Quality control method for anti-influenza a virus np protein antibody and application thereof

Abstract

The application relates to the technical field of immunodetection methods, and discloses a quality control method for anti-influenza A virus NP protein antibodies and application, wherein a quality control antigen is a recombinant influenza A virus H3N2 subtype NP protein, the amino acid sequence of which is shown as SEQ ID NO:1, and the antigen is prepared through an inclusion body renaturation process. The NP protein of the specific subtype (H3N2) prepared through the inclusion body renaturation process can more sensitively'recognize' the antibody performance changes caused by slight disturbances (such as too high cell passage number, culture process suspension, low cell viability, etc.) in the production process, compared with the NP proteins prepared through other preparation modes (such as cytosolic soluble expression) or other subtypes (such as H1N1).

Description

Technical Field

[0001] This invention relates to the field of immunoassay methods, specifically to a quality control method and application for anti-influenza A virus NP protein antibodies. Background Technology

[0002] Influenza viruses are classified into three types: A, B, and C. Influenza A virus, due to its high variability, is the primary pathogen causing influenza pandemics. The nucleoprotein (NP) of influenza A virus is the core structural protein of the virus and is highly conserved. It is the core detection target for the vast majority of clinical immunodiagnostic reagents (such as colloidal gold assay kits). The performance of these assay kits is highly dependent on the specificity and stable batch-to-batch consistency of the anti-NP protein antibody used.

[0003] Currently, the preparation processes for anti-NP protein antibody raw materials, whether through hybridoma-induced mouse ascites or recombinant expression in mammalian cells, are limited by the inherent complexity of the biological system (such as individual mouse differences, fluctuations in cell growth and metabolism, cell passage number, and perturbations in culture parameters), resulting in significant batch-to-batch performance inconsistencies. For example, excessive cell passage, unexpected culture interruption, and decreased cell viability can all lead to decreased antibody activity or conformational changes, thus affecting the stability of the final detection reagent. Conventional physicochemical property detection methods (such as SDS-PAGE purity testing and concentration determination) have poor correlation with key performance characteristics such as antibody functionality and immunomodulation, making it difficult to effectively identify antibody batches with performance defects. Therefore, developing a quality control method that is highly correlated with antibody performance and can sensitively distinguish between batch-to-batch differences is crucial for ensuring the robustness of the performance of influenza A virus NP protein immunoassay reagents. Summary of the Invention

[0004] The purpose of this invention is to provide a quality control method and application for anti-influenza A virus NP protein antibodies, particularly a quality control antigen for distinguishing the performance of anti-influenza A virus NP protein antibodies under different production conditions and its application, so as to solve the problem in the prior art that it is difficult to effectively distinguish the differences in antibody performance under different production conditions.

[0005] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a quality control method for antibodies against influenza A virus NP protein, comprising the following steps: S1. Provide a quality control antigen, wherein the quality control antigen is a recombinant influenza A virus H3N2 subtype NP protein prepared by inclusion body refolding process; S2. Contact the monoclonal antibody against the NP protein of influenza A virus to be tested with the quality control antigen, and detect the binding activity of the antibody to be tested with the quality control antigen; S3. Based on the detected binding activity, determine the performance of the antibody to be tested; if the binding activity detection result is weakly positive or negative, determine that the performance of the antibody to be tested is unqualified or has defects; if the detection result is strongly positive, determine that the performance of the antibody to be tested is qualified.

[0006] In some embodiments, the amino acid sequence of the recombinant influenza A virus H3N2 subtype NP protein is shown in SEQ ID NO:1.

[0007] Specifically, the amino acid sequence is shown in SEQ ID NO:1: MASQGTKRSYEQMETDGDRQNATEIRASVGKMIDGIGRFYIQMCTELKLSDHEGRLIQNSLTIEKMVLSAFDERRNKYLEEHPSAGKDPKKTGGPIYRRVDGKWMRELVLYDKEEIRRIWRQAN NGEDATSGLTHLMIWHSNLNDATYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGIGTMVMELIRMIKRGINDRNFWRGENGRKTRSAYERMCNILKGKFQTAAQKAMVDQVRESRNPG NAEIEDLIFLARSALILRGSVAHKSCLPACAYGPAVSSGYDFEKEGYSLVGIDPFKLLQNSQIYSLIRPNENPAHKSQLVWMACHSAAFEDLRLLSFIRGTKVSPRGKLSTRGVQIASNENMDNM GSSTLELRSGYWAIRTRSGGNTNQQRASAGQTSVQPTFSVQRNLPFEKSTIMAAFTGNTEGRTSDMRAEIIRMMEGAKPEEVSFRGRGVFELSDEKAANPIVPSFDMSNEGSYFFGDNAEEYDN.

