Hepatitis c antibody detection antigen compositions and uses, kits and methods of detection

Abstract

The application provides a hepatitis C antibody detection antigen composition and application, kit and detection method thereof, relates to the antibody detection technical field, and the hepatitis C antibody detection antigen composition comprises a labeled recombinant antigen and a coated recombinant antigen, the amino acid sequence of the labeled recombinant antigen includes Trx and hepatitis C Core region, NS3 protein, NS4b protein dominant epitope and His tag, and the full-length sequence is shown as SEQ ID NO. 1. The labeled recombinant antigen used in the application is obtained by mutating 7 cysteines (C) into alanine (A) by amino acid mutation on the NS3 protein, reducing the aggregation and precipitation caused by disulfide bond of the recombinant protein, and improving the protein stability. When colloidal gold is used for antigen labeling, a secondary indirect labeling method is adopted, which is beneficial to the exposure of antigen epitopes, can improve the reaction sensitivity, and reduces the antibody missed detection.

Description

Technical Field

[0001] This invention relates to the field of antibody detection technology, and more specifically, to a hepatitis C antibody detection antigen composition, its application, kit, and detection method. Background Technology

[0002] Hepatitis C, commonly known as HBV, is an infectious disease caused by the hepatitis C virus. It has a high incidence rate and can be transmitted through close contact, blood transfusions, and needle injection. Hepatitis C has an insidious onset, with few or no early symptoms. As the disease progresses, it can lead to cirrhosis, ascites, liver fibrosis, and even liver cancer, posing a significant threat to the patient's life. Hepatitis C antibodies, especially colloidal gold antibody testing, are commonly used in the diagnosis of hepatitis C.

[0003] The methodological principle of colloidal gold assay for hepatitis C antibody detection is mainly based on the combination of immunochromatography and colloidal gold labeling. Its core is to achieve detection through the specific binding reaction of antigen and antibody, combined with the colorimetric properties of colloidal gold. While existing colloidal gold double-antigen sandwich hepatitis C antibody detection kits improve the specificity of the reaction compared to the indirect colloidal gold method, false positives occasionally occur due to the non-specific adsorption of proteins and serum components other than HCV antibodies by colloidal gold particles and NC membranes, or due to non-specific reactions caused by impurities in the antigens used for coating or labeling. Furthermore, because colloidal gold particles have a relatively large diameter (typically 10-40 nm), direct coupling to the surface of the antigen may cause steric hindrance, hindering the binding of antigen and antibody (especially when the antibody binding site is located in a recessed area on the antigen surface), thus reducing detection sensitivity and making it easy to miss samples during the window period. Summary of the Invention

[0004] The purpose of this invention is to provide a hepatitis C antibody detection antigen composition, its application, a kit, and a detection method to improve the aforementioned problems. To achieve the above objective, the technical solution adopted by this invention is as follows:

[0005] In a first aspect, this application provides a hepatitis C antibody detection antigen composition, comprising a labeling recombinant antigen and a coating recombinant antigen, wherein the amino acid sequence of the labeling recombinant antigen is shown in SEQ ID NO.1.

[0006] Optionally, the amino acid sequence of the coating recombinant antigen is shown in SEQ ID NO.2.

[0007] This application also provides the use of the above-described hepatitis C antibody detection antigen composition, wherein the use is in the preparation of products for detecting hepatitis C antibodies.

[0008] Optionally, the product includes a colloidal gold detection kit, an immunofluorescence chromatography kit, or a latex microsphere immunochromatography kit.

[0009] This application also provides a colloidal gold-based hepatitis C antibody detection kit based on secondary indirect labeling, comprising the hepatitis C antibody detection antigen composition described in this application, and further comprising colloidal gold particles, a polyclonal antibody against mouse anti-Trx antibody, and mouse anti-Trx antibody.

[0010] Optionally, the polyclonal antibody against the mouse anti-Trx antibody may include a goat polyclonal antibody or a rabbit polyclonal antibody.

[0011] Optionally, the recombinant antigen used for labeling and the biological material associated with the recombinant antigen used for coating are any of the following:

[0012] (a) A nucleic acid molecule encoding the recombinant antigen as described in claim 2;

[0013] (b) Expression cassette containing the nucleic acid molecules in (a);

[0014] (c) A recombinant vector containing the nucleic acid molecule in (a) or the expression cassette in (b);

[0015] (d) Recombinant prokaryotic cells containing the nucleic acid molecule in (a), the expression cassette in (b), or the recombinant vector in (c);

[0016] (e) Recombinant eukaryotic cells containing the nucleic acid molecule in (a), the expression cassette in (b), or the recombinant vector in (c).

[0017] Optionally, the amino acid sequence of the recombinant antigen used for labeling includes Trx and the hepatitis C Core region, NS3 protein, NS4b protein dominant epitope, and His tag;

[0018] The NS3 protein uses the NS3 protein sequence of the 1a subtype HCV strain, and at least one segment of the NS3 protein has been mutated with amino acid mutations, changing cysteine ​​to alanine.

[0019] Optionally, the amino acid sequence of the coating recombinant antigen includes an N-terminal His tag, an NS3 fragment, a Core fragment, an NS4B dominant epitope, and a C-terminal His tag;

[0020] The coating is obtained by cascading recombinant antibody from various sequence fragments, or by finally mixing and coating protein sequence fragments that express each epitope.

