MXPA00009956A - Improved immunodiagnostic assays using reducing agents - Google Patents

Abstract

The present invention relates to a solid phase immunoassay comprising on said solid phase an antigen in the presence of a reducing agent. The present invention also relates to a method for purifying a cysteine containing recombinantly expressed protein comprising at least 2, preferably 3 or 4 and even more preferably all of the following steps:(a) sulphonation of a lysate from recombinant host cells or lysis of recombinant host cells in the presence of guanidinium chloride followed by a subsequent sulphonation of the cell lysate, (b) treatment with a zwitterionic detergent, preferably after removal of the cell debris, (c) purification of the sulphonated version of the recombinant protein or purification of the sulphonated version of the recombinant protein with subsequent removal of the zwitterionic detergent, with said purification being preferably chromatography, more preferably a Ni-IMAC chromatography with said recombinant protein being a His-tagged recombinant protein, (d) desulphonation of the sulphonated version of the recombinant protein, preferably with a molar excess of DTT, (e) storage in the presence of a molar excess of DTT. The present invention also relates to novel HCV NS3 sequences as depicted in Figures 1-8.

Description

IMPROVED INMUNOD IAGNÓS T I CO TESTS THAT USE REDUCING AGENTS FIELD OF THE INVENTION The present invention relates to the diagnosis and treatment of HCV infection. More particularly, the present invention relates to NS3 helicase of HCV and its uses. The present invention also relates to improved immunodiagnostic tests.

BACKGROUND OF THE INVENTION Hepatitis C viruses (HCV) are a genus within the Flavi viridae, with homologies closer to the hepatitis G and GB viruses, and Pestivirus.

The positive-strand RNA genome encodes at least 9 proteins. The nucleus, El, and E2 constitute the structural proteins. NS2, NS3, NS4A, NS4B, NS5A and NS5B are non-structural proteins (NS). The isolates of HCV show high levels of sequence heterogeneity, allowing classification in at least 11 types and 90 subtypes (Maertens and Stuyver, 1997). Infection of the human liver with HCV is often clinically benign, with benign icterus in the Ref. 123787 acute phase. The disease still could not be noticed in some cases of acute hepatitis C determination. However, in the majority of cases (> 70%), HCV infection leads to chronic or persistent infection, often with complications of liver cirrhosis and autoimmune diseases. Hepatocellular carcinoma could occur after approximately 20 to 35 years (Saito et al., 1990), sometimes even without the intermediate phase of cirrhosis. Prophylaxis is not currently available and treatment with interferon-alpha (IFN-a) leads only to long-term resolution in approximately 4 to 36% of treated cases, depending on the HCV genotype (Maertens and Stuyver, 1997).

Since HCV-producing culture methods are not commonly available, and since only small amounts of HCV antigens circulate in the infected patient, the direct determination of HCV particles can not be performed routinely, and only indirect diagnosis is possible. using complicated amplification techniques for the detection of HCV RNA. Unlike other viral infections, in general, HCV particles persist in the blood, liver and lymphocytes despite the cellular and humoral immune response to most HCV proteins. Antibodies to HCV can be conveniently detected by Elisa techniques, which allow high screening rates in blood banks and clinical laboratories. Supplemental antibody testing is required, and is now mandatory in most countries. The reactivity of the true HCV is thus discriminated from the false reactivity, which could be caused by non-specific links of serum or plasma immunoglobulins or anti-i-typosite components with the coating or blocking reagents, or by contaminants present in the preparations. of HCV antigen, or even by fusion of non-specific parts or regions of the same recombinant antigens (McFarlane et al., 1990). The detection of HCV RNA by PCR or segmented DNA techniques (bDNA) has recently been introduced to monitor chronic HCV disease, especially during therapy. Surprisingly, the detection of HCV RNA is sometimes used to confirm screening tests for HCV Ab, despite the fact that only -70-94% of samples from patients positive for HCV Ab are repeatedly positive by serial PCR (Marin et al., 1994). Of the positive HCV Ab donors, which usually present with more moderate forms of the disease and low HCV RNA levels, confirmation by serial PCR is usually of the order of -40% (aumans et al., 1993; Stuyver et al., 1996). Band-based tests therefore provide the only reliable alternative to HCV Ab confirmation. Even in the case of an indeterminate result in the confirmatory test, the serological one increases the effect per patient's action more than the detection of HCV RNA is indicated (Di Bisceglie et al., 1998). Since native HCV antigens are not available in sufficient amounts, such confirmatory tests incorporate synthetic peptides and / or recombinant HCV protein fragments. One of the most critical problems in the confirmation of antibodies constitutes the reactivity of the NS3 protein (Zaijer et al., 1994). The NS3 antibodies often appear first in the seroconversions series and the reactivity of the NS3 protein appears to be different in the different commercial tests currently available.

The ingenetics introduced the concept of band technology, in which a combination of synthetic peptides and recombinant proteins are applied as discrete lines in an orderly and easy reading form. The INNO-LIA HIV Ab tests have proven to be superior to the western blots routinely used (Pollet et al., 1990). The Online Immuno Test allows multiparame testing and thus allows the incorporation of cuts and other classification systems, as well as testing for false reactivity to non-HCV proteins used as carriers or fusion pattern required by some antigens in the Elisa test. In principle, the test format allows combining antigens from different etiological agents or phenotically linked conditions in a simple test.

The INNO-LIA of HCV Ab III is a 3rd generation Immuno Online Test, which incorporates antigens from the HCV derived from the Central region, the hypervacurable E2 region (RHV), the NS3 helicase region, and the NS4A, NS4B and NS5A regions. In the third generation test, the highly purified recombinant NS3 subtype Ib and the E2 peptides allowed for superior sensitivity, while safeguarding the reliable specificity that is characteristic of peptide-based tests.

(Peeters et al., 1993). Perhaps one of the most important characteristics of this test is its unprecedented correlation with being positive to HCV RNA (Claeys et al., 1992, De Beenhouwer et al., 1992).

The antigens are covered as 6 discrete lines in a nylon band with plastic backing. In addition, four control lines are covered in each band: anti-streptavidin, positive control 3+ (ant i-human, Ig), positive control 1+ (human IgG), and line ± cut (human IgG). A diluted test sample is incubated in a tub together with the LIA III band. If present in the sample, the HCV antibodies will bind to the HCV antigen lines in the band. Subsequently, an anti-human goat conjugate IgG (H + L) labeled with affinity-purified alkaline phosphatase is added and reacted with specific HCV antigen / antigen antibodies if they are previously formed. The incubation with the substrate of the enzyme produces a brown color, the intensity of which is proportional to the amount of HCV-specific antibody captured from the sample in any given line. The development of color stops with sulfuric acid. If HCV-specific antibodies are not present, the conjugate binds only to the control lines ±, 1 +, and 3+. If the addition of the sample is omitted, only the control lines ± and 1+ will be stained.

DEFINITIONS The following definitions serve to illustrate the different terms and expressions used in the present invention.

The term "HCV NS3 protein" refers to a polypeptide or an analog thereof (eg mimotopes) comprising an amino acid sequence (and / or amino acid analogs) that define at least one epitope of either the NS3 helicase or protease of the HCV.

The term 'hepatitis C virus envelope protein refers to a polypeptide or analog thereof (eg mimotopes) comprising an amino acid sequence (and / or amino acid analogs) that defines at least one HCV epitope already be the region El or E2 (see WO 96/04385 of which the content is incorporated herein by reference).

It should also be understood that the isolates (biological samples) used in the example section of the present invention are not intended to limit the scope of the invention, and that any HCV isolate belonging to type 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10, 11, 12 or any other new genotype of HCV is an adequate source of the. HCV sequence for the practice of the present invention.

The HCV antigens used in the present invention could be full-length viral proteins, especially full-length versions thereof, or functional fragments thereof (eg, fragments that do not lack the essential sequence for the formation or retention of a epitope). further, the HCV antigens of the present invention could also include other sequences that do not block or prevent the formation of some conformational epitope of interest. The presence or absence of a conformational epitope can be easily determined even when the antigen of interest is screened with an antibody (polyclonal or monoclonal serum antibody) and its reactivity is compared to that of a denatured version of the antigen that retains only linear epitopes ( if there are any) . When using such screened polyclonal antibodies, it may be advantageous to first adsorb the polyclonal serum with the denatured antigen and see if it retains antibodies to the antigen of interest.

The term 'fusion polypeptide' is intended for a polypeptide in which the antigen (s), in particular HCV antigen (s), are part of a single continuous chain of amino acids, this chain does not occur naturally. The HCV antigens could be connected directly to one another via peptide bonds, or separated by amino acid spacer sequences. The fusion polypeptides could also contain amino acid sequences exogenous to HCV.

