous peptide.   43. The method of claim 42, wherein the heterologous peptide comprises 1 cysteine residue, 1 lysine residue, and at least 2 glycine residues.   41. The method of claim 40, wherein the linker comprises a branched amino acid polymer. Said linker is:

50. The method of claim 49, comprising a structure represented by: (1) the peptide has the amino acid sequence of SEQ ID NO: 1;
(2) the carrier is a carboxylated magnetic microsphere;
(3) the linker is SMCC and a heterologous peptide, the heterologous peptide having the amino acid sequence of GGCK;
(4) the heterologous peptide is attached to the peptide at the C-terminus of the peptide; and
(5) The step (b) is performed by flow cytometry.
43. The method of claim 42.   52. The method of claim 51, wherein the linker further comprises a heterologous protein BSA directly attached to the carrier, wherein SMCC is attached to BSA and to the peptide via the heterologous peptide. (1) The peptide has an amino acid sequence consisting of amino acids 5 to 32 of SEQ ID NO: 1;
(2) The carrier is a carboxylated magnetic microsphere:
(3) the linker includes SMCC and a heterologous peptide, the heterologous peptide having an amino acid sequence of GGGGCK;
(4) the heterologous peptide is attached to the peptide at the C-terminus of the peptide; and
(5) The step (b) is performed by flow cytometry.
43. The method of claim 42.   54. The method of claim 53, wherein the linker further comprises a heterologous protein BSA that is directly attached to the carrier, wherein SMCC is attached to BSA and to the peptide via the heterologous peptide. (1) The peptide has an amino acid sequence consisting of amino acids 9 to 32 of SEQ ID NO: 1;
(2) the carrier is a carboxylated magnetic microsphere;
(3) the linker includes SMCC and a heterologous peptide, the heterologous peptide having an amino acid sequence of GGGGCK;
(4) the heterologous peptide is bound to the peptide at the C-terminus of the peptide;
(5) The step (b) is performed by flow cytometry.
43. The method of claim 42.   56. The method of claim 55, wherein the linker further comprises a heterologous protein BSA that is directly attached to the carrier, wherein SMCC is attached to BSA and to the peptide via the heterologous peptide. (1) The peptide has an amino acid sequence consisting of amino acids 9 to 32 of SEQ ID NO: 1;
(2) the carrier is a carboxylated magnetic microsphere;
(3) The linker includes SMCC and a heterologous peptide, the heterologous peptide having an amino acid sequence of KCGGGGG;
(4) the heterologous peptide is attached to the peptide at the N-terminus of the peptide; and
(5) The step (b) is performed by flow cytometry.
43. The method of claim 42.   58. The method of claim 57, wherein the linker further comprises a heterologous protein BSA that is directly attached to the carrier, wherein SMCC is attached to BSA and to the peptide via the heterologous peptide. (1) The peptide has an amino acid sequence consisting of amino acids 9 to 32 of SEQ ID NO: 1;
(2) the carrier is a carboxylated microsphere;
(3) the branched amino acid polymer further comprises a short peptide of CK, wherein the short peptide is directly linked to the terminal K residue of the branched amino acid polymer via its C residue; And
(4) Step (b) is performed by flow cytometry.
51. The method of claim 50.

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