Method for determining hcv-antigen/antibody complex

FIELD: medicine.

SUBSTANCE: method involves determining complex formed between HCV anticore antigen and NS3/4a-antibody capable of recognizing both HCV- antigens and antibodies available in sample when using common solid base and solid substrates usable in immunoassay.

EFFECT: high accuracy and sensitivity of hepatitis C diagnosis.

47 cl, 8 dwg, 10 tbl

 

The scope of the invention

The present invention relates in General to detecting viruses. In particular, the invention relates to the determination of complex antigen/antibody for accurate diagnosis of infection with hepatitis C.

Background of the invention

Hepatitis C virus (HCV) is a major cause of parenteral hepatitis non-a, non-b (NANBH), which is primarily transmitted through blood and sexual contact. The virus is present in 0.4 to 2.0% of blood donors. Chronic hepatitis develops in about 50% of those infected, and about 20% of them develop cirrhosis, which sometimes leads to hepatocellular carcinoma. Therefore, the study and control of this disease are important for medicine.

HCV was first identified and characterized as the cause of NANBH by Hitena with co-authors (Houghten et al.). Cm. International Publication number WO 89/04699, WO 90/11089 and WO 90/14436. HCV genome has presented 9,5 TPN sense, single-chain RNA and is a member of the virus family Flaviridae. At least six separate, though related genotypes of HCV were identified on the basis of phylogenetic analyses (Simmonds et al., Tomashevskaya et al (1993) 74: 2391-2399). The virus encodes only polyprotein with more than 3000 amino acid residues (Choo et al., Science (1989) 244:359-362; Choo et al., Proc. Natl. Acad. Sci. USA (1991) 88:2451-2455; Han et al., Proc. Natl. Acad. Sci USA (1991) 88: 1711-1715). During and PEFC is broadcast this protein is converted as in the structural, so in non-structural (NS) proteins.

In particular, as shown in figure 1, HCV genome encodes several proteins. The order and nomenclature of the products of the cleavage of polyprotein HCV following: NH2-C-E1-E2-p7-NS2-NS3-NS4a-NS4b-NS5a-NS5b-COOH. Initial cleavage polyprotein is catalyzed by proteases of the host, which releases three structural protein, N-terminal nucleocapsid protein (denoted "cor") and two envelope glycoproteins, "E1" (also known as "E") and "E2" (also known as E2/NS1"), as well as non-structural (NS) proteins containing viral enzymes. NS region marked NS2, NS3, NS4 and NS5. NS2 is an integral membrane protein with proteolytic activity. NS2 as one, and together with N33, splits NS2-NS3 weak link, which, in turn, creates N-end NS3 and releases a large polyprotein, which has activity as a serine protease and RNA-helicase. NS3-protease converts the remaining polyprotein. The end of the maturation polyprotein is initiated by autocatalytic cleavage connection NS3-NS4a catalyzed NS3 serine protease. Subsequent cleavage of polyprotein HCV, NS3-mediated most likely include the detection of fissile relations polyprotein molecule N33 another polypeptide. In these reactions, NS3 releases NS3 cofactor - (NS4a), two proteins (NS4b and NS5a) and RNA head of the independent RNA polymerase (NS5b).

Described a number of General and specific polypeptides derived from polyprotein HCV, suitable as immunological and diagnostic reagents. For example, Houghton et al., European publication No. 318,216 and 388,232; Choo et al., Science (1989) 244:359-362; Kuo et al., Science (1989) 244:362-364; Houghton et al., Hepatology (1991) 14:381-388; Chien et al., Proc. Natl. Acad. Sci. USA (1992) 89:10011-10015; Chien et al., J. Gastroent. Hepatol. (1993) 8-S33-39; Chien et al., International publication number WO 93/00365, Chinen D. Y. international publication number WO 94/01778. These publications offers considerable promise for studies of HCV in General and for the production and use of immunological reagents for HCV polypeptide.

Sensitive, specific methods of selection and identification of carriers of HCV and blood and blood products infected with HCV, will create significant progress in medicine. Post-transfusion hepatitis occurs in approximately 10% of patients undergoing transfusion and HCV marked up to 90% in these cases. The treatment of patients, and the prevention and transmission of HCV by blood and blood products or by close personal contact requires reliable diagnostic and prognostic tools. Thus, we developed several tests for serodiagnosis of HCV infection. For example, see Choo et al., Science (1989) 244:359-362; Kuo et al.. Science (1989) 244,: 362-364; Choo et al., Br. Med. Bull. (1990) 46:423-441; Ebeling et al., Lancet (1990) 335:982-983; van der Poel et al. Lancet (1990) 335:558-560; van der Poel et al., Lancet. (1991) 337:317-319; Chien, D.Y. international publication number WO 97/01178; Valenzuela et al., International publication number WO 97/44469; and Kashiwakuma et al., U.S. Patent No. 5,871,904.

In some analyses on the basis of serum there was a serious problem because of the large interval between infection and detection of the virus, which often exceed 80 days. This interval may create a large risk to recipients of blood. To overcome this problem have been developed tests based on nucleic acids (NAT), which directly determine the viral RNA, and tests for HCV core antigen, which is a viral antigen, instead of the reaction of the antibody. See, for example, Kashiwakuma et al., U.S. patent No. 5,871,904; Beld et al. Transfusion (2000) 40:575-579.

Nevertheless preserved the need for sensitive and accurate diagnostic and prognostic tools that allow both to arrange appropriate treatment of patients and to prevent transmission of HCV by blood and blood products or by close personal contact.

Brief description of the invention

The present invention is based in part on the discovery that respond to HCV serum antibodies are typically antipoverty and anti-NS3 (helicase). Accordingly, the invention involves a determination of complex measles antigen HCV NS3 and antibodies, which gives the opportunity to discover how HCV antigens and antibodies in the sample, using the CTD is inogo solid matrix.

Thus, in one of the embodiments, the subject invention is a solid substrate for immunoassay comprising at least one HCV-antikarov antibody and at least one selected NS3/4a-HCV epitope associated with it. Antibody and NS3/4A-epitope can be any of the molecules described here. In addition, the solid substrate may include any of the described herein fused antigens with multiple epitopes, such as fused antigen with multiple epitopes comprising amino acid sequence depicted in FIGU-7F.

In certain embodiments of the implementation, the solid substrate comprises at least two associated HCV-antikarov antibodies. In addition, antikarov antibody may be a monoclonal antibody. Moreover, NS3/4A-epitope can be a conformational epitope, such as conformational NS3/4a-epitope containing amino acid sequence depicted in FIGU-4D.

In another embodiment, the invention relates to a solid substrate for immunoassay containing at least two HCV-antikarov monoclonal antibody and at least one conformational epitope NS3/4a HCV containing the amino acid sequence depicted in FIGU-4D associated with it.

In the following embodiment, the image is giving relates to a method for determination of HCV infection in a biological sample. The method includes: (a) obtaining a solid substrate for immunoassay, as described above; (b) combining a biological sample with a solid substrate under conditions which allow HCV antigens and antibodies, when they are present in the biological sample, contacting at least one antiquorum antibody and NS3/4a-epitope, respectively; (C) adding to the solid substrate from step (b) under complex-forming conditions (i) a first labeled antibody, where the first detectable labeled antibody is antiquorum HCV antibody with a detectable label, where labeled antikarov antibody directed against a different epitope of measles HCV than at least one antikarov antibody associated with the solid substrate; (ii) antigen that interacts with HCV-antibody from the biological sample, reacting with NS3/4a-epitope and (iii) a second labeled antibody, where the second antibody with a detectable label can react with antigen (ii); and (d) determination of complexes formed between antibodies and antigens, if present, as an indication for the presence of HCV infection in a biological sample. NS3/4a-epitope can be a conformational epitope, such as conformational epitope represented by a sequence of NS3/4a, depicted in figures 4A-4D.

In another embodiment, the invention is tositsa to,a method of determining HCV infection in a biological sample. This method includes: (a) obtaining a solid substrate immunoassay with at least two related anticorrosie HCV antibodies, as described above; (b) adding a biological sample to a solid substrate under conditions that allow antigen and HCV antibodies in case of their presence in the biological sample, contacting at least two anticorrosie antibodies and NS3/4a-epitope, respectively; (C) adding to the solid substrate from step (b) complexing the conditions (i), the first antibody with a detectable label, where this is the first antibody with a detectable label observed detectable way antiquorum antibody HCV, where labeled antikarov antibody directed against a different epitope of measles HCV than antikarov antibody associated with the solid substrate; (ii) epitope from the area SS polyprotein HCV connected with hSOD amino acid sequence; and (iii) a second antibody with a detectable label, where the second antibody with a detectable label reacts with hSOD amino acid sequence; and (d) determination of complexes formed between antibodies and antigens, if present, as an indication of HCV infection in a biological sample. NS3/4a-epitope can be a conformational epitope, such as conformational epitope represented by a sequence of NS3/4a, from what is given in figures 4A-4D.

In any of the above embodiments, antikarov antibody may be directed against the N-terminal region of HCV measles antigen, such as amino acids 10-53 HCV, numbered relative to the HCV1 polyprotein sequence, and/or detectable-labeled HCV antikarov antibody may be directed against the C-terminal region of HCV crustal antigen, such as amino acids 120-130 HCV, numbered relative to the HCV1 polyprotein sequence. In addition, the antigen that reacts with HCV-angitola from a biological sample may be from the NS3 region, for example, the epitope of SS area polyprotein HCV, and may be merged with the amino acid sequence of human superoxide dismutase (hSOD). In this embodiment, the second antibody with a detectable label reacts with the amino acid sequence hSOD.

In another embodiment, the invention is directed a method of determination of HCV infection in a biological sample. The method includes: (a) obtaining a solid substrate for immunological analysis, which includes two HCV antikarov monoclonal antibodies and conformational epitope comprising amino acid sequence depicted in figures 4A-4D; (b) adding a biological sample to the solid substrate, under conditions that allow the antigens to antibodies, HCV, when they are present in the biological sample, contacting at least two antigravity antibodies and NS3/4A conformational epitope, respectively; (C) adding to the solid substrate from step (b) complexing the conditions (i) the first antibody with a detectable label, where the antibody with a detectable label detectable labeled HCV antiquorum antibody, and where it antikarov antibody with a detectable label directed against another epitope of measles HCV than at least two antikarov antibody associated with the solid substrate; (ii) epitope from the area SS polyprotein HCV connected with amino acid sequence hSOD; and (iii) a second antibody with a detectable label, where the second antibody with a detectable label reacts with the mentioned hSOD amino acid sequence; (d) determination of the formed complexes between antibodies and antigens, if present, as an indication for the presence of HCV infection in a biological sample.

In some embodiments, the implementation of at least two antikarov antibodies directed against the N-terminal region of measles antigen HCV, for example, against amino acids 10-53 HCV, numbered relative to polyprotein HCV1, and HCV-antibrane antibody with a detectable label directed against the C-terminal region of HCV measles antigen, nab is emer, against amino acids 120-130 HCV, numbered relative to the HCV1 sequence of polyprotein.

In the following embodiment, the invention relates to a method of determining HCV infection in a biological sample. The method includes: (a) obtaining a solid substrate for immunological analysis, which includes fused antigen with multiple epitopes; (b) combining a biological sample with a solid substrate under conditions that allow antigen and HCV antibodies when they are present in the biological sample, contacting at least one antiquorum antibody NS3/4a-epitope and fused antigen with multiple epitopes; (C) adding to the solid substrate from step (b) complexing the conditions (i) the first antibody with a detectable label, where the first antibody with the detectable label is antiquorum HCV antibody with a detectable label, where it is labeled antikarov antibody directed against different HCV epitope of measles than at least one antikarov antibody associated with the solid substrate; (ii) first and second antigens that react with HCV-antibody from the biological sample, reacting with NS3/4a-epitope, and fused antigen with multiple epitopes, respectively; (iii) a second antibody with a detectable label capable of reacting with an what iwanami from (ii); (d) determination of the complexes formed between antibodies and antigens, if present, as an indication for the presence of HCV infection in a biological sample.

Antikarov antibody may be directed against the N-terminal region of HCV-crustal antigen, and said first HCV-antikarov antibody with a detectable label may be directed against the C-terminal region of HCV-crustal antigen, as described above. In addition, the first antigen-reactive HCV-antibody from the biological sample can include an epitope of the field SS polyprotein HCV and may be merged with the amino acid sequence hSOD. Thus, the second detectable labeled antibody reacts with the amino acid sequence hSOD. In addition, the second antigen-reactive HCV-antibody from the biological sample can include an epitope of C22 area polyprotein HCV, such as an epitope comprising amino acids Lys10-Ser99polyprotein HCV, with a deletion of AGD and replacement of Trp to Leu at position 44, numbered relative to the sequence of polyprotein HCV1. The epitope can be fused with the amino acid sequence hSOD. In this case, a second antibody with a detectable label reacts with the amino acid sequence hSOD. Fused antigen with multiple epitopes may include linakis is now the sequence depicted in figures 7A-7F.

In another embodiment, the invention relates to a method of determining HCV infection in a biological sample, and the method includes: (a) obtaining a solid substrate for immunoassay, which includes two antikarov monoclonal antibodies HCV, NS3/4a-conformational epitope of HCV comprising the amino acid sequence depicted in figures 4A-4D, and fused antigen with multiple epitopes containing the amino acid sequence depicted in figures 7A-7F associated with it; (b) combining a biological sample with a solid substrate under conditions that allow antigens and antibodies to HCV when they are present in the biological sample, contacting at least two anticorrosie antibodies, NS3/4A conformational epitope and fused antigen with multiple epitopes, respectively; (C) adding to the solid substrate stage (b) under complex-forming conditions (i) the first antibody with a detectable label, where the first antibody with a detectable label is HCV antiquorum antibody with a detectable label, where labeled antikarov antibody directed against different HCV-crustal epitope than at least two antikarov antibody associated with the solid substrate; (ii) epitope from the area SS polyprotein HCV, if the CSOs with hSOD amino acid sequence, and epitope from the area of C22 polyprotein HCV, merged with hSOD amino acid sequence; and (iii) a second antibody with a detectable label, where the aforementioned second antibody with a detectable label capable of reacting with said amino acid sequences hSOD; (d) determination of the complexes formed between antibodies and antigens, if present, as an indication for the presence of HCV infection in a biological sample.

In this embodiment, at least two antikarov antibodies may be directed against the N-terminal region of HCV-crustal antigen, such as amino acids 10-53 HCV, numbered relative to HCVl-polyprotein, and HCV-antikarov antibody with a detectable label directed against the C-terminal region of HCV-crustal antigen, such as amino acids 120-130 HCV, numbered according to the sequence polyprotein HVC1. Moreover, the epitope of the region 22 may include amino acids Lys10-Ser99, polyprotein HCV with a deletion of AGD and replacement of Trp to Leu at position 44, numbered in accordance with the sequence polyprotein HVC1.

In other embodiments, the implementation, the invention relates to the production of immunodiagnostics test set that includes a solid substrate for immunoassay described above, and instructions for conducting them is nanodiagnostics test.

In another embodiment, the invention relates to methods of producing a solid substrate for immunoassay comprising: (a) obtaining a solid substrate; and (b) linking with it at least one HCV-anticorodal antibodies, for example one, two, or more, and at least one selected NS3/4a-HCV epitope, and, optionally, fused antigen with multiple epitopes. Antikarov antibodies, NS3/4a-epitopes and fused antigens with multiple epitopes described above.

In additional embodiments, the implementation, the invention relates to fused antigen with multiple epitopes, which includes in itself the amino acid sequence depicted in figures 7A-7F, or amino acid sequence identical to at least 80%, for example, identical to 90% or more and, in addition, with a specific reaction to anti-HVC antibodies in a biological sample from an HCV-infected subject. In certain embodiments of the implementation, fused antigen with multiple epitopes consists of the amino acid sequences depicted in figures 5A-5F.