[0008] In some embodiments, the inclusion body refolding process includes: separating the recombinantly expressed H3N2 subtype NP protein from the host cell in the form of inclusion bodies, dissolving it in a buffer solution containing a denaturing agent, and refolding it by gradually decreasing the concentration of the denaturing agent.

[0009] In some embodiments, the host cell is Escherichia coli; the denaturing agent is urea; and the refolding is performed using gradient dialysis.

[0010] In some embodiments, the detection of binding activity in step S2 is performed on an immunochromatographic platform.

[0011] In some embodiments, the immunochromatographic platform is a colloidal gold immunochromatographic test strip.

[0012] In some implementations, in step S3, a strong positive result is defined as a test strip detection line (T line) showing a deeper color, and a weak positive result is defined as a test strip detection line (T line) showing a lighter color.

[0013] The core of this invention lies in the fact that NP proteins of a specific isotype (H3N2) prepared through inclusion body refolding technology, compared to NP proteins prepared by other methods (such as cytoplasmic soluble expression) or other isotypes (such as H1N1), can more sensitively "identify" changes in antibody performance caused by subtle interferences during the production process (such as excessive cell passage number, culture pause, low cell viability, etc.). This difference, which cannot be reflected by conventional physicochemical tests (such as SDS-PAGE purity testing), can be clearly presented by the method of this invention, thus providing an efficient and intuitive quality control method for batch-to-batch consistency of antibody raw materials. This method is particularly suitable for monoclonal antibodies targeting conformationally sensitive epitopes of NP proteins.

[0014] Secondly, the present invention provides the use of recombinant influenza A virus H3N2 subtype NP protein in the preparation of quality control standards for evaluating batch-to-batch consistency of anti-influenza A virus NP protein antibodies, wherein the amino acid sequence of the recombinant influenza A virus H3N2 subtype NP protein is shown in SEQ ID NO:1, and it is prepared by inclusion body refolding process.

[0015] Specifically, the recombinant influenza A virus H3N2 subtype NP protein prepared by inclusion body refolding process is tested on an immunochromatographic platform by contacting the test antibody against the anti-influenza A virus NP protein monoclonal antibody to determine the performance of the antibody.

[0016] The recombinant H3N2 NP protein provided by this invention is prepared through an inclusion body refolding process. Compared with cytoplasm-soluble recombinant H3N2 NP protein, inclusion body-refolded recombinant H1N1 NP protein, and cytoplasm-soluble recombinant H1N1 NP protein, it can specifically reflect the performance differences of anti-influenza A virus NP protein antibodies under different production conditions. Experiments show that all four proteins can be recognized by commercially available mainstream colloidal gold reagent kits, indicating that they all have basic binding ability with anti-NP antibodies. However, in the detection system assembled with antibodies obtained under production condition disturbances (such as abnormal production conditions simulated by poor expression conditions such as excessively high cell passage number, culture interruption, and excessively low cell viability, but not limited to these three abnormal situations), only the inclusion body-refolded recombinant H3N2 NP protein of this invention shows a significantly weakened binding signal (weak positive), while the other three comparison proteins still show strong positive results. This indicates a synergistic effect between the specific amino acid sequence of the H3N2 subtype NP protein and the inclusion body refolding process, which together maintain a specific conformational epitope that is highly sensitive to antibody quality. This technical effect cannot be predetermined by those skilled in the art based on existing technology.

[0017] Based on the above technical solution, the embodiments of the present invention can produce at least the following technical effects: (1) High discrimination: The quality control method provided by the present invention can effectively distinguish the differences in antibody performance caused by disturbances in production conditions, while conventional purity detection and other methods cannot achieve this distinction.

[0018] (2) Simple and fast: This method can be implemented on an immunochromatographic platform (such as colloidal gold test strips), without the need for complex equipment, is easy to operate, and the results are visible to the naked eye. It is suitable as a rapid initial screening tool for antibody batch-to-batch differences.

[0019] (3) Low cost: The quality control antigen used is recombinant expression of Escherichia coli, which has low preparation cost, easy process scale-up, and good industrial application prospects.