The term 'solid phase' or 'solid support' indicates a solid body to which the HCV antigens or the fusion polypeptide, containing HCV antigens, are linked covalently or by non-covalent means such as by hydrophobic adsorption. Examples of solid phases are microtiter plates, membrane strips such as nylon strips or. nor t rocellulose, and silicon particles.

The term "biological sample" is intended for a fluid or tissue of a mammalian individual (e.g., an anthropoid, a human) that commonly contains antibodies produced by the individual, more particularly antibodies against HCV. The fluid or tissue may also contain HCV antigen. Such components are known in the art and include, without limitation, blood, plasma, serum, urine, spinal fluid, lymphatic fluid, secretions from the respiratory, intestinal or genitourinary tracts, tears, saliva, milk, lymphocyte cells and myelomas. The bodily components include biological fluids. The term 'biological fluid' refers to a fluid obtained from an organism. Some biological fluids are used as a source of other products, such as coagulation factors (e.g. Factor VIII), serum albumin, growth hormone and the like. In such cases, it is important that the source of biological fluid be free of virus contamination such as HCV.

The term "immunological reagent" indicates that the antigen in question will react specifically with anti-HCV antibodies present in a body component of an individual infected with HCV or an immunized individual.

The term "immune complex" is intended for the combination formed when an antibody binds to an epitope on an antigen.

The terms El and E2 as used herein are fully described in WO 96/04385, of which the content is incorporated by reference in the present description.

The term 'purified' as applied herein to proteins refers to a composition wherein the desired protein contains at least 35% of the total protein component in the composition. The desired protein preferably contains at least 40%, more preferably at least about 50%, more preferably at least about 60%, even more preferably at least about 70%, even more preferably at least about 80%, even more preferably at least about 85%, even more preferably at least about 90%, and more preferably at least about 95% of the total protein component. The composition could contain other compounds such as carbohydrates, salts, lipids, solvents, and the like, without affecting the percent purity determination as used herein. An 'isolated' HCV protein is intended for an HCV protein composition that is at least 35% pure.

The term 'essentially purified proteins' refers to proteins purified in such a way that they can be used for in vitro diagnostic methods and as a therapeutic compound. These proteins are substantially free of cellular proteins or DNA, proteins derived from vector or DNA, or other viral components of HCV. These proteins are usually purified at homogeneity (at least 80% pure, preferably 85%, more preferably 90%, more preferably 95%, more preferably 97%, more preferably 98%, more preferably 99%, even more preferably 99.5%, and more preferably the contaminating proteins should be indet ectable by conventional methods such as SDS-PAGE and silver staining).

The term 'recombinantly expressed' used within the context of the present invention relates to the fact that the proteins of the present invention that are produced by means of recombinant expression methods are in prokaryotes, or lower or higher eukaryotes as discussed with detail later.

The term 'lower eukaryote' refers to host cells such as yeast, fungi and the like. Lower eukaryotes are generally (but not necessarily) unicellular. The lower eukaryotes are preferred are yeasts, particularly species within Saccharomyces, Klyveromyces, Pichia (e.g. Pichi pastoris), Hansenula (e.g. Hansenula polymorpha), Yarowia, Shwanniomyces, Zygos to echa romyees and the like. Saccharomyces cerevisiae, S. car Isbergens is and K. lactis are the most commonly used yeast hosts.

The term 'prokaryotes' refers to hosts such as E. coli, Lactobacillus, Lactococcus, Salmonella, Streptococcus, Bacillus subti lis or Streptomyces.

These guests are also contemplated within the present invention.

The term 'higher eukaryote' refers to host cells derived from higher animals, such as mammals, reptiles, insects, and the like. Currently the upper eukaryote host cells are derived from Chinese hamster (eg CHO), monkey (eg COS and Vero cells), baby hamster kidney (BHK), swine kidney (PK15), rabbit kidney 13 cells (RK13) , the human bone osteosarcoma 143 B cell line, the HeLa human cell line and the Hep G2 human hepatoma cell lines, and insect cell lines (eg spodoptera frugiperda). Cell lines could be provided in suspension or flask cultures, tissue cultures, organ cultures and the like. Alternatively, the host cells could also be from transgenic animals.

The term "polypeptide" refers to a polymer of amino acids and does not refer to a specific length of the product, thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. ao excludes modifications post-expres ion of the polypeptide, eg, g 1 icosi 1 ations, acetylations, phosphorylations and the like Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including , for example, non-natural amino acids, PNS, etc.), polypeptides with substituted bonds, as well as other modifications known in the art, both those that occur naturally and those that occur unnaturally.

The term 'recombinant polynucleotide or nucleic acid' is intended for a polynucleotide or nucleic acid of genomic origin, .DNA, semisintensible or synthetic, which by virtue of its origin or manipulation: (1) is not associated with all or a portion of a polynucleotide with which it associates in nature, (2) binds to a polynucleotide different from that which binds in nature, or (3) does not occur in nature.

The term 'recombinant host cells', 'host cells', 'cells', 'cell lines', 'cell cultures', and other terms denoting microorganisms or higher eukaryotic cell lines cultured as unicellular entities, refers to cells that can used or used as a receptor for a recombinant vector or other transfer polynucleotide, and include the progeny of the original cell that has been harvested. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental or deliberate mutation.

The term 'replicon' is any genetic element, e.g., a plasmid, a chromosome, a virus, a cosmid, etc., which behaves as an autonomous unit of polynucleotide replication within a cell; i.e., capable of replication under its own control The term 'vector' is a replicon further comprising sequences that provide replication and / or expression of a desired open reading frame.

The term "control sequence" refers to polynucleotide sequences that are necessary to effect the expression of coding sequences to which they are linked. The nature of such control sequences differs depending on the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and terminators; in eukaryotes, in general, such control sequences include promoters, and could include enhancers. The term 'control sequences' is intended to include, at a minimum, all components whose presence is necessary for expression, and could also include additional components whose presence is advantageous, for example, leader sequences that govern secretion.

The term 'promoter' is a nucleotide sequence that is composed of consensus sequences that allow the binding of the RNA polymerase to the DNA template, in such a way that the production of mRNA is initiated at the normal transcription initiation site for the adjacent structural gene.

The expression 'operably linked' refers to a juxtaposition where the components so described are in a relationship that allows them to function in their planned form. A control sequence 'operably linked' to a coding sequence is ligated in such a way that expression of the coding sequence is obtained under conditions compatible with the control sequences.

An "open reading frame" (ORF) is a region of a polynucleotide sequence that encodes a polypeptide and does not contain stop codons in the selected reading frame; this region could represent a portion of a coding sequence or a total coding sequence.

A 'coding sequence' is a polynucleotide sequence that is transcribed into mRNA and / or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The limits of the coding sequence are determined by a start codon of the translation in the 5'- term and a stop codon of the translation in the 3'- terminus. A coding sequence can include but is not limited to mRNA, viral RNA, DNA (including cDNA), and recombinant polynucleotide sequences.

As used herein, "epitope" or "antigenic determinant" denotes an amino acid sequence that is immunoreactive. In general, an epitope consists of at least 3 to 4 amino acids, and more usually, consists of at least 5 to 6 amino acids, sometimes the epitope consists of about 7 to 8, or even about 10 amino acids. As used herein, an epitope of a designated polypeptide denotes epitopes with the same amino acid sequence as the epitope on the designated polypeptide, and immunological equivalents thereof. Such equivalents include specific variants of strain, subtype (= genotype), or type (group), e.g. of the commonly known sequences or strains belonging to the genotypes la, Ib, le, Id, le, lf, 2a, 2b, 2c, 2d, 2e, 2f, 2g, 2h, 2i, 3a, 3b, 3c, 3d, 3e, 3f, 3g ', 4a, 4b, 4c, 4d, 4e, 4f, 4g, 4h, 4j, 4k, 41, 5a, 5b, 6a, 6b, 6c, 7a, 7b, 7c, 8a, 8b, 9a , 9b, 10a, lia, 12a or any other (sub) type of recently defined HCV. It will be understood that the amino acids that constitute the epitope do not need to be part of a linear sequence, but could be scattered by one or more series of any number of amino acids, thus forming a conformational epitope.

The term 'immunogenic' refers to the ability of a substance to cause a humoral and / or cellular response, either alone or when binding to a carrier, in the presence or absence of an adjuvant. 'Neutralization' refers to an immune response that blocks the infectivity, either partially or totally, of an infectious agent. A 'vaccine' is an immunogenic composition capable of attracting protection against HCV, either partial or complete. A vaccine could also be useful for the treatment of an individual, in this case it is called a therapeutic vaccine.

The term 'therapeutic' refers to a composition capable of treating HCV infection.

