CZ695A3 - Method of detecting antibodies of c hepatitis virus and a set for detection hcv antibodies - Google Patents

Abstract

Immunoassay methods utilizing HCV envelope antigens that contain conformational epitopes reactive with antibodies in serum from infected individuals are useful for screening and diagnosis. These antigens detect antibodies that are not detected by denatured HCV envelope antigens. In addition, these HCV envelope antigens comprised of conformational epitopes are more immunologically reactive than a number of other HCV antigens. This is the first evidence that conformational epitopes may be involved in the immunologic response to HCV antigens.

Description

Technical field

The present invention relates to materials and methodologies for managing the expanded CV (HCV) Y 'Z pupil. used as immunological agents suitable for the detection, prevention and treatment of HCV infections as well as the use of immunological tests and devices.

Prior art

He first identified and characterized HCV as the primary cause of post-transfusion non-A ane-B hepatitis (NANBH) Houghton et al. In addition to providing essential information regarding HCV, Houghton and co-workers found a number of general and^:cifick-ý-c-h-i-m-un-o 1 o-g i ckých- '-r ea-ge nebí — a'-nre tod'. ' Vů z '' napH-k-lad ^ Hou-ght o n a coll. published applications of the judgment Ε P O number 318216 and number 388232; Chco et al. Science (1989) 244, p. 359 to 362; Kuo et al., Science (1989) 244, pp. 362-364; Takeuchi et al., J. G e η. V, i rol. (1990) 71, pp. 3027 to 3033; Takeuchi et al. Gene (1990) 91,. pp. 287-291; Takeuchi et al. Nucl. Acids.Res.

(190) 18, pp. 4626; Miyamura et al. Why. Nati. Acad. Sci. USA (1990) 87, pp. 983-987; Saito et al. Why. Nati. Acad.

Sci. USA (190) '87, pp. 6547-654; Choo et al. Why. Nati. Acad. Sci. USA (1990) 88, pp. 2451-2455; Han et al. Why. Nati. Acad. Sci. USA {1990} 88, pp. 1711-1715; Houghton et al. Hepatology (1991) 14, pp. 381-338; Weiner et al. Why. Nati. Acad. Sci. USA (1990) 89, pp. 3468-3472. These publications form an extensive basis on the state of the art about HCV in general, as well as informing about the production and use of HCV polypeptide immunological reagents. For the sake of brevity, these publications are only pointed out here.

The work of Houghton et al. promptly applied and disseminated

other researchers. See, e.g., Highfield et al., British Published Application No. 2,239,445 (The We 1 come Foundation Ltd.); Wang Published EPO Application No. 442394 (United Biomedical Inc.); Leung et al. EPO Published Application No. 445423 (Abbott Laboratories); Habit et al., EPO Publication No. 451891 (Akzo N.V.); Reyes et al., POT Pub'l, WO 91/15516 (Oenelabs et al., Mákl et al., EPO Publication No. 468657 (Tonen Corp.); and Karnada et al., EPO Publication No. 469348 (Shionogi Seiyaku KK) See also Matsuura et al (1992) J. Virology 66, 1425, Kato et al Proc Natl Acad Sci USA (1990) 87, 9524-9528, Takamizawa et al., J. Virol.

1113; Chiba et al., Why. Nati. Acad. Sci. USA (1991) 88, pp. 4545; Harada et al., J. Virol. (1991) 65, pp. 3015-3021; Hijikata et al., .Why. Nat1. Acad. Sci, USA (1991), 88, pp. 557-55151; Okamoto et al. Jpn. J.Exp.Med. (1990) 60,

177; Yuasa et al., J.Gen. Virol. (1991) 72,

Watanabe et al. Int.J. Cancer (1991) 48, pp. 11-5-464 1 pp. 167-2021-2024; pp. 340 to 343.

Sensitive, specific methods for the detection and identification of carriers of HCV and HCV contaminated blood or blood derivatives are significant advances in medicine. Posttransfusion hepatitis (PTH) occurs in 10% of patients after transfusion, and HCV accounts for 90% of these cases. The main problem with this disease is the frequent incidence of chronic liver damage (25 to 55%) compared to other jaundice, such as type B.

Patient care, such as the prevention of HCV transmission by blood and blood derivatives or close personal contact, requires reliable diagnostics and prognostic tools, such as HCV polypeptides, to detect HCV-related antibodies. Such polypeptides are also useful as vaccines and immunotherapeutic agents for the prevention and / or treatment of disease. As HCV is a relatively new agent, there is a continuing need to define additional immunological agents that will allow further study of the clinical course of the disease and the epidemiology of HCV in the population.

The essence of the invention

A method of detecting hepatitis G virus (HCV) antibodies in a mammalian body component suspected of containing such antibodies is to contact such body components with an HCV antigen component containing mammalian antibodies. The HCV under the conditions allowing the immunological reaction between any antibodies and the antigen is determined and the presence of a possible immune complex of these antibodies and these antigens is determined. .

The invention is based on the fact that further serological studies of HCV antigens have been performed and that it has been found that immunological assays using envelope HCV antigens that retain conformational epitopes are more effective in detecting anti-HCV. antibodies than assays using the same antigens with linear epitopes.

The invention also relates to a method wherein the antigens are encoded in the domains. E1 and / or E2 or are E1 and / or E2 antigens. In some embodiments, the antigens may be displaced, expressed from vaccinia vectors or. In addition, according to some embodiments of another invention, a second antigen consisting of a feline epitope is incorporated.

The invention also relates to a method of screening for blood components or blood for HCV prior to the use of such blood or blood components for the preparation of blood components or compositions having the following degrees:

a) the body component is reacted from a potential donor with an HCV antigen containing a conformational epitope from the E1 or E2 domains of HCV under conditions allowing an immunological reaction between any antibodies in the body component and the antigen;

b) detecting the complex formed between the antigen and any antibodies, and discarding any high blood or blood deposits from the donor which have been detected according to step b).

The invention also relates to a kit for detecting HCV antibodies comprising an HCV antigen formed by a formulation. epitope from HCV E1 or E2 domains and control standards, packaged in suitable vials and instructions for use of the kit components.

The invention is further elucidated by the following description and the accompanying drawings:

Figure 1 schematically indicates putative HCV polyprotein domains.

Fig. 2 is a diagram showing some features of the pSC39 vector.

Giant. 3A, 3B and 3C show the percentage of patients in group ί ,. II and IIT reactive in ELISA assays for individual HCV antigens.

An HCV antigen is a polypeptide or polypeptide analog (e.g., mimitope) comprising an amino acid sequence (and / or amino acid analog) defining at least one HCV epitope. Generally, sequences defining an epitope correspond to the amino acid sequence of the HCV protein (either identically or by substituting analogs of ^a native amino acid residue that does not disrupt the epitope). In general, the sequence defining the epitope will be 5 or more amino acids in length, usually 8 and preferably 10 or more amino acids. With respect to concomitant epitopes, the length of the sequence may be defining

0 * spi top ... pod§. _r both naj | ir o.ký.m ... o..b.mě n., á m., .. nebo f _ _r se ... má ... zato.ž.e _tyuto .. e.-.

pitopes are formed by a three-dimensional antigen (e.g. folding folds). Thus, the epitope-defining amino acids may be few in number, but may be widely dispersed along the length of the molecule (or even on different molecules in the case of dimers, etc.) brought into the correct epitope conformation by folding. The portions of the antigens between the epitope-defining residues may not be critical to the conformational structure of the epitope. For example, deletions or substitutions of these intervening sequences may not affect the conformational epitope, provided that sequences decisive for both the ep conformation are retained (e.g., cysteines involved in the disulfide bond of the glycosylation site, etc.).