[0021] This application also provides a colloidal gold method for detecting hepatitis C antibodies based on secondary indirect labeling, wherein the method uses the kit described in this application, comprising:

[0022] The colloidal gold particles were labeled with a polyclonal antibody against mouse anti-Trx antibody to obtain the first labeled product;

[0023] The mouse anti-Trx antibody was labeled with the first marker to obtain the second marker;

[0024] The third marker is obtained by labeling the recombinant antigen with the Trx tag with the second marker;

[0025] The sample to be tested is added to the sample pad and forms a first complex with the third marker. As the sample migrates to the detection line by chromatography, the sample at the detection line is captured by the recombinant antigen, forming a second complex. The detection result is obtained based on the displayed color.

[0026] The beneficial effects of this invention are as follows:

[0027] The recombinant antigen used in this invention is a fusion protein in which Trx is tandemly expressed with the dominant epitopes of the hepatitis C Core region, NS3 protein, NS4b protein, and a His tag. The addition of hydrophilic linker peptides between the epitopes facilitates epitope exposure, improves reaction sensitivity, and reduces antibody false negatives. Amino acid mutations are performed on the NS3 protein, replacing seven cysteine ​​(C) residues with alanine (A), reducing aggregation and precipitation caused by disulfide bonds in the recombinant protein and improving protein stability. The secondary indirect labeling method used when labeling colloidal gold with the antigen further facilitates epitope exposure, improves reaction sensitivity, and reduces antibody false negatives.

[0028] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing embodiments of the invention. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments.

[0030] Antibody detection kits typically use recombinant proteins or synthetic peptides as coating or labeled antigens to mimic antigenic epitopes on viral proteins, thereby capturing specific antibodies in patient serum. Commonly used proteins include:

[0031] Core protein:

[0032] Diagnostic significance: Anti-core antibodies are usually among the first antibodies to appear and also among the antibodies that persist the longest after infection. They are a key component of antibody detection kits.

[0033] NS3 protein:

[0034] Location and function: Non-structural protein 3, possessing protease and helicase activities. Its C-terminal portion (approximately 27 kDa) is often used as an antigen.

[0035] Diagnostic significance: Anti-NS3 antibodies usually appear in the early stages of infection and are one of the important diagnostic markers. They have high immunogenicity.

[0036] NS4 protein:

[0037] Location and function: Non-structural protein 4, often subdivided into NS4A and NS4B. NS4A is a cofactor of the NS3 protease, while NS4B is involved in the formation of the viral replication complex.

[0038] Diagnostic significance: Anti-NS4 antibodies usually appear slightly later than anti-core and anti-NS3 antibodies. Recombinant proteins or specific synthetic peptides (such as C100-3, derived from NS4) are often used as antigens. Anti-NS4 antibodies are an important component of antibody detection and help improve the specificity of the test.

[0039] NS5 protein:

[0040] Location and function: Non-structural protein 5 is the viral RNA-dependent RNA polymerase, responsible for viral RNA replication. It is often subdivided into NS5A and NS5B.

[0041] Diagnostic significance: Anti-NS5 antibodies appeared relatively late. NS5B (polymerase) or specific regions of NS5 (such as NS5 c200 and c33c antigens) are often used as antigens. They are an important component of third-generation and later kits.

[0042] Modern anti-HCV detection kits (especially third- and fourth-generation kits) do not use a single protein, but rather a combination of multiple antigens (recombinant proteins or synthetic peptides) derived from the core protein, NS3, NS4, and NS5. This multi-antigen combination significantly improves the sensitivity (reducing false negatives, especially in early infection or in immunocompromised patients) and specificity (reducing false positives).

[0043] Example 1

[0044] This embodiment provides a hepatitis C antibody detection antigen composition, comprising a recombinant antigen for labeling and a recombinant antigen for coating. The amino acid sequence of the recombinant antigen for labeling is shown in SEQ ID NO.1; the recombinant antigen for labeling is designated C01, and its protein sequence is as follows:

[0045] Trx + Hepatitis C Core region + NS4B protein dominant epitope + NS3 protein (1a subtype, 7C→7A) + hydrophilic sequence + His;

[0046] Trx: The commonly used hydrophilic protein tag thioredoxin (Trx protein) was selected as a tag protein that is beneficial for the expression of recombinant protein supernatant and indirect labeling.

[0047] Core: Hepatitis C core protein (Core) was used for antigenic epitope analysis. Regions with concentrated antigenic epitopes were selected, and non-specific binding sites were removed. 79aa was selected as the Core epitope.

[0048] Core antigen epitope amino acid sequence:

[0049] STNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPG;

[0050] NS4B: Antigenic epitope analysis was performed on the NS4B protein of hepatitis C. Regions with concentrated antigenic epitopes were selected, and amino acid mutations were performed in combination with other subtypes. 43aa was selected as the NS4B epitope.

[0051] NS4B antigenic epitope amino acid sequence:

[0052] PGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTQILSS;

[0053] NS3 (subtype 1a, 7C→7A): The NS3 protein uses the NS3 protein sequence of the 1a subtype HCV strain, and at least one segment of the NS3 protein has undergone amino acid mutation, mutating cysteine ​​to alanine. Specifically, in this embodiment, hepatitis C NS3 protein was used for antigenic epitope analysis, selecting regions with concentrated antigenic epitopes, and combining with other subtypes for amino acid mutation, selecting 264aa as the NS3 epitope. Amino acid mutation of the NS3 protein, changing 7 cysteines (C) to alanine (A), reduces aggregation and precipitation caused by disulfide bonds in the recombinant protein, improving protein stability.

[0054] NS3 (subtype 1a, 7C→7A) epitope amino acid sequence: LETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGVDPNIRTGVRTITTGSPITYSTYGKFLADGGASGGAYDI I IADEAHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVSHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFAHSKKKADELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVSTDALMTGFTGDFDSVIDANTAVTQTVDFS;

[0055] Hydrophilic sequence: Random hydrophilic amino acids increase the hydrophilicity of the protein, while also facilitating the exposure of the His tag and its binding to affinity chromatography packing material, thereby improving protein purification efficiency.