The term "effective amount" refers to an amount of polypeptide, which carries epitope, sufficient to induce an immunogenic response in the individual to whom it is administered, or on the contrary immunoreaction detectable in its intended system (e.g., immuno-test). Preferably, the effective amount is sufficient to effect the treatment, as defined above. The exact amount needed will vary according to the application.

The term 'antibody' refers to polyclonal or monoclonal antibodies. The term 'monoclonal antibody' refers to an antibody composition having a homogeneous antibody population. The term is not limited with respect to the species or source of the antibody, it is not intended to be limited by the way it is done. It should also be noted that humanized antibodies, single chain antibodies or any other fragment thereof that has retained much of the specificity of the antibody are covered by the present invention.

As used herein, the term "humanized antibody" means that at least a portion of the regions of the structure of an immunoglobulin are derived from human immunoglobulin sequences.

As used herein, the term 'single chain antibody' refers to antibodies prepared by determining binding domains (both heavy and light chain) of a binding antibody, and providing a binding portion that allows the preservation of the function of link.

As used herein, the term 'fragments (of antibodies)' refers to Fab, F (ab) 2, Fv, and other fragments that retain the binding function and antigen specificity of the parent antibody.

OBJECTIVES OF THE INVENTION It is an object of the present invention to provide components for the improved diagnostic test for HCV and therapeutic proteins.

More particularly it is an object of the present invention to provide improved preparations of HCV NS3 protein for use in the diagnosis of HCV antibody and / or HCV treatment.

It is a further object of the present invention to provide a method for increasing the reactivity of HCV antibodies with the recombinant or synthetic NS3 helicase protein or part thereof present in a solid phase It is also an object of the present invention to provide a novel method for purifying recombinant proteins containing cysteine, more particularly recombinant HCV proteins.

It is also an object of the present invention to provide novel sequences encoding the HCV NS3 protein.

It is also an object of the present invention to provide novel sequences encoding HCV NS3 protein, of which the product does not react with falsely positive HCV samples.

It is also an object of the present invention to provide a method for detecting the nucleic acids of the invention.

It is also an object of the present invention to provide probes and primers for the detection of the nucleic acids of the invention.

It is also an object of the present invention to provide a diagnostic kit for the detection of the nucleic acids of the invention.

It is another object of the present invention to provide novel NS3 HCV polypeptides.

It is another object of the present invention to provide novel HCV NS3 polypeptides that do not react with falsely positive HCV samples.

It is another object of the present invention to provide a pharmaceutical composition for preventing or treating HCV infection.

It is another object of the present invention to provide a method for the detection of the polypeptides of the invention.

It is another object of the present invention to provide antibodies to the polypeptides of the present invention, for use in passive immunization and / or therapy.

It is another object of the present invention to provide a method for the production of the polypeptides of the invention.

All the objectives of the present invention are considered to have been met by the modalities as set forth below.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates more particularly to a solid-phase immuno-test which comprises an antigen in the solid phase in the presence of a reducing agent. As demonstrated in the Examples section, the present inventors have found that the presence of a reducing agent such as DTT, in addition to an antigen coating a solid phase, makes an antigen coupled to the solid phase immunoprote more reactive with the antibodies directed to the antigen. Also in solution, the antigen becomes more reactive by reduction.

A reducing agent according to the present invention is any agent that achieves the reduction of disulfide S-S bridges. The reduction of the disulfide bridges 'S-S' is a chemical reaction by means of which the disulfides are reduced to thiol (-SH). The agents that break the disulfide bridge and the methods described in WO 96/04385 are incorporated herein by reference in the present description. The reduction of 'S-S' can be obtained by (1) enzymatic routes in cascade, or by (2) reducing compounds. T iorredoxin, glut arredoxin type enzymes are known to be included in the in vivo reduction of disulfides, and have also been shown to be effective in reducing the disulfide 'S-S' bridges in vitro. The disulfide bonds are broken rapidly by thioredoxin reduced to pH 7.0, with a second order apparent velocity that is about 104 times larger than the corresponding rate constant for the reaction with DTT. Reduction kinetics can be increased dramatically • by preincubation of the protein solution with DTT 1 mM dihydrolipoamide (Holmgren, 1979).

The thiol compounds capable of reducing the disulfide bridges of the protein are, for example, Dithiothreitol (DTT), Dithioerythritol (DTE), ß -me rcapt oe t anol, thiocarbamates, bi s (2 -rnercapt oe ti 1) sulfone and N, N '-bis (mercaptoacet il) hydrazine, and sodium dithionite.

Reducing agents without thiol groups such as ascorbate or stannous chloride (SnCl2), which have been shown to be very useful in the reduction of disulfide bridges in monoclonal antibodies (Thakur et al., 1991), could also be used for the reduction of NS3. Treatment with sodium borohydride has been shown to be effective for the reduction of disulphide bridges in peptides (Gailit, 1993). Tris (2-carboxyethyl) phosphine (TCEP) is able to reduce disulfides at low pH (Burns et al., 1991). Selenol catalyzes the reduction of disulfide to thiols when DTT or sodium borohydride is used as a reducing agent. The first one, a commercially available diselenide, was used as a precursor to catalysis (Singh and Kats, 1995).

The present invention relates more particularly to a method for producing an immuno test as defined above, wherein the reducing agent is added to the solid phase during the steps of coating, blocking and / or fixing the antigen to the solid phase.

The present invention also relates to a method for carrying out an immuno test as defined above, wherein the reducing agent is added to the pretreatment step of the solid phase The coating conditions can vary widely as known to the person skilled in the art, and involves applying the protein to a solid phase and allowing a reaction to occur resulting in the binding of the protein to the solid phase. The link may be, but not restricted to, covalently hydrophobic or ionic bonds, Van Der Waels forces or hydrogen bonds. Different dampers known to the person skilled in the art could be used for this step, including but not limited to carbamate and phosphate buffers.

Blocking can be carried out by any method known in the art and can also be performed for example using albumin, whey proteins, polyvinylpyrrolidone (PVP), detergents, gelatins, polyvinylalcohol (PVA) or casein.

The fixation may be presented according to some method known in the art.

Additional examples of blocking, clamping and coating conditions are given in the section of E j ploses The present invention still more particularly relates to a method as defined above, wherein the reducing agent is added to the solid phase during the step of coating the antigen to the solid phase. Examples of coating dampers are given in the Examples section. The other coating dampers known in the art are also part of the present description.

The present invention also relates to a method as defined above, wherein the reducing agent is added to the solid phase during the step of blocking the solid phase, which contains the antigen that has been applied thereto in the presence or absence of a reducing agent. Examples of blocking dampers are given in the Examples section. The other blocking dampers known in the art are also part of the present description.

The present invention also relates to a method as defined above, wherein the reducing agent is added to the solid phase during the step of binding the antigen of the coating to the solid phase containing the antigen that has been applied thereto. presence or absence of a reducing agent. The fixation step could also have been preceded by a blocking step in the presence or absence of a reducing agent. Examples of fixation dampers are given in the Examples section. The other fixing dampers known in the art are also part of the present description.

The present invention also relates to a method for carrying out an immuno test as defined above, wherein the reducing agent is added during the pretreatment step of the solid phase before the addition of the sample. The pretreatment of the plates can be done with plates that have been treated with a reducing agent in the coating step, blocking and / or fixing or with plates that have not been previously treated with a reducing agent.

Finally, the reducing agent could also be added during any of the additional steps carried out in enzyme immunoprotes, as part of the present invention, possibly after the application of a reducing agent, in one or more of the above 4 steps of coating , blocking, fixing and / or pretreatment. Such additional steps include, but are not limited to, incubation of the antibodies, detection of bound antibodies and development of color.

The present invention preferably relates to a method as defined above, wherein the reducing agent is DTT, DTE or TCEP.

The present invention also relates to a method as defined above, wherein the reducing agent is used in a concentration range of 0.1 mM to 1 M, more particularly 0.5 mM to 500 mM, even more particularly from 1 mM to 250 mM, more particularly from 1 to 50 mM. Some applications may require ranges of 0.5 to 50 mM, 1 to 30 mM, 2 to 20 mM, 5 to 15 mM, or approximately 10 mM of the reducing agent. Other applications require DTT concentrations of 50-500 mM, 100-300 mM or 200 mM. DTT is particularly preferred as a reducing agent.

The present invention also relates to a method as defined above, wherein the antigen is a NS3 protein of HCV. More particularly a NS3 helicase from HCV. An HCV envelope protein such as the El and / or E2 protein is also preferred.

Also any other protein known in the art could react better with antibodies against the protein when the protein is added to the solid phase in the presence of DTT, or treated with DTT thereafter.