In subsequent versions of the implementation, the invention relates to polynucleotide, which includes the coding sequence for the above-mentioned fused antigen with multiple epitopes, rivers is minantly vectors, includes polynucleotide, cell host transformed with the recombinant vectors, and methods of producing recombinant fused antigen with multiple epitopes, comprising: (a) obtaining a population of above-mentioned host cells; and (b) culturing the population of cells under conditions in which is expressed fused antigen with multiple epitopes encoded by the coding sequence in the recombinant vector.

These and other aspects of the present invention will become apparent when reading the following detailed description and the accompanying drawings.

A brief description of the drawings.

Figure 1 is a graphic representation of the HCV genome, representing different areas of polyprotein, of which the reagents for the present analysis (proteins and antibodies).

Figure 2 is a schematic drawing illustrative of the complex antigen/antibody according to the invention.

The figure 3 depicts the amino acid sequence of illustrative NS3/4a conformational antigen, for use in these analyses. Bold alanine at position 182 is replaced by the natural serine, normally held in this position.

In figures 4A-4D depict the DNA and corresponding amino acid sequence of another and lustrating NS3/4a conformational epitope for use in these analyses. Amino acids in positions 403-404 figures 4A-4D represent a replacement on Thr Pro Ser to Ile in the natural amino acid sequence of HCV-1.

Figure 5 - diagram of the structure pd.HCV1a.ns3ns4aPI

In figure 6 - graphical representation of the MEFA 12.

In figures 7A-7F shows the DNA sequence and corresponding amino acid sequence MEFA 12.

Figure 8 is a schematic drawing illustrative of immunological analysis according to the invention using MEFA 12.

Detailed description of the invention

In the practical application of the present invention will be used, unless otherwise indicated, conventional methods of chemistry, biochemistry, methods of recombination DNA and immunology, is within the skills of specialists. Such techniques are fully represented in the literature. See, for example. Fundamental Virology, 2nd Edition, vol. I & II (B.N.Fields and D.M.Knipe, eds.); Handbook of Experimental Immunology, Vols. I-IV (D.M.Weir and C.C.Blackwell eds., Blackwell Scientific Publications); ..Creighton, Proteins: Structures and Molecular Properties (W.H.Freeman and Company, 1993); A.L.Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S.Colowick and N.Kaplan eds.. Academic Press, Inc.).

It should be noted that in this description and the attached claims forms in the singular should be understood in the plural, unless the content clearly does not dictate otherwise. For example, the "antigen" which includes a mixture of two or more antigens, and the like.

The text uses the following abbreviations amino acids:

Alanine: Ala (A)Arginine: Arg (R)
Asparagine: Asn (N)Aspartic acid: Asp (D)
Cysteine: Cys (C)Glutamine: Gin (Q)
Glutamic acid: Glu (E)Glycine: Gly (G)
Histidine: His (H)Isoleucine: Ile (I)
Leucine: Leu (L)Lysine: Lys (K)
Methionine: Met (M)Phenylalanine: Phe (F)

Proline: Pro (P)Serine: Ser (S)
Threonine: Thr (T)Tryptophan: Trp (W)
Tyrosine: Taut (Y)Valine: Val (V)

Definition

The following are terms used in the description of the present invention, and their definitions.

The terms "polypeptide" and "protein" refer to polymers of amino acid residues and is not limited by the minimum length of the product. Thus, peptides, oligopeptides, dimers, multimer and the like included in this definition. As a full-sized proteins and fragments thereof are also included in this definition.

The terms also include postexplosion modifications of the polypeptide, for example glycosylamine, acetylation, phosphorylation and the like. In addition, for the purposes of this invention, the term "polypeptide" refers to a protein that contains a modification in the natural sequence, such as deletions, additions and substitutions (generally conservative in nature), as long as the protein maintains the desired activity. These modifications can be created intentionally, for example, when site-specific mutagenesis, or by accident, for example, when mutations in the host, producing proteins, or error-PCR amplification.

The HCV polypeptide is a polypeptide, as defined above, derived from polyprotein HCV. It is not necessary that this polypeptide is physically descended from HCV, it can be obtained by synthesis or recombination. In addition, the polypeptide may be derived from different HCV strains, such as strains of HCV 1, 2, 3 or 4. These strains are known a number of conserved and variable regions, and in General amino acid sequences of epitopes originating from these areas will have a high degree of sequence homology, more than 30%, preferably more than 40%, when the two sequences are aligned. For example, the term " polypeptide "NS3/4a" refers to natural NS3/4a any of the various HCV strains, as well as analogs, NS3/4a, mutiny and immunogenic fragments, as determined by either the E. The full genotypes of many of these strains. See, for example. U.S. patent No. 6150087 and GenBank receipt No. AJ 238800 and AJ 238799.

The terms "analog" and "mutein" refer to biologically active derivatives of the basic molecules or fragments of such derivatives, that retain desired activity, such as immunoreactivity in the analyses described here. Usually the term "analog" refers to compounds that have a natural polypeptide sequence and structure with one or more amino acid insertions, substitutions (generally conservative in nature) and/or divisions, compared with the natural molecule, as long as these modifications do not violate the immunogenic activity. The term "mutein" refers to peptides having one or more peptide simulations (peptides), such as described in International publication no WO 091/04282. Preferably, the similar or mutein has at least the same immunoactivity as natural molecule. How to create analogues and Malinov polypeptides known in this field and are described below.

The most preferred analogs include substitutions that are conservative in nature, i.e., such changes that occur within a family of amino acids, related their side chains. In particular, amino acids are generally divided into four families: (1) acid is s - aspartate and glutamate; (2) basic - lysine, arginine, histidine; (3) nonpolar alanine, valine, leucine, isoleucine, Proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar glycine, asparagine, glutamine, cysteine, series, threonine, tyrosine. Phenylalanine, tryptophan and tyrosine are sometimes classified as aromatic amino acids. For example, it is predictable that an isolated replacement of a leucine for isoleucine or valine, an aspartate to glutamate, a threonine in series, or a similar conservative replacement amino acids on structurally related amino acid will not have a major effect on biological activity. For example, the polypeptide of interest, can carry approximately 5-10 conservative and non-conservative amino acid substitutions, or even up to about 15-25 conservative or non-conservative substitutions, or any integer between 5 to 25, provided that the desired function of the molecule remains intact. The person skilled in the art can easily identify areas of interest to the molecule, allowing replacement of addressing schemes Hopp/Woods and Kyte-Doolittle, well known in this field.

The term "fragment" denotes a polypeptide consisting of only parts of the sequence and structure of the whole, full-length polypeptide. This fragment can include a C-terminal deletions and/or N-terminal deletions natural in the of peptide. "Immunogenic fragment" HCV protein, in particular, typically includes at least about 5-10 contiguous amino acid residues of the full-length molecule, preferably at least 15-20 contiguous amino acid residues of the full-length molecule, and most preferably not less than about 20-50 or more contiguous amino acid residues of the full-length molecule, determining the epitope, or any integer between 5 amino acid residues to the full sequence, provided that the fragment retains the immunoreactivity in the analyses described here. For example, the preferred immunogenic fragment includes, though not limited to, crustal fragments of HCV, which contain, for example, amino acids 10-45, 10-53, 67-88 and 120-130 of polyprotein, epitope 5-1-1 (NS3-region of the viral genome), as well as certain epitopes derived from regions E1, E2, SS (NS3), c100 (NS4), NS3/4a and NS5 of polyprotein HCV), as well as any of the various epitopes identified in HCV-polyprotein. For example, see Chien et al., Proc. Natl. Acad. Sci. USA (1992) 89:10011-10015; Chien et al., J. Gastroent. Hepatol. (1993) 8:S33-39; Chien et al., International publication number WO 93/00365; Chien, D. Y.,. International publication number WO 94/01778; U.S. Patent No. 6150087 and 6121020.

The term "epitope", as used herein, refers to a sequence comprising at least from about 3-5 amino acids, before occhialino, approximately 5-10 or 15, and not more than about 1000 (or any integer in this range), which define a sequence that by itself or as part of a larger sequence binds to an antibody generated in response to this sequence. There is no critical upper limit to the length of the fragment, which could comprise nearly the full-size protein sequence, or even a protein comprising two or more epitopes from polyprotein HCV. The epitope for use in the object of the invention is not limited to a polypeptide having the exact sequence of the part of the parent protein from which it was obtained. Indeed, viral genomes are in a constant state of change and contain several variable domains, which show high levels of variability between isolates. Thus, the term "epitope" includes a sequence that is identical to the natural sequence, and modification of natural sequence, such as deletions, insertions and substitutions (generally conservative nature).

The scope of this polypeptide containing the epitope can be identified using any number of methods of epitope mapping are well known in this field. For example, see an epitope Mapping Protocols in Methods in Molecular Bioliy, Vol. 66 (Glenn E. Morris, Ed., 1966) Humana Press, Totowa, New Jersey. In particular, the linear epitopes can be determined using, for example, simultaneous synthesis of large numbers of peptides on solid substrates and mapping of these peptides with parts of the protein molecule and the reaction of these peptides with antibodies, when the peptides remain bound to the substrate. Such methods are known in this field and are described, in particular, U.S. Patent 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002; Geysen et al. (1985) Proc. Natl. Acad. Sci. USA 82:178-182; Geysen et al (1986) Molec. Immunol. 23:709-715. Using this technique, it was determined the number of HCV epitopes. For example, see Chien et al., Viral Hepatitis and Liver Disease (1994) pp. 320-324, and further below. Also conformational epitopes can be easily determined when determining spatial conformation of amino acids using, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. For example, see an epitope mapping protocol, supra. Antigenic region of the protein can also be determined using standard graphics of antigenicity and hydropathy, such as a computed program Omiga, version 1.0, available in Oxford Molecular Group. In this computer program uses the method of Hopp/Woods, Hopp et al, Proc. Natl. Acad. Sci USA (1981) 78:3824-3828 to determine profiles of antigenicity, and the method of Kyte-Doolittle, Kyte et al., J. Mol. Biol. (1982) 152:105-132 for graphs hydropathy.

The term "conformational epitope", as used here, means h is here full of protein, or similar, or mutein with' natural structural properties of amino acid sequences that encodes the epitope full of natural protein. Natural structural properties include, but are not limited to, glycosylation, and the three-dimensional structure. The length of the sequence defining the epitope can be widely varied, as it is believed that the epitopes are formed three-dimensional shape of the antigen (e.g., folding). Thus, the amino acids defining the epitope may be relatively small but widely dispersed along the length of the molecule (or even different molecules, in the case of the dimers and the like) and to assemble in the correct conformation of the epitope due to folding. Part of the antigen between residues that define the epitope may be irrelevant to a conformational epitope patterns. For example, deletion or replacement of these intermediate sequences may not affect sequence, creating a conformational epitope that is required to maintain the conformation of the epitope (e.g., cysteine involved in a disulfide bond; areas glycolythiourea etc).

Conformational epitope located in the region of NS3/4a, is reliably detected using the methods discussed above. In addition, the presence or absence of a conformational epitope in the polypeptide can be easier in order to identify screening antigen of interest with an antibody (polyclonal serum or monoclonal to the conformational epitope), and compare its reactivity with denatured version of the antigen, which preserves only the linear - imitators (if any). In such screening using polyclonal antibodies can initially absorb polyclonal serum denatured antigen and show that there are antibodies to a specific antigen. In addition, in the case of NS3/4a, a molecule that retains the natural conformation will also have enzymatic activity of the protease, and, optionally, helicase. These activities can be determined using enzymatic assays, as described below.

Preferably, the conformational epitope is produced by recombination, and it is secreted by the cell, from which it can be extracted under conditions that maintain its desired structural features, i.e. without denaturation of the epitope. These cells include cells of bacteria, yeast, insects and mammals. Selection and isolation of recombinant conformational epitopes from polyprotein HCV are described, in particular, in International publication number WO 96/04301, WO 94/01778, WO 95/33053, WO 92/08734. Alternatively, you can select the antigens and then denaturiruet protein after separation. It is also clear that chemical synthesis also allows you to get mimotope conformational antigen that cross-react with conformat the district epitope "natural" antigen.

The term "fused antigen with multiple epitopes", or "MEFA"used herein means a polypeptide in which multiple HCV antigens are part of a single, continuous chain of amino acids, which does not occur in nature. HCV-antigens can be connected directly to each other by peptide bonds or may be separated by intermediate amino acid sequences. Fused antigens may also contain a sequence that is exogenous with respect to polyprotein HCV. Moreover, the present sequence of HCV can occur from a variety of genotypes and/or isolates of HCV. Examples of specific MEASURE for use in the present immunological analyses are detailed, for example, in International publication no WO 97/44469 and described below.

"Antibody" means a molecule that by chemical or physical reasons specifically binds to interest the polypeptide. Thus, HCV-korovou antibody is a molecule that specifically binds to HCV crustal protein. The term "antibody", as used here, includes antibodies obtained from both polyclonal and monoclonal preparations, as well as the following: hybrid (chimeric) antibody molecules (see, for example. Winter et al. (1991) Nature 349: 293-299; U.S. Patent No. 4816567); F(ab')2 and F(ab)fragments, Fv molecules of the non-covalent heterodimer, see, for example, Inbar et ai. (1972) Proc Nati Acad Scl USA 69:2659-2662; and Ehrlich et al. (1980) Biochem 19:4091-4096); single-chain Fv molecules (sFv) (see,for example, Huston et ai. (1988) Proc Nati Acad Sci USA 85:5879-5883); dimeric and make-up designs fragments of antibodies, Minitel (see, for example. Pack et al. (1992) Blochem 31:1579-1584; will CUMBER et al. (1992) J Immunology 1498:120-126); humanitarian molecule antibodies (see, for example, Riechmann et al. (1988) Nature 332: 323-327; Verhoeyan et al. (1988) Science 239:1534-1536; and Patent publication England No. GB 2276169, published on September 21, 1994); and any functional fragments obtained from such molecules, which retain the immunological binding properties of the parent molecule antibodies.

The term "monoclonal antibody", as used here, refers to a composition of antibodies with a homogeneous population of antibodies. This term is not limited by the type or source of the antibody or the manner of receiving them. Thus, this term includes antibodies derived from murine hybridomas, as well as monoclonal antibodies obtained using human rather than murine hybridomas. For example, see, Cote, et al. Monclonal Antibodies and Cancer Therapy, Alan R. Liss, 1985, p. 77.

"Recombinant" protein is a protein that retains the desired activity and obtained by the methods of DNA recombination, as described here. In General, interest gene clone and then Express in the transformed organism is, as is described below. The host organism expresses the foreign gene to produtsirovaniya protein in terms of expression.

When referring to the polypeptide, under "dedicated" means that the indicated molecule is selected and separated from the whole organism, in which the molecule is found in nature, or it is largely in the absence of other biological macromolecules of the same type. The term "isolated" in respect of polynucleotide means a nucleic acid molecule, devoid, in whole or in part, of sequences normally associated with it in nature; or a sequence, such as it exists in nature, but includes a heterologous sequence to which it is associated; or a molecule, dissociating with the chromosome.

Under "equivalent antigenic determinant" should be understood antigenic determinant from a different subspecies or strain of HCV, such as HCV strains 1, 2 or 3. More specifically, known epitopes, such as 5-1-1, and these epitopes vary among strains 1, 2 and 3. Thus, the epitopes 5-1-1 from three different strains are equivalent antigenic determinants and, thus, are "copies", even if their sequence and not identical. In General, the amino acid sequence equivalent antigenic determinants will have a high degree of homology is posledovatelnosti, for example, the amino acid sequence homology of more than 30%, preferably more than 40%when two sequences are aligned.