[0056] Hydrophilic amino acid sequence:

[0057] GSTQQNTGGPQTTSGGQQNGYPTSQ;

[0058] Multi-antigen combinations significantly improve detection sensitivity and reduce false negatives. The sequence of epitopes in the C01 recombinant protein is crucial. The Trx protein tag, located at the N-terminus, facilitates its binding to the Trx monoclonal antibody, resulting in the correct spatial arrangement on the colloidal gold particle surface. The NS3 protein, located at the C-terminus, facilitates the exposure of the antigenic epitope, enhancing its activity and thus detecting the NS3 antibody, which is most abundant in generally positive serum. The Core and NS4B protein epitopes, situated in the middle of the antigen amino acid sequence, serve to supplement the antigenic epitope.

[0059] SEQ ID NO.1:

[0060] MSDKI IHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAK

[0061] LNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKEFLDANLAMAGGSTN

[0062] PKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRR

[0063] QPIPKARRPEGRTWAQPGGGTSPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAAR

[0064] VTQILSSGGEFLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQ

[0065] GYKVLVLNPSVAATLGFGAYMSKAHGVDPNIRTGVRTITTGSPITYSTYGKFLADGGA

[0066] SGGAYDI I IADEAHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVSHPNIE

[0067] EVALSTTGEIPFYGKAIPLEVIKGGRHLIFAHSKKKADELAAKLVALGINAVAYYRGL

[0068] DVSVIPTSGDVVVVSTDALMTGFTGDFDSVIDANTAVTQTVDFSGSTQQNTGGPQTTSGGQQNGYPTSQLEHHHHHH.

[0069] The amino acid sequence of the recombinant antigen used for coating is shown in SEQ ID NO.2; the recombinant antigen used for coating is denoted as CO2; the CO2 protein sequence is: N-terminal His + NS3 fragment + Core fragment + NS4B dominant epitope + C-terminal His;

[0070] His: The antigen has a His tag with 6 histidine residues at both ends, which is beneficial for protein purification.

[0071] NS3: Antigenic epitope analysis was performed on the NS3 protein of hepatitis C. Regions with concentrated antigenic epitopes were selected, and amino acid mutations were performed in combination with other subtypes. 265aa was selected as the NS3 epitope.

[0072] NS3 antigen white epitope amino acid sequence:

[0073] METTTRSPVFTDNSSPPAVPQTFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPSVRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDSTTILGIGTVL DQAETAGARLVVLATATPPGSVTVPHPNIQEVALSNTGEIPFYGKAIPIEAIRGGRHLIFCHSKKKCDELAAKLSSLGLNAVAYYRGLDVSVIPSSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFS;

[0074] Core: Hepatitis C core protein (Core) was used for antigenic epitope analysis. Regions with concentrated antigenic epitopes were selected, and non-specific binding sites were removed. 79aa was selected as the Core epitope.

[0075] NS4B: Antigenic epitope analysis was performed on the NS4B protein of hepatitis C. Regions with concentrated antigenic epitopes were selected, and amino acid mutations were performed in combination with other subtypes. 43aa was selected as the NS4B epitope.

[0076] The sequence of epitopes in the CO2 recombinant protein is crucial. The His tag, located at the N-terminus and C-terminus, facilitates exposure and binding to affinity chromatography materials, improving protein purification efficiency. The NS3 protein's proximity to the N-terminus promotes exposure of the antigenic epitope, enhancing its activity and thus detecting the NS3 antibody, which is most abundant in generally positive serum. The Core and NS4b protein epitopes, located at the C-terminus of the antigenic amino acid sequence, serve to supplement the antigenic epitope sequence.

[0077] SEQ ID NO.2:

[0078] MHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMETTTRSPVFTDNSSPPAVPQTFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPSVRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDI IICDECHSTDSTTILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIQEVALSNTGEIPFYGKAIPIEAIRGGRHLIFCHSKKKCDELAAKLSSLGLNAVAYYRGLDVSVIPSSGDVVVVATDALMTGFTGDFDSVIDCNTCVT QTVDFSASGGSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGGGTSPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTQILSSGGLEHHHHHH.

[0079] The coating antigen CO2 can also be obtained by tandemly connecting various sequence fragments to express each epitope, or by finally mixing and coating protein sequence fragments that express each epitope.

[0080] Example 2

[0081] This embodiment provides the application of the hepatitis C antibody detection antigen composition of Example 1, wherein the application is in the preparation of products for detecting hepatitis C antibodies.

[0082] As an optional implementation, the product includes a colloidal gold detection kit, an immunofluorescence chromatography kit, or a latex microsphere immunochromatography kit.

[0083] Example 3

[0084] This embodiment discloses a colloidal gold-based hepatitis C antibody detection kit based on secondary indirect labeling, comprising the hepatitis C antibody detection antigen composition described in Example 1, and further comprising colloidal gold particles, a polyclonal antibody against mouse anti-Trx antibody, and mouse anti-Trx antibody.

[0085] Optionally, the polyclonal antibody against the mouse anti-Trx antibody may include a goat polyclonal antibody or a rabbit polyclonal antibody.

[0086] The method for synthesizing the recombinant antigen for labeling includes:

[0087] The amino acid sequences of the Core antigenic epitope, NS4b antigenic epitope, NS3 (1a subtype, 7C→7A) antigenic epitope, and hydrophilic amino acid sequences were tandemly linked, and an NcoI restriction site was added at the N-terminus and an XhoI restriction site was added at the C-terminus to obtain the synthetic gene C01-pUC.