[0020] (4) Great application potential: The quality control antigen and quality control method of the present invention can not only be used for the quality control of antibody raw materials, but also have the potential to be developed into a performance verification standard for influenza A virus detection kits, thereby improving the overall quality of the detection kits. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0022] Figure 1 This is an SDS-PAGE detection spectrum of monoclonal antibodies against influenza A virus NP protein prepared under normal and simulated abnormal production conditions according to the present invention. In the diagram: Lane 1, 19C7-0; Lane 2, 27H4-0; Lane 3, 19C7-1; Lane 4, 19C7-2; Lane 5, 19C7-3; Lane 6, 27H4-1; Lane 7, 27H4-2; Lane 8, 27H4-3.

[0023] Figure 2 This diagram shows the color development of the recombinant H2N3 NP protein against the antibody test strips of Ab27H4-0, Ab27H4-1, Ab27H4-2, and Ab27H4-3 according to the present invention; in the figure, S: sample well; C: control line; T: test line.

[0024] Figure 3 This diagram shows the color development of the recombinant H2N3 NP protein against the antibody test strips of Ab19C7-1, Ab19C7-2, and Ab19C7-3 according to the present invention; in the figure, S: sample well; C: control line; T: test line. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0029] Example 1: Preparation of Recombinant H3N2 NP Protein from Inclusion Body Refolding S1. Gene synthesis and expression vector construction: The company Sangon Biotech (Shanghai) Co., Ltd. was commissioned to synthesize the nucleotide sequence (amino acid sequence as shown in SEQ ID NO:1) encoding the H3N2 NP protein, and cloned it into the pET-28a(+) expression vector to construct the recombinant expression plasmid pET-28a(+)-H3N2.

[0030] S2. Induction of Expression: The above plasmid was transformed into *E. coli* BL21(DE3) competent cells. Single colonies were picked and inoculated into LB medium containing 50 μg / mL kanamycin and cultured overnight at 37°C and 220 rpm. 1.5 mL of the overnight culture was transferred to 50 mL of LB liquid medium with a final concentration of 50 μg / mL kanamycin and cultured for 6 h at 37°C and 220 rpm. 15 mL of the 6-h culture was transferred to 500 mL of LB medium with a final concentration of 50 μg / mL kanamycin and cultured at 37°C and 230 rpm with constant temperature shaking. When the OD600 reached 0.6-0.8, 500 μL of 1 mol / L isopropyl-β-D-thiogalactoside (IPTG) was added, and the cells were induced at 37°C and 220 rpm for 5-6 h.

[0031] S3. Inclusion Body Purification and Renaturation: Bacterial cells were collected by centrifugation, resuspended in 50 mM, pH 7.4 phosphate buffer, and then sonicated. The inclusion body precipitate was collected by centrifugation. The precipitate was washed with 50 mM, pH 7.4 phosphate buffer containing 1 M NaCl and 2 M urea to obtain inclusion bodies. The washed inclusion bodies were dissolved in 50 mM, pH 7.4 phosphate buffer containing 8 M urea. The dissolved protein was re-renatured using a gradient dialysis method: sequentially dialyzed in buffers containing 4 M, 2 M, 1 M, 0.5 M, and 0 M urea (50 mM, pH 7.4 phosphate buffer containing 150 mM NaCl) at 4°C, with the dialysate changed every 12 hours. After dialysis, the supernatant was collected by centrifugation, which is the recombinant H3N2 NP protein (Ib-H3N2) of this invention.