"Homology" refers to the percentage of similarity between two polynucleotide or two polypeptide sequences. Two DNA, or two polypeptide sequences are "substantially homologous" to each other when the sequences exhibit at least about 50%, preferably at least about 75%, more preferably at least about 80-85%, and most preferably at least about 95-98% sequence similarity over a defined length of the molecules. "Substantially homologous" here also refers to sequences showing complete identity with specific DNA or polypeptide sequences.

Usually "identity" refers to an exact nucleotide-nucleotide or amino acid-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. The percent identity can be determined by direct comparison of sequence information between two molecules using sequence alignment, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter is sledovatelnot and multiplying by 100.

To aid in the analysis of similarity and identity you can use readily available computer programs such as ALIGN, Dayhoff, M.O. in Atlas of Protein Sequence and. Structure M.O. Dayhoff ed., 5 Suppi. 3:353-358, National biomedical Research Foundation, Washington, DC, which adapts the algorithm of the local homology of Smith and Waterman (Smith &Waterman, Advances in Appl. Math. 2: 482-489, 1981) for the analysis of peptides. Program to determine the similarity and identity of nucleotide sequences available in the Wisconsin Sequence Analysis Package, version 8 (available from Genetics Computer Group, Madison, WI), for example, the program BESTFIT, FASTA, GAP, which also used the algorithm of Smith and Waterman. These programs are easily used with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package, mentioned above. For example, a percent similarity of a specific nucleotide sequence with the control sequence can be determined using the homology algorithm of Smith and Waterman installed by default evaluation table, and a negative score for a period of six nucleotide positions.

Another way of establishing the percentage of similarity in the context of the present invention is to use the MPSRCH package of programs, copyrighted by the University of Edinburgh, developed by John F.Collins and Shane S.Sturrok and distributed by IntelliGenetics, Inc. (Mountain View, CA). This set of programs and is gorithm Smith and Waterman used the default settings, established for evaluation table (for example, a negative score -12 an open interval, a negative score - 1 in the extended period and the period of 6 positions). In the received data value is "Match" reflects "sequence similarity". Other suitable programs for calculating the percent identity or similarity between sequences are generally known in this field, for example, another equalization program is the program BLAST, used with default parameters. For example, BLASTN and BLASTP can be used with the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + Swiss protein + Spupdate + PIR. The details of these programs can be found at the following Internet address: http://www.ncbi.nlm.gov/cgi-bin/BLAST.

Alternatively, homology can be determined by hybridization of polynucleotides in conditions, when the formation of stable duplexes between homologous regions, followed by digestion adnotatione-specific nuclease (nucleases) and determining the size of digested fragments. Substantially homologous DNA sequences can be determined by experiment by southern blot hybridization, for example, stringent conditions as defined in this specific with the system. Defining appropriate hybridization conditions is within the skill of experts in this field. For example, see Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra.

"Coding sequence" or a sequence which "encodes" a selected polypeptide, is a nucleic acid molecule which is transcribed (in the case of DNA) or transmitted (in the case of mRNA) into a polypeptide in vivo or in vitro, being placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence defined by start codon at the 5'(amino) end and a stop codon broadcast on the 3'(carboxy) end. Sequence, stopping transcription, may be in the range from 3' end to the coding sequence.

"Operatively linked" refers to the arrangement of elements in which the components described thus arranged so as to perform the desired function. Thus, this promoter, operatively linked to the coding sequence, can affect expressiona coding sequence, when present in the proper translational factors, etc. the Promoter does not have to border on the coding sequence, because its function is to control its expression. For example, PR is interstitial untranslated, although transcribed, the sequence may be located between the promoter sequence and the coding sequence, such as the transcribed introns and promoter sequence may nevertheless be considered to be "operatively linked" to the coding sequence.

The term "regulatory element" refers to a polynucleotide sequence that facilitates expression of the coding sequences with which it is associated. The term includes promoters, sequences, transcription termination, upstream regulatory domains, polyadenylation signals, untranslated region, including the 5'-UTR and 3'UTR, and, under appropriate conditions, leader sequences and enhancers, which collectively determine the transcription and translation of the coding sequences in the cell of the host.

The term "promoter"as used here, refers to a regulatory region of DNA capable of contact with RNA polymerase in the cell host and initiate the transcription of downstream (in the direction 3') coding sequence, operatively associated with it. For the purposes of the present invention promoter sequence comprises the minimum number of bases or elements necessary to initiate transcription of a gene, representing in the EPEC, at levels detectable above background. The promoter sequence contains the site that initiates the transcription as well as protein binding domains (consensus sequences)responsible for the Assembly of RNA polymerase. The promoters of eukaryotic often, but not always, contain "TATA"boxes and "SAT"boxes.

The control sequence directs transcription of" coding sequence in a cell when RNA polymerase will attach to the promoter sequence and transcribing the coding sequence into mRNA, which then translated into the polypeptide encoded by the coding sequence.

Cassette expression or expressing the construct" refers to a collection of sequences that are capable of driving expression of interest sequences (sequences) or a gene (genes). The expression cassette includes regulatory elements, as described above, such as a promoter that is operatively linked to (in order to control their transcription) of interest sequence (sequences) or a gene (genes), and often also includes a polyadenylated sequence. In some embodiments of the invention described here, the expression cassette can be located inside the plasma is IGNOU design. In addition to the components of the expression cassette plasmid construction may also contain one or more selectable markers, a signal which allows the plasmid constructs exist as single-stranded DNA (e.g., the replication origin M13), at least one multiply-cloning site, and the replication origin "mammal" (e.g., SV40 or the replication origin of adenovirus).

The term "transformation", as used here, refers to the insertion of exogenous polynucleotide cells of the host, regardless of the method used to insert: for example, transformation by direct uptake, transliterowany, infection, etc. On a separate transfection methods, see below. Exogenous polynucleotide can be in the form of reintegrating vector, for example, as the formation of the episome, or - as an alternative - can be integrated into the host genome.

"A host cell" is a cell which is transformed or is capable of transformation by exogenous DNA sequence.

"Conventional solid substrate" means an individual firm basis from which HCV-polypeptides, used for immunological assays involve covalent bond or non-covalent means such as hydrophobic adsorption.

"Immunologically reactive" means considered an shall Egan, which specifically reacts with anti-PSD-antibodies, present in the biological sample obtained from HVC-infected subject.

"Immune complex" means a complex formed when an antibody binds to the epitope of the antigen.

The term "biological sample", as used here, refers to a sample of tissue or fluid obtained from a subject, including but not limited to, for example, blood, plasma, serum, fecal matter, urine, bone marrow, bile, spinal fluid, lymph, skin samples, external allocation of the skin, respiratory, intestinal and genitourinary tracts, tears, saliva, milk, blood cells, organs, biopsies and also samples of components of cell culture in vitro, including, but not limited to, air-conditioned environment resulting from the growth of cells and tissues in culture medium, for example, recombinant cells and cell components.

The terms "label" and "detectable label" refer to a molecule that can be detected, including, but not limited to: radioactive isotopes, fluorescent, chemiluminescent agents, chromophores, enzymes, substrates, enzymes, cofactors of enzymes, enzyme inhibitors, chromophores, dyes, metal ions, sols of metals, ligands (such as Biotin, streptavidin or haptens) and the like. The term "phosphor" refers to a substance or its dose, which is able to fluoresce and registered range. Examples of labels that can be used in this invention include, but are not limited to: a horseradish peroxidase (HRP), fluorescein, FITC, rhodamine, dansyl, umbelliferone, dimethylcarbamoyl ester (DMAE), Texas red, luminal, NADPH and α-β-galactosidase.

II. Embodiments of the inventions

Before a detailed description of the present invention should be understood that the invention is not limited to private formulations or process parameters, because they, of course, can vary. Also, it should be clear that the terminology used here only to describe specific embodiments of the present invention, and should not be interpreted as restrictive.

Although in the practical implementation of the present invention can be used in various compositions and methods similar or equivalent to the one described here, the text describes the preferred materials and methods.

As noted above, the present invention is based on the discovery of new diagnostic methods, accurate early detection of HCV infection. Methods based on the identification and use of highly immunogenic antibodies and HCV antigens, which are present at an early article which dealt with HCV-seroconversion, thus increasing the accuracy and reducing the proportion of false positives. These methods can be easily applied in a single analysis.

Specifically, the analysis is carried out on a solid podoski, which are sewn with one or more HCV-antikarov antibodies (directed against either the same or different HCV-core epitopes), and epitope derived from the NS3/4a-area polyprotein HCV. Examples of specific antikarov antibodies that are applicable in the present invention include, but are not limited to, molecules are antibodies, such as monoclonal antibodies directed against epitopes in the cow area found between amino acids 10-53, amino acids 10-45, amino acids 67-88, amino acids 120-130, or antibodies directed against any of crustal epitopes that are installed, for example, in: Houghton et ai., U.S. patent No. 5350671; Chien et al., Proc. Natl. Acad. Sci. USA (1992) -89:10011-10015; Chien et al., J. Gastroent. Hepatol. (1993) 8:S33-39; Chien et al., International publication number WO 93/00365; Chien, D. Y., international publication number WO 94/01778; Patent application U.S. with one successor. Ser. No. 08/403590 and 08/444818.

Described NS3/4a-area polyprotein HCV, and amino acid sequence and the complete structure of a protein is described in, for example, Yao et al., Structure (November 1999) 7:1353-1363; Sali et al., Blochezn. (1998) 37:3392-3401; and Bartenschlager, R., J.Viral Hepat. (1999) 6:165-181. Also see Dasmahapatra et al., U.S. patent No. 5843752. The application of immunological the analyses use at least one conformational epitope derived from the NS3/4a-region found in natural HCV particle or infective product, as evidenced by the preservation by or - optional - helicase activity, normally demonstrated gene product NS3/4a and/or immunoreactivity of the antigen with the antibody in a sample obtained from HCV-infected subject, and loss of immunoreactivity epitope by denaturation of the antigen. For example, a conformational epitope can be destroyed by heat, pH changes in the direction of very acidic or alkaline values or adding known organic denaturation, such as dithiothreitol (DTT), or a suitable detergent. For example, see Protein Purification Methods, a practical approach (E.L.V.Harris and S.Angal eds., IRL Press), and the denatured product compared to a product that is not processed, as described above.

Protease and helicase activity can be determined using standard enzymatic assays, well known in this field. For example, protease activity can be determined using assays that are well known in this field. For example, Takeshita et al. Anal. Biochem. (1997) 241:242-246: Kakiuchi et al., J. Biochem. (1997) 122:749-755; Sali et al., Biochemistry (1998) 37:3392-3401; Cho et al., J. Virol. Meth. (1998) 72:109-115; Cerretani et al., Anal. Biochem. (1999) 266:192-197; Zhang et al., Anal. Biochem. (1999) 270:268-275; Kakiuchi et al., J. Virol. Meth. (1999) 80:77-84; Fowler et al., J. Blomol. Screen. (2000) 5:153-158; and Kim et al., Anal. Biochem. (2000) 284:42-48. Specific suitable analysis is to test by activity described in the examples below.

Similarly, analyses helicase activity are well known in this field and helicase activity of NS3/4a-epitope can be identified using, e.g., ELISA, as described, for example, Hsu et al., Biochem. Blophys. Res. Common. (1998) 253:594-599; system analysis scintillation similarity, as described by Kyono et al., Anal. Biochem. (1998) 257;120-126; analyses based high throughput screening, as it is written, for example, Hicham et al., Antiviral Res. (2000) 466:181-193 and Kwong et al., Methods Mol. Med. (2000) 24:97-116; as well as using other analytical methods known in this field. For example, Khu et al., J. Virol. (2001) 75:205-214; Utama et al.. Virology (2000) 273:316-324; Paolini et al., J. Gen. Virol. (2000) 81;1335-1345; Preugschat et al., Biochemistry (2000) 39:5174-5183; Preugschat et al., Methods Mol. Med. (1998) 19:353-364; and Hesson et al., Biochemistry (2000) 39:2619-2625.

The length of this antigen sufficient to contain immunoreactive conformational epitope. Often the polypeptide-containing antigen used can be a full size, however, the polypeptide can be shortened, for example, to increase the solubility or to improve secretion. Typically, the conformational epitope found in NS3/4a, is expressed in the cell as a recombinant polypeptide, and the polypeptide forms an epitope in the required form, as will be described in detail below.

Illustrative amino acid sequence of the NS3/4a polypeptides shown in figure 3 and figures 4A-4D. Granted the military fatty alanine, position 182 in figure 3, replaces the natural serine found in this position in order to prevent autocatalysis molecules, which otherwise may occur. Amino acid sequence shown in position 2-686 in figures 4A-4D, corresponds to amino acid positions 1027-1711 HCV-1. The initiating codon (ATG), encoding Met, shown in position 1. In addition, Thr, normally occurring at position 1428 HCV-1 (amino acid position 403 in figure 4) is changed to Pro, and Ser, normally occurring at position 1429 of HCV-1 (amino acid position 404 in figure 4) is changed to Ile. However, either natural sequence, with or without a N-terminal Met, shows similar, with or without a N-terminal Met, or other analogs or fragments can be used in the claimed analyses, provided that the epitope is obtained using a method that preserves or restores it to its natural conformation, such as protease activity, and is not necessarily stored helicina activity. In both patents: Dasmahapatra et al., U.S. patent No. 5843752 and Zhang et al. U.S. patent No. 5990276 - described analogs of NS3/4a.

NS3-protease of the NS3/4a was found approximately in the provisions 1027-1207, numbered respectively HCV-1, the provisions 2-182 figure 4. The structure of the NS3 protease and its active center is known. For example, see De Francsco et al., Antivir. Ther. (1998) 3:99-109; Koch et al., Biochemistry (2001) 40:631-640. Normal changes are allowed in the natural sequence that are outside the active center of the molecule. In particular, it is desirable to keep amino acids 1-St or 2-nd-155-Yu (figure 4), with a small number of substitutions or only conservative substitutions. Amino acids after 155th allow bóbigger changes. In addition, if you use fragments of the sequences of NS3/4a, depicted in figure 4, these fragments should typically include at least amino acids 1 or 2-155, preferably amino acids 1 or 2-175, and most preferably amino acids 1 or 2-182, with N-terminal Met or without him. Domain helicase found in the scope of the provisions 1193-1657 HCV-1 (position 207-632, figure 4). Thus, if necessary helicina activity, this part of the molecule must be saved with little or only conservative changes. Based on the known structure of the NS3/4a the person skilled in the art will readily identify other areas in which the changes are valid.

The solid substrate may also include other antigens. For example, fused antigens with multiple epitopes (marked "MEFA"), as described in International publication WO/44469, which can be grafted to a solid substrate for use in the claimed analyses. Such MEFA contain multiple EP is tops, derived from two or more different viral regions, shown in figure 1 and in table 1. In particular, as shown in figure 1 and table 1, the splitting of polyprotein HCV constitutes at least 10 separate products in order NH2-Kop-E1-E2-p7-NS2-NS3-NS4a-NS4b-NS5a-NS5b-COOH. The core polypeptide is within the provisions 1-191, numbered relative to HCV-1 (see Choo et al. (1991) Proc. Natl. Acad. Sci. USA 88:2451-2455, for HCV-1 genome). This polypeptide further processed to obtain HCV from about amino acids 1-173. The polypeptides of the membrane E1 and E2 are approximately the provisions 192-383 and 384-746, respectively. Domain P7 found in roughly provisions 747-809. NS2 is an integral membrane protein with proteolytic activity found in the provisions of polyprotein 810-1026. NS2, both single and in combination with NS3 (about provisions 1027-1657) splits the weak link NS2-NS3, which, in turn, forms the N-end NS3 and releases a large polyprotein, including activity as a serine protease and RNA-helicase. The NS3 protease, found in the provisions 1027-1207, handles the remaining polyprotein. Helicina activity is detected in the area of provisions 1193-1657. The completion of maturation polyprotein is initiated by autocatalytic cleavage of communication NS3-NS4a catalyzed by serine protease NS3. Found that NS3-about orestone splitting polyprotein HCV include recognition of degradable links polyprotein molecule NS3 another polypeptide. In this reaction NS3 releases cofactor NS3 (NS4a, 1658-1711), two proteins (NS4b, noted about the provisions 1712-1972, and NS5a, about the provisions 1973-2420) and RNA - dependent RNA polymerase (NS5b, about the provisions 2421-3011).