[0088] C01-pUC was ligated into the pET30a-Trx expression vector via NcoI / XhoI double enzyme digestion to construct a recombinant plasmid;

[0089] The recombinant plasmid was transformed into E. coli clones, single spots on plates were picked for colony PCR, positive spots were screened and plasmids were extracted.

[0090] The extracted plasmid was transformed into E. coli expression bacteria, and E. coli expression was induced to obtain expressed E. coli sludge;

[0091] The expressed E. coli bacterial sludge was resuspended and ultrasonically disrupted. After centrifugation and sedimentation, the supernatant was collected and purified to obtain the purified recombinant antigen for labeling. The specific procedures are as follows:

[0092] S110 and C01 gene synthesis;

[0093] The amino acid sequences of the Core antigenic epitope, NS4b antigenic epitope, NS3 (1a subtype) antigenic epitope, and hydrophilic amino acid sequences were tandemly linked, and an NcoI restriction site was added at the N-terminus and an XhoI restriction site was added at the C-terminus to obtain the synthetic gene C01-pUC.

[0094] S120, double enzyme digestion;

[0095] The expression vector used is pET30a-Trx, which has a Trx sequence at its N-terminus, making the final expressed protein a Trx-tagged protein at its N-terminus.

[0096] The C01-pUC plasmid and pET30a-Trx plasmid were double-digested with NcoI and XhoI to form the backbone. The digestion system was as shown in Table 1, and the digestion was carried out at 30℃ for 30 min. The restriction enzymes used in this example were purchased from Beijing TransGen Biotech Co., Ltd., and were their Flycut series rapid digestive enzymes.

[0097] Table 1 Double enzyme digestion system

[0098]

[0099]

[0100] S130, Construct recombinant plasmids;

[0101] The enzyme digestion system was subjected to agarose gel electrophoresis and DNA purification and recovery. The gel-recovered product was ligated through a ligation system for 15-18 hours to obtain the ligation product.

[0102] The gel-recovered products were ligated for 16 hours according to the ligation system shown in Table 2. The DNA ligase Solution I was purchased from TAKARA.

[0103] Table 2 Connection System

[0104]

[0105] The ligation product was transformed into *E. coli* Trans5α competent cells to obtain the recombinant plasmid. *E. coli* Trans5α competent cells were purchased from Beijing TransGen Biotech Co., Ltd.

[0106] Completed amino acid sequence:

[0107] MSDKI IHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKEFLDANLAMAGGSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKA RRPEGRTWAQPGGGTSPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTQILSSGGEFLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGVDPNIRTGVRTITTGSPITYSTYGKFLADGGASGGAYDI I IADEAHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVSHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFAHSKKKADELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVSTDALMTGFTGDFDSVIDANTAVTQTVDFSGSTQQNTGGPQTTSGGQQNGYPTSQLEHHHHHH*.

[0108] S140, positive spot screening and plasmid extraction;

[0109] Single spots of white *E. coli* were observed growing on plates containing transformed clones. These single spots were selected for colony PCR. The PCR primers were pET-fw: CACTATAGGGGAATTGTGAGCGGATAAC; pET-rv: CTCAGCTTCCTTTCGGGCTTTGTTAG. The components of the colony PCR reaction system are shown in Table 3. The PCR reaction program was as follows: pre-denaturation: 94℃ for 5 min; cycles: 94℃ for 30 s, 60℃ for 30 s, 72℃ for 150 s, for a total of 30 cycles; extension: 72℃ for 5 min. The theoretical size of the PCR product fragment from C01 positive spots is 1846 bp.

[0110] Table 3 Colony PCR Reaction System

[0111] Template DNA 0.5μl pET-fw(20μM) 1μl pET-rv (20μM) 1μl 2×TaqMix 10μl ddH2O 7.5μl Total volume 20μl

[0112] The selected positive plaques were cultured overnight, and plasmids were extracted the following day using the OMEGA plasmid mini-prep kit. The obtained C01 plasmid was identified as positive by double digestion with NdeI and XhoI, and then sent to the Beijing sequencing department of Sangon Biotech Co., Ltd. for sequencing. The sequence was completely consistent with the design and could be used to transform E. coli expression bacteria.

[0113] S150 and C01 plasmids were transformed into Escherichia coli expression bacteria;

[0114] Take 1 μl of C01 plasmid and add it to 50 μl of *E. coli* BL21(DE3) competent cells. Incubate on ice for 30 min, heat shock at 42°C for 90 s, incubate on ice for 2 min, add 500 μl of LB medium (antibiotic-free), and incubate at 37°C and 200 rpm for 1 hour. Spread 60 μl of the culture medium onto a plate (containing 50 μg / ml kanamycin sulfate). The *E. coli* BL21(DE3) competent cells used in this example were purchased from Beijing TransGen Biotech Co., Ltd.

[0115] The successfully transformed expression bacteria were preserved in sterile glycerol to obtain glycerol bacteria;

[0116] The glycerol-containing strain of the expression bacteria was inoculated into 300 ml of culture medium (containing 50 μg / ml kanamycin sulfate) and cultured in a shake flask at 37°C and 200 rpm until OD reached. 600 =0.8, add IPTG to induce expression (final IPTG concentration 0.7mM), and continue culturing for 4 hours. Centrifuge the bacterial culture at 6500rpm for 10min, discard the supernatant, and collect the bacterial sludge.