The HCV antigens of the invention form conformational epitopes from domains. . (both hunted) E1 and / or E2 HCV. The E1 domain, which is thought to correspond to the viral envelope, of the protein, is commonly considered to be the linker amino acids 192-383 of the HCV polyprotein (PCT Publication No. WO91 / 15771, Fig. 1). According to expression in the CHO (glycosylated) system, it is considered to have an approximate molecular weight of 35 Kd as determined by SDS-PAGE. The E 2 protein, formerly called Ji Ξ1, is thought to associate amino acids 384-800 of the polyprotein, and is also a coat protein. According to expression in the CHO (glycosylated) system, it is believed to have an apparent "g.e.l.o_vo.u.mo.le" to a bulk density of approximately 72 Kd. It's bright. not that these protein endpoints are approximate (for example, the carboxy terminus of E2 could lie somewhat in the amino acid region

750-820). It is also clear that the prototype sequence of the HCV1 isolate in said PCT published application is cited for illustration only, and that any HCV isolate (see for example references cited in the prior art) is a suitable source of the E1 and / or E2 sequence for carrying out the method of of the invention.

The E1 and E2 antigens used in the invention may be full-length viral proteins, essentially full-length versions thereof, or functional fragments thereof (e.g., fragments lacking the sequence necessary to form or preserve a conformational epitope). In addition, the HCV antigens of the invention may also include other sequences that do not block or prevent the formation of the conformational epitope of interest. The presence or absence of a conformational epitope is possible from the same screen. ed.o-ya n-ého ..a.nt.igenu ... s..proti.-. substance (polyclonal serum or monoclonal to conformational -epitope) - and by comparison. reactants with reactivity of the denatured version of dhťVgemiT ^ -iřťě ^ ry- ^ žů ^ dT-žOui-é-ýí-en— ^ T-í-pa-d-ryé ^ line ^ -r-hví ^ e-pú Topical screening using monoclonal antibodies, it may be convenient to absorb the polyclonal serum first with the denatured antigen and determine if it captures antibodies to the antigen of interest.

The HCV antigens of the invention can be prepared in a conventional manner that provides the conformational epitope of interest. For example, recombinant expression of mammalian or insect cells is a preferred way to provide secreted glycosylated E1 and / or E2 antigens in a native conformation. However, as is known for proteins, it may be possible to express antigens in another recombinant host and renature the protein after recovery. It will also be appreciated that chemical synthesis may also provide conformational antigenic mimitopes that interact with the native conformational epitone of the antigen.

Preferred antigens are also E1 and / or E2 complexes (also called aggregates) containing more than one E1 or E2 monomer. E1: E1 dimers, E2, E2 dimers, and E1: E2 heterodimers are all antigens within the scope of this invention. The aggregates may also include larger formations and may have molecular weights in excess of 800 kD.

The term linked polypeptide. (fusion is determined) is a polypeptide in which at least one HCV antigen is part of a single contiguous chain of amino acids, such chain not occurring in nature. HCV antigens may be linked directly to each other by peptide bonds, or may be separated by intervention of amino acid sequences. The fused polypeptides may also contain HCV exogenous amino acid sequences.

The term common solid matrix refers to a solid to which individual HCV antigens or linked polypeptides are bound by covalent or non-covalent bonds, such as hydrophobic adsorption.

The body component determines the fluid or tissue of the mammalian body (e.g., the anthropoid human body) which usually contains the antibodies produced by the individual. Such body components are known in the art and include, for example, blood, plasma, serum, spinal fluid, lymphatics, respiratory or urogenital tract secretions, tears, saliva, milk, white blood cells, and bone marrow. Body components include biological fluids. The term biological liquid refers to a fluid obtained from an organism. Some biological fluids are used as sources of other products, such as clotting factors (e.g., factor VIII: C), serum albumin, and growth hormone. In such cases, it is important that the source of the biological fluid be free of contamination by a virus such as HCV.

The term immunologica 11y reactive means that the antigen of interest will react specifically with anti-HCV against substances present in the body component of an HCV-infected individual. The term immune complex refers to the combination formed when an antibody binds to an epitope on an antigen.

As used herein, the term E1 means a protein or polypeptide that is expressed between the first 400 amino acids of the HOV polyprotein, sometimes referred to as the E-protein or S-protein.

In its natural form, it is a 35 ki) glycoprotein which is strongly associated with the membrane. In most HCV strains, the E1 protein is encoded in a viral polyprotein following the C (core) protein. The E1 protein extends from about amino acid (aa) 192 to about aa 383 of the full length polyprotein. As used herein, the term E1 also includes analogs and distorted forms that are immunologically cross-reactive with natural E1.

As used herein, the term E2 means a protein or polypeptide that is expressed between the first 900 amino acids of the HOV polyprotein, sometimes referred to as the NS1 protein. In its natural form, it is a 72 k D glycoprotein that is strongly associated with the membrane. In most HCV strains, the E2 protein is encoded in a viral polyprotein following the E1 protein. The E2 protein extends from about amino acids aa 384 to about aa 820. As used herein, the term E2 also includes analogs and distorted forms that are immunologically cross-reactive with native E 2.

As used herein, the term aggregate (aggregate) means the complex of E1 a / n-eho ·· -E5-. -content j-íc-í v íc.e _n.e ž Je.d.en monome r_Ej. ..or _E2, Dimers E1: E.1, dimers E2: E2, a. heterodimers E1: E2 are all aggregates within the scope of this definition. The aggregates may also include larger 7'<v ~ - ř-y - and -'- mo-ho-n - m-í-t ·· -m-ad -e: k: i! D: Q V- é ^ hmo: t: no.S: ti .-- V-ě-t š-í — ne-ž -.8-0-0.-kD ..- ..._.....

The term ptirified in reference to proteins refers to compositions wherein the desired protein comprises at least 35 total protein components in the composition. The desired protein is preferably at least 40X, more preferably at least about 50X, even more preferably at least about 60X, even more preferably at least about 70X, even more preferably at least about 95X by weight of the total protein component. The composition may contain other ingredients, such as carbohydrates, salts, lipids, and solvents, without affecting the purity determination used herein. The term isolated protein means an HCV composition that is at least 35% pure.

The term isolated polypeptide means a polypeptide that has more preferably been at least about 80 X 90 X, and most preferably about substantially free of other HCV viral components, particularly an HCV genomic polynucleotide. A polypeptide composition is substantially free of another component if the weight of the polypeptide in the composition is at least 70% by weight of the polypeptide and other combined components, more preferably at least about 89, even more preferably about 90%, and most preferably 95% or more percent. __

The immunoassay methods of the invention use HCV antigens from the E1 and E2 domains and those that contain conformational epitopes recognized by antibodies in the serum of HCV-infected individuals. Each of the antigens has at least one conformational epitope that exists in a naturally occurring HCV particle or infectious product thereof, as seen from the munor reactivity of antigens with antibodies in the body component of an HCV-infected individual and the loss of immunoreactivity of the epitope after antigen denaturation. . The length of the antigens is sufficient to maintain the immunoreactive conformational epitope associated with that epitope. It is within the scope of the invention to have more than one conformational epitope on antigens. Often, native envelope antigens may have immunological tests, but may be distorted, for example to increase solubility. or to improve secretion (e.g., by omitting the connective membranes). The invention also encompasses the use of either individual antigens or combinations or aggregates thereof. Thus, immunoassays may use E1 epitopes, E2 epitopes, aggregates, or combinations of native E1 and E2. It is also within the scope of the invention to use antigens with linear epitopes in combination with envelope conformational epitopes of HCV.