The present invention also relates to a method as described above, wherein the solid phase immunoprotene comprises a combination of antigens of different etiological agents or phenotylated and linked conditions.

The present invention also relates to a solid phase immunoprotection produced by a method as defined above. More particularly, a kit containing at least one solid phase such as a microtitre plate, a strip of membrane or silicon particle, which contain an antigen in the presence of a reducing agent.

More particularly, the present invention relates to an ELISA produced by a method as defined above.

In a preferred embodiment, the present invention relates to an ELISA produced by a method as defined above, wherein the reducing agent is preferably added in the coating and / or fixing steps. In a preferred embodiment, the reducing agent can be applied in the coating step. In another preferred embodiment, the reducing agent can be applied in the fixation step. In a particularly preferred embodiment, the reducing agent is added both in the coating step and in the setting step.

In another preferred embodiment, the present invention relates to an ELISA produced by a method as defined above, wherein the reducing agent is added during pretreatment of the plates prior to the addition of the sample. The pretreatment of the plates can be done with plates that have been treated with a reducing agent in the coating and / or fixing step, or with plates that have not been previously treated with a reducing agent. The reducing agent could also be added during any additional step carried out in enzyme immunoprotes. Such additional steps include, but are not limited to, incubation of antibodies, detection of bound antibodies and development of color.

The present invention also relates to an Online Immuno Test (LIA) produced by a method as defined above.

In a preferred embodiment, the present invention relates to an Online Immuno Test (LIA) produced by a method as defined above, wherein the reducing agent is preferably added in the blocking step and / or washing step. The reducing agent could also be added during any of the additional steps to produce or carry out enzyme immunoprotes. Such additional steps include but are not limited to fixation, pretreatment, incubation of the antibodies, detection of bound antibodies and color development.

The present invention also relates to a QUICK test produced by a method as defined above.

In a preferred embodiment, the present invention relates to a QUICK test produced by a method as defined above, wherein the reducing agent is preferably added during the coating of the antigen on the layer. The QUICK test is a lateral flow test in which the antigens cover the strips by atomizing them. In this test, the reducing agent is preferably added to the spray solution. The reducing agent could also be added during any of the additional steps to produce or perform enzyme immunoprotes. Such additional steps include but are not limited to blocking, fixing, pretreatment, incubation of the antibodies, detection of bound antibodies and development of color.

The present invention also relates to the use of a test as defined above for in vitro diagnosis of antibodies raised against an antigen, as defined above.

The present invention also relates to a HCV NS3 protein treated by a method comprising the steps of suifonation and subsequent desulphonation.

Suifonation and desulphonation is a reaction by means of which the -S03 groups are introduced or removed respectively from the protein.

Suifonation is defined as a process where thiol (SH) groups in proteins (R) and disulfide bonds are converted to S-Sulfonates, according to the following reactions: RSH > RS-SO3- (3 RS-SR + 2-SO3- + H20> 2 RS-SOA + 2 OH "(2) The products of the reactions are S-Sulfoprot and they are usually stable at neutral pH. Reaction (1) can be obtained by incubating the protein solution with tetrathionate at pH>. 7 (Inglis and Liu, 1970). Reaction (2) proceeds to completion in the presence of copper ions (Cole, 1967). Chan (1968) has shown that the treatment of the protein with sodium sulfite and catalytic amounts of cysteine in the presence of oxygen gives sulfo-pro teins.

Desulphonation can be obtained (1) by an excess of competitive -SH (thiol) groups, (2) by reducing agents or (3) by incubation under non-neutral pH conditions.

RS-SO3- + R'SH > RSH + R'S-S03_ RS-SO3"+ reducing agent> .RSH Competitive thiol groups could be obtained from low molecular weight compounds or from proteinaceous -SH groups.

Examples of compounds containing mono- or dithiol are: cysteine, cysteamine, reduced glutathione, N-acetyl cysteine, homocysteine, β-mercaptoethanol, thiocarbamates, bis (2-mercapt oet il) sulfone (BMS) and N, N'-bis ( mercaptoacet il) hydrazine (BMH), 5, 5 '-di ti obi s - (2-nitrobenzoic acid) (DTNB or Elman's reagent), Dithiothreitol (DTT) and Di t ioerythritol (DTE).

The present invention further relates to a HCV NS3 protein as defined above, which is further treated with a zwitterionic detergent. Empigen is known as, betaine • and is a particularly preferred example of a zwitterionic detergent. Other suitable detergents are known to those skilled in the art and are also reviewed in WO 96/04385.

The present invention further relates to a method for purifying a recombinantly expressed protein containing cysteine, comprising at least 2, preferably 3 or 4, and most preferably all of the following steps: (a) suifonation of a lysate of recombinant host cells or lysis of recombinant host cells in the presence of guanidinium chloride (preferably 6M Gu.HCl) and suifonation of the cell lysate, (b) treatment with a z-t eriónico detergent, preferably after the removal of cellular debris, (c) purification of the sulfonated recombinant protein, or purification of the sulfonated recombinant protein with subsequent removal of the detergent z wi tt er ionico, with purification which is preferably chromatography, more preferably Ni-IMAC chromatography with the recombinant protein that is a recombinant protein labeled with His, (d) desulfonation of the sulfonated recombinant protein, preferably with a molar excess of a reducing agent such as DTT, (e) storage in the presence of a molar excess of DTT.

The empigene is a particularly preferred example of a zwitterionic detergent. The inclusion of such a zwitterionic agent and DTT was found to improve the purification protocol for HCV NS3 helicase and HCV envelope proteins.

The present invention also relates to a HCV polynucleic acid encoding an HCV NS3 polyprotein as shown in Figure 1 (SEQ ID NOs 3-18) or a unique part of a HCV polynucleic acid having a sequence that is represents in Figures 2 -1, 3-1, 4-1, 5-1, 6-1, 7-1 and 8-1 (SEQ ID NOs 19, 21, 23, 25, 27, 29 and 31).

The present invention also relates to a polynucleic acid of HCV as defined above, characterized in Figures 2-1, 3-1, 4-1, 5-1, 6-1, 7-1 and 8-1 and by the fact that their product does not react with false positive HCV samples, or a part of them that encode NS3 epitopes that do not react with false positive HCV samples. It was particularly surprising that the proteins encoded by the clones represented by SEQ ID NOs 19, 21, 23, 25, 27, 29 and 31 have the property of not reacting with false positive HCV samples, were still able to react with the majority of the known samples positive for the NS3 antibody after treatment with DTT.

The present invention further relates to a recombinant vector comprising a polynucleic acid as described.

The present invention further relates to a host cell comprising a vector of the invention.

The present invention also relates to a method for detecting a nucleic acid of the invention. This detection method can be any method known in the art, as described in detail in WO 96/13590 by Maertens & Stuyver More particularly, the present invention relates to a method for detecting a nucleic acid of the invention comprising: contacting the nucleic acid with a probe; - determine the complex formed between the nucleic acid and the probe.

Accordingly, the present invention relates to an isolated nucleic acid as described above or a fragment thereof for use as a probe or a primer.

The present invention further relates to a diagnostic kit for the detection of a nucleic acid sequence as described above, comprising at least one primer and / or at least one probe according to the invention. Reference is made to WO 96/13590 for a detailed description of a general idea of these applications.

In addition to the reactivity gained by reduction, the reactivity of NS3 is also determined rigorously by the sequence of the NS3 antigen.

Therefore, the present invention also relates to a HCV polypeptide having part or all of the amino acid sequences as shown in Figures 1, 2-2, 3-2, 4-2, 5-2, 6- 2, 7-2 and 8-2 (SEQ ID NOs 20, 22, 24, 26, 28, 30, 32). The present invention also relates to an NS3 helicase protein of HCV as described in Figure 1 (SEQ ID NOs 1-18) or a single part thereof.

The present invention also relates to an HCV NS3 helicase protein or part thereof containing either S1200, A1218, A1384, P1407, V1412, P1424, or F1444, or a combination of these amino acids with any of the following amino acids L1202 , S1222, 11274, S1289, T1321, A1323, T1369, L1382, V1408, A1409, or F1410. The numbering is according to the numbering system commonly accepted for HCV amino acids.