[0117] S160, E. coli, ultrasonically broken up;

[0118] The expressed *E. coli* bacterial sludge was resuspended in 10 mM Tris-HCl (pH 8.0) at a ratio of 1 g of sludge to 40 ml of buffer. The cells were then sonicated for 20 min at 200 W for 2 seconds followed by a 2-second pause. The completely sonicated bacterial solution was clear and showed minimal precipitation after centrifugation at 11000 rpm for 10 min. 30 μl of the supernatant was collected as a sample for SDS-PAGE analysis to detect the expression of the target protein. C01 protein expression was detected in the supernatant. All supernatant was used for subsequent experiments.

[0119] Purification of S170 and C01 proteins;

[0120] Recombinant antigens were purified using a Ni-NTA affinity column, following the instructions of the Ni-NTA Sefinose™ Resin Kit. The procedure generally included the following steps: E. coli supernatant was added to the affinity column; the target protein bound to Ni ions and remained in the column, while other proteins flowed through. Non-specifically adsorbed proteins were removed by passing the column through a solution of 20 mM Tris-HCl (pH 8.0) + 150 mM NaCl + 50 mM imidazole. The target protein was then eluted with 20 mM Tris-HCl (pH 8.0) + 150 mM NaCl + 200 mM imidazole to obtain the protein solution, i.e., the CO1 protein. The purified target protein was then subjected to SDS-PAGE to determine its purity; the purity was ≥80%.

[0121] S200, Synthesis of recombinant antigens for coating;

[0122] The method for synthesizing the recombinant antigen used for coating is basically the same as the method for synthesizing the recombinant antigen used for labeling, except that:

[0123] The synthesis of gene fragments coated with recombinant antigens includes: tandemly connecting the amino acid sequences of the Core antigen epitope, the NS4b antigen epitope, and the NS3 antigen epitope, and adding an NcoI restriction site at the N-terminus and an XhoI restriction site at the C-terminus to obtain the synthetic gene CO2-pUC.

[0124] The expression vector used is pET30a, which has His tags at the N-terminus and C-terminus, so that the N-terminus and C-terminus of the final expressed protein are His tags.

[0125] This embodiment also provides a method for preparing mouse anti-Trx antibody:

[0126] S310, Immunity;

[0127] The Trx protein was mixed with an equal volume of Freund's adjuvant to prepare a water-in-oil emulsion.

[0128] The mice used for monoclonal antibody preparation are derived from BALB / c mice. The initial immunization is best performed at 8-12 weeks of age, via subcutaneous injection at multiple sites on the back, followed by 3-4 immunizations. After the final immunization, a booster immunization is administered via intraperitoneal injection of 50 μg of antigen. The spleen is then harvested three days later for fusion.

[0129] S320, integration;

[0130] Mice were euthanized, and spleen cells were collected and prepared into a cell suspension. 1×10⁻⁶ cells were then added to the suspension. 8 Spleen cells and 1×10 7SP2 / 0 fusion of myeloma cells. 100 μl of 20% fetal bovine serum-1640HAT medium was dispensed into 96-well cell culture plates with CO2, and the plates were then incubated at 37°C in a 5% CO2 incubator.

[0131] S330, Filtering;

[0132] 100 μl of cell supernatant was aspirated into a 96-well plate, and an antibody-labeled gold strip was inserted for detection. Positive polyclonal cells were then subjected to subcloning. The cloning method was limiting dilution, and positive cells were selected through multiple subcloning processes.

[0133] S340, Expanded cultivation;

[0134] When the cell coverage reaches 80%, it can expand to 110 cm. 2 Place the culture flask in a 37℃, 5% CO2 incubator and incubate statically.

[0135] S350, preparation of monoclonal antibodies;

[0136] Mice were sensitized with paraffin oil, and each mouse was injected intraperitoneally with 0.2 ml of the solution. Cell inoculation could be performed 7-15 days later. Collected monoclonal cells were injected intraperitoneally into each mouse with 1 ml of the solution. The growth of inoculated mice was monitored regularly; if the abdomen became rounded and the mice were in poor condition, ascites fluid was promptly aspirated. Ascites fluid should be collected within 15 days after cell injection and purified.

[0137] S360, purification;

[0138] S361. Ascites pretreatment;

[0139] Slowly add an equal volume of saturated (NH4)2SO4 to the collected ascites fluid. After completion, continue stirring at room temperature for 2 hours, then incubate at 2-8℃ for 12-16 hours. Centrifuge, discard the supernatant, dissolve the precipitate in 10 mmol / L PBS and dialyze for 24 hours, changing the solution every 2 hours. Remove the dialyzed antibody, centrifuge, determine its volume and concentration, and calculate the protein content. Store at 2-8℃ for later use or for further purification.

[0140] S362, CNBr-activated Sepharose 4B affinity chromatography purification;

[0141] Column packing: Pack CNBr-activated Sepharose4B packing material coupled with ligand (Trx protein) into the chromatography column.

[0142] Detection: Connect to an online ultraviolet detector, select 280nm wavelength, 0.5A setting.

[0143] Equilibration: After packing the CNBr-activated Sepharose 4B column, equilibrate it thoroughly with 20 mmol / L PBS (pH 7.2–7.4). Use a linear flow rate of 60 cm / h. Inject the sample only after the detector baseline has stabilized.

[0144] Sample loading: After equilibration, inject the sample at a linear flow rate of 60 cm / h and collect the breakthrough solution. After injection, flush the tubing and column with 20 mmol / L PBS (pH 7.2-pH 7.4) until the detector baseline is stable.

[0145] Elution: After leveling, the column was eluted with 0.1 mol / L glycine solution (pH 2.6) to remove the antibody sample attached to the column.

[0146] Collection: Collection of target peaks.