Methods for detecting the presence of conformational epitopes are known in the art, and some of them are further illustrated in the examples.

Based on the putative amino acids encoded in the HCV1 nucleotide sequence and other knowledge, the possible protein domains of the encoded HCV protein, as well as approximate interfaces, are as follows:

Expected domain

C (nucleocapsid protein) E1 (viral envelope protein)

Approximate interface (amino acid numbers)

1-191

192-383

E2 / NS1 (cover?) NS2

NS3 (protease?) MS4

384-800

800-1050

1050-1650

1651-2100

NS5 (polymerase)

21Ό-3-311 (termination) However, these domains are experimental. For example, the interface E1-NS2 is probably in the range 750-810 and the interface N S 3 - N S 4 is approximately 1640-1650. It is also noted that versions 191a and C are precursors that are further fused (e.g., to a length of approximately 170 aa), and that the NS2, NS4, and NS5 proteins are each further fused to two perfect proteins. The relative position of the domains is shown in. Fig. 1.

Methods of antigen preparation. E1 and E2, including antigens with internal conformations, are described in Spaste R et al., Virology (1999) 1888, pp. 819-830 and WO92 / O8734. Generally, cells are selected. .u.m.o.žn í ... vy t vá_varení .na t vní í c h k nformačních. e.p.itopes in expressed envelope proteins; these hosts. the cells may be, for example, animal cells, insect cells

Eukaryotic hosts include yeast and mammalian _T cell cultures. The most commonly used yeast hosts are Saccharomyces cerevisiae and Saccharomyces carlsbergensis and are suitable fungal hosts. Yeast-compatible vectors carry tags that allow the selection of successful transformants conferring prototropy to auxotropic mutants or heavy metal resistance in wild-type strains. The yeast compatible age Tor may · ooužívat 2 micron origin of replication (Broach, ^in, et al. (1983) Meth. Enz. 101, pp. 307), the combination of CEN3 and ARSl ¹ or other means for assuring replication, such as sequences which lead to the incorporation of the relevant fragment into the genome of the host cell. The control sequences for the yeast vector? 4900), including the 3 phosphoglycerate kinase promoter (Hitzeman (180) J. Biol. Chem. 2 5 5, p. 2073). Terminators may also be included, such as terminators derived from the enolase gene (Holland (1981) J. Biol. Chem. 256, p. 1385). Particularly suitable control systems are those which contain a glyceraldehyde-3 phosphate debyrogenase (GAPDH) promoter or a controllable alcohol dehydrogenase (ADH) promoter, terminators also derived from GAPDH and, if secretion is desired, a yeast α-factor leader sequence. In addition, the transcription control region and the transcription initiation region that are operably linked may be such that, naturally, they are not related to a wild-type organism. These systems are described in detail in EPO 120551, published 3. October 1984, EPO 116201, published August 22, 1984, and EPO 164556, published December 18, 1985.

The range of mammalian cells suitable for expression includes many immortalized cell types from the American Type C u1111 re Collection (A TC0), including Hela cells, Chinese hamster ovary (CHO) cells, young hamster kidney cells ( BHK) and many other mammalian cell lines. Suitable nrcv are known in the art and include viral 40 (SV40) (Flers (1978) Mature mammalian cell motors are also promoters such as from Simian Virus 273, p. 113) Rous sarcomas virus (RSV), adenovirus (ADV) and bovine tumor virus (bovine papilloma virus -BPV). Mammalian cells may also require terminator sequences and pole A sequences; Facilitating expression enhancing sequences may also be included, and sequences that cause gene amplification may also be desirable. These sequences are known in the art.

Vectors suitable for mammalian cell replication are known in the art and may include a viral replica of an icon, or a sequence ensuring the integration of appropriate sequences encoding NANBV epitopes into the host genome.

A vector which is used to express foreign DNA and which can be used to prepare v a k c i n y. j e V a c c i n i a virus. In this case, the heterologous DNA is integrated into the Vaccinia genome. The technique of incorporating foreign DNA into the vaccinia virus genome is known in the art and uses, for example, homologous recombination. Heterologous DNA is generally incorporated into a gene that is by nature redundant, such as the thymidine kinase (tk) gene, which also provides a selectable label. Plasmid vectors that greatly facilitate the construction of recombinant viruses are described in the literature (see, e.g., Mackett et al., (1984) J. Virol. 49, p. 857; Chakrabarti et al., (1985) Mol. Cell). Biol. 5, p. 3403; Moss (1987 in Gene Transfer Vectors for Mammalian Cells (Miller and Calos, Cold Spring Harbor Laboratory, Cold Spring Harhor, NY), p. 10). occurs in cells or in individuals who have been immunized with live recombinant vaccine virus.

The HCV CDNA segment encoding the desired sequence is inserted into the Vaccinia vector. The sequence encoding the polypeptide may be attached to the leader sequence. The sequence leader may be a sequence for a tissue plasminogen gene (TPA) from another source, for example a sequence for β-glohin. The heterologous sequence may be

- ·, ....... zac-le-ň-n, .- d.o ..., v..a.c.cú n í.a. v.e.kioru, .k.t.erý .. ..j.e _ver_z_í..p.SCl 1 .., _d____ad_ad set a linker sequence (polylinker) which contains a cloning site.

In order to determine whether HCV polypeptides are expressed from the expression of HCV polypeptides: b: ω.t-bjuňkv-BSC-1 -—- i-nfe-kt-ov-in-y -.- S-re.comb.incombinant vector and grown on a microscope slide under conditions allowing expression. The cells can then be fixed with acetone and immunofluorescence assays performed with serum known to contain anti-HCV antibodies to polypeptides encoded in the region of the HCV genome from which the HCV segment was recombinantly expressed in the vector.

Other systems of eukaryotic or viral genomes include insect cells and vectors suitable for use in such cells. These systems are known in the art and include, for example, insect expression transfer vectors derived from the baculovirus Autography californica nuclear polyher virus (A c Ν Ρ V), which is a helper-dependent viral expression vector. Expression vectors, derived from the 12th hotosystem, typically use a strong viral post-lyhedrin gene promoter to control the expression of heterologous genes. A commonly not commonly used transfer vector for the incorporation of foreign genes into AcNPV is p, Ac373. Many other vectors known to those skilled in the art have also been designed to improve expression. These include, for example, pVL985 (which changes the initial lyhedron new codon from ATC to ATT, and which introduces the base pair of SamHI cloning site 32 downstream of ATT; see Luckow and Summers (1989) Virology 17, p. 31). unrelated foreign proteins usually requires foreign genes that ideally have a short leader sequence containing appropriate signals to initiate translation preceding the initial ATC signal.The plasmid also contains a polyhedrin polyadenyla signal and an ampicillin resistance (amp) gene and an origin of replication for selection and propagation in E. coli .