The present invention further relates to a pharmaceutical composition comprising a polypeptide of the invention or any variant or fragment thereof functionally equivalent. The term "a pharmaceutical composition" refers to a composition or medicament (both terms may be used interchangeably) comprising a polypeptide of the present invention and a pharmaceutically acceptable carrier or excipient (both terms may be used interchangeably). This pharmaceutical composition can be used as a medically, this pharmaceutical composition can be used as a medicament for the treatment or prevention of HCV infection. The suitable vehicles or excipients known to the expert in arre are saline, Ringer's solution, dextrose solution, Hank's solution, fixed oils, ethyl oleate, 5% dextrose in saline, substances that increase isotonicity and stability chemistry, shock absorbers and preservatives. Other suitable vehicles include any vehicle that does not itself induce the production of antibodies harmful to the individual receiving the composition such as proteins, olisaccharides, pyridic acids, polyglycolic acids, polymeric amino acids and amino acid copolymers. The "pharmaceutical composition" or "medicament" could be administered by any suitable method within the knowledge of the expert. The preferred route of administration is parenterally or a vaccine. In parenteral administration or vaccine, the medicament of this invention will be formulated in an injectable unit dosage form such as a solution, suspension or emulsion, in association with the pharmaceutically acceptable excipients as defined above. For vaccine applications or for the generation of anti-bodies / anti-serum polyclonal, for example, the effective amount could vary depending on the species, age, and in general condition of the individual, the severity of the condition to be treated, the particular polypeptide selected and its mode of administration, etc. It is also believed that the effective amounts will be within a relatively large, non-critical range. An appropriate effective amount can be determined quickly using only routine experimentation. The preferred ranges of NS3 and / or El and / or E2 and / or E1 / E2 alone or oligomeric envelope proteins specific for HCV disease prophylaxis are 0.01 to 1000 μg / dose, preferably 0.1 to 100 μg / dose. dose, more preferably 1 to 50 μg / dose. Several doses may be needed per individual to obtain a sufficient immune response and subsequent protection against HCV disease. In the case of a therapeutic vaccine, the number of doses required could amount to more than 10. Continuous infusion could also be used. If so, the medicament could be given in infusion at a dose between 5 and 20 μg / kg / minut or, more preferably between 7 and 15 μg / kg / minut or. It should also be clarified that the pharmaceutical composition of the present invention could comprise a variant or functionally equivalent fragment of the sequences given in SEQ ID NOs' 3-18, 20, 22, 24, 26, 28, 30, 32. The latter terms refer to a molecule containing the complete protein sequence of the polypeptide of the invention or part of the protein sequence of the polypeptide of the invention, to which certain modifications have been applied, and which retains all or part of the "biological properties" of the polypeptide of the invention Such modifications include but are not limited to the addition of polysaccharide chains, the addition of certain chemical groups, the addition of lipid portions, the fusion with other peptide or protein sequences and the crosslinking. intramolecular.

The present invention also relates to an immuno test comprising a HCV polypeptide as defined above. The immunoprotein can be of any type of format known in the art (see for example WO 96/13590 and Coligan et al., 1992). In particular, the present invention relates to a method for detecting a polypeptide of the invention, which comprises: - contacting the polypeptide with a ligand that binds to the polypeptide; - determining the complex formed between the polypeptide and the ligand.

Accordingly, the present invention also relates to a ligand that binds to a polypeptide according to the invention. The term "a ligand" refers to any molecule capable of binding the polypeptides of the present invention. The latter term refers specifically to polyclonal and / or monoclonal antibodies specifically cultivated (by any method known in the art) against the polypeptides of the present invention and also encompasses any antibody-like construction, and others, as described in detail in EP 97870092.0 of Lorré et al. Such antibodies could be very useful for the detection of antigen in biological fluids. Antigen detection can be done by any immunopreceptor known in the art, such as biotin and avidin or streptavidin tests, ELISA and immunoprecipitation, immunohistochemical techniques and agglutination tests. A detailed description of these tests is given in WO 96/13590 which is incorporated herein by reference.

In addition, the antibodies could be very useful for HCV therapy or other diseases, and therefore could be humanized if generated in a non-human host. Accordingly, the present invention relates to compositions of these antibodies in a pharmaceutically acceptable excipient, for use as a medicament.

The present invention also relates to any method for producing and using the polyproteins of the invention. Methods for producing and using the HCV polyproteins are described in WO 96/13590. The uses include not only diagnostic uses, but also therapeutic and prophylactic uses. The NS3 proteins of the invention are also particularly suitable for incorporation into vaccine compositions. The vaccine composition may contain, in addition to the active ingredient, any type of adjuvant known in the art. The content of WO 96/13590 is incorporated herein by reference of the present disclosure. The NS3 proteins of the present invention could also be used in any application where it is applicable to use an NS3 helicase, such as for drug screening purposes.

LEGENDS OF THE FIGURES Figure 1. Amino acid sequence of NS3 NS3 clones isolated from serum infected with subtype la and Ib of HCV.

Figure 2-1. DNA coding sequence of the fusion protein mTNFH6NS3 clone 19b. The sequence shown in bold is not the NS3 sequence. This sequence encodes the fusion partner mTNF, the hexahistidine tag and part of the multilayer.

Figure 2-2. The amino acid sequence of the fusion protein mTNFH6NS3 clone 19b. The sequence shown in bold is not the NS3 sequence. This sequence contains the fusion partner mTNF, the hexahistidine tag and part of the zyranthem.

Figure 3-1. DNA coding sequence of the fusion protein mTNFH6NS3 clone B9. The sequence shown in bold is not the NS3 sequence. This sequence encodes the fusion partner mTNF, the hexahistidine tag and part of the zyranthem.

Figure 3-2. The amino acid sequence of the fusion protein mTNFH6NS3 clone B9. The sequence shown in bold is not the NS3 sequence. This sequence encodes the fusion partner mTNF, the hexahistidine tag and part of the zyranthem.

Figure 4-1. DNA coding sequence of the fusion protein mTNFH6NS3 Type 3a clone 21. The sequence shown in bold is not the NS3 sequence. This sequence encodes the fusion partner mTNF, the hexahistidine tag and part of the zyranthem.

Figure 4-2. The amino acid sequence of the fusion protein mTNFH6NS3 Type 3a clone 21. The sequence depicted in bold is not the NS3 sequence. This sequence encodes the fusion partner mTNF, the hexahistidine tag and part of the multienolver.

Figure 5-1. DNA coding sequence of the fusion protein mTNFH6NS3 Type 3a clone 32. The sequence shown in bold is not the NS3 sequence. This sequence encodes the fusion partner mTNF, the hexahistidine tag and part of the multilayer.

Figure 5-2. The amino acid sequence of the fusion protein mTNFH6NS3 Type 3a clone 32. The sequence depicted in bold is not the NS3 sequence. This sequence encodes the fusion partner mTNF, the hexahistidine tag and part of the multilayer.

Fig. 6-1. DNA coding sequence of the mTNFH6N? 3 type 2a fusion protein. The sequence shown in bold is not the NS3 sequence. This sequence encodes the fusion partner mTNF, the hexahistidine tag and part of the zyranthem.

Figure 6-2. Amino acid sequence of the fusion protein of mTNFH6NS3 type 2a. The sequence shown in bold is not the NS3 sequence. This sequence encodes the fusion partner mTNF, the hexahistidine tag and part of the multilayer.

Figure 7-1. DNA coding sequence of the fusion protein mTNFH6NS3 type 2b. The sequence shown in bold is not the NS3 sequence. This sequence encodes the fusion partner mTNF, the hexahistidine tag and part of the zyranthem.

Figure 7-2. Amino acid sequence of the fusion protein of mTNFH6NS3 type 2b. The sequence shown in bold is not the NS3 sequence. This sequence encodes the fusion partner mTNF, the hexahistidine tag and part of the zyranthem.

Figure 8-1. DNA coding sequence of the mTNFH6NS3 type 2c fusion protein. The sequence shown in bold is not the NS3 sequence. This sequence encodes the fusion partner mTNF, the hexahistidine tag and part of the multilayer.

Figure 8-2. Amino acid sequence of the fusion protein of mTNFH6NS3 type 2c. The sequence shown in bold is not the NS3 sequence. This sequence encodes the fusion partner mTNF, the hexahistidine tag and part of the multilayer.