[0147] Sample preparation: Elute the sample and dialyze it with 10 mmol / PBS buffer (pH 7.2-pH 7.4) for 24 h, changing the dialysate twice, every 2-3 hours.

[0148] Storage: The protein concentration and volume of the collected antibody were determined, and the protein content was calculated to obtain mouse anti-Trx antibody. It was then stored by adding 0.02% NaN3.

[0149] This embodiment also provides a method for preparing goat polyclonal antibody (A01) of mouse anti-Trx antibody:

[0150] S410, immunized sheep;

[0151] The immunogen was mouse anti-Trx monoclonal antibody (B01).

[0152] For the initial immunization, Freund's complete adjuvant is used, and the bottle needs to be inverted several times to resuspend BO1 evenly. Use a 1000 μl pipette to draw the required volume of Freund's complete adjuvant, antigen, and physiological saline into a syringe. For subsequent immunizations, Freund's incomplete adjuvant is used, and the procedure is the same as for the initial immunization (the first immunization is the initial immunization, and all subsequent immunizations are subsequent immunizations, with an interval of 3-4 weeks between immunizations).

[0153] S420, Blood draw;

[0154] Weekly small-sample blood tests for polyclonal antibodies are conducted in sheep. Using disposable lancets and disposable vacuum blood collection tubes, 2-3 ml of blood is collected from each sheep. The blood collection tubes are centrifuged at 3000 rpm for 30 minutes, and the serum is then aspirated to determine the serum titer. If the serum titer is above L5, a larger-sample blood draw can be performed. If the test results do not meet the requirements, the sheep should be immunized again three to four weeks after the last immunization date. Small-sample tests should be conducted again in the second week after immunization.

[0155] Sheep serum centrifugation method: Place the drawn sheep blood into a primary centrifuge cup, set the centrifuge speed to 8500 rpm and the temperature to 4℃, and centrifuge for 20 min; after centrifugation, prepare a secondary centrifuge cup, place the serum from the primary centrifugation of two sheep of the same breed into the same secondary centrifuge cup and label it, set the centrifuge speed to 8500 rpm and the temperature to 4℃, and centrifuge for 20 min to obtain sheep polyantiserum.

[0156] S430, purification;

[0157] S431, serum pretreatment;

[0158] Thaw the goat polyantiserum at 2-8℃ and allow it to thaw completely. Centrifuge the serum at 15000g for 15 min at 4℃, filter the supernatant, and discard the precipitate. While stirring, slowly add saturated ammonium sulfate to the supernatant to a final concentration of 50% (V / V), continue stirring for 1-2 h, and incubate at 2-8℃ for 18-20 h to ensure all proteins precipitate. Centrifuge at 4℃ for 15 min, discard the supernatant, and dissolve the precipitate thoroughly in 10 mmol / L PBS (pH 7.2-pH 7.4) buffer. Dialyze for 24 h, changing the dialysis buffer 5-6 times, every 2-3 hours. Remove the dialyzed antibody, centrifuge at 15000g for 15 min, filter, determine the protein concentration and volume, and calculate the protein mass. Store at 2-8℃ for later use or continue purification.

[0159] S432, CNBr-activated Sepharose 4B affinity chromatography purification;

[0160] Column packing: Pack CNBr-activated Sepharose4B packing material coupled with ligand (B01 protein) into the chromatography column.

[0161] Detection: Connect to an online ultraviolet detector, select 280nm wavelength, 0.5A setting.

[0162] Equilibration: After packing the CNBr-activated Sepharose 4B column, equilibrate it thoroughly with 20 mmol / L PBS (pH 7.2–7.4). Use a linear flow rate of 60 cm / h. Inject the sample only after the detector baseline has stabilized.

[0163] Sample loading: After equilibration, inject the sample at a linear flow rate of 60 cm / h and collect the breakthrough solution. After injection, flush the tubing and column with 20 mmol / L PBS (pH 7.2-pH 7.4) until the detector baseline is stable.

[0164] Elution: After leveling, the column was eluted with 0.1 mol / L glycine solution (pH 2.6) to remove the antibody sample attached to the column.

[0165] Collection: Collection of target peaks.

[0166] Sample preparation: Elute the sample and dialyze it with 10 mmol / PBS buffer (pH 7.2-pH 7.4) for 24 h, changing the dialysate twice, every 2-3 hours.

[0167] Storage: Determine the protein concentration and volume of the collected antibody purified product, and calculate the protein content. Store in 0.02% NaN3.

[0168] Example 4

[0169] This embodiment provides a colloidal gold method for detecting hepatitis C antibodies based on secondary indirect labeling. The method uses the kit described in Example 3, comprising:

[0170] S510, Colloidal gold labeling (preparation of colloidal gold-A01-B01-C01 complex);

[0171] S511. Colloidal gold particles were labeled with goat polyclonal antibody against mouse anti-Trx antibody to obtain the first label;

[0172] Add 10 μl of 0.2 mol / L K2CO3 solution to each 1 ml of colloidal gold solution, mix thoroughly, then add 25 μg of A01 protein, mix thoroughly, and react for 15 min. Add 10 μl of 20% BSA solution to each 1 ml of colloidal gold solution, mix thoroughly, and react for 10 min. Centrifuge at 10000 rpm for 10 min, discard the supernatant, and resuspend the precipitate in 1 / 10 volume of TB9 solution to obtain the first label (colloidal gold-A01).

[0173] S512. Label the mouse anti-Trx antibody with the first marker to obtain the second marker;

[0174] Add 25 μg of B01 protein to every 100 μl of resuspension, mix thoroughly, and react for 10 min; centrifuge at 10000 rpm for 10 min, discard the supernatant; resuspend the precipitate in 1 / 10 volume of TB9 solution of the original colloidal gold solution. The second label (colloidal gold-A01-B01) is obtained.