Methods for introducing heterologous DNA into a desired site in a baculovirus are known in the art. (See Summer and Smith, Texas A g r i -

c u 1 a r Experiment Station Bulletin number 1 5 5 5: ; J u et al. (1987); Smith et al. (1 9 83) Mo.l. & Cell B and 1. 3, p. 2156 to 2165; a. Lucl íow and Sum.m.e.rs (198.9). V i. R o. 1 o.gy .1.7, .._ , s tr .... 31) .. For example. mu.ze.

be incorporated into a gene such as the polyhedrin new gene by philological recombination; the incorporation may also be into a restriction enzyme site directed to the desired baculovirus gene. Incorporated sequences can be sequences that encode all or aberrant segments of a polyprotein or other orfs that encode viral polypeptides.

Signals for post-translational modifications, such as peptide cleavage, proteolytic cleavage, and phosphorylation, appear to be recognizable by insect cells. The signals required for secretion and nuclear accumulation also appear to be conserved among invertebrate and vertebrate cells. Examples of vertebrate cell signal sequences that are effective in invertebrate cells are known in the art, for example, the human interleukin-2 signal (IL-2), a signal for transport from the cell, is recognized and neatly cleared in insect cells. .

Structure of immunological tests

The HGV ant1 and E2 genes of the present invention can be used in generally any structure that uses known antigens to detect antibodies. However, structures that deform the HCV conformational epitope of interest need to be eliminated or modified. A common feature of all of these assays is that the antigen is contacted with a body component suspected of containing HCV antibodies under conditions that allow the antigen to bind to any such antibody present in the component. Such conditions are typically physiological temperature. pH and ionic strength using an excess of antigen. Incubation of the antigen with the sample is followed by detection of the immune complexes given by the antigen.

The planning of immunoassays is subject to considerable change, and many structures are known in the art. For example, protocols may include solid supports or immunoprecipitation. Most assays use labeled antibodies or polypeptides; the labeling can be, for example, by means of enzymatic molecules, which are fluorescent. che iii i luminescent, radioactive or color Assays that amplify signals from the immune complex are also known; its examples ...... la _: d.em. .j.sou. These assays use enzymes, labeled enzymes, and enzyme-labeled enzymes, mediated by immunoassays such as ELISAs.

Immunological assays can be, but are not limited to, heterogeneous or: H-G'-m ^: o ^: g-'Snn-í- · - sLŤ .'- r..irk-.t..n; rn-zaT -.-..-- m: ph: oji-_h: ý: t- :. sta-nda-rd-í-hQ - n-ej3_0 — koňku r-eč-n-íh.Qtvpu. In heterogeneous structures, the polypeptide is typically bound to a solid matrix or carrier to facilitate separation from the polypeptides after incubation. Examples of solid supports that can be used are nitrocellulose (e.g. in the form of membrane or microtiter wells), polyvinyl chloride (e.g. in leaves or microtiter wells) polystyrene-latex (e.g. in bead or microtiter plates, oolyvinylidinium fluoride - 2 known as Imm union®), di azotized paper, nylon membranes, activated beads and Protein A beads. In the case of heterogeneous structures, for example, Dynatech Immunlon ™ 1 or Immunlon ™ 2, microtiter, can be used. plates or polystyrene beads with a diameter of 6.35 mm (Precision Plastic 3all). The solid support containing the antigenic polypeptide is typically washed after separation from the test sample and before detection of bound antibodies. Both standard and competitive structures are known in the art.

For homogeneous structures, the test sample is incubated with a combination of antigens in solution. This may be, for example, under conditions that exclude any antigen-antibody complexes that form. Both standard and competitive structures of these assays are known in the art.

human) antibodies will bind by formation

For the standard structure, the amount of HCV antibodies in the antigen-antibody complex is directly recorded. This can be done by determining whether labeled anti-xenotic agents (e.g., those that recognize an epitope on HCV complex antibodies. In a competitive structure, the amount of HCV antibodies is judged by monitoring the competitive effect on binding a known amount of labeled antibody (or another competing ligand) in the complex.

The complexes formed containing anti-HCV antibodies (or in the case of competing assays, the amount of competing antibody) are determined by a number of known techniques, depending on the structure. For example, detection of unlabeled HCV antibodies in a complex can be accomplished by conjunction of antigenotic γ in a complex with a label (e.g., an enzyme label).

In an immunoprecipitation or agglutination assay, reactions between HCV antigens and antibodies form a network that precipitates from solution or suspension to form a visible coating or film of the preciptate. If no HCV antibody is present, no visible overflow is formed.

There are commonly three specific types of partial agglutination tests (ΡΛ). These assays are used to detect antibodies to various antigens, even when coated on a support.

haemagglutination tests

One type of such red blood cells (RBCs) that are sensitized by passive adsorption of antigen (or antibody) to the RBCs. The addition of specific antimony antibodies, optionally present in the body component, causes the RBCs to be coated with the purified antigen to agglutinate.

To rule out potential non-specific reactions in the haemagglutination test, two artificial carriers must be used instead of RBCs in PA. Of these, latex particles are the most common. However, gelatin particles can also be used. Assays using any of these carriers are based on passive agglutination of particles coated with purified antigens.

HCV E1 and E2 conformational epitope antigens are typically packaged into kits used in these immune assays. The kit normally contains native HCV antigen in separate packages, control antibody formulations cost the antibody if required (e.g., enzyme substrate), (positive and / or negative), know the structure, and signal reagents if the label does not generate a signal directly. The native HCV antigen can be bound to the solid matrix or separately with the matrix binding reagents. The kit usually includes instructions (e.g., written, tape, and CDROM) for performing the test. ♦

Immunological assays using native HCV antigens are useful for screening blood to prepare a pool from which potentially infectious HCV is excluded. The process for the preparation of a blood supply consists of a plurality of steps, for reacting a component, preferably blood or a blood component. from individual donor blood with native antigen. HCV Ε1 or native HCV E2 antigen to allow immunoglobulin-g-řc ^ k-é ^: “-p ^ -3k: c« = - · β. · γ · οt-i - 1-á-tk-am -i- -HC-V-. - -a, ._ a.ntá.genem_H CV_i ...

Determination of whether the reaction has formed complexes of anti-HCV antibody and HCV antigens results from the reaction. Blood added to the bloodstream. Λ- · k., »If. II _Λ -fc ', .ΛΙ *, (-. [· »,. -, T,. * Fc- ₊ i reindeer are from donors who do not show antibodies to native HCV E1 or E2 antigens.

In cases of positive reactivity to HCV antigens, it is advantageous to repeat the immunoassay to rule out the possibility of false positives. In addition, when screening blood on a large scale for the production of blood derivatives (e.g. blood by transfusion, plasma, factor

Box tests are typically structured to increase sensitivity (to ensure that no contaminating blood passes) at the expense of specificity; This is an increase in the rate of false positives. Thus, it is typically only to refrain from further testing of those donors who or more

They are intended to be repeatedly reactive, that is, positive in one of the immunoassays of the donated sample.

The following examples illustrate the invention without limiting it in any way.

Examples of embodiments of 11

Example 1

Construction of PSC59 Poles

The HCV sequence used for the construction was isolated from plasmid p C 5 P -1 as a Stul fragment partially / BglII. This fragment extends from the first methionine HCV-1 polyprotein to aspartic acid at position 966. The domains involved are the nucleocapsid, C, both putative glycoprotein envelopes, E1 and E2, and the distorted NS2 form. In addition, the fragment also contains about 60 bp corresponding to portions of the 5'-untranslated region of the HCV genome.