Table 1
DomainApproximate boundaries*
(KOR)1-191
E1192-383
E2384-746
P7747-809
NS2810-1026
N331027-1657
NS4a1658-1711
NS4b1712-1972
NS5a1973-2420
NS5b2421-3011
* Numbered accordingly HCV-1. Cm. Choo et al. (1991) Proa. Natl. Acad. Sci. USA 88:2451-2455.

Multiple HCV antigens are part of a single, continuous chain of amino acids, which does not occur in nature. Thus, the linear order of the epitopes different from their linear order in the genome in which they occur. Preferably, the linear order of the sequences MEFA, used here, was arranged for optimal antigenicity. Preferably, the epitopes would be and more than one strain of HCV, thus providing an additional opportunity to detect different strains of HCV in a single analysis. Thus MEFA used herein may contain various immunogenic region obtained from the above polyprotein. Moreover, protein, obtained by shifting the reading frame in the cow area polyprotein, such as described in International publication no WO 99/63941, can be used to MEFA. Optionally, at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more of one or more epitopes derived from HCV-polyprotein, can be fused protein.

For example, epitopes, derived in particular from surveyable region of E2, such as the region spanning amino acids 384-410 or 390-410, can be included in the MEFA-antigen. Especially effective epitope of E2 is one of those, which includes a consensus sequence derived from this area, such as the consensus sequence Gly-Ser-Ala-Ala-Arg-Thr-Thr-Ser-Gly-Phe-Val-Ser-Leu-Phe-Ala-Pro-Gly-Ala-Lys-Gln-Asn, which is a consensus sequence of amino acids 390-410 genome of HCV type 1. Illustrative E2-epitope presented in MEFA according to the invention may contain hybrid epitope spanning amino acids 390-444. Such hybrid E2-epitope may include a consensus sequence representing the amino acids 390-410, SL is made with the natural amino acid sequence 411-444 of HCV E2.

In addition, the antigens can be obtained from different strains of HCV. There are many viral strains of HCV, and fused protein can be used epitopes derived from these strains. It is well known that any kind of organisms varies from one individual to another and, moreover, that every organism, including a virus, can have a number of different species (strains). For example, as shown above, HCV includes at least 6 genotypes. Each of these genotypes includes equivalent antigenic determinants. More specifically, each strain contains a number of antigenic determinants that are found in all strains of the virus, but slightly vary from one virus strain to another. For example, HCV contains antigenic determinant, known as a 5-1-1 (see figure 1). This specific antigenic determinants found in three different forms in three different viral strains of HCV. Accordingly, in a preferred embodiment of the present invention, all three forms are 5-1-1 in the merged antigen with multiple epitopes used in the claimed immunological assays. Similarly, there may be an equivalent antigenic determinants of crustal regions of different strains of HCV. Usually an equivalent antigenic determinants have a high level of homology in relation to amino acid sequences, the level of homology, when they are aligned, is usually 30% or more, preferably 40% or more. Multicopying epitope according to the invention may also include multiple copies, which is the exact copy of the same epitope.

Illustrative MEFA, for use in assays according to the invention described in international publication no WO 97/44469. Additional illustrative MEFA used here include MEFA 12, MEFA 13 and MEFA 13.1. You have to understand that these MEFA shown only as examples, and other epitopes derived from the HCV genome, can also be used in the data analyses and can be embedded in these or other MEFA.

The DNA sequence and corresponding amino acid sequence MEFA 12 shown in figures 7A-7F. The General structural formula MEFA 12, shown in Fig.6, is as follows: hSOD-E1(Type1)-E2 HVR consensus(type 1A)-E2 HVR consensus(types 1 and 2)-SS short(type 1)-5-1-1(type 1)-5-1-1(type 3)-5-1-1(type 2)-C100(type 1)-NS5(type l)-NS5(type 1)-cor(types 1 and 2) - cor(types 1 and 2). This multicopying the epitope comprises the following amino acid sequence, numbered respectively HCV-1 (the numbering of the amino acids given below follows the numbering given Choo, et al. (1991) Proc. Natl. Acad. Sci. USA 88:2451-2455, where the amino acid No. 1 is the 1st methionine, which is encoded by the coding sequence of a cow field): amino acids 1-6 of superoxide dismutase (SOD, used to enhance recombinant protein expression); amino acids 303-320 polyprotein from the area E1; amino acids 390-410 polyprotein, representing the consensus sequence of the hypervariable region of E2 of HCV-1a, amino acids 384-414 polyprotein of the field E2, which represents the consensus sequence hypervariable regions of E2 from HCV-1 and HCV-2; amino acids 1211-1457 of HCV-1-polyprotein determining helicase; three copies of the epitope of 5-1-1, amino acids 1689-1735, one of the HCV-1, one of the HCV-3 and one of the HCV-2, whose copies are equivalent antigenic determinants of three different viral strains of HCV; HCV-polypeptide C100 from HCV-1, amino acids 1901-1936 polyprotein; two exact copies of the epitope from the NS5 region of HCV-1, each with amino acids 2278-2313 HCV-polyprotein, and two copies of the three epitopes of the cow area, two of HCV-1 and one of the HCV-2, whose copies are equivalent antigenic determinants represented by the amino acid 9-53 and 64-88 HCV-1 and 67-84 HCV-2.

Table 2 shows the position of the amino acids of different epitopes in the MEFA 12 with reference to figa-7F. The numbering in the table given accordingly HCV-1. Cm. Choo, et al. (1991) Proc. Natl. Acad. Scl. USA 88:2451-2455. MEFA 13 and 13.1 share a common formula above for MEFA 12, with modifications as shown in tables 3 and 4, respectively.

According to one analysis, the sample is combined with the solid substrate, as described below. If the sample is infected with HCV, core antigen as well as antibodies to epitopes located on the solid substrate will be connected with the components of the solid substrate. Then add antikarov antibody with a detectable label. Labeled antikarov antibody directed against the epitope different from the one that is associated with the solid substrate. This antikarov antibody is associated with crustal antigen captured anticorrosie antibodies on a solid substrate.

Also add the antigen-reactive captured HCV-antibody from the biological sample, where the captured antibody from the sample can react with the epitope NS3/4a. Preferably the antigen is an epitope derived from the NS3 region of polyprotein HCV. This antigen binds to the captured HCV-antibody from the sample. The number of antigens, including such epitopes are known and include, not limited to, antigens derived from areas SS and C100, as well as fused proteins, including NS3-epitope, such as C25. These and other NS3-epitopes can be used in these analyses, they are known in this field and are described, for example, Houghton et al., U.S. patent 5350671; Chien et al., Proc. Natl. Acad. Scl. USA (1992) 89:10011-10015; Chien et al., J. Gastroent. Hepatol. (1993) 8:S33-39; Chien et al., International publication No. O 93/00365; Chien, D. Y. international publication number WO 94/01778; and Patent application US with one successor No. 08/403590 and 08/444818.

Add the second labeled antibody against the above-described antigen. This antibody can be directed against any epitope in the antigen. For example, the antibody can be directed against the NS3 region, located in the antigen; on the Contrary, if the antigen is expressed as an integral protein, the second labeled antibody may be directed against a merge partner. The analysis can be added for more antigens and antibodies, especially if the solid substrate includes MEFA. Such analyses are described below.

Illustrative analysis according to the invention are depicted in figure 2. As shown in the figure, the solid substrate contains two antiqueóglobal monoclonal antibodies, designated C11-3 and c11-7. These antibodies are directed against an epitope located in the N-terminal region of measles-containing protein, amino acids 10-53, numbered according to the sequence polyprotein HCV1. Solid substrate also includes the epitope to NS3/4a. The biological sample is added to a solid substrate. HCV-core antigen as well as antibodies directed against the epitope NS3/4a together are present in the sample will bind reagents seizing on a solid substrate.

Then d is balali labeled with horseradish peroxidase (HPR) antiqueó global monoclonal antibody c11-14, directed against the C-terminal region of the cortex, the amino acid occupying position 120-130, numbered according to the sequence polyprotein HCV1. As the second HPR-labeled antibodies directed against SOD-part of the fused protein, add a protein comprising the sequence of SOD person (hSOD) and the epitope of the field SS. Merge SOD-SS contact with the anti-NS3-specific antibody and anti-SOD-antibody, in turn, will bind protein SOD-SS. Detection of the label indicates the presence of HCV infection.

Another illustrative analysis according to the invention is depicted in Fig. Configuration analysis using antibodies represents an analysis capture sandwich antigen-antibody-antigen using NS3/4a and MEFA 12. The solid substrate includes the above two antikarov monoclonal antibodies, the epitope to NS3/4a, as well as illustrative MEFA, MEFA12, which include a shortened version of SOD person. As in the case of the above analysis, the solid substrate is added to the biological sample. HCV-browy antigen as well as antibodies directed against the NS3/4a-epitope and epitope MEFA present in the sample will bind reagents seizing on a solid substrate. Add two antigen, one capable of reacting with antibodies specimen is, which bind NS3/4a (as described above) and one capable of reacting with the antibodies of the sample that bind MEFA 12. In figure 8 the antigen-reactive complex MEFA 12/antibody sample is a merge between the molecule SOD and c22ks Δ47-L44W. Antigen c22ks comes from a cow field and contains amino acid polyprotein Lys10-Ser99as well as present in normal deletions AGD and replacement of Trp to Leu at position 44. Detected conjugate antibodies is the second HPR-labeled monoclonal anti-SOD-antibody described above.

The above analyses are by definition complex antigen/antibody particularly successful, because HCV-core antigen, and antibodies to NS3/4a and/or bark can be detected using the same substrate in the same analysis. Moreover, in order to cover other non-structural HCV epitopes, in the combined cocktail can be used, as described above, additional HCV-epitopes, such as SOD merged with C100, 5-1-1, NS5 antigens, as well as protein, resulting in a shift of the reading frame in the cow area polyprotein, such as described in International publication no WO 99/63941.

For a better understanding of the present invention is given below for a more detailed discussion of obtaining antibodies used in the claimed immunological analyses, obtaining polypeptides used in immunological assays, the methods of carrying out the immunological assays.

Antibodies for use in HCV-immunological tests

As shown above, this analysis uses a variety of antibodies that are associated with a solid substrate (e.g., one or more antikarov antibodies), and thus detect the complexes of antigen/antibody formed, if the pattern is present HCV infection. These antibodies can be polyclonal or monoclonal antibody preparations, the monospecific antisera, antibodies of the person, or can be a hybrid or chimeric antibodies such as humanitarian antibodies, altered antibodies, F(ab')2fragments, F(ab)fragments, Fv fragments, single domain antibodies, dimeric or make-up design fragments of antibodies, Minitel or their functional fragments that bind to the antigen.

Antibodies obtained using techniques well known to specialists in this field and disclosed, for example, in U.S. Patents№4011308, 4722890, 4016043, 3876504, 3770380 and 4372745. For example, polyclonal antibodies are produced by immunization with an antigen of interest, a suitable mammal, such as mouse, rat, rabbit, sheep or goat. To enhance the immunogenicity of the antigen can be associated with the media prior to immunization. Such carriers are well known to specialists in this field. Immunization is usual done by mixing or emulsification of antigen in saline, preferably Freund, such as full beta-blockers, and injecting the mixture or emulsion (generally subcutaneously or intramuscularly). The animal is usually subjected to a secondary injection of antigen through 2-6 weeks, one or more injections of antigen in saline solution, preferably using the incomplete beta-blockers. Antibodies can also be obtained through immunization in vitro, using methods known in this field. Then from the immunized animal get polyclonal anticigarette. To describe receiving anti-HCV-polyclonal antibodies see, for example, Houghton et al., U.S. patent No. 5350671.

Monoclonal antibodies are usually obtained using the method of Kohler and Milstein (Kohler, Milstein; 1975, Nature 256: 495-497, or its modification. In a typical case, a mouse or a rat subjected to immunization, as described above. However, instead of taking the blood sample for separation of serum, it is better to remove the spleen (or, optionally, several large lymph nodes) and divided into individual cells. If you want, spleen cells can be sorted (after removal of the nonspecific-adhesive cells) by applying a cell suspension to a tablet or well coated with the antigen. In cells expressing the specific antigen is associated with membrane immunoglobulin, will contact with the tablet and will not see the bear, along with the remnants of the suspension. The remaining b-cells, or all dissociatively cells of the spleen, and then to induce fusion with myeloma cells for the formation of a hybrid, and then cultured in the selective medium (for example, gipoksantin, aminopterine, timeinfo environment, "HAT"). The resulting hybridoma placed on the tablet with limited dilution and screened for production of antibodies that specifically associated with immunizing antigen (and which is not associated with foreign antigens). Then selective monoclonal, secreting antibodies of hybridoma cultivated in vitro (e.g. in the bottle for tissue culture or reactors with hollow fibers) or in vivo (e.g., ascites mice).

Receiving various anti-HCV-monoclonal antibodies are described, for example, Houghton et al. U.S. patent No. 5350671, Chien et al. International publication number WO 93/00365, Patent application US with one successor No. 08/403590 and 08/444818 and Kashiwakuma et al. U.S. patent No. 5871904.

As shown above, fragments of antibodies which retain the ability to recognize interest antigen, will also find use in the claimed immunological assays. In this area there are a number of fragments of antibodies, which contain the antigen-binding sites, which can exhibit immunological binding properties of the whole molecule antibodies. For example, we can expect the performance of functional antibody fragments by splitting constant regions of the antibody molecules, not responsible for binding to the antigen, using, for example, pepsin, to obtain F (ab')2-fragments. These fragments will contain two antigen-binding site, but they will not be part of the constant region of each of the heavy chains. Also if you want to, Fab fragments, including single antigen-binding site can be obtained, for example, by digestion of polyclonal or monoclonal antibodies with papain. Functional fragments containing only the variable regions of the heavy and light chains can also be obtained using standard techniques, such as obtaining recombinants or election. proteolytic cleavage of immunoglobulin molecules. These fragments are known as Fv. For example, see Inbar et al. (1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976) Blochem 15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.

Single-chain polypeptide. Fv ("sFv"or "scFv") covalently bound to VH-VL-heterodimer, which is expressed fused gene comprising VHand VL-coding genes associated peptide-encoding linker. Huston et al., Proc. Nat. Acad. Scl. USA 85:5879-5883. Described a number of methods of discernment and improvement of chemical structures (linkers)that transforms naturally aggregated but chemically separate heavy and light chains of the field V-antibodies, sFv-molecule, which is formed in a three-dimensional structure very similar to the structure of the antigen-szaniawska site. For example, see U.S. Patent No. 5091513, 5132405 and 4946778. The sFv molecules can be obtained using the techniques described in this field. For example, see Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85:5879-5883; U.S. Patent No. 5091513, 5132405 and 4946778. Design criteria include the determination of appropriate length, occupying the distance between the end of one chain and the N-end of the other, where the linker is usually formed small hydrophilic amino acid residue, which has no tendency to curl or the formation of secondary structures. These methods are described in this area. For example, see U.S. Patent No. 5091513, 5132405 and 4946778. Suitable linkers typically contain polypeptide chains of alternating groups of residues glycine and serine and may include inserting residues of glutamic acid and lysine to increase solubility.