[0032] The amino acid sequence of the H3N2 NP protein is shown in SEQ ID NO:1: MASQGTKRSYEQMETDGDRQNATEIRASVGKMIDGIGRFYIQMCTELKLSDHEGRLIQNSLTIEKMVLSAFDERRNKYLEEHPSAGKDPKKTGGPIYRRVDGKWMRELVLYDKEEIRRIWRQAN NGEDATSGLTHLMIWHSNLNDATYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGIGTMVMELIRMIKRGINDRNFWRGENGRKTRSAYERMCNILKGKFQTAAQKAMVDQVRESRNPG NAEIEDLIFLARSALILRGSVAHKSCLPACAYGPAVSSGYDFEKEGYSLVGIDPFKLLQNSQIYSLIRPNENPAHKSQLVWMACHSAAFEDLRLLSFIRGTKVSPRGKLSTRGVQIASNENMDN MGSSTLELRSGYWAIRTRSGGNTNQQRASAGQTSVQPTFSVQRNLPFEKSTIMAAFTGNTEGRTSDMRAEIIRMMEGAKPEEVSFRGRGVFELSDEKAANPIVPSFDMSNEGSYFFGDNAEEYDN Example 2: Comparative Preparation of Recombinant Protein 1. Preparation of cytoplasm-soluble recombinant H3N2 NP protein (Sol-H3N2) S1. Using the same expression vector pET-28a(+)-H3N2 as in Example 1, transform *E. coli* BL21(DE3) competent cells. Pick single colonies and inoculate them into 5 mL of LB medium containing 50 μg / mL kanamycin, and culture overnight at 37°C and 220 rpm. Transfer 1.5 mL of the overnight culture to 50 mL of LB liquid medium with a final concentration of 50 μg / mL kanamycin, and culture for 6 h at 37°C and 220 rpm. Transfer 15 mL of the 6 h culture to 500 mL of LB medium with a final concentration of 50 μg / mL kanamycin, and culture at 37°C and 230 rpm with constant temperature shaking. When the OD600 reaches 0.6-0.8, add 500 μL of 1 mol / L isopropyl-β-D-thiogalactoside (IPTG), and induce for 16 h at 16°C and 220 rpm. After culture, collect the cells by centrifugation.

[0033] S2. After resuspending the cells in 50mM phosphate buffer (pH 7.4) containing 500mM NaCl and 20mM imidazole, the cells were sonicated to disrupt the cell structure. The supernatant was collected by centrifugation and purified by Ni-NTA affinity chromatography. Chromatography packing material: NiSepharose6FastFlow (Cytiva), column height 10cm.

[0034] Equilibration buffer: 50mM phosphate buffer, pH 7.4, containing 500mM NaCl and 20mM imidazole.

[0035] Wash buffer: 50mM phosphate buffer, pH 7.4, containing 500mM NaCl and 50mM imidazole.

[0036] Elution buffer: 50mM phosphate buffer, pH 7.4, containing 500mM NaCl and 250mM imidazole.

[0037] Flow rate: 200-300 cm / h. Equilibrate with 3-5 column volumes of equilibration buffer before loading the sample. After loading, wash with 3-5 column volumes of washing buffer, followed by 3-5 column volumes of elution buffer. Collect the separated components based on UV absorption changes.

[0038] The purified fraction was preserved by ultrafiltration or dialysis in 50mM phosphate buffer containing 150mM NaCl at pH 7.4, which is the cytoplasm-soluble recombinant H3N2 protein.

[0039] 2. Preparation of inclusion body-refolded recombinant H1N1NP protein (Ib-H1N1) S1. Following the method of Example 1, but replacing the H3N2 sequence with the H1N1 NP protein sequence (amino acid sequence as shown in SEQ ID NO:2), prepare inclusion body-renatured recombinant H1N1 NP protein: The amino acid sequence of the H1N1 NP protein is shown in SEQ ID NO:2: MASQGTKRSYEQMETGGERQDTTEIRASVGRMIGGIGRFYIQMCTELKLSDYDGRLIQNSITIERMVLSAFDERRNKYLEEHPSAGKDPKKTGGPIYRRIDGKWTRELILYDKEEIRRVWRQAN NGEDATAGLTHIMIWHSNLNDATYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGVGTIAMELIRMIKRGINDRNFWRGENGRRTRVAYERMCNILKGKFQTAAQRAMMDQVRESRNPG NAEIEDLIFLARSALILRGSVAHKSCLPACVYGLAVASGHDFEREGYSLVGIDPFKLLQNSQVVSLMRPNENPAHKSQLVWMACHSAAFEDLRVSSFIRGKKVIPRGKLSTRGVQIASNENVET MDSNTLELRSRYWAIRTRSGGNTNQQKASAGQISVQPTFSVQRNLPFERATIMAAFSGNNEGRTSDMRTEVIRMMESAKQEDLSFQGRGVFELSDEKATNPIVPSFDMSNEGSYFFGDNAEEYDN Construct the recombinant expression plasmid pET-28a(+)-H1N1.

[0040] Induction of expression: The above plasmid was transformed into *E. coli* BL21(DE3) competent cells. Single colonies were picked and inoculated into LB medium containing 50 μg / mL kanamycin and cultured overnight at 37°C and 220 rpm. 1.5 mL of the overnight culture was transferred to 50 mL of LB liquid medium with a final concentration of 50 μg / mL kanamycin and cultured for 6 h at 37°C and 220 rpm. 15 mL of the 6-h culture was transferred to 500 mL of LB medium with a final concentration of 50 μg / mL kanamycin and cultured at 37°C and 230 rpm with constant temperature shaking. When the OD600 reached 0.6-0.8, 500 μL of 1 mol / L isopropyl-β-D-thiogalactoside (IPTG) was added, and the cells were induced at 37°C and 220 rpm for 5-6 h.