The fragment was treated with Klenow polymerase to form blind ends and then cloned into the Stul site of the vaccine vector, PSC59 (obtained from Dr. B. Moss of the National Institutes of Health, Bethesda, MD). The vector is shown in Figure 2, V due to ligation into the polylinker sequence of the vector, the C'-terminus of the NS2 region contains an additional Pro-Tyr sequence.

Example 2

Preparation of Vaccinia Virus stocks by encoding an HCV polyprotein fragment comprising E1 and E2

Screening for recombinant Vaccinia virus is performed essentially as described by Mackett et al. in DNA Cloning Volume II (published by D.M., Glover, IRL Press, Oxford, England, 1985, pp. 191-211). More specifically, the wild-type Vaccinia virus (WR-strain) is infected with a confluent monolayer (6 cm dish) of African green monkey kidney cells, BSC40, at a multiple infection (MOI) of 0.05. After incubation for two hours at 37 DEG C., the cells were transfected with 25 .mu.l of PSAC59 poles of DNA by the calcium phosphate method. After four hours of incubation, the medium is changed to normal and the cells are incubated for another 48 hours at 37 ° C. Cells are harvested by scraping from the dish and the virus is released for 3 freeze-thaw cycles and the released virus is stored in cell well at -80 ° C.

For screening. of the recombinant virus, the continental monolayer of human TK 143 cells is infected with cell lysate for two hours in 10-fold serial dilutions. After removing the inoculum, 1% agarose in serum medium containing 25 pg / ml 5-bromodeoxyutidine was added and the cells were incubated at 37 ° C for 72 hours. The plates are visible from the phone. by coating the cell layer with 1% aga dew plus 0.01% neutral red and incubating the cells overnight at 37 ° C. The Ase is then carefully removed and the cell layer is aspirated through a nitrocellulose matrix filter (Schleir and Schuell, BA85, 0.45 μ-m). A replica of the matrix filter was obtained and labeled with a ³² P HCV sequence hybridization probe. Positive plates were isolated from the matrix sequin, placed in 0/5 ml of serum-free medium, and plated twice for 30 seconds. The screening process is repeated twice to clear the virus.

......... K. 10 dishes (150 cm ² ) of BSC40 cells are infected with the stock virus at Μ0Ι 0.5 in Vaccinia _v iru. The infection is carried out for two hours at 37 DEG C. and the viral stock is replaced by the same. .-7-2 :. -h od-i-ná-ch · --se-. cells— sh-rom ážd.í -, - Su.spendu .j.í. 10 .mu.m of Tris / acid to hydrochloric acid, pH 9.0 and homogenized in a Wheaton fine tissue grinder. The cell debris was collected by centrifugation, the supernatants were trypsinized, and the quota of the virus suspension was stored at -80 ° C.

Example 3

Production of antigens E1 / E2 'One liter of Hela S3 (spinr.er) cells grown in a rotating flask (Spinner Flask) to a density ^of 10 cells per ml. Cells were infected with recombinant Vaccinia virus encoding the polys of the HCV protein fragment using MCI 1.0, incubated overnight, harvested, and stored in cell pellets at -80 ° C.

The E1 / E2 expression product is purified by lysogenizing the cells in hypotonic buffer, followed by extraction of the nonionic detergent contained in the buffer. The cell extract was subjected to lectin (ONA) agarose column chromatography. The desired proteins are eluted from the column with methyl-α-D-mannopyranoside (Sigma C o r p '.). The eluted fractions are monitored for E1 and E2 Western blots with specific antiserum to E1 or E2. Fractions containing antigens were pooled and concentrated on an S-Sepharose column (Pharmacia). The purity of the final product is approximately 70%;

Example 4

Use of EliE2 immune tests

Patients Serum samples. collected from randomly won paid blood donors. ' z.eastern, western, midwestern and southwestern parts. United States, are obtained from both the Lines and Globe Research Corporation (Resolutions, ICA). Serum samples provided by H. Tong (Huntington Memorial K ospita 1 in Pasadema, California), transfusion patients related to a group of 39 drug users were obtained from group 38 chronic ΝΛΝΒΗ and from another I V with chronic NA Ν Β H.

In these 77 patients, there was no other liver disease, anti-HAV and anti-Hbsag were non-reactive, and anti-nuclear antibodies (ANA) were normal. From. H ·. Alter (the National Institute of Health, Bethesda, Maryland) Ca of Serologicals Incorporation in Clarkston, Georgia are obtained. three seroconversion transfusion panels related to HC V. Samples of acute degradation HC V were provided by the Max von Petlenkofer-Institute, University of Munich, Germany. 19 cases of acute degradation NANBH were diagnosed · on the basis of a lavish examination and on the basis of long-term study (2 to 11 years) of normal amino transfer levels (ALT).

Expression and purification of recombination on HCV antigens

C22 antigens are expressed in S. cerevisiae yeast

I119aa), E1 (130aa), E2 (251aa), NS5 (942aa) and chimeric C25 (also called c200 / c22) (858aa), as C-terminal fusions with human superoxid dismutase (SOD) by the methods described first by G et al., Science (1989) 244, pp. 362-364 and Cousens L. et al., Gene (1987) 61, p. 26-272, The C33'C antigen (266aa) is expressed as an internal SOD-linked polypeptide in E. coli by methods described previously for 5-1-1 antigen synthesis (see PCT WO89 / O46699, EPO Pub. No. 318216 and Hougton Science 244, 359 (1989) After cell comminution and centrifugation, insoluble SOD coupled or 1% SDS is obtained from the cell pellet by extraction and purified with either polypeptides using 5 M urea by gel filtration or a combination of ion exchange chromatography. graphics (Q and S-sepha'rose) and gel filtration chromatography (Sephacryl).

HCV native antigen E1 and E2 [also called rVV (el &. E2)], the reticulum of infected re (rvv) cells, which contain the full length of Stt, is purified from the endoplastic combined Vaccinia virus of the HCV E1 and E2 genes. Purification is indicated by affinity chromatography, followed by exchange chromatography from undenaturing conditions. These methods are described in WO92 / O8734.

Na-tivni.-antigen-.H.CV E2.,. .CHO-e 2. In particular, according to Spaeta et al., Virology (199 1) 188, pp. 819-830. ν; · Η-ΰ · ' _ί -ι ^ζ ~ ο-Η-η · η-Η · ^ι . ^ι . ~ -ι-í-c -í: sek-V: e, n: C: i --- HLGj.lr - l -.- -ko dui-í-c-í-- · A La 3 -8-3. on,. .GXU 6.1.1 _ »_

The plasmid is then transfected into CHO cells to form a stable line e x transferring the full length e 2 antigen (also called e 2 / n s1). High-expressing clones are selected and expanded in cylinders in growth in DMR./H21 with 10% dialyzed fetal calf serum and the usual supplements plus 1.6 μΜ Methotrexate. The culture medium supernatant is collected and used to purify ECHO-e2 antigen. The purification scheme involves affinity and flash chromatography under non-denaturing conditions.