"Mini-antibodies"or "mini body" will also find use in the present invention. Mini body are circuits sFv-polypeptide, which include oligomerization domains at its C-end, separated from the sFv area of the loop. Pack et al. (1992) Biochem 31:1579-1584. The oligomerization domain includes samoassotsiiruyutsya α-helix, for example, lacinova "lightning", which in the future may be more stable disulfide what vasami. Oligomerization domain must be compatible with the vector packaging across membranes - a process designed to promote spatial packing of the polypeptide in vivo in a functional binding protein. Usually Minitel get using recombination methods, well known in this field. For example, Pack et al. (1992) Biochem 31:1579-1584; will CUMBER et al. (1992) J Immunology 149B:120-126.

The production of antigens for use in immunological assays HCV

As shown above, the molecules according to the invention are usually obtained through recombination. So, polynucleotide, encoding the HCV antigens, for use in this invention may be obtained using standard molecular biology techniques. For example, the polynucleotide sequence encoding the above-described molecules can be obtained using recombination methods, such as cyclic screening DNA libraries of genes from cells ekspressiruyushchikh this gene, or removing a gene from the vector, which obviously contains it. Moreover, the desired gene can be distinguished directly from the nucleic acid molecules of the virus, using methods known in this field, such as Houghton et al. U.S. patent No. 5350671. The gene of interest, it is better to synthesize than clone. You can build molecules with the corresponding codon for a specific sequence. the ATEM full sequence is assembled from overlapping oligonucleotides, prepared according to standard methods and assembled into a complete coding sequence. For example, see Edge (1981) Nature 292:756; Nambair et al. (1984) Science 223:1299; and Jay et al. (1984) J. Biol. Chem. 259:6311.

Thus, a specific nucleotide sequence can be obtained from vectors containing the desired sequence, or synthesize, fully or partially, using, when appropriate, various methods of synthesis of oligonucleotides known in this field, such as site-specific mutagenesis and polymerase chain reaction (PCR). For example, see Sambrook, supra. In particular, there is one method of obtaining nucleotide sequence that encodes a desired sequence by annealing complementary sets of overlapping synthetic oligonucleotides obtained in standard automated polynucleotide synthesizer, followed by legirovaniem corresponding DNA ligase and amplification legirovannoi nucleotide sequence by PCR. For example, see Jayaraman et al. (1991) Proc. Natl. Acad. Sci. USA 88:4084-4088. In addition, in the framework of the invention it is possible to use direct oligonucleotide synthesis (Jones et al. (1986) Nature 54:75-82), nucleotide-directed mutagenesis preexisting nucleotide regions (Riechmann et al. (1988) Nature 332:323-327 and Verhoeyen et al. (1988) Science 239:1534-1536), and enzymatic filling in of gaps in oligonucleotid the Ah using DNA polymerase T4 (Queen et al. (1989) Proc. Natl. Acad. Sci. USA 86:10029-10033) to obtain molecules having modified or enhanced the antigen-binding properties and/or reduced immunogenicity.

When the coding sequences obtained or selected, such sequences can be cloned into a suitable vector or replicon. Specialists in this area known for numerous cloning vectors, and selection of a suitable cloning vector is a matter of choice. Suitable vectors include, but are not limited to, plasmids, phages, transposons, Comedy, viral chromosome, is capable of replication in Association with suitable control elements.

Then, the coding sequence is placed under control of suitable control elements, depending on the system used for expression. Thus, the coding sequence can be placed under the control of the promoter region binding to ribosomes (for bacterial expression) and, optionally, the operator, so that the DNA sequence of interest, transcarbamoylase in RNA suitable transformants. The coding sequence may contain (or may not) contain a signal peptide or leader sequence, which can then be removed by the owner in posttranslational processing. For example, see U.S. Patent No. 4431739, 442537 and 4338397.

In addition to control sequences, it is desirable to add regulatory sequences which allow to regulate the expression of the sequences in accordance with the growth of the host cell. Regulatory sequences are known to specialists in this field, and among them examples of those which cause or off gene expression in response to physical or chemical stimuli, including the presence of a regulatory compound. Other types of regulatory elements may also be present in this vector. For example, it is possible to use the enhancer elements to increase levels of expression constructs. Examples include early gene enhancer SV40 (Dijkema et al., (1985) EMBO J. 4:761), the enhancer/promoter derived from the long terminal repeat (LTR) of the rous sarcoma virus (German et al. (1982) Proc. Natl. Acad. Sci. USA 79:6777), and elements derived from the human CMV (Boshart et al. (1985) Cell 41:521), such as the items And sequence of CMV-intron (U.S. Patent No. 5688688). The expression cassette may additionally contain the origin of replication for Autonomous replication in a suitable cell host, one or more selectable markers, one or more restriction sites, the potential for a large number of copies, and a strong promoter.

Expressing the vector is constructed so that a specific encoding the selected was located in the vector with the appropriate regulatory sequences, and the location and orientation of the coding sequence relative to the control sequence was such that the coding sequence was transcarbamoylase under "control" of the control sequences (i.e., RNA polymerase which binds to the DNA control sequences, Transcriber coding sequence). To achieve this may require modification of the sequence encoding the molecule of interest. For example, in some cases it may be necessary to modify the sequence so that it could be attached to the control sequence having a certain orientaciyu, i.e. maintaining the reading frame. Escape sequences and other regulatory sequences can be legirovanyh with the coding sequence to be inserted into the vector. Either the coding sequence may be cloned directly into an expression vector which already contains the control sequences and the corresponding restriction site.

As shown above, may also be desirable to obtain mutants or analogs of the antigen of interest. This is especially true in the case of NS3/4a. Methods to accomplish this are described, for example, Dasmahapatra et al. U.S. patent No. 5843752, and Zhang et al., P is the awning U.S. No. 5990276. Mutants or analogs or other HCV proteins for use in the claimed analyses can be obtained by deletion of parts of the sequence that encodes the desired polypeptide, insertion sequences, and/or substitution of one or more nucleotides in the sequence. Methods of modification of nucleotide sequences, such as site-directed mutagenesis, and the like, well known to specialists in this field. For example, see Sambrook et al., supra; Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. USA (1985) 82:448; Geisselsoder et al. (1987) BioTechnlques 5:786; Zoller and Smith (1983) Methods Enzyiaol. 100:468; Dalbie-McFarland et al. (1982) Proc. Natl. Acad. Sci USA 79:6409.

Molecules can be expressed in a variety of systems, including systems for the expression of insects, mammals, bacteria, viruses and yeast, also known in this field.

For example, expression system, cells of the insect, such as a baculovirus system, known to specialists in this field and are described, for example, Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and methods in the form of a kit for gene-expression system is the baculovirus/insect cell available for purchase, inter alia, Invitrogen, San Diego, California (Mahvash"). Also expression systems based on bacterial cells and mammalian cells are well known in this field and are described, for example, in Sambrook et al., above. Yeast is istemi expression is also known in this field and described, for example, in Yeast Genetic Engineering (Barr et al., eds., 1989) Butterworths, London.

A number of suitable host cells for use in these systems is also known. For example, mammalian cell lines are known in this field and include immortalized cell lines available in the American type culture Collection (ATSC), including, but not limited to; cells Chinese hamster ovary (Cho), HeLa cells, kidney cells baby hamster (KSS)cells, monkey kidney (COS)cells, human kidney embryonic cells, hepatocellular carcinoma person (for example, ner G2)cells medinas-Derby (Madin-Darby) bovine kidney ("MDBK"), and many others. Bacterial hosts such as E. coli, Bacillis subtilis and Streptococcus spp., will be used in expressing these constructs. The hosts yeast that can be used in the present invention include, among others, Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces. lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarroma lipolytica. The insect cells used in baculovirus expressing vectors include, inter alia, Aedes aegypti, Autographs californica, Bombyx mori, Drosophila melanoqaster, Spodoptera frugiperda, and Trichoplusiani.

Molecule of nucleic acid comprising the nucleotide sequence of interest can be stably integrated into the genome of the cell is ozaena or be maintained in a stable episomal element in a suitable cell host, using a variety of methods of gene delivery, well known in this field. For example, see U.S. Patent No. 5399346.

Depending on the expression system, and the selected host, when the growth of cells transformed described above expressing vector, receive molecule under conditions in which the protein is expressed. Then, the expressed protein is isolated from the host cells and purified. If the expression system secretes the protein into the culture medium, the product is distilled directly from this environment. If secretion does not occur, the product can be isolated from cell lysates. Selection of suitable growing conditions and methods of recovery is within the skills of specialists in this field.

Describes the obtaining of various HCV antigens using recombination. For example, see Houghton et al., U.S. patent No. 5350671, Chien et al. J. Gastroent. Hepatol. (1993). 8:S33-39; Chien et al. International publication number WO 93/00365, Chien, D.Y., international publication number WO 94/01778.

Immunodiagnostics tests

Immediately after getting referred to antikarov antibodies and NS3/4a-antigens is placed on an appropriate solid substrate for use in the claimed immunoassays. Solid substrate for the purpose according to the invention can be any material that can be insoluble base with rigid or semi-rigid surface. A typical t is Erda substrate includes, not limited to such substrates as nitrocellulose (e.g., in the form of a membrane or holes for micrometrology), PVC skin (for example, in the form of sheets or holes for micrometrology), polystyrene latex (e.g., in the form of beads or tablet for micrometrology), polyvinylidenfluoride, diazotized paper, nylon membranes, activated beads, beads for magnetic stirrers, etc. are special cases of substrates include tablets, pellets, disks, capillaries, hollow fibers, pins, needles, solid fibers, cellulose beads, porous glass beads, silica gels, polystyrene beads, optional Poperechnaya with divinylbenzene beads of the grafted copolymer, polyacrylamide beads, latex beads, dimethylacrylamide balls, optional Poperechnaya with N, N'-bis-acryloylmorpholine, and glass particles coated with hydrophobic polymer.

If desired, the molecules attached to a solid substrate, can be easily attached to the function of education styrene or acrylate fragments, thus creating the possibility of embedding molecules in polystyrene, polyacrylate and other polymers, such as polyimide, polyacrylamide, polyethylene, polyvinyl, polydiacetylene, polyphenylen-vinile, polypeptide, polysaccharide, polysulfone, polypyrrole, polyimides, polythiophene, polyester, E. the oxide resin, quartz glass, silica gel, siloxane, polyphosphate, hydrogel, agarose, cellulose, etc.

In this context, the solid substrate is initially reacts with HCV-anticorrosie antibodies and NS3/4a-epitope (together referred to here as the "solid components") and, optionally, with one or more MEFA, under suitable conditions, stitching, in which the molecules are sufficiently immobilizovana on the substrate. Sometimes immobilization on the substrate can be further enhanced with the first linkage of antigen and/or antibody with a protein having the best properties of solid-phase binding. Suitable binding proteins include, but are not of ogranichivaya them, such macromolecules as serum albumin, including bovine serum albumin (BSA), hemocyanin marine saucer, immunoglobulin molecules, thyroglobulin, egg albumin and other proteins well known to specialists in this field. Other reagents that can be used to bind molecules to the substrate, include polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, etc. Such molecules and methods of linking these molecules antigens are well known to specialists in this field. For example, see Brinkley, M.A. (1992) Blo conjugate Chem. 3:2-13; Hashida et al. (1984) J. Appi. Biochem. 6:56-63; and Anjaneyulu and Staros (1987) International J. of Peptide and. Protein Res. 30:117-124.

After shimodate solid substrate with a solid-phase components of all nemobiinae solid-phase components are removed from the substrate by washing, and then the components associated with the substrate come into contact with a biological sample suspected in the presence of antibodies or antigens of HCV (together referred to here as the "ligand molecules") in the appropriate binding conditions. After washing to remove all unbound ligand molecules in an appropriate binding conditions add the second antikarov antibody directed against an epitope that is different from anticorodal the antibodies bound to the substrate. Added antikarov antibody includes a detectable label, as described above, and connects any anticorodal antigen that may be present in the sample, which was reacted with antiquorum antibody bound to the substrate. Also add one or more antigens that can react with the audience in the sample with the antibodies that, in turn, reacted with NS3/4a-epitope. As explained above, this antigen is usually derived from the NS3 region of HCV-polyprotein and, in particular, from SS-region of HCV. This area and derived from it the epitopes described by Houghton et al. To describe this area, and derived from it epitopes, see U.S. Patent No. 5350671; Chien et al., Proc. Nat. Acad. Sci. (1989) 89:10011-10015; international publication number WO 93/00365 and Patent application US with one successor No. 08/403590 and 08/444818. Also add a labeled antibody directed against the Antiga is and. The antibody then binds to the antigen that reacted with anti-NS3-antibodies in the sample. Epitope SS can be easily obtained for this purpose as a merger between SS and superoxide dismutase person (hSOD), obtained through recombination, for example, by the method described in Hough-ton et al., US Patent No. 5350671. Nucleotide and amino acid sequence of the SOD man known and published in Haliewell et al., U.S. patent No. 5710033. Therefore, the labeled antibody directed against SOD person, can be used to detect the presence of complexes formed between NS3/4A-epitope, any antibodies in the sample which react with the epitope, and HCV-polypeptides, which, in turn, bind to the antibody in the sample.

If MEFA is present on the solid substrate, the analysis can also add one or more additional antigens that react with antibodies from a biological sample associated with the antigens presented in the MEFA. Particularly useful in this context, the antigen is derived from cow region of HCV, and, in particular, of the antigen C22, which includes 119 N-terminal toóglobal amino acids polyprotein HCV. This antigen derived from C22 is c22ks Δ47-L44W, which includes amino acid polyprotein Lys10-Ser99as in the present norm businessman who Yu AGD and replacement of Trp to Leu at position 44. As with the above-described epitope SS, this antigen can be obtained as a fusion with hSOD, and the same labeled antibody directed against SOD person, can be used to detect the presence of complexes formed between the antibody present in the sample, and NS3/4A-epitope and/or MEFA, whose complexes are also associated with HCV-antigens (for example, SS and C22).

In particular, you can use the ELISA method, in which wells for micrometrology coated solid-phase components. Then in the coated wells type of biological sample that contains or presumably contains the ligand molecules. After a period of incubation sufficient to ligand molecules contacted immobilizerturning solid-phase components, the tablet (tablets) may be washed to remove unbound fragments and add a second link molecule with a detectable label (labeled antikarov antibody), NS3-epitope-containing molecule and an antibody directed against NS3-hepatopancrease molecules. These molecules are given time to react with any captured antigen and antibody from the sample, washed tablet, and determine the presence of labeled antibodies using techniques well known in the field.

The reagents for the analysis described above, including a solid substrate for immunological anal is for antibodies and antigens, as well as antibodies and antigens to react with the captured sample can be supplied as sets, with the appropriate instructions and other necessary reagents to conduct immunoassays as described above. Depending on the specific immunoassay kit can include appropriate labels and other packaged reagents and materials (e.g., wash buffer, and the like). With these sets you can perform the above standard immunological tests.

III. Experimental part

The following are examples of specific embodiments of the present invention. These examples are offered for illustrative purposes only and do not pursue the goal to limit the scope of the present invention in any way.

There have been some efforts to ensure the accuracy of the used digits (e.g., amounts, temperature, etc), but you should consider the possibility of some experimental errors and deviations.

EXAMPLE 1

Immunological analysis of complex formation HCV antigen/antibody.