[0175] S513. Label the recombinant antigen with the Trx tag using the second marker to obtain the third marker;

[0176] Add 0.1-0.4 μg of C01 protein to each 100 μl resuspension, mix thoroughly, and react for 10 min; centrifuge at 10000 rpm for 10 min, discard the supernatant; resuspend the precipitate in 1 / 10 volume of TB9 solution of the original colloidal gold solution. The third label (colloidal gold-A01-B01-C01) is obtained.

[0177] After each labeling step, a thorough washing process can be performed to preferentially remove unbound polyclonal antibodies and free monoclonal antibodies, avoiding false positives caused by residues; at the same time, it reduces the possibility of false positives caused by the electrostatic adsorption of positively charged impurities in the sample by the negative charge on the surface of colloidal gold particles.

[0178] S514, the colloidal gold-A01-B01-C01 resuspension was reconstituted with 9 / 10 volume of the original colloidal gold solution in DOA solution, and the solution was evenly coated onto a glass cellulose membrane (1200 glass fiber), 42ml / sheet.

[0179] S515. Freeze-dry or oven-dry the gold pads. After drying, pack the gold pads into aluminum foil bags, add desiccant, vacuum-pack them, and store them at 4-30℃.

[0180] S520, Coating CO2;

[0181] CO2 protein was diluted to 1.5 mg / ml with HCV coating buffer and uniformly coated onto an NC membrane (13502, 2.0 cm). The NC membrane (nitrocellulose membrane) was then dried in a drying room (temperature 24±4℃, humidity <25%) for 1-2 hours. After drying, the NC membrane was placed in an aluminum foil bag, desiccant was added, and the membrane was vacuum-packed and stored at 4-30℃.

[0182] S530, Assemble colloidal gold test strips;

[0183] Assemble the marked gold pad, coated nitrocellulose membrane, absorbent paper, PVC base plate, sample pad, and other auxiliary materials into a test strip. 7.35cm plate, 2.8cm absorbent paper, 4mm x 2 gold leaf, whole blood indirect method SP, 3.4mm wide strips.

[0184] S540. The sample to be tested is added to the sample pad and forms a first complex with the third marker. As the sample moves to the detection line by chromatography, the sample at the detection line is captured by the recombinant antigen to form a second complex. The detection result is obtained based on the displayed color.

[0185] Comparative Example 1

[0186] S610, preparation of control group;

[0187] Control group with colloidal gold labeling (preparation of colloidal gold-BO1-CO1 complex):

[0188] Add 10 μl of 0.2 mol / L K₂CO₃ solution to each 1 ml of colloidal gold solution, mix thoroughly, then add 25 μg of BO1 protein, mix thoroughly, and react for 15 min. Add 10 μl of 20% BSA solution to each 1 ml of colloidal gold solution, mix thoroughly, and react for 10 min. Centrifuge at 10000 rpm for 10 min, discard the supernatant, and resuspend the precipitate in 1 / 10 volume of TB9 solution from the original colloidal gold solution. This yields colloidal gold-BO1.

[0189] Add 4 μg of CO1 protein to every 100 μl of resuspension, mix thoroughly, and react for 10 min; centrifuge at 10000 rpm for 10 min, discard the supernatant; resuspend the precipitate in 1 / 10 volume of TB9 solution of the original colloidal gold solution. This yields colloidal gold-BO1-CO1.

[0190] The colloidal gold-B01-C01 resuspension was reconstituted with 9 / 10 volume of the original colloidal gold solution in DOA solution; the solution was uniformly coated onto a glass cellulose membrane (1200 glass fiber), 42 ml / membrane.

[0191] Freeze-dry or oven-dry the gold pads. After drying, pack the gold pads into aluminum foil bags, add desiccant, vacuum-pack them, and store them at 4-30℃.

[0192] Then, CO2 was coated and assembled into colloidal gold test strips using the same method as in Example 4, serving as a control kit.

[0193] S620, Sensitivity Test;

[0194] Sensitivity test samples: three types of positive sera: HCV-HP (strongly positive), HCV-MP (moderately positive), and HCV-COV (weakly positive);

[0195] The samples were tested using the kit from Example 4 and the control kit. The color development time and the final color intensity at 15 minutes were recorded. The color intensity was determined according to the color development card, which ranged from L0 to L10, with L0 being the lightest color and L10 being the darkest. The color development results are shown in Table 4.

[0196]

[0197]

[0198] Based on the above experimental data, the reagent kit group (using a two-step indirect labeling method) of this application has a shorter color development time and a deeper final color intensity compared with the control reagent kit group (using a one-step indirect labeling method). Therefore, it can be concluded that the sensitivity of the reagent kit group is higher than that of the control group.

[0199] S630, Specificity test;

[0200] Specific samples: 10 positive samples from enterprise trays (P1-P10), 20 negative samples from enterprise trays (N1-N20), and 1000 random clinical samples. The tests were performed using a kit, and the results are shown in Table 5.

[0201] Table 5. Results of Specificity Tests

[0202]

[0203] Sample #59 tested negative using the HCV antibody confirmatory kit, indicating that the clinical specificity of the kit group in this application is superior to that of the control kit group.

[0204] S640, Stability Test

[0205] The same batch of reagent kits from Example 4 was divided into two portions. One portion was placed in a 4°C refrigerator, and the other portion was placed in a 37°C constant temperature incubator for 6 days. After 6 days, it was taken out and placed in a 4°C refrigerator for overnight equilibration. Stability was tested using sensitivity samples, and the results are shown in Table 6.