[0041] Inclusion body purification and renaturation: Bacterial cells were collected by centrifugation, resuspended in 50 mM, pH 7.4 phosphate buffer, and then sonicated. The inclusion body precipitate was collected by centrifugation. The precipitate was washed sequentially with 50 mM, pH 7.4 phosphate buffer containing 1 M NaCl and 2 M urea to obtain inclusion bodies. The washed inclusion bodies were dissolved in 50 mM, pH 7.4 phosphate buffer containing 8 M urea. The dissolved protein was renatured using a gradient dialysis method: sequentially dialyzed in buffers containing 4 M, 2 M, 1 M, 0.5 M, and 0 M urea (50 mM, pH 7.4 phosphate buffer containing 150 mM NaCl) at 4°C, with the dialysate changed every 12 hours. After dialysis, the supernatant was collected by centrifugation, which is the recombinant H1N1 NP protein (Ib-H1N1) of this invention.

[0042] 3. Preparation of cytosolic recombinant H1N1 NP protein (Sol-H1N1) for expression. Following the method of Example 2, but replacing the H3N2 sequence with the H1N1 NP protein sequence (SEQ ID NO:2), a soluble form of recombinant H1N1 NP protein was prepared. Construct the recombinant expression plasmid pET-28a(+)-H1N1.

[0043] Transformed into *E. coli* BL21(DE3) competent cells. Single colonies were picked and inoculated into 5 mL of LB medium containing 50 μg / mL kanamycin, and cultured overnight at 37°C and 220 rpm. 1.5 mL of the overnight culture was transferred to 50 mL of LB liquid medium with a final kanamycin concentration of 50 μg / mL, and cultured for 6 h at 37°C and 220 rpm. 15 mL of the 6-h culture was transferred to 500 mL of LB medium with a final kanamycin concentration of 50 μg / mL, and cultured at 37°C and 230 rpm with shaking. When the OD600 reached 0.6-0.8, 500 μL of 1 mol / L isopropyl-β-D-thiogalactoside (IPTG) was added, and induction was performed at 16°C and 220 rpm for 16 h. Cells were collected by centrifugation after the culture was complete.

[0044] Cells were resuspended in 50mM phosphate buffer (pH 7.4) containing 50mM NaCl and 20mM imidazole, then sonicated to disrupt the cells. The supernatant was collected by centrifugation and purified by Ni-NTA affinity chromatography. Chromatography packing material: NiSepharose6FastFlow (Cytiva), column height 10cm.

[0045] Equilibration buffer: 50mM phosphate buffer, pH 7.4, containing 500mM NaCl and 20mM imidazole.

[0046] Wash buffer: 50mM phosphate buffer, pH 7.4, containing 500mM NaCl and 50mM imidazole.

[0047] Elution buffer: 50mM phosphate buffer, pH 7.4, containing 500mM NaCl and 250mM imidazole.

[0048] Flow rate: 200-300 cm / h. Equilibrate with 3-5 column volumes of equilibration buffer before loading the sample. After loading, wash with 3-5 column volumes of washing buffer, followed by 3-5 column volumes of elution buffer. Collect the separated components based on UV absorption changes.

[0049] The purified fraction was preserved by ultrafiltration or dialysis in 50mM phosphate buffer containing 150mM NaCl at pH 7.4, which is the cytoplasm-soluble recombinant H1N1 protein.

[0050] Example 3: Preparation and Preliminary Characterization of Normal and Abnormal Antibodies Normal-quality monoclonal antibodies against influenza A virus NP protein, Ab19C7-0 and Ab27H4-0, were prepared according to the following monoclonal antibody sequences and methods (described in CN120795134B and CN120795135B). The monoclonal antibody sequences are as follows: The 19C7 sequence is as follows: Heavy chain (SEQ ID NO:3): EVQLQQSGPELVKPGASVKMSCTASGYTFTSYTVHWVRQKPGQGLEWIGYILPYNDGTKYNEKFKGKATLTSDRSSSTAYMELNSLTSEDSAVFYCARWGWDGFDYWGQG TTLTISSASTKGPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