This immunological analysis of complex formation HCV antigen/antibody compared with other HCV-analyses to test the limits of detection of seroconversion and to compare these bounds with those obtained in other available for when is bretania analyses, as follows.

A. Materials and methods

Blood samples: Used sampling available for purchase human blood. These sampling is available, for example, in Boston Biomedica, Inc., West Bridgewater, MA (BBI); Bioclinical Partners, Franklin, MA (BCP); and North American Biologies, Inc., BocoRatan, FL (NABI). The days shown in tables 5 and 6 indicate the days when the blood was collected from donors.

Monoclonal antibodies: Monoclonal antibodies C11-3, C11-7, and c11-14 were obtained from Ortho Clinical Diagnostics, Raritan, New Jersey. Antibodies C11-3 and C11-7 directed against the N-terminal part of the crust (amino acids 10-53, numbered respectively HCV1-polyprotein). Monoclonal antibody c11-14 directed against the C-terminal part of the crust (amino acids 120-130, numbered respectively HCV1-polyprotein). Antibody c11-14 conjugatively with horseradish peroxidase (HPR) using standard methods.

Monoclonal antibody 5A-3 and anti-SOD-antibody directed against amino acids 1-65 SOD, were obtained using standard techniques. Antibody conjugatively (HPR), as described above.

Century Antigens:

Antigen SS (266 amino acids, amino acids 1192-1457 HCV1-polyprotein) expressed as internal SOD-fused polypeptide in E. coli, using the techniques described for the synthesis of 5-1-1 antigen. (Choo, et al. Science (1989) 244:359-362). Recombinant antigen was purified and described Chien, et al., Proc. Natl. Acad. Sci. (1989) 89:10011-10015. See also the gender Protocol is implemented SOD-SS at Houghton et al. U.S. patent No. 5350671.

Epitope NS3/4a used in the analysis is a conformational epitope having the sequence shown in figure 3.

C. Formats immunological tests:

Analysis Abbot PRISM (Abbott Laboratories, Abbott Park, IL), is available for purchase by the analysis based on antibodies. Analysis was performed using the manufacturer's instructions.

Test system version 3.0 ELISA ORTHO HCV (denoted here Ortho 3.0 analysis, Ortho Clinical Diagnostics, Raritan, New Jersey) is available for purchase by the analysis based on PCR. Analysis was performed using the manufacturer's instructions.

Analysis of the Roche Amplicor (Roche, Pleasant, CA) is available for purchase PCR test. Analysis was performed using the manufacturer's instructions.

Analysis of the Gen-Probe TMA (San Diego, Ca) is available for purchase transcription-mediated amplification assay. Analysis was performed using the manufacturer's instructions.

Antigenic analysis Ortho (Ortho Clinical Diagnostics, Raritan, New Jersey) is an analysis based on antibodies. Analysis was performed using the manufacturer's instructions.

Declared immunoassay education complex HCV antigen/antibody was carried out as follows. Purified monoclonal antibodies (4 mg/ml each) SP-7 and SP-3 1x fosfato-salt solution (PBS), pH 7.4, were United, stirring well. In covering the same buffer was added to 90 ng recombine the spas antigen NS3/4a. Before coating the solution was stirred for 30 minutes. Was added to this solution in the wells of 96-well microtiter plate to bind environment Costar (Corning, Inc.), 200 µl per well. The plates were incubated at 15-30°C for 16-24 hours. Tablets twice washed with distilled H2O, followed by addition of 300 µl/well poslepechatnogo buffer (1% bovine serum albumin (BSA)in 1x PBS) for one hour and 300 µl/well of a stabilizing buffer (1x PBS, 1% BSA, mannitol, polyethylene glycol (PEG), gelatin), for 1 hour. The liquid from the plates was removed, and dried them with 4°within 24 hours liofilizadora. The tablets were placed in bags with desiccant pack.

For carrying out immunological analysis in the tablet was added 100 μl of reinforcing lysis buffer (1% N-laurylsarcosine, 0.65 M NaCl, 50 mg/ml mouse IgG, technical quality (Sigma, St. Louis, MO), 1% modified by sulfhydryl BSA (Bayer), 0.1% casein). Then added 100 μl of the sample. All this was incubated on a shaker at 40°within hours. The tablets were washed six times with 1x PBS and 0.1% Tween-20 at the sink Ortho Plate Washer. 200 μl of conjugate solution (c11-14-HPR in a dilution of 1:75 with SOD-SSSs 250 ng/analysis, plus anti-SOD-HPR mouse, diluted 1:5000 in diluent for sample HCV 3.0 (ORTHO HCV version 3.0 ELISA test system, Ortho Clinical Diagnostics, Raritan, New Jersey) without extract SOD, all prepared 30 minutes before adding). The solution Incubus is listed with shaking for 45 minutes at 40° C. and Then washed six times, as shown above, and add 200 µl of substrate solution (1 tablet ODP/10 ml). Tablet ODP available in Sigma, St.Louis, MO, contains o-phenylenedimethylene and hydrogen peroxide for staining reaction of horseradish peroxidase. It was incubated for 30 minutes at 15-30°in the dark. The reaction was stopped by adding 50 ml of 4n H2SO4and the tablets were read at 492 nm, relative to the absorption at 690 nm as a control.

D. Results:

The results of the various analyses are shown in tables 5 and 6, which shows two separate experiment the blood samples infected with HCV, as shown. Gray shows the detection of a virus. As shown below, the analysis of the combination antigen/antibody of Cheiron (Chiron) was revealed seroconversion in all samples, whereas all other analyses are based on antigens and antibodies failed to detect seroconversion in any of the samples. In particular, none of the analyses with antibodies revealed no seroconversion at least until the 18th day (table 5). Table 6 shows that none of the tests based on antibodies did not reveal the presence of HCV infection on day 22. Moreover, after 85 days of analysis, based on Ortho-antigen was not able to detect seroconversion.

Thus, on the basis of the above results it is clear that a new analysis of the combination antigen/antibody, umenshaetsa false negative results, obtained using other standard analyses based on antigens and antibodies.

Table 5

HCV seroconversion
DaysAbbott PRISMOrtho 3.0Roche AmplicorGen-Probe TMAOriho AgChironAg/Ab
00.10.0>5×1059.2518.62.8
40.10.0>5×1059.2919.03.1
70.10.0>5×1059.5222.31.5
130.30.1>5×1059.5926.21.7
181.30.4>5×1059.7015.91.2
212.21.0>5×1059.3911.31.5
1644.24.44×1049.280.112.5

Table 6

HCV seroconversion
DaysAbbott PRISMOrtho 3.0Roche AmplicorGen-Probe TMAOrthoAgChironAg/Ab
00.10.0BLD0.110.5
130.10.0>5×10544.03.0
200.10.0>5×10524.21.3
220.30.0>5×10529.21.6
855.44.7BQR0.061.1
1314.34.7BQR0.091.0
1354.64.73×1030.091.2
1385.54.7BLD0.081.2
1465.94.7BLD 0.112.1
1525.24.7BQR0.071.8

EXAMPLE 2

Obtaining conformational epitope NS3/4a with substitutions on Thr Pro Ser to Ile

Conformational epitope NS3/4a was prepared as follows. This epitope has the sequence shown in figures 4A-4D and differs from the natural sequence at positions 403 (amino acid 1428 full sequences of HCV-1) and 404 (amino acid 1429 full sequences of HCV-1). More precisely, Thr normal in 1428 natural sequence mutated to Pro, a Ser, met at position 1429, mutated to Ile.

In particular, we used the above-described yeast expressing vector RV. Plasmid pd.hcvla.ns3ns4aPL, which encodes an illustrative epitope NS3/4a used in the present immunoassays, was prepared as follows. Used a two-stage methodology. First ligated following DNA segments: (a) synthetic oligonucleotides that form a 5' HindIII cloning site, followed by a sequence of AAAAAAAA, the initiator ATG and the codons for HCVIa beginning with amino acids 1027 and continuing until the BglI site at amino acids 1046; (b) restriction fragment 683 bp BglI-ClaI (encoding amino acids 1046-1274) from pAcHLTns3ns4aPI; is (C) the vector pSP72 (Promega, Madison, WI, GenBank/EMBL Accession Number X65332), which was digested HindIII and ClaI, the dephosphorylated and purified by gel-filtration. Plasmid pAcHLTns3ns4aPI was obtained from pAcHLT expressing the baculovirus vector, are commercially available from BD Pharmigen (San Diego, California). In particular, was received vector pAcHLT EcoRI- > PST, as well as the following fragments: EcoRI-AlwnI, 935 mon, corresponding to amino acids 1027-1336 genome of HCV-1; AlwnI-SacII, 247 mon, corresponding to amino acids 1336-1419 genome of HCV-1; HinfI-BglI, 175 mon, corresponding to amino acids 1449-1509 genome of HCV-1; BglI- > PST, 619 mon, corresponding to amino acids 1510-1711 genome of HCV-1; plus codon termination of transcription. Synthesized 91 mon SacII fragment-HinfI, corresponding to amino acids 1420-1448 genome of HCV-1 and containing PI mutation (Thr-1428 replaced with Pro, Ser-1429 replaced by Ile), ligated with 175 mon-HinfI fragment-BglI, and 619 mon-BglI fragment- > PST, described above, and was subcloned into the vector pGEM-5Zf(+), SacII digested and > PST. pGEM-5Zf(+) is available for purchase by the E. coli vector (Promega, Madison, WI, GenBank/EMBL Accession Number X65308). After transformation of competent cells NW, ministeringangel analysis of individual clones and verification sequence, the fragment 885 mon Sac II-Pstl from pGEM5. PI clone 2 was purified by gel filtration. This fragment is ligated with a fragment of EcoRI-AlwnI 935 mon, AlwnI fragment-SacII 247 mo and vector pAcHLT EcoRI- > PST, described above. The resulting design is Ktsia was named pAcHLT Tns3ns4aPI.

The above lilacina mixture was transformed into HBlOl-competent cells and placed on agar tablets Luria containing 100 μg/ml ampicillin. Miniprep analysis of individual clones led to the determination of false positives, two of which were amplified. Plasmid DNA pSP72 1aHC of the clones #1 and #2, were obtained using the kit Qiagen Maxiprep and sequenced.

Then, the following fragments were legirovanyh with each other: (a) 761 mon fragment HindIII-CalI from RAS #1 (pSP72.laHC was obtained by ligating to each other: pSP72, which was digested HindIII and ClaI, synthetic oligonucleotides, which form the cloning site 5' HindIII, followed by a sequence of AAAAAAAA, the initiation codon ATG and the codons HCVIa beginning with amino acids 1027 and continuing to amino acids 1046 BglII site, and restriction fragment BglII-ClaI in 683 mo (encoding amino acids 1046-1274) from pAcHLTns3ns4aPI); (b) a fragment of BamHI-HindIII in 1353 mo hybrid promoter of the yeast ADH2/GAPDH; (C) a fragment of the ClaI-SalI in 1320 mo (encoding amino acids HCVIa 1046-1711 with Thr 1428, replaced by a Pro, and Ser 1429, Not replaced by) from pAcHLTns3ns4aPI; and (d) expressing the yeast vector pBS24.1, which was digested BamHI and SalI, the dephosphorylated and purified using gel filtration. Lilacina mixture was transformed into HB101 competent cells and placed on agar tablets Luria containing 100 μg/ml ampicillin. Miniprep-anal the threat of individual clones led to the determination of individual clones with the expected insert BamHI-SalI in 3446 mon, which consisted of promoter ADH2/GAPDH, the initiating codon ATG and NS3/4a from HCVIa consisting of amino acids 1027-1711 (shown as amino acids 1-686 on figa-4D) with Thr 1248 (amino acid position 403 to figa-4D)is replaced by Pro, and Ser (amino acid position 404 to figa-4D), replaced by No. The design called pd.HCVla.ns3ns4aPI (see figure 5).

The S.cerevisiae strain AD3 transformed pd.HCVla.ns3ns4aPI and after depletion of glucose in the medium were examined for the expression of individual transformants. In yeast was marked by high level expression of recombinant protein, as was shown by the dye Kumasi blue and confirmed by analysis of Western blot turns using polyclonal antibodies to helicase domain N33.

EXAMPLE 3

Cleaning conformational epitope NS3/4a

Conformational epitope NS3/4a was purified as follows. Mentioned cells S. cerevisae, expressing the epitope NS3/4a, collected as described above. Cells suspended in lyse buffer (50 mm Tris pH 8.0; 150 mm NaCl; 1 mm EDTA; 1 mm PMSF; 0.1 ám pepstatin; 1 μm leupeptin) and literally in a Dyno-Mill (Wab Willy A. Bachofon, Basel, Switzerland) or similar apparatus using glass beads in a ratio of 1:1:1 cells:buffer:0.5 mm glass beads. The lysate was centrifuged at 30100 × g for 30 minutes at 4°and the residue, containing the insoluble fraction of protein, it is time to relax is whether to wash buffer (6 ml per gram of initial weight of cell paste) and mixed by stirring at room temperature for 15 minutes. Wash buffer consisted of 50 nm NaPO4pH 8.0; 0.3 M NaCl; 5 mm β-mercaptoethanol, 10% glycerol, 0.05% octylglucoside, 1 mm EDTA; 1 mm PMSF, 0.1 ám of pepstatin; 1 μm leupeptin. Cell fragments were removed by centrifugation at 30100 × g throughout 30 minutes at 4°C. the Supernatant was poured and left a residue.

Protein of sediment was prepared as follows. Added 6 ml/g of extracting buffer and mixed by stirring at room temperature for 15 minutes. Extracting buffer consisted of 50 nm Tris pH 8.0; 1 M Nad, 5 mm β-mercaptoethanol, 10% glycerol, 1 mm EDTA; 1 mm PMSF, 0.1 ám of pepstatin; 1 μm leupeptin. All this was centrifuged at 30100 × g for 30 minutes at 4°C. Retain the supernatant was added ammonium sulfate to 17.5%using the following formula: volume of supernatant (ml) multiplied by x% ammonium sulphate/(1 - x% ammonium sulphate) = ml 4.1 M of saturated ammonium sulfate, which should be added to the supernatant. Ammonium sulfate was added dropwise under stirring on ice, and the solution was stirred on ice for 10 minutes. The solution was centrifuged at 17700 × g for 30 minutes at 4°and a residue was left and kept at 2-8°With up to 48 hours.

Sediment resuspendable and passed through a column of Poly U (Poly U-Sepharose 4B, Amstersham Pharmacia) at 4°as follows. Sediment resuspendable Poly U-balancing buffer - 6 ml per gram weight of sediment. Equilibrating buffer consisted of 25 mm HEPES pH 8.0, 200 mm NaCl, 5 mm DTT (added fresh), 10% glycerol, 1,2 octylglucoside. The solution was mixed by shaking at 4°C for 15 minutes and centrifuged at 30100 × g throughout 30 minutes at 4°C.

Prepared Poly U-column (1 ml resin per gram of initial weight of sediment). The linear velocity was 60 cm/hour and the speed at stuffing was 133% of 60 cm/h. The column was equilibrated balanced buffer, and the supernatant resuspending ammonium sulfate precipitate was loaded onto equilibrated column. The column was washed to its original status equilibrating buffer and protein was suirable sequential elution of the following Poly U-eluting buffer: 25 mm HEPES pH of 0.8; 1 M NaCl, 5 mm DTT (added fresh), 10% glycerol, 1,2 octylglucoside. The eluate from the column was passed through SDS-PAGE (colored Kumasi), and aliquots were frozen and kept at -80°C. the presence of the epitope NS3/4a was confirmed using Western blot analysis, using polyclonal antibodies directed against the NS3-proteasome domain, and monoclonal antibodies against 5-1-1-epitope (HCV 4A).