To disrupt native putative e2 conformational epitopes, denatured CH0-e2 was prepared by the addition of DL-dithiothreitol (DTT) to a final concentration of 10 mM, 0.2% sodium dodecyl sulfate (SDS) and boiled at 100 ° C for 5 minutes. All recombinant HCV antigens are considered to be ζ.ϊ at least 90% pure by SDS polyacrylamide gel analysis and Coomassie blue staining.

Tests .ELISA

RVV antigens (el & é?

OHO-e 2 and denatured CH0-e2 were diluted to the optimal concentration in phosphate buffered saline (PBS) (pH 7.4) and plated on Immunlon ™ 1 plates (Dynatech, Chantily, FA). ELISAs were performed as follows. : The assay samples on the plate are diluted 40-fold in the sample diluent and incubated for one hour at 37 ° C and then washed with wash buffer. Horseradish peroxidase (HRPO) conjugated goat anti-human IgG polyclonal antibody (H + L) is added to each well. The plates were incubated for one hour at 37 ° C and then washed with o-phenylenediamine 2 HCl (OPD) and hydrogen peroxide was added to induce color. The results are read using a plate reader at 492/630 nm. Final ELISA values O.D. proantigenic RVV (el & e2). SOD, 02 5, 022, 0330 and ns-3 are 0.4 plus O.D. from the center of negative values. The final values of C H 0-e 2 and d e n a t u r gv a n é h o OHO-e2 are 0.4 O.D. plus O.D. from the corresponding negative regulation of antigens. Final value. anti-0100-3 is 0.45 O.D. plus its center of negative control O.D.

Immunological tests

The results of ELISA tests using the indicated antigens lc detection of anft-HCV antibodies in blood components from a group of American patients are shown in Table T.

tč \ O na ί * ι θ 'u

<1>

and τ »

IMM

Ϊ.

ta a

H «· ts

OJ O n cm wi

Ol u

W * l «Λ

OF

UΓΊ <* l υ

you? oj o <* i oj

C 0 »oj - tí? n $ ttí? O Cn f * l - \ ΰ «λ ct σ '

ĚŘ

C o \ o o - es o σ \ o n - tí?

θ '- \ O NO ΓΊ θ' es

04 m oo es r- o ΓΊ '• ώ o o o · n - <

VI

3>

s- <

C a

'3

N

Tubu1ku i

OJ

4>

>

r ·, garden * 1 w

ί * ta \ í £

> L

CL fe *

O CN ΓΊ - * tí?

Ό «Ί n c>

eS o o \ o m -

Ol

4)

About £

And yes

4Í

4)>

OJ

U> y o

X

Hive

O Ol pí - ιη ΐ £ - θ 'θ' ΓΊ θ '00 ΓΊ - v>

ijl · ‘« ta □ □ 3 y <- * 3 y

• H

T íR «ΊΊ η θ '- <Λ ΓΊ (Λ

ΓΊ 00 ΓΊ ιη.-Íl — 6? Sep:

«Oo ΓΊ η Οι oo

Pi i

Hive

V u> 3 y

m z

tí σ> ο

ΡΊ - cn ρί - m es

Γ Tř - ττ σ 'ο η - θ'

ΡΊ ca τ 'tí

OF ?

> N σ »o

and c

£>

C

See £

<s

Ό

Λ .C

O

X i

<

In β

tí 71 D '3 o3. \ w

£ 2

Of those of

íl 'τ

Ul a

> N

3.

.3

N vs □

>

at

Jtl

M 3 y the y y T> C- the the '[AND C 7) 11 and and 0 y 5 and y £ ? y a "the y and y ►ta •the . Λ Hive the

Ul '3 o3 ™ 4

r.

y

the the and w Nta y with the the 3 -the y 3 Nta C? with L the 3 3 * ** y and and y '3 3 *the 3. ..N. _and ·· ta Ν ' ' ^: w' and J3 σ> 0

3 '. .y „

-a'3

N • JS-1 and -jj—; y i o! N?

^v y>

τ o y y iy nv

- w

Ul □

lil ·.

□

E-. ., B.

>

Φ

L '- jy ^>

= H ‘>

y n 2í τ

1 'p

> y i!

c y

P Z y o a ui ca

The results in Table I show that the yeast-produced envelope antigens HCV si a «2 are. significantly less immunoreactive than recombinant antigens produced from Vacci ni and R.W virus (e 1 & e2) and in CHO, CHO-e2 cells. ϋ Each of the test groups had only 2/3 or less of the activity of the yeast and α1 antigens produced by the yeast than the antigens produced by the animal cells. It is noteworthy that the yeast antigens e1 and e2 produced in yeast cells have been purified under denaturing conditions under these a n t i g e n e c h. R V ’/ {a 1 5 ··

It is therefore thought that even near epitopes. On the rosette _g 2) and CKO - s2 cheeks ·; vany podr, í nok,! r '· t, · »- n 1

S. £ combi on n t? .5 native antigen ',

El and Ξ 2 · in cells · appear to be the same as munoreacti in and perhaps C25 and immunoraacti external than any? 1 /. -j ·, L L U • r. ,, n o o r; 3 λ Q 1. /! HCV genes. C'25 is a chimeric polyprotein consisting of

MS ids connected according to FIG.

., ·, ...... i. ... ''. r

OZV '.

i. L ϋ Li Π t / '·

I «ΐ; ·. · Test 5 s r o k o n v r 3 η í c h HCV panels. The results in the table of activity parameters of both external activities are HCV, R. V V {e. and

CHOva three 3 e r o k o r. V a r s η í o h panels. Each sample in the panels represents a subsequent blood sample of an individual with transfused HAMSH. As can be seen from the results in Table TI, HCV natal antigens, i.e. pitopa antigens, detect antibodies. which · hu infection, tested • n comparison n :. irony HCV \ * · »Tí

·. pr 1 r cd η. ί n s; <c · o · 'r ·'? ϊ appears pomsr ^ S early 7

·. om i which c? I find full genes can be very exciting to be used in immunological tastes to diagnose HCV infection

- 23 Comparison of CHO-e2. rW <e * i or a; inu 1 tiant i <ienní ELISY on seroconversion panels ll ’)

SŠ8

O 2j © «2 8 ^c d <Λ n« <O §2 8.-SJ w α <on «□ π 3 ϋ Su»

N 50 PC IS O

UJ w íM «

'*' <O r- <2 o uJ w ® 4 o όϋ> 8 x 23 «> v’> «i

ST yours

Λ 'δ;

.1Φ; c <rt

1 ’©

and:

O

ÍH

4!

n 01 © Lf) 04 D O) 1— IT IT n © rf 01 Cl n F*) CD O O CM IT Γ- OJ tn it what IT © · - © TO 9 OJ ω (O CM © OJ IT F" H- © IT © CM <OI IT tn O* OJ OJ —1 © 00 o r> © 01 r- OJ what «Η © O CM IT O O O_ O n 7 · © ^ O O As ¢ 0 *with. , ¾ CM O O" 1-T pT what O O* O O cmT '© * O" O O O O O O O —1 D

P7 Cl CO CM - o O CM P) © o o o ©

PÍ © TO P) PÍ CM cm - ~ O-—.