EXAMPLES Example 1. Expression of HCB type Ib NS3 clone 19b in E. col ± 1. 1 Cloning of genes 19a and 19b of HCV type Ib NS3 clones The NS3 helicase domain (amino acids 1118-1465 was amplified by RT-PCR of the IG8309 serum of HC subtype Ib (Innogenetics, Ghent, Belgium) using the synthetic oligonucleotide primers HCPr59 (51-GGGCCCCACCATGGGGGTTGCGAAGGCGGTGGACTT-3 ') (SEQ ID NO 1) ) and HCPr60 (5 '-CTATTAGCTGAAAGTCGACTGTCTGGGTGACAGCA-3') (SEQ ID NO 2) This yielded a PCR fragment 19 which was cloned into E. coli The sense primer HCPr59 introduces an Apal restriction site that includes an artificial methionine. The antisense oligonucleotide HCPr60 introduces a stop codon after 1465 aa.The PCR fragment was subsequently cut with Apal and the resulting 833 bp Apal fragment was cloned into the expression vector pmTNFHRP cut in Apal (Innogenetics, 'Ghent, Belgium) Four hepatitis C clones (HCCI) were sequenced: HCCI19a and HCCI19b (see the deduced amino acid sequence given in Figure 1 and Figure 2-2) The clone HCCI19b (pmTNFHRPHCCI 19b) was retained for additional subcloning. 1. 2 Construction of the expression plasmid pEmTNFMPHHCCI19b Starting from vector pmTNFHRPHCCI 19b the coding sequence 19b of clone NS3 was isolated as a Ncol fragment of 900 bp and inserted into the Ncol cut expression vector pEmTNFMPH (Innogenetics, Ghent, Belgium) resulting in the vector pEmTNFMPHHCCI19b. This plasmid expresses clone 19 b of HCV NS3 as an N-terminal fusion protein with the N-terminus of 25 aa of murine TNF followed by a purification mark of hexahistidine and a cut-off site of formic acid (SEQ ID NO: 19) and 20; Figure 2). 1. 3 Expression of 1 clone 19b of NS3 of HCV in E. col ± The MC1061 cells (pAcI) of the E strain. co l i (Wertman et al., 1986) were transformed with the plasmid pEmTNFMPHHCCI19b. The MC1061 (pAcI) cells harboring pEmTNFMPHHCCI 19b were grown overnight in Luria broth (LB) supplemented with 10 μg / mL tetracycline at 28 ° C. The cultures were diluted 20 times in fresh LB, then grown at 28 ° C to an OD600 of 0.2, after which the temperature was increased to 42 ° C. At 2 to 3 hours post-induction, the cells were harvested. Expression of the fusion protein of HCV NS3 clone 19b was analyzed by Western blot using the monoclonal specific antibodies and human HCV positive serum.

Example 2. Expression of the B9 clone of NS3 of HCV in E. col ± 2. 1 Cloning of the B9 gene from the NS3 type NS3 clone The NS3 helicase domain (amino acids 1118-1465) was amplified by RT-PCR of the IG21054 serum of HC subtype Ib (Innogenetics, Ghent, Belgium) using the synthetic oligonucleotide primers HCPr59 (51-GGGCCCCACCATGGGGGTTGCGAAGGCGGTGGACTT-3 ') (SEQ ID NO. 1) and HCPr60 (5 '-CTATTAGCTGAAAGTCGACTGTCTGGGTGACAGCA-3') (SEQ ID NO 2). This produced a PCR B fragment that was cloned in E. col i. The sense primer HCPr59 introduces an Apal restriction site that includes an artificial methionine. The antisense oligonucleotide HCPr60 introduces a stop codon after 1465 aa. The PCR fragment was subsequently cloned into the vector pGEM-T (Promega, Madison, Wl, US). Four clones were sequenced: B7, B9, B12 and B14 (see the amino acid sequences deduced in Figure 1 and Figures 3-2). Clone B9 (pGEMTNS3B9) was retained for further subcloning. 2. 2 Construction of the expression plasmid pIGFHlllNS3B9 Starting from vector pGEMTNS3B9, the coding sequence of clone B9 was isolated as a Ncol / Spel terminated fragment of 850 bp and inserted into the expression vector pIGFHIII of the Ncol / Stul cut (Innogenetics, Ghent, Belgium), resulting in the vector PIGFH111NS3B9. This plasmid expresses HC9 NS3 clone B9 as a fusion protein with the N-terminus of 25 aa of murine TNF followed by a purification mark of hexahistidine and a cut-off site of formic acid (SEQ ID Nos. 21 and 22). Figure 3). 2. 3 Expression of HC9 NS3 clone B9 in E. coli The MC1061 cells (pAcI) of the E strain. col i (Wertman et al., 1986) were transformed with the plasmid PIGFH111NS3B9. The MC1061 (pAcI) cells harboring pIGFHl 11NS 3B9 were grown overnight in Luria broth (LB) supplemented with 10 μg / ml tetracycline at 28 ° C. The cultures were diluted 20 times in fresh LB, then grown at 28 ° C to an OD600 of 0.2, after which the temperature was increased to 42 ° C. At 2 to 3 hours post-induction, the cells were harvested. Expression of the fusion protein of HCV NS3 clone 19b was analyzed by Western blot using the monoclonal specific antibodies and human HCV positive serum.

Example 3. Expression of the A26, C16 and D18 clones of the NS3 type of HCV in E. coli Clones A26, C16 and D18 were isolated from HC21051, IG17790 and infected IG21068 subtype HCV subtype, respectively, in a similar manner as described for clone B9 using the primers HCPr59 and HCPr60. Initially, clones A5, A26, Cl, C3, C4, C12, C16, D17, D18 and D19 were cloned and sequenced (see the deduced amino acid sequences given in Figure 1). Clones A26, C16 and D18 were maintained for additional subcloning.

Example 4. Expression of NS3 type 3a clones 21 and 32 of HCV in E. coli 4. 1 Cloning of HC3 NS3 type 3a genes 3 and 3a The NS3 helicase domain (amino acids 1118-1465) was amplified by RT-PCR of serum IG21349 and IG20014 of subtype 3a of HCV (Innogenetics, Ghent, Belgium) using the synthetic oligonucleotide primers 403 (5 'GGGCCCCACCATTAGGTGTAGCAAAAGCCCTACAGTT-3') (SEQ ID NO 33) and 404 (5 * -CTATTAGCTGAAGTCAACGTACTGTTCAACAGC-3 ') (SEQ ID NO 34). This produced in both cases a PCR fragment of approximately 850 bp which was subsequently subcloned into the pGEM-T vector (Promega, Madison, Wl, US). Of each of the clones from several cloned PCR fragments were sequenced, but each serum was only tested one cloned fragment that was completely correct in the sequencing. This was clone 21 (pGEM-TNS3T3a .21) for clone IG21349 and clone 32 (pGEM-TNS3T3a.32) for serum IG20014 (Figures 4 and 5). 4. 2 Construction of the expression plasmids pIGFHlllNS3T3a.21 and pIGFHlllNS3T3a .32 Starting from vectors pGEM-TNS3T3a .21 and pGEM-TNS3T3a .32, the coding sequences of clone 21 and 32 were isolated as Ncol / Sall fragments of 850 bp and inserted into the expression vector pIGFHIII cut in Ncol / Sall (Innogenetics, Ghent, Belgium), resulting in the vectors pIGFHl 11NS 3T3a .21 and pIGFHlllNS3T3a .32, respectively. These plasmids express HCV NS3 type 3a clones 21 and 32 as N-terminal fusion proteins of murine TNF 25 aa followed by a hexahistidine purification tag and a formic acid cleavage site (SEQ ID NOs 23) -26; Figures 4 and 5). 4. 3 Expression of clones 21 and 32 of type 3a of NS3 of HCV in E. coli The MC1061 cells (pAcI) of the E strain. col i (Wertman et al., 1986) were transformed with the plasmids pIGFHl 1 lNS3T3a .21 and pIGFHl 11NS 3T3a .32, respectively. The MC1061 (pAcI) cells harboring pIGFHlllNS3T3a .21 or pIGFH 111NS 3T3 to .32 were grown overnight in Luria broth (LB) supplemented with 10 μg / ml tetracycline at 28 ° C. The cultures were diluted 20 times in fresh LB, then grown at 28 ° C to OD6cc of 0.2, after which the temperature was increased to 42 ° C. At 2 to 3 hours post-induction, the cells were harvested. The fusion protein pressure of HC3 NS3 type 3a clones 21 and 32 was analyzed by Western blot using monoclonal specific antibodies and human serum HCV positive Example 5. Expression of NS3 type 2a clone 3 of HCV in E. coli . 1 Cloning of the HC3 NS3 type 2a clone 3a gene The NS3 helicase domain (amino acids 1118-1465) was amplified by RT-PCR of an Ig21342 serum of HCV subtype 2a (Innogenetics, Ghent, Belgium) using the synthetic oligonucleotide primers 412 (5'-GGGCCCCACCATGGGCGTGGCCAAGTCCATAGACTT-3 ') (SEC ID No. 35) and 413 (5 '-CTATTAGCTGAAGTCTACAACTTGAGTGACCGC-3') (SEQ ID NO 36). This produced a PCR fragment of approximately 850 bp which was subsequently subcloned into the pGEM-T vector (Promega, Madison, Wl, US). Several clones were sequenced and clone 3 (pGEM-TNS 3T2a) was retained for further subcloning (Figure 6). . 2 Construction of the expression plasmid pIGFHlllNS3T2a Starting from vector pGEM-TNS3T2a, the coding sequence of clone 3 was isolated as Ncol fragments of 850 bp and inserted into the expression vector pIGFHIII cut in Ncol (Innogenetics, Ghent, Belgium), resulting in vector pIGFHlllNS3T2a. This plasmid expresses HC3 NS3 type 2a clone 3a as an N-terminal fusion protein of 25 aa of murine TNF followed by a hexahistidine purification tag and a formic acid cleavage site (SEQ ID NOs. 28, Figure 6). . 3 Expression of type 3 clone 3 of NS3 of HCV in E, coli The MClOdl (pAcI) cells of the E. co l i (Wertman et al., 1986) were transformed with the plasmid pIGFHlllNS3T2a. The MC1061 (pAcI) cells harboring pIGFH1 1 lNS3T2a were grown overnight in Luria broth (LB) supplemented with 10 μg / ml tetracycline at 28 ° C. The cultures were -diluted 20 times in fresh LB, then grown at 28 ° C to OD600 of 0.2, after which the temperature was increased to 42 ° C. At 2 to 3 hours post-induction, the cells were harvested. The expression of the NS3 NS3 clone 3 type 2a fusion protein was analyzed by Western blot using the monoclonal specific antibodies and human HCV positive serum.