Additionally, the purification process was monitored protease enzymatic activity as follows. The NS4A peptide (KKGSVVIVGRIVLSGKPAIIPKK) and the sample containing the conformational epitope NS3/4a, astoral in 90 μl of reaction buffer (25 mm Tris, pH 7.5; 0.15 mm NaCl, 0.5 mm EDTA, 10% glycerol, 0.05 for n-dodecyl-B-D-maltoside, 5mm DTT) and allowed to mix for 30 minutes at room temperature. In the tablet for micrometrology (Costar, Inc., Corning, NY) were added to 90 µl of the mixture, and 10 ál of HCV substrate (AnaSpec, Inc., San Jose, California). The contents of the tablet was stirred and read on the reader-tablets Fluostar. The results were expressed in units of relative fluorescence (RFU) per minute.

When using these methods, the product extraction with 1M NaCl had an activity of 3.7 RFU/min, the precipitate of ammonium sulfate had an activity of 7.5 RFU/min, the product Poly U cleaning had activity 18,5 max/min

Example 4

Competitive research.

Were carried out following a competitive assessment study whether the conformational epitope NS3/4a to identify other antibodies, compared with other HCV-antigens. In particular, the antigen NS3/4a was compared with the C200 antigen as follows.

0.5 μg and 0.1 μg NS3/4a, obtained as described above, or C200 (Hepatology (1992) 15; 19-25, available in the ORTHO HCV Version 3.0 ELISA Test System, Ortho Clinical Diagnostics, Raritan, New Jarsey) was mixed with 20 μl of the sample PHV914-5 (sample of early seroconversion obtained from the blood of an infected subject) in total volume of 220 μl (1 × PBS). The mixture is incubated for one hour in microlance at 37°C. the Tablets were washed and perform the analysis as follows.

1 µg of antigen C200 was added to 10 µg about what Azza PHV914-5 in total volume of 220 μl. The mixture is incubated for 1 hour in microlance at 37°S, and 200 µl was transferred to a tablet covered with NS3/4a (100 ng/assay), and incubated for 1 hour at 37°C. the Tablets were washed five times with 1 × PBS, 0,1 Tween-20. Added 200 μl of conjugate solution (described above), the plates were incubated, and perform the analysis. Control frequency, which consisted of PHV914-5 and 1 × PBS (without antigen), were treated in the same way.

The results are shown in table 7. Results the percentage of inhibition is shown in column 4, was calculated as follows: column 3 minus column 2 divided by column 3 and multiplied by 100). As you can see, the data show that NS34a neutralizing antibodies early seroconversion and C200 - no. Achieved a strong signal when antibodies in PHV914-5 SS of sampling early seroconversion reacted with NS34a applied to the microplate. The C200 antigen was not neutralized by these antibodies. This is shown in the upper part of table 7. When NS34a was mixed with sample PHV914-5, he was neutralized, and therefore in the sample did not have antibodies that react with NS34a applied to the microplate. Data show that NS34a could recognize the class of antibodies that are different from those that recognize the C200.

Competitive research showing that NS34a antigen recognized the other antibody in the sample specimen of the early SS ser the conversion. compared to the C200 antigen

Table 7
1234
Control
C200+PHV914-51×PBS% inhibition
SS
1ug1.4501.64512
1ug1.5451.6878
0.5ug1.5571.91319
0.5ug1.7191.8045
NS3/4a+PHV914-5
ss
1ug0.0541.59997
1ug0.0371.67798
0.5ug0.0661.67296
0.5ugNA1.524NA

EXAMPLE 5

Stability studies of the conformational epitope NS34a.

To evaluate the role of stability of NS3/4A-epitope in the analysis was carried out following the research that determines the dependence of immunoreactivity NS3/4a from the time when the room temperature. Small aliquots available NS3/4a was kept at room temperature and frozen at intervals shown in table 8. All the bubbles were covered simultaneously and tested on two samples of specimens of early seroconversion NS3.

As can be seen from table 8, the stock NS3/4a unstable and immunoreactivity decreases with time. In addition to the preservation of immunoreactivity it is necessary to maintain the conformation of NS3/4a.

Further stability studies were set as follows. Two conformational monoclonal antibodies against NS3/4a, created using standard procedures, was replaced with a sample specimen of the early anti-HCV seroconversion. Bubbles NS3/4a from the main stock was stored at room temperature, increasing the storage time intervals of 3, 6 and 24 hours. NS3/4a from the frozen bubbles (90 ng/ml) was applied and tested using the above method. The results indicate that both monoclonal antibodies were really conformational and their reactivity is sensitive to manipulirovanie NS3/4a-antigen from the main reserve at room temperature. Reactivity of monoclonal antibodies in positive control was not changed.

EXAMPLE 6

Immunoreactivity conformational epitope NS3/4a in comparison with denatured NS3/4a.

Immunoreactivity conformational epitope NS3/4a, obtained as described above was compared with the NS3/4a, which was are denatured by adding SDS to the drug conformational epitope NS3/4a to a final concentration of 2%. Denatured NS3/4a conformational NS3/4a was put on tablets for micrometrology, as described above. The C200 antigen (Hepatology (1992) 15:19-25 available in the ORTHO HCV Version 3.0 ELISA Test System, Ortho Clinical Diagnostics, Raritan, New Jarsey), also put on tablets for micrometrology. The C200 antigen, which is used for comparison, is conformational due to present his recipe is a reducing agent (DTT) and detergent (SDS).

Immunoreactivity was tested against two sample specimens of early HCV seroconversion PHV 904 and PHV 914 (available for purchase samples of human blood from Boston Biomedica, Inc., West Bridgewater, MA). The results are shown in table 9. Dunn is e show that denatured or linearized form of NS3/4a (as well as C200) does not recognize a sampling of early seroconversion as early as NS3/4A-conformational epitope.

Immunoreactivity conformational epitope also tested using monoclonal antibodies to NS3/4a obtained by standard methods. Monoclonal antibodies were then tested in the ELISA format against NS3/4a, denatured NS3/4a antigen C200. Data show that anti-NS3/4A-monoclonal antibodies similarly react with NS3/4a and denatured NS3/4a on seroconversion panels, shown in table 10. This result is further evidence that the NS3/4a is conformational in nature, as well as monoclonal antibodies that can be produced with similar reactivity to samples samples early SS seroconversion.

Table 10
Tablet
NS3/4adNS3/4ac200
Lookonline acticalODODOD
W/EZ1:1001.8200.6160.369
1:10001.3970.3800.246
1;100000.8640.1730.070
1:200000.6070.1160.085
5B7/D71:1002.8850.8980.436
1:10002.8660.5410.267
1:100001.6720.2150.086
1:200001.0530.1240.059
A/N21:1001.0200.1690.080
1:10000.9210.1010.043
1:100000.6530.0370.013
1:200000.3370.0270.011

Accordingly, new methods of analysis for the detection of HCV. On the basis of the above it becomes clear that although specific embodiments of this invention have been described for illustrative purposes, can be made a variety of changes without betraying the Oia, the nature and scope of this invention.

1. Solid substrate for immunoassay to determine infection of hepatitis C virus containing the associated at least one antikarov antibody against hepatitis C virus (HCV) and at least one selected conformational NS3/4a-HCV epitope.

2. Solid substrate for immunoassay according to claim 1, containing at least two related HCV-antikarov antibodies.

3. Solid substrate for immunoassay according to claim 1, where the aforementioned at least one antikarov antibody directed against the N-terminal region of crustal HCV antigen.

4. Solid substrate for immunoassay according to claim 3, where the aforementioned at least one antikarov antibody directed against amino acids 10-53 HCV, numbered according to the sequence polyprotein HCV1.

5. Solid substrate for immunoassay according to claim 1, where the aforementioned at least one antikarov antibody is a monoclonal antibody.

6. Solid substrate for immunoassay according to claim 1, where the specified NS3/4A-epitope contains the amino acid sequence depicted in FIGU-4D.

7. Solid substrate for immunoassay according to claim 1, additionally containing an associated slit antigen with multiple epitopes.

8. Solid substrate for immunoassay according to claim 7, where the specified fused antigen with multiple epitopes contains the amino acid sequence is lnost, depicted on figa-7F.

9. Solid substrate for immunoassay to determine infection of hepatitis C virus containing the associated two antikarov monoclonal antibodies against hepatitis C virus (HCV) and conformational NS3/4a-HCV epitope containing amino acid sequence depicted in FIGU-4D.

10. Solid substrate for immunoassay according to claim 9, where the two antikarov antibodies directed against the N-terminal region of crustal HCV antigen.

11. Solid substrate for the immunoassay of claim 10, where the two antikarov antibodies directed against amino acids 10-53 HCV, numbered according to the sequence polyprotein HCV1.

12. Solid substrate for immunoassay to determine infection of hepatitis C virus containing the associated two antikarov monoclonal antibodies against hepatitis C virus (HCV) and conformational NS3/4a-HCV epitope containing amino acid sequence depicted in FIGU-4D, and fused antigen with multiple epitopes containing the amino acid sequence depicted in FIGU-7F.

13. The method for detecting infection of hepatitis C virus (HCV) in a biological sample, and the method includes

(a) obtaining a solid substrate for immunoassay according to claim 1;

(b) connection of a biological sample from a decree of the Noah solid substrate under conditions which allow the antigen and HCV antibodies, if present in the biological sample, to communicate with the specified at least one antiquorum antibody and the specified NS3/4A-epitope, respectively;

(c) adding to the solid substrate from step (b) under conditions of complex formation (i) the first antibody with a detectable label, where the aforementioned first antibody with a detectable label is HCV-antiquorum antibody with a detectable label, where the specified labeled antikarov antibody directed against the crustal HCV epitope that is different from at least one anticorrosive antibody associated with the solid substrate; (ii) antigen that reacts with HCV-antibody from the biological sample, is capable of reacting with the specified conformational NS3/4A-epitope; and (iii) a second antibody with a detectable label where the specified second antibody with a detectable label capable of reacting with the antigen (ii);

(d) detecting complexes formed between antibodies and antigens, if any, as an indication of HCV infection in a biological sample.

14. The method according to item 13, where the aforementioned at least one antikarov antibody directed against the N-terminal region of crustal HCV antigen, as specified antikarov HCV antibody with a detectable label directed against the C-terminal region of the crustal HCV.

15. The method according to 14, where the aforementioned at least one antikarov antibody directed against amino acids 10-53 HCV, numbered according to the sequence polyprotein HCV1, as specified HCV-antikarov antibody with a detectable label directed against amino acids 120-130 HCV, numbered according to the sequence polyprotein HCV1.

16. The method according to item 13, where the specified antigen-reactive HCV-antibody from the biological sample contains the epitope of SS-area polyprotein HCV.

17. The method according to clause 16, where SS-epitope fused with the amino acid sequence of human superoxide dismutase (hSOD), a second antibody with a detectable label capable of reacting with the specified amino acid sequence hSOD.

18. The method according to item 13, where the specified conformational NS3/4a-epitope contains the amino acid sequence depicted in FIGU-4D.

19. The method for detecting infection of hepatitis C virus (HCV) in a biological sample, and the method includes

(a) obtaining a solid substrate for immunoassay according to claim 2;

(b) connection of a biological sample with a solid substrate under conditions that allow antigen and HCV antibodies, if present in the biological sample, to communicate with the specified at least two anticorrosie antibodies and specify the output conformational NS3/4a-epitope, respectively;

(c) adding to the solid substrate from step (b) under conditions of complex formation (i) the first antibody with a detectable label, where the aforementioned first antibody with a detectable label is HCV-antiquorum antibody with a detectable label, where the specified labeled antikarov antibody directed against HCV-crustal epitope that is different from at least two antikarov antibody associated with the solid substrate; (ii) epitope from SS-area polyprotein HCV, merged with hSOD amino acid sequence; and (iii) a second antibody with a detectable label, where the aforementioned second antibody with a detectable label capable of reacting with the specified amino acid sequence hSOD;

(d) detecting complexes formed between antibodies and antigens, if any, as an indication of HCV infection in a biological sample.

20. The method according to claim 19, where the specified conformational S3/4A-epitope contains the amino acid sequence depicted in FIGU-4D.

21. The method for detecting infection of hepatitis C virus (HCV) in a biological sample, and the method includes

(a) obtaining a solid substrate for immunoassay in accordance with claim 9;

(b) connection of a biological sample with a specified solid substrate under conditions that allow the antigens to which the antibodies of HCV, if they are present in the biological sample, to communicate with the specified at least two anticorrosie antibodies and specified conformational NS3/4a-epitope, respectively;

(C) adding to the solid substrate from step (b) under conditions of complex formation (i) the first antibody with a detectable label, where the aforementioned first antibody with a detectable label is HCV-antiquorum antibody with a detectable label, where the specified labeled antikarov antibody directed against HCV-crustal epitope that is different from at least two antikarov antibody associated with the solid substrate; (ii) epitope from SS-area polyprotein HCV, merged with hSOD amino acid sequence; and (iii) a second antibody with a detectable label, where the aforementioned second antibody with a detectable label capable of reacting with the amino acid sequence hSOD;

(d) detecting complexes formed between antibodies and antigens, if any, as an indication of HCV infection in a biological sample.

22. The method according to item 21, where at least two antikarov antibodies directed against the N-terminal region of HCV-crustal antigen, as specified HCV-antikarov antibody with a detectable label directed against the C-terminal region of crustal HCV antigen.

23. The method according to item 22, where the specified at least two an igorovich antibodies directed against amino acids 10-53 HCV, numbered according to the sequence polyprotein HCV1, as specified HCV-antikarov antibody with a detectable label directed against amino acids 120-130 HCV, numbered according to the sequence polyprotein HCV1.

24. The method for detecting infection of hepatitis C virus (HCV) in a biological sample, and the method includes

(a) obtaining a solid substrate for immunoassay according to claim 7;

(b) connection of a biological sample with a specified solid substrate under conditions that allow antigen and HCV antibodies, if present in the biological sample, to communicate with the specified at least one antiquorum antibody, specified conformational NS3/4a-epitope and the specified fused antigen with multiple epitopes, respectively;

(c) adding to the solid substrate from step (b) under conditions of complex formation (i) the first antibody with a detectable label, where the aforementioned first antibody with a detectable label is HCV-antiquorum antibody with a detectable label, where the specified labeled antikarov antibody directed against HCV-crustal epitope that is different from at least one anticorrosive antibody associated with the solid substrate; (ii) first and second antigens that react with HCV-antibody of Biologicheskaya, capable of reacting with the specified NS3/4A-epitope and the specified fused antigen with multiple epitopes, respectively; and (iii) a second antibody with a detectable label, where the aforementioned second antibody with a detectable label capable of reacting with antigens (ii);

(d) detecting complexes formed between antibodies and antigens, if any, as an indication of HCV infection in a biological sample.

25. The method according to paragraph 24, where the aforementioned at least one antikarov antibody directed against the N-terminal region of HCV-crustal antigen and the specified first HCV-antikarov antibody with a detectable label directed against the C-terminal region of HCV-crustal antigen.

26. The method according A.25, where the aforementioned at least one antikarov antibody directed against amino acids 10-53 HCV, numbered according to the sequence polyprotein HCV1, as specified HCV-antikarov antibody with a detectable label directed against amino acids 120-130 HCV, numbered according to the sequence polyprotein HCV1.

27. The method according to paragraph 24, where the first antigen-reactive HCV-antibody from the biological sample contains the epitope of SS-area polyprotein HCV.

28. The method according to item 27, where SS-epitope fused with the amino acid sequence of human superoxide dismutase (hSOD), a second EN is Italo with a detectable label capable of reacting with the specified amino acid sequence hSOD.