«« # L o o o o o o cm - p> r> o r * ® rs to s oooooooooo - - «w ta - fc. ta ta, 'ta ta »ta

O O O O O 0-0 “O-O-Oσ» o

- CM p «© _ Ο ~ Ο« · ta ta ta »«

O OO O CM

- CM «-ή CM CO) O cm iň m · ** >> _?» > o -r pi <

ř- Ν # N (O

O O PÍ V © ta ta ta · · »

O © OO CM r- -, -. -in her. to '©, ί \ pJ tM_ <D _x O * O —T CM CM - 05 PÍ ta - cm. fc

0—1

p. to 01 CD _n CD CO CM Φ ° cm nv ©

- O CO li‘l Sú

CM ÍM - - - ©

9 * ta * * s C * ta

O O O O O - oo - oo co © to o - o cm oo h * * A *

O O O O O V

TO CO TO © QOtf o ^ 9; ° t. oooo ^Ύ

CM - pi tm - Vl ta

Λ «A

OOO —l fj-íncosaiOrs® '* 00000 0 - 000 o ¹ o ** o “o'o“ o cTo “o“ o “σίττσιο® - oi - cm r> MT TO

OOO - O - OCM »ta. ta ta. ta »ta 2 *. X oooooooooo

CM

PÍ w

©

CM CM τ, Ά

CM CO

PÍ ©. © ._ (* · CM © O PII eo CM - O O Ο '-' Ό ^ · ρ) Ρν-Ν O O O O O O O — f - cm © CM © CM CM cm π co v ©

P.COCM © TOPta - CMe-l ° o o o © o - © -j * »* · * * \ **» * · »·% 4

OOOOOOOO _and -f

TOTOTO ^ TTOVO ^ Ol o o o ---- - © H oooooooo — O ta

CM _CM © TO © «tr« - ® ©

Φ b »! C

WHAT 00 IT IT O tw IT IT the IT IT IT © © © © © © © Φ <0 ω QD oo IT CD IT IT IT LIMB · *. IT IT IT N Ρ » —W Pfc fs P> P; pfc Γ * 0 · r * WHAT tn © O IN IT © <n H · IT IT Φ ΊΓ © O CM IT Ρ » IT Π O Γ * IT C CM CM OJ O O CM C O fc * CM O O r- O OJ OJ P> what IT IT 01 w Λ, Pfc IT 00 O) IT IT (D b. fcfc p. p · IT •"the. what «. IT IT cn σ> Ο) O O O O O. m ai O O O O O O -C .O O O O O O O O Ό ο

v ií

O u

- © p. TO TO JI <<4 <<ř / 1

N - (O

U U

TO Ol U U

O

CJ o

«W

O>

c o

o o

Φ co

- 24 <o * £ £ 2 S ^C m »» - OO - rs - r> eo O r * * - <ro co oo coi co O

O O O ifiTrn - «r» v © CNfNfNin - o * c? ůo * of

Table II <continued>

<O

Š £ o

-tíw u <o © w u © 3 = ť o UJ W

N <O n 52 o ^{υ ji} w

No

«3 <O

S ° tí ω>

>

φ <Q ó 52 α i úí «u> □ .W * · ► w,. »'* \ I- | íC> I * r *

1 ’and

in'

N>

C3cd O co oo w o - o - · ~ »s“ * o o o o * r · »tn ©

O 'O * o tn © © © © Ο, - co ro * «* * a

O O O eo © tn © © O rt 'ř ©

-. r. O O O Η ©

° N tn * es © © © X - v rc s

ta c

<o

Ui

C

N> 1 '_c c

ii> u ta ta n

\

C

VI

VI

Ui

Ta ta ta c

v u

1 '' Ji

what © G0 what what © 00 what **. CD © O what ** CN n <N -IN. r- Φ what © O O O O

©

U J 24 VI ů!

>

c r i \

rn £ D

O - CN © o tn © m ·: Table 5 shows a comparison of native antigens from the E1 and E2 genes with comparable denatures and acute genes! i š s n o u W A ří Β H a j a p o; .,, 4 i ...

-ř: j 7

HCV is derived from scanning antigens. The table was monitored depending on n o r e a k ί t-3

r o v η á - a s and he doesn't r a a k ti v 1 to u p-3 bulky 3 you p 1 lives , show antigens, which contain how P..VV (el & e2) ξ CHO-e than ant t o p y e 1 a e2 ( produced in

balls

The overall view is that the immunoractivation of H C V s a z v4 v i e 8 - * to 9 - · k r a t u jen a; i 2 times in patients 5 chrc s s concerns k o n f o r m a é η í c h e p i t o pů, z a tr

uniformly distributed rne z i konf T; / i o studios represented in u her first tops:? can c ýk ·? t immunological 'iů H · ’-O

i t a No those outer packaging o vých a n t i - furnace i a n t ů 'g a k u t .η í r o z. 1 i 5 e n o u a ; ρ τ. 'J He knows MA ^ SH. Apparently -j 3 t s d y that o n t 0!.! WITH r o z 1 i 5 e n o u f -j r m o u WANBH rne o at acute forms is at closer r ma ch í and linear ep itopy

knowledge, conformational spi 'on HCV α ho-s ťi tel skáhb .Tjbul .-:.: IU ......., ...... ....... ...... ...... ........... _v ... .

The predominance of anti-HCV antigens by bs and its conformational ones. 'epitopes versus antigens containing only linear epitopes in differentiated and chroPcst (¾) anti-HCV positive after transfusion D i and gnz z z Ak ut ni ro z' i šsné Η Λ ΜβH C h r o n ické Ν A Ν Β H p o b j n z.

transfusion. C-5 months 6-17 months> 12 months

An.tige? .Y HCV

C2 5 : 9 / ’9 * 19/19 * 13/19 ’ o / o · i i oct; (100¾) (95¾) (100¾) r VV ('$. ·.? 2) 16/19 ’ 17/13 ’ 16/19 ’ g / g * (34¾) (33¾) (3 4¾) (100¾) CHO-e 2 ! 1/19 ’ 12/19 ’ 13/19 ’ 9/9 ’ (53¾) (53¾) (53¾) (100¾)

? 2

T /; 9 * 3/1 9 * 4/13 * / Q / σ ψ a ·, ' ^{, n} j (1 5¾) f n 1 a * v \ / (c ς = v \ - »« _ · 1/19 * 1/19 * 4/19 * 4/9 (5 ?,) (5¾) (21¾) (4 4 2. 2/19 * 4/19 * 4/19 * 4/9 (*} (21¾) (21¾) (44¾

* Number of positive ch / conceived tested ** Diagnosa 'acute differentiated NANBH (predominant majority small-scale cresistsnc ·.? ^L. Ra no amin í ;.

on the basis of biopsy .insured) and the difference e- r. i. tč; muno competent (Á ~. J? ** Ή 1 (2 l *. η - ² “* i;

k t? ' i i s o u i i ~ a k τ m u n o log; c k ý t ·? 5 t El / E 2: Pero v n a η i p a c i 3 i mvr.o s op r3;, r; í c h

O Γ) T

1ÍT ’: -v - - t i 1 i - ky ιΎ - -zi-u HIV. CH:; ·;! · “. Believes 2C by chronic infection H C '' í.íok or.o u fr an 3 f u z. Rávi. All p o c; n t i yr positive n ant i-HCV and HCV PNA. Ska.pt nu II consists of 15 patients per h.? mo d: a · ~ J3 ΐ Eleven of them are anti-HCV positive on intake, one .me during trans? 'antation of the kidney. Five patients have become anti-HC III form i? recipients 1? dv: novyc is HCV and HCV R NA positive n.

income. Seven s? became anti-HCV positive in the course of which dv. was; first themselves HCV HA positive ;. The two pne ion i were HCV R.NA s;

ti v.-, i na η ř ('j mu, o ba from Ct-3 ta 1 i HCV S NA po z. t 1 v η 1, a' positive during. Group of transp1 ahtátů. .fi -HCV n 2 ga t: va i during HCV RMA emergency in orbit.