[0206] Table 6. Results of stability test

[0207]

[0208]

[0209] The reagent kit of this application, when placed at 37℃ for 6 days, showed consistent sensitivity interpretation with the initial test kit placed at 4℃, indicating good stability.

[0210] Performance evaluation:

[0211] The above experiments show that the reagent kit of this application has higher sensitivity and specificity than the control group, and its stability is satisfactory. Therefore, it can be concluded that the two-step indirect labeling method is more effective than the one-step indirect labeling method.

[0212] Indirect labeling is like adding an "arm" between nanoparticles and labeled antigens, which is beneficial for exposing antigen epitopes. Secondary indirect labeling is like adding another "arm", which makes the exposure more complete and reduces the spatial steric interference between antigens, thus improving sensitivity.

[0213] The binding of the tag antibody to the tag protein and the binding of the tag antibody to the tag antibody are both specific bindings. The antigen is equivalent to undergoing a second specific screening, which reduces the possibility of non-specific impurity proteins being labeled onto the nanoparticles and can improve specificity.

[0214] Compared to primary indirect labeling, secondary indirect labeling results in a larger particle size and deeper color development of the colloidal gold-labeled complex, thus improving sensitivity. It is suitable for the highly specific detection of low concentrations of antibodies in complex samples (such as serum).

[0215] This invention can reduce the amount of recombinant antigen used for labeling and avoid the post-zone effect (hook effect, also known as the HOOK effect, refers to the phenomenon of false negatives caused by an inappropriate antigen-antibody ratio. Specifically, when there is an excess of antibody, it is called the pre-zone effect, and when there is an excess of antigen, it is called the post-zone effect) caused by excessive antigen.

[0216] The kit provided by this invention is easy to operate, requires no instruments, and results can be manually interpreted simply by adding whole blood (serum) samples. The detection time is short, requiring only 15 minutes for result interpretation. The labeled antibodies used are mouse anti-Trx monoclonal antibody (B01) and goat polyclonal antibody against mouse anti-Trx monoclonal antibody (A01). The labeled antigen used is a fusion protein of Trx with hepatitis C Core region, NS3 protein (1a subtype, 7C→7A), NS4b protein dominant epitope, and His tag tandemly expressed. The addition of hydrophilic linking peptides between the epitopes facilitates epitope exposure, improves reaction sensitivity, and reduces antibody false negatives. The coating antigen used is a fusion protein of hepatitis C NS3 protein, Core region, NS4b protein dominant epitope, and His tag tandemly expressed, which facilitates protein purification, reduces the influence of contaminating proteins on the reaction, and allows for simultaneous detection of multiple epitope antibodies, further improving reaction sensitivity and reducing antibody false negatives.

[0217] This method employs a colloidal gold secondary indirect labeling technique. Leveraging the high specificity of monoclonal antibodies, it ensures complete exposure of the active epitopes of the detected antigen, avoiding epitope masking or conformational changes caused by direct labeling, thus effectively improving detection specificity. Thorough washing after each labeling step prioritizes the removal of unbound polyclonal antibodies and free monoclonal antibodies, preventing false positives caused by residues. It also reduces the possibility of false positives caused by electrostatic adsorption of positively charged impurities in the sample due to the negative charge on the colloidal gold particle surface. Polyclonal and monoclonal antibodies act as "flexible connectors," reducing steric hindrance. The cascade of goat polyclonal antibodies amplifies the detection signal, improving detection sensitivity. It also reduces the amount of recombinant antigen used for labeling, avoiding false negatives caused by the after-band effect due to excessive antigen. This method exhibits high sensitivity and specificity.

[0218] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0219] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A hepatitis C antibody detection antigen composition, characterized in that, It includes a recombinant antigen for labeling and a recombinant antigen for coating, wherein the amino acid sequence of the recombinant antigen for labeling is shown in SEQ ID NO.

1.

2. The hepatitis C antibody detection antigen composition according to claim 1, characterized in that, The amino acid sequence of the coating recombinant antigen is shown in SEQ ID NO.

2.

3. The application of the hepatitis C antibody detection antigen composition according to any one of claims 1-2, characterized in that, The application is in the preparation of products for detecting hepatitis C antibodies.

4. The application of the hepatitis C antibody detection antigen composition according to claim 3, characterized in that, The product includes at least one of colloidal gold detection kit, immunofluorescence chromatography kit, or latex microsphere immunochromatography kit.

5. A colloidal gold-based hepatitis C antibody detection kit based on secondary indirect labeling, characterized in that, The hepatitis C antibody detection antigen composition according to claim 2 further includes colloidal gold particles, a polyclonal antibody against mouse anti-Trx antibody, and mouse anti-Trx antibody.

6. The colloidal gold-based hepatitis C antibody detection kit according to claim 5, characterized in that, The polyclonal antibodies against mouse anti-Trx include goat polyclonal antibodies or rabbit polyclonal antibodies.

7. The hepatitis C antibody detection kit based on secondary indirect labeling using colloidal gold as described in claim 5, characterized in that, The biomaterials associated with the labeling recombinant antigen and the coating recombinant antigen are any one of the following: (a) A nucleic acid molecule encoding a labeled recombinant antigen or a coating recombinant antigen as described in claim 2; (b) Expression cassette containing nucleic acid molecules from (a); (c) A recombinant vector containing either the nucleic acid molecule in (a) or the expression cassette in (b); (d) Recombinant prokaryotic cells containing the nucleic acid molecule in (a), the expression cassette in (b), or the recombinant vector in (c); (e) Recombinant eukaryotic cells containing the nucleic acid molecule in (a), the expression cassette in (b), or the recombinant vector in (c).

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