29. The method according to paragraph 24, where the specified second antigen-reactive HCV-antibody from the biological sample contains an epitope of the field of C22 polyprotein HCV.

30. The method according to clause 29, where the epitope of the region C22 contains amino acids Lys10-Ser99polyprotein HCV with a deletion of AGD and replacement of Trp to Leu at position 44, numbered according to the sequence polyprotein HCV1, where the specified epitope fused with the amino acid sequence of human superoxide dismutase (hSOD), and the second antibody with a detectable label capable of reacting with the specified amino acid sequence hSOD.

31. The method according to paragraph 24, where the specified fused antigen with multiple epitopes contains the amino acid sequence depicted in FIGU-7F.

32. The method for detecting infection of hepatitis C virus (HCV) in a biological sample, and the method includes

(a) obtaining a solid substrate for immunoassay in accordance with clause 12;

(b) connection of a biological sample with a specified solid substrate under conditions that allow antigen and HCV antibodies, if present in the biological sample, to communicate with the specified at least two anticorrosie antibodies indicated conformational NS3/4A-epitope and the specified fused antigen with multiple epitopes, respectively;

(c) adding to the solid substrate from step (b) under conditions of complex formation (i) the first antibody with a detectable label, where the aforementioned first antibody with a detectable label is HCV-antiquorum antibody with a detectable label, where the specified labeled antikarov antibody directed against HCV-crustal epitope that is different from at least two antikarov antibody associated with the solid substrate; (ii) epitope from SS-area polyprotein HCV, merged with hSOD amino acid sequence and epitope from C22-area polyprotein HCV, merged with hSOD amino acid sequence; (iii the second antibody with a detectable label, where the aforementioned second antibody with a detectable label capable of reacting with the amino acid sequence hSOD;

(d) detecting complexes formed between antibodies and antigens, if any, as an indication of HCV infection in a biological sample.

33. The method according to p where indicated at least two antikarov antibodies directed against the N-terminal region of HCV-crustal antigen and specified HCV-antikarov antibody with a detectable label directed against the C-terminal region of HCV-crustal antigen.

34. The method according to p where indicated at least two antikarov antibodies directed against amino acids 10-53 HCV, numbered, respectively, is sledovatelnot polyprotein HCV1, and specified HCV-antikarov antibody with a detectable label directed against amino acids 120-130 HCV, numbered according to the sequence polyprotein HCV1.

35. The method according to p, where the epitope of the region C22 contains amino acids Lys10-Ser99polyprotein HCV with a deletion of AGD and replacement of Trp to Leu at position 44, numbered according to the sequence polyprotein HCV1.

36. Immunodiagnostics test set containing solid substrate for immunoassay to determine infection of hepatitis C virus according to any one of claims 1 to 12, and instructions for conducting immunodiagnostics test.

37. A method of obtaining a solid substrate for immunoassay to determine infection of hepatitis C virus, including

(a) obtaining a solid substrate; and

(b) linking with it at least one anticorrosive antibodies against hepatitis C virus (HCV) and at least one selected NS3/4a-conformational epitope of HCV.

38. The method according to clause 37, further comprising linking at least one fused antigen with multiple epitopes with a solid substrate.

39. A method of obtaining a solid substrate for immunoassay to determine infection of hepatitis C virus, including

(a) obtaining a solid substrate; and

(b) linking to it on minicamera two antikarov antibodies against hepatitis C virus (HCV) and selected conformational NS3/4a-HCV epitope.

40. The method according to § 39, further comprising linking at least one fused antigen with multiple epitopes with a solid substrate.

41. Fused antigen with multiple epitopes containing the amino acid sequence depicted in FIGU-7F, or an amino acid sequence having with it at least 80% identity, which specifically reacts with anti-HCV antibodies present in a biological sample from an HCV-infected individual.

42. Fused antigen with multiple epitopes on paragraph 41, where the specified fused antigen with multiple epitopes contains the amino acid sequence depicted in FIGU-7F, or an amino acid sequence having with it at least 90% identity, which specifically reacts with anti-HCV antibodies present in a biological sample taken from HCV-infected individual.

43. Fused antigen with multiple epitopes on p where specified fused antigen with multiple epitopes consists of the amino acid sequence depicted in FIGU-5F.

44. Polynucleotide containing the coding sequence fused antigen with multiple epitopes on any of PP-43.

45. Recombinant expressing a vector containing

(a) polynucleotide ACC is accordance with item 44;

(b) and control elements operatively associated with the specified polynucleotide, whereby the coding sequence can be transcribed or translated in a cell-master.

46. Cell line, transformed with the recombinant vector according to item 45.

47. A method of obtaining a recombinant fused antigen with multiple epitopes on paragraph 41, which includes

(a) obtaining a population of a cell line according to § 46; and

(b) culturing the specified population of cells under conditions in which is expressed fused antigen with multiple epitopes encoded by the coding sequence present in the indicated recombinant vector.

Priority points and features:

15.06.2000 - claims 1 to 5, 7, 13-17, 19, 24-28, and 37-39;

02.04,2001 - PP, 9-11, 18 and 20-23;

14.06.2001 - PP, 12, 29-35 and 40-46;

15.06.2000 - p - signs that are dependent on claims 1 to 5 and 7;

02.04.2001 - signs-dependent PP, 9-11;

14.06.2001 - signs-dependent PP and 12.



 

Same patents:

FIELD: virology, biotechnology, medicine, pharmacy.

SUBSTANCE: invention proposes the envelope HCV protein (hepatitis C virus). Protein comprises 80% of glycosylated sites. Such protein is more preferable for diagnostic, prophylactic and therapeutic using. Also, invention proposes a method for preparing such protein, and a drug, vaccine and different pharmaceutical compositions also comprising such protein. Proposed group of inventions can be used in medicine for diagnosis, treatment and prophylaxis of HCV infection and to prediction of clinical effectiveness of treatment.

EFFECT: improved preparing method, valuable medicinal properties of protein.

37 cl, 77 dwg, 16 tbl, 29 ex

FIELD: medicine, clinical virology.

SUBSTANCE: method involves assay of antibodies raised against some antigen sites of two surface proteins (E1 and E2) by using the following 5 synthetic peptides as antigens: 1) NH2-SGHRMAWDMMMNWSP-CONH2; 2) NH2-STLTSLPTPGASG-CONH2; 3) NH2-RPYAWHYAPRPAGIVPASQV-CONH2; 4) NH-TTDRIFGAPTYSWGENETDVL-CONH2; 5) NH2-TWIMNSTGFTKTAGGPPANIGGVGNNT-CONH2. The favorable finish of disease is prognosticated by detection of antibodies for the first 3-4 weeks of disease. The advantage of the invention involves the prognosis for the disease finish in the early stage of disease. Invention can be used in clinics of infectious diseases for aims of early prognosis of acute hepatitis C finish.

EFFECT: improved method for prognosis of hepatitis C.

6 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: the present innovation deals with detecting serumal concentrations of immunoglobulins A, M and E and, also, cytokins: interleukin 1-beta, interleukin 8, tumor necrosis factor of alpha- and gamma-interferon and at immunoglobulin A values being below 0.93 g/l, immunoglobulin M below 0.77 g/l, immunoglobulin E below 44 kU/l interleukin 1-beta below 300 pg/ml, interleukin 8 below 124 mg/ml, tumor necrosis factor below 140 pg/ml and gamma-interferon below 21 pg/ml in case of acute hepatitis B one should predict the development of lingering hepatitis, and at values of immunoglobulin A below 0.95 pg/ml, immunoglobulin E below 240 kU/l, interleukin 8 below 76 pg/ml, interleukin 1-beta below 280 pg/ml and gamma-interferon below 80 pg/ml in case of acute hepatitis C one should predict the development of chronic hepatic flow.

EFFECT: higher accuracy of prediction.

3 ex, 4 tbl

FIELD: medicine, virology, immunology.

SUBSTANCE: invention proposes a set that involves combination of recombinant antigens obtained on the basis of amino acid sequences of the most immunoreactive epitopes of hepatitis C proteins of different genotypes immobilized on surface of solid-phase carrier. Invention provides the development of new set used for assay of antibodies raised against hepatitis C virus with higher sensitivity and specificity. Invention can be used for assay of antibodies raised against hepatitis C virus.

EFFECT: valuable immunological properties of set.

2 tbl, 17 ex

FIELD: medicine, diagnostics.

SUBSTANCE: as immunological values one should detect microsomal and mitochondrial autoantibodies in blood serum and at their increased content by 25% and more it is possible to predict the flow of chronic hepatitis C with complications.

EFFECT: increased accuracy of prediction.

2 ex

FIELD: medicine, gastroenterology.

SUBSTANCE: in patient's blood serum it is necessary to detect the level of lipid peroxides (LP) and total antioxidizing activity (AOA) and at the level of LP being 83.9 and higher, AOA value being 15.5 and lower one should detect high activity of chronic hepatitis, at LP value ranged 83.8 to 74.5, AOA value ranged 24.3 to 15.6 - moderate degree of activity and at LP value ranged 74.4 to 66.0 and AOA value ranged 24.4 to 33.5 - inactive degree of activity should be detected. The method enables to accelerate and simplify testing in patients and detect indications for therapy and evaluate efficiency of therapy conducted.

EFFECT: increased information value and accuracy of detection.

3 ex, 2 tbl

FIELD: biotechnology, vaccines.

SUBSTANCE: invention proposes an oligomeric particle that is able to induce immunity against hepatitis C virus. Particle consists of purified proteins or their parts, hepatitis C virus envelope (HCV) E1, E1s and E2. Proposed particle has diameter from 5 to 100 nm. Also, invention proposes a method for preparing such particle. Method involves purification of proteins HCV in solution, formation of oligomeric particles by replacing purified proteins with detergent solution or salt solution and purification of oligomeric particles. Also, invention proposes a composition for inducing immunity against HCV that comprises the proposed particle, antibody specific to particle, sets for detection of antigens HCV, immunological analysis for detection of antibodies to HCV, and vaccine against hepatitis C virus (HCV) that comprises the oligomeric particle. Proposed inventions provide carrying out the induction of specific humoral and cellular immunity to HCV envelope proteins and to carry out diagnosis of HCV. Invention can be used in medicine.

EFFECT: valuable medicinal properties of particle, improved preparing methods.

71 cl, 27 dwg, 6 tbl, 14 ex

FIELD: medicine, biochemistry.

SUBSTANCE: in blood serum one should detect the level of lactoferrin and biliary acids. At their ratio being equal to 5-17 it is necessary to detect chronic hepatitis of high activity.

EFFECT: higher accuracy of detection.

3 ex

Diagnostic analysis // 2228530
The invention relates to medicine, in particular to the analysis on the basis of nucleic acids for detection of hepatitis b virus
The invention relates to medicine, in particular for immunochemical analysis, and can be used to improve analytical and technological characteristics of immunochemical analysis

FIELD: biotechnology, immunology, microbiology.

SUBSTANCE: freeze-dried 10-40 % cell slurries are treated singly or twice with 25-35 % iron sulfate solution. After repeat freeze drying treatment with 20-25 % ammonium aqueous solution is carried out. Then obtained magnetic cells are activated with 5 % glutaric aldehyde for 4 h. Further cells are treated with species-specific competent immunoglobulins from lemic, tularemia, or brucellosis serum containing 10 mg/ml of antibodies for 24±2 h. Method of present invention makes it possible to detect lemic bacteria, tularemia, and brucellosis in concentration of 103-104 m.c./ml. Obtained biomagnetic immunosorbents may be stored for 2 years at 4-8° without losses of specific activity.

EFFECT: biomagnetic immunosorbents of improved sensitivity and prolonged storage-time.

5 cl, 9 ex

FIELD: immunochemistry, sorbents.

SUBSTANCE: method for preparing immunosorbent involves modifying aerosil with chitosan solution in acetic acid in common with iron formate followed by oxidation and immobilization of the protein ligand. Invention can be used in diagnosis for detection of specific response antigen-antibody with using immunoenzyme analysis and immunofluorescence reaction.

EFFECT: improved preparing method.

1 tbl, 4 ex

FIELD: medicine, clinical laboratory diagnosis.

SUBSTANCE: method involves using carboxylated polystyrene latex spherical particles suspension with immobilized specific polyclonal antibodies raised against antithrombin III on their surface and wherein milk α-casein (antithrombin III-latest) is added as an inert component. Method for assay involves successive fractional plasma dilutions and the concentration of antithrombin III in the parent sample is calculated by the formula. Method provides rapid determination of antithrombin III concentration in blood plasma. Method can be used for diagnosis of pathological states in the blood coagulation system.

EFFECT: improved assay method.

2 tbl, 2 ex

FIELD: pharmaceutical chemistry.

SUBSTANCE: invention relates, in particular, to a composition for interaction of ligands wherein the composition comprises a noncovalent associate of multiple separate conjugates being each of that comprises a head group and a tail group wherein tail groups of conjugates form hydrophobic aggregate. Conjugates are mobile within the associate and in the presence of ligand at least two head groups are places by a method corresponding to the epitope formation that is able to interact with ligand stronger as compared with each separate head group. Invention provides applying conjugates in combinatory approach for detecting effective combinations to induce the desirable interaction in binding in receptor-specific treatment of patients.

EFFECT: valuable properties of epitopes.

31 cl, 2 dwg, 4 ex

FIELD: medicine.

SUBSTANCE: method involves making interaction of biological sample with solid phase reagent covered with catching antibodies showing affinity to component A binding to produce the first reacted particle. Making the particle to come in contact with labeled antibodies showing specific binding affinity with respect to A and B components results in the second reacted particle production. Labels are measured on the second reacted particle by applying flowing cystometry method.

EFFECT: wide range of functional applications in early stage detection of infections like AIDS or hepatitis C.

19 cl, 2 dwg

FIELD: medicine.

SUBSTANCE: method involves applying ELISA. When doing it, antigen or antibody is immobilized on activated substrate. Microwave radiation is applied at frequency taken within the limits of 2300 and 2500 MHz at each stage from 5 to 200 s. Device has loading chamber, reaction chamber having magnetron, air-exhauster and unit for focusing light, chamber for washing and drying base plate or unit, detection chamber, platform for transferring base plates and computer means.

EFFECT: accelerated analysis method.

14 cl, 8 tbl

FIELD: medicine, immunology.

SUBSTANCE: method involves synthesis of magnetic latex in the presence of colloidal-dispersed ferromagnetic particles of iron oxide wherein iron oxide is added into microspheres of polyacroleinic sorbents after their synthesis. Method provides simplifying the technology in preparing the serological test-system.

EFFECT: improved preparing method.

2 ex

The invention relates to biotechnology

FIELD: biotechnology, molecular biology, medicine.

SUBSTANCE: invention discloses amino acid sequences of human obesity polypeptide (OB) two isoforms possessing capacity for modulation of animal body mass, their signal peptide-containing precursors and analogues. Polypeptide isoforms are prepared as result of insertions, deletions and amino acid changes that retain activity typical for nonmodified forms of OB-polypeptides, and polyclonal and monoclonal antibodies interaction specifically with new agents modulating the body mass value also. Invention describes DNA sequences encoding these polypeptides and their analogues, vector structures comprising these sequences used for preparing recombinant forms of OB-polypeptides. Invention proposes using new polypeptides and their analogues as an active component in pharmaceutical compositions. Using this invention can promote to solving the problem for providing medicine, veterinary science and animal husbandry with effective agent used for decreasing the body mass value. Invention can be used in medicine for diagnosis and treatment of pathological states associated with disturbance of regulation of human body mass, and in animal husbandry and veterinary science.

EFFECT: valuable biological, medicinal and veterinary properties of polypeptide.

23 cl, 71 dwg, 12 tbl, 17 ex

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