Sera 'deliberately collected during April and July 1C5C 2 .3 ng would ulež ENAV 1 .3 ^{1 The} quotas ec h charger -70 C ^J - R 2 C 3 F 7 CTO -r · m. · 0 to four samples 5hro máždéns at intervals of 5 to 12 months; tested prc- patients of group I. Two samples collected in the interval of 1.5 to 2 years were tested for all patients of groups II and III; additional samples in the intervention period and a continuous sample; were also tested for patients who changed from anti-HCV and / or HCV RNA in the second sample.

í k ΐ 3du 4 In tests N 2 S a on ligament C Pl í m va cc i n t ζ · '7? t u ř- thinner • τ 7 · y. ^of i i) r \ ÍJ í itím vacci r. í onh r 0 1 Íι í C S _r v z 0 r k ů. giant 3 A d r 3C. Only £ c r # ago and 1 0 na a nt i ge * »17 ^l *? 'r- ·. J rp i c h n and “S ·.? i 2 n · '1 r and C; c 5 :'IN and £ ·. {' 0? ·; ·: · Λι né v -

’’ FS /.·* 'Γ <i kýslsdky are shown on

ELI SA tests

HCV antigens were prepared as in Example 4 and using t? S · £ <e? with; protein;

'i) additive in 3 coini 3 bv v e c e éh i • st IQ ij 5 i! ⁴ F · 'ki * -' -ί Γ '- ^L - ί Γι f č Π t! 'ÍJ 2 Π η kontrol 1 η í chvzork ů.

5K i mu no kc * τι ρ ^Ώ t ^Q nt η í r t- r - ipntr sknoír ^ raa π n *. ra 1 π π sn ť í π pvo 1 γε sb í e 2 5 x ρ za oova η í in yeast; r * · <; / r _: '': / -> -t: I 3 .Ί * r: Λ * '<7 ./ iru. Jo ko ui mzno k 5npe te nt η ích patient, by ': v č t ό: λ'brmzd; C

-l-yzn í-ch- • .. • pac-i-eďit-ů. skuo-i n, y. ..IX. .. r.sa.kti y.ri.Lch .. ... na ..... an tigz; y L.2 5., _ C.2 .. 'a N e.> S <s2. A significantly lower proportion of patients responded to -s.ClOO-3. C332C,. NS-5,. el and e2. Compared with immunocompetent patient. ¹

- ('sa-gO-v-sR-G - ^ - ý-z ^ rAaynpjě-md-n-š ^ p ^ ízrjzsTm ^ rT; -a: nS: piáíii-72di = S = rŽ2kí! JŽJť ny III against all tested HCV antigens.

Lice c hn ii muno k cmps tent η í pa cisntirea oc v3 1 i na '225, 2 2 2 C33C; 90% responded to HS-5 'and 30 / to V100-3. Only 5% reacted with yeast-derived s1 & e2 antigens, but all responded to vaocinia expressed by N1 & e2 antigens, indicating that they are formal epitopes. configuration and glycosis 1. 55 n .. to 90 dia 1 ya o ^J ; iny r 3 agtf; 7 .τ 0.!

% to 5 0 r ea g c - 1 va 1 o at 725, C? 2 and H?! O? 2, rd.?:·· emergency on 071 100-3, 0370 and>! 3 -5. Cmonšcní or loss r-ja k t i ·· i t y na ar. t i g s n hepatitis 0 virus b y 1 and observed after transplantation 1 2 d in 1 y. or bone marrow, with C25 and M sl S e2 being less affected.

j s o u k o- n f o r ma č η í a r e a k 11 vita vč i even with these 3 p i t o pům is at least o v1 i vn and to suppress i munty compared to other HCV antigens.

P „C.S TV A .. 1..9..7.Á ........ '<y i ί 1 t ..? ...! ... 9., θ 9 í,

Immunoassay methods using HCV envelope antigens that contain conformational epitopes reactive with antibodies in the serum of infected individuals are suitable for screening and diagnosis. These antigens detect antibodies that are not detected by HCV antigen antigens. In addition, these envelope antigens

HCV, formed by k '• 3 Z t I 7 li e J f v (, -4 / ty Zfy-fí

Claims (1)

PATENTS N A R. O K V Method for the detection of hepatitis C virus (KCV) antibodies in the body component of said cells, which is suspected to contain these antibodies, with 5 s of the body component containing HCV antigen containing a confirmatory epitope from the E1 or E2 domains of HCV under conditions allowing an immunological reaction between the possible antibodies and the antigen, and the presence of a possible immunocomponent of these antibodies and these antigens is determined. 2ρ as ob according to claim 1, wherein the HCV envelope antigen is encoded by me; o enom u H CV. The method of claim 1, wherein the HCV envelope antigen is E1. m, Ί. · Method according to claim, c h a _((r a _c ή casing above the <Niiza - Ho is -inv Domain E 2 The method of claim 1, wherein the HCV and E2 envelope antigen. gsnomu HCV 6. The method of claim 1, further comprising the second HCV envelope antigens t f 0 rma č η í m a ρ i t -o p <? m. n é hc i m, The method according to claim 1, three. HCV native packaging antigens are expressed from recombinant - ů.c ó p c d 1 «n á r o k u 5, v y z ·: \ a u E2. is expressed in CHO cells. 9. A method of screening blood components for HCV prior to use / é k r v n η θ b c k 'e v η í ingredients for sour cream k · o v ηί c h der i you t 0 '-i, t; v - know C C j í o s s t í m, ž s a) s e Π • 3 c b »2 Γ *“ ;; ov3 t body component from potential ηí ho gift p P 3 “| * 1 _ J i 1 t i g s n 9 m HCV t vc ř e n é m conformational spi - S H am domain El n s bo Ξ2 HCV 2 3 pO dm i 3ί 3 k, ' - - Ή, · Α vo 1 ii j ť immunologi c k 3 T s. ·? k c; m 3 of ί P Ϊ́ ΐ n Q rj n o u p r c t i 1 í t * L> r · ·, 4 ~ X} 5. ί. · I u 3. 3 A Ó 5 ! o ž k? / a? n t: g e n e if! , b) se perform d 31 e k c a complexes, vyt vc between ant í g sne ΙΪΙ ¢ 3 · p ř í ~ strong and against 1 á t k a m i and c) training 1 o her in = š k e r á blood or blood in s beds from the donor t when z j and 3 t ě η í t. é kovýc h complexes in step in b 1 0 _ _{Λ r} . -3 73 R detection against 1á flow Opu _r . - '- r S ·. . · .. · r _r . in:: 1 Λ · Q v y z F. 3 Č Q : c í s s t í m, of • e o b s 3 h'1; ; and n t i qe - [J 6 V formed 7 k c n f o m ·? ň n í m so i t o o em from domains C i nabc £ 2 HCV, , 2 ko π t r o 1 - n i n t a n d s r d y, c d -dii ď in suitable amp ! ká C b 2 i n S t * i k C 9 pr £ D C 'í ž i -