Example 6. Expression of type 9b NS3 clone 9b VHC in E coli 6. 1 Cloning of the NS3 NS3 type 2b clone 9 gene The NS3 helicase domain (amino acids 1188-1465) was amplified by RT-PCR of an IG20192 serum of HCV subtype 2b (Innogenetics, Ghent, Belgium) using the synthetic oligonucleotide primers 401 (51-GGGCCCCACCATGGGCGTAGCCAAATCCATTGACTT-3 ') (SEQ. NO 37) and 402 (5 '-CTATTAGCTGAAGTCTACAATTTGAGAGACCGC-3') (SEQ ID NO 38). This produced a PCR fragment of approximately 850 bp which was subsequently subcloned into the vector pGEM-T (Promega, Madison Wl, US). Several clones were sequenced and clone 9 was retained for further subcloning (Figure 7). 6. 2 Construction of the expression plasmid pIGFHlllNS3T2b Starting from vector pGEM-TNS3T2b, the coding sequence of clone 9 was isolated as a Ncol fragment of 850 bp and inserted into the expression vector pIGFHIII cut in Ncol (Innogenetics, Ghent, Belgium), resulting in the ector pIGFHlllNS3T2b . This plasmid expresses HCN NS3 type 2b clone 9b as an N-terminal fusion protein of 25 aa of murine TNF followed by a purification mark of hexahistidine and a cut-off site of formic acid (SEQ ID NOs 29- 30, Figure 7). 6. 3 Expression of HCB type 2b clone 9 NS-3 in E. coli The MC1061 cells (pAcI) of the E strain. co l i (Wertman et al., 1986) were transformed with the plasmid pIGFHlllNS3T2b. The MC1061 (pAcI) cells harboring pIGFH1 1 lNS3T2b were grown overnight in Luria broth (LB) supplemented with 10 μg / ml tetracycline at 28 ° C. The cultures were diluted 20 times in fresh LB, then grown at 28 ° C to an OD600 of 0.2, after which the temperature was increased to 42 ° C. At 2 to 3 hours post-induction, the cells were harvested. The expression of the NS3 NS3 type 2b clone 9 fusion protein was analyzed by Western blot using the monoclonal specific antibodies and human HCV positive serum.

Example 7. Expression of type 2c clone 14c of NS3 in HCV. coli 7. 1 Cloning of the gene of type 2c clone 14c of HCV NS3 The NS3 helicase domain (amino acids 1188-1465) was amplified by RT-PCR of an IG20031 subtype 2c HCV serum (Innogenetics, Ghent, Belgium) using the synthetic oligonucleotide primers 401 (5'-GGGCCCCACCATGGGCGTAGCCAAATCCATTGACTT-3 ') (SEC IDN037) and 402 (5 '-CTATTAGCTGAAGTCTACAATTTGAGAGACCGC-3') (SEQ ID NO 38). This produced a PCR fragment of approximately 850 bp which was subsequently subcloned into the vector pGEM-T (Promega, Madison Wl, US). Several clones were sequenced and clone 14 (pGEM-TNS3T2c) was retained for further subcloning (Figure 8). 7. 2 Construction of the expression plasmid pIGFHlllNS3T2c Starting from vector pGEM-TNS3T2c, the coding sequence of clone 14 was isolated as a Ncol fragment of 850 bp and inserted into the expression vector pIGFHIII cut in Ncol (Innogenetics, Ghent, Belgium), resulting in vector pIGFHlllNS3T2c . This plasmid expresses clone 14 of type 2c of HCV NS3 as an N-terminal fusion protein of 25 aa of murine TNF followed by a purification mark of hexahistidine and a cut-off site of formic acid (SEQ ID NOs. 32, Figure 8). 7. 3 Expression of clone 14 of type 2c of NS3 of HCV in E. coli The MClOdl (pAcI) cells of the E. co l i (Wertman et al., 1986) were transformed with the plasmid pIGFHlllNS3T2c. The MC1061 (pAcI) cells harboring pIGFH1 1 lNS3T2c were grown overnight in supplemented Luria broth (LB). with 10 μg / ml of tetracycline at 28 ° C. The cultures were diluted 20 times in fresh LB, then grown at 28 ° C to an OD600 of 0.2, after which the temperature was increased to 42 ° C. At 2 to 3 hours post-induction, the cells were harvested. The expression of the HC3 NS3 type 2c clone 14c fusion protein was analyzed by Western blot using the monoclonal specific antibodies and human HCV positive serum.

Example 8. Purification of the domain of the NS3 protein helicase Nine volumes of 8M guanidinium hydrochloride (Gu.HCl) and 1 volume of NaHPO. 0.2M were added to each gram equivalent of E cell paste. co l i and the solution was homogenized by continuously vortex formation. Solid N S4Oc was added to the solution to a final concentration of 65 and 360 mM, respectively. CuS04 (reserve solution: 0.MI in 25% NH3) was added to a final concentration of 100 μM. The solution was stirred overnight in the dark at room temperature and after incubation at -70 ° C it was clarified by centrifugation at 4 ° C (30 min, 20,000 rpm, JA20 rotor).

Empigen BBTK (Albright &Wilson Ltd., Okibury, UK) and imidazole were added to the supernatant to a final concentration of 1% (w / v) and 20 m, respectively. The pH was adjusted to 7.2 with IN HCl. A sample corresponding to the cell culture equivalent of 3 L was loaded at 2 mL / min in a Ni-IDA Sepharose FF of 25 mL (column XK 16/20, Pharmacia, Upsala, Sweden), which had been equilibrated with the buffer A containing 20 mM imidazole (buffer A: 50 mM phosphate, 6M Gu.HCl, 1% Empigen, pH 7.2). The Ni-IDA Sepharose column was washed consecutively with: A buffer containing 20 mM imidazole buffer A containing 35 mM imidazole A buffer containing 50 mM imidazole buffer B containing 50 mM imidazole (buffer B: 50 mM phosphate, 6M Gu.HCl, pH 7.2) B buffer containing 200 mM imidazole Each washing step was maintained during chromatography until the absorbance at 280 nm reached the baseline level. The column was regenerated with 50 mM EDTA, 500 mM NaCl, pH 7.0.

Fractions were analyzed by SDS-PAGE using non-reducing conditions and stained by silver. The fusion protein mTNF-NS3 B.9 was recovered in the elution of 200 mM imidazole. Western blot using rabbit anti-human TNF (1 μg of NS3 / lane) and anti-£. rabbit col i (10 μg of NS3 / lane) showed that NS3 exhibited a purity of approximately after this simple chromatography step.

Imidazole elution fractions of 200 mM were poured out and desalted.

A sample of Ni-IDA eluate was loaded at 10 mL / min on a 300 mL Sephadex G25 column (XK 50, Pharmacia, Upsala, Sweden), which had been equilibrated with 50 mM phosphate, 6M ureo, 1 mM EDTA, pH 7.2. Fractions of 10 mL were collected and the concentration of the protein was determined by the micro BCA method (Pierce, Rockford, IL, US). The concentration of the protein was adjusted to 500 μg / mL with the desalting buffer before desulphonation. and reduction. The overall yield was 50-55 mg of the equivalent of the NS3 fusion protein / culture equivalent L.

Finally, DDT (reserve solution: 100 mM in distilled water) was added in a 100-fold molar excess against the content of cysteine in the NS3 antigen (e.g. NS3 19b 'contains 7 cysteines). The solution was flushed with nitrogen and incubated for 1 h at 28 ° C.

The NS3 sample was subsequently diluted in the appropriate buffer for the ELISA and LIA coating.

Example 9. Reactivity of NS3 antibody helicase tested in LIA To test the reactivity of the NS3 helicase antibody, a 50 μg / ml line of NS3 antigen solution in phosphate buffered saline on nylon membrane strips. The strips were dried for at least 1 hour at a temperature between 18-24 ° C and subsequently blocked with PBS / casein in the presence (10 mM) or absence of the reducing agent DDT. The strips were subsequently washed with PBS containing Tween 20 and either without DDT or 10 mM DDT and with water containing either no DDT or 10 mM DDT and 1 mM EDTA. The membranes were dried for 30 minutes and cut into strips to test different samples of the patient.

The results of an experiment where the strips were incubated with the PHV903 anti-HCV sero-conversion panel (Boston Biomedica Inc., Boston, US) are given in Table 1.

Example 10. Reactivity of the NS3 antibody helicase tested in ELISA To test the reactivity of the NS3 helicase antibody, the ELISA plates were coated with the purified NS3 antigens as in Example 4 in the following manner.

The wells of the microtitre plate were coated with the NS3 protein at a concentration of 0.3 μg / ml of the NS3 protein in the coating buffer containing 50 mM carbonate buffer, either 20 mM DDT or without DDT and 1 mM EDTA . The microtiter plates were incubated for 18 hours at 20 ° C, and blocked with 300 μl of PBS / casein buffer per well. Plates were incubated for 2 hours at 20 ° C and subsequently fixed with 300 μl of fixation buffer, which contained either 200 mM or non-DDT DDT, and 1 mM EDTA for 2 hours at 20 ° C.

The results are shown in Tables 2 and 3. Table 2 gives the signal-to-noise values of the tests that include NS3 coated or fixed with or without DDT, with the sero-conversion ion panels from BBI PVH901 to PHV912. Table 3 shows a summary of the number of days in which HCV antibodies can be detected earlier by the test incorporating DDT. Clearly, a total of 34 days of pre-screening, in 12 sero-HCV conversions can be obtained by incorporating DDT into the test.

Table 1. BBl panels tested in LIA coated with HCV NS3 as described in Example 9.

-: Without reaction; -positive reaction; the intensity levels are given in comparison with the different cut lines atomized in the same strip.

Table 2: BBl panels tested in ELISA coated with NS3 of HCV as described in example 10.

Table 3: Overview of the panels BBl number of days with the previous detection.

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It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, the content of the following is claimed as property.

Claims (35)

1. The solid phase immuno test, characterized in that it comprises an antigen in the solid phase in the presence of a reducing agent.

2. The method for producing or carrying out an immunoprotection according to claim 1, characterized in that the reducing agent is added to the solid phase during the steps of coating, blocking and / or fixing the antigen to the solid phase or during the pretreatment of the the solid phase.

3. The method according to claim 2, characterized in that the reducing agent is added to the solid phase during the step of coating the antigen to the solid phase.

4. The method according to claim 2, characterized in that the reducing agent is added to the solid phase during the step of blocking the solid phase comprising the antigen applied thereto in the presence or absence of a reducing agent.

5. The method according to the rei indication 2, characterized in that the reducing agent is added to the solid phase during the step of fixing the solid phase comprising the antigen applied thereto in the presence or absence of a reducing agent.

6. The method according to claim 2, characterized in that the reducing agent is added during the pretreatment of the solid phase comprising the antigen applied thereto in the presence or absence of a reducing agent.

7. The method according to any of the rei indications 1 to 6, characterized in that the reducing agent is DTT, DTE or TCEP.

8. The method according to any of claims 1 to 7, characterized in that the reducing agent is used in a concentration range of 0.1 mM to 1 M, more particularly 0.5 mM to 500 mM, even more particular from 1 mM to 250 mM , some applications may require ranges of 0.5 to 50 mM, 1 to 30 mM, 2 to 20 mM or 5 to 15 mM or approximately 10 mM.

9. The method according to any of claims 2 to 8, characterized in that the antigen is a NS3 protein of HCV.

10. The solid phase immuno test, characterized in that it is produced by a method according to any of claims 2 to 9.

11. ELISA, characterized in that it is produced by a method according to any of claims 2 to 9.

12. ELISA according to claim 11, characterized in that the reducing agent is added in the coating and / or fixing steps.

13. The QUICK test, characterized in that it is produced by a method according to any of claims 2 to 9.

14. The QUICK test, according to claim 13, characterized in that the reducing agent is added in the blocking step.

15. The Online Immuno Test, characterized in that it is produced by a method according to any of claims 2 to 9.

16. The Online Immuno Test according to claim 15, characterized in that the reducing agent is added in the blocking step.

17. The use of a test according to claims 10 to 16, characterized in that it is used for the in vitro diagnosis of antibodies raised against an antigen as described in claim 1.

18. The NS3 protein of HCV, characterized in that it is treated by a method comprising the steps of suifonation and subsequent desulphonation.

19. The NS3 protein of HCV according to claim 18, characterized in that it is further treated with a z-wit ionic detergent, preferably Empigen.

20. The method for purifying a cysteine containing the recombinantly expressed protein, characterized in that it comprises at least 2, preferably 3 or 4 and even more preferably all the following steps: (a) suifonation of a lysate of the recombinant host cells or cell lysis recombinant host in the presence of guanidinium chloride followed by a subsequent suifonation of the cell lysate, (b) treatment with "a zwitterionic detergent, preferably after the removal of cellular debris, (c) purification of the sulfonated version of the recombinant protein or purification of the sulfonated version of the recombinant protein with the subsequent removal of the zwitterionic detergent, with the purification being preferably by chromatography, more preferably a Ni-IMAC chromatography with the recombinant protein which is the His-brand recombinant protein. (d) desulphonation of the sulfonated version of the recombinant protein, preferably with a molar excess of DTT. (.e) storage in the presence of a molar excess of DTT, or immediate use in a test

21. An HCV polynucleic acid, characterized in that it encodes a polypeptide as depicted in Figure 1 (SEQ ID NOS 3-18) or a unique part of a HCV polynucleic acid, more particularly a polynucleic acid having a sequence as depicted in Figures 2-1, 3-1, 4-1, 5-1, 6-1, 7-1 or 8-1 (SEQ ID NOs 19, 21, 23, 25, 27, 29 and 31).

22. A polynucleic acid of HCV according to the indication rei 21 as shown in Figures 2-1, 3-1, 4-1, 5-1, 6-1, 7-1 or 8-1 and characterized by the fact that your product does not react with falsely positive HCV samples, or a part of it that encodes an NS3 epitope that does not react with the falsely positive HCV samples.

23. A recombinant vector, characterized in that it comprises a polynucleic acid according to claim 21 or 22.

24. A host cell, characterized in that it comprises a vector according to claim 23.

25. A method for detecting a nucleic acid sequence according to claim 21 or 22, characterized in that it comprises: - contacting the nucleic acid with a probe - determine the complex formed between the nucleic acid and the probe.

26. An isolated nucleic acid according to claim 21 or 22 or a fragment thereof, characterized in that it is used as a probe or a primer for the detection of a nucleic acid according to claim 21 or 22.

27. A diagnostic kit for the detection of a nucleic acid sequence according to claim 21 or 22, characterized in that it comprises at least one primer and / or at least one probe according to claim 26.

28. A HCV polypeptide, characterized in that it has part or all of the amino acid sequences of a polypeptide encoded by a polynucleic acid according to claim 21 or 22.

29. An HCV NS3 protein helicase or part thereof, characterized in that it contains either S1200, A1218, A1384, P1407, V1412, P1424 or F1444 or a combination of these amino acids with any of the following amino acids L1201, 11274, S1289, T1321, A1323, T1369, L1382, V1408, A1409, F1410.

30. A pharmaceutical composition, characterized in that it comprises a polypeptide according to claims 28 or 29, or any variant or functionally equivalent fragment thereof.

31. A pharmaceutical composition, characterized in that it comprises a polypeptide according to claim 28 or 29, or any variant or functionally equivalent fragment thereof for use as a medicament for preventing or treating HCV infection.

32. A method for detecting a polypeptide according to claim 28 or 29, characterized in that it comprises: - contacting the polypeptide with a ligand that binds to the polypeptide to determine the complex formed between the polypeptide and the ligand.

33. A ligand, characterized in that it binds to a polypeptide according to claim 28 or 29.

34. A composition, characterized in that it comprises at least one ligand according to claim 33, in an acceptable pharmaceutical excipient, for use as a medicament.

35. A method for the production of a polypeptide according to claim 28 or 29, characterized in that it is used for diagnostic or therapeutic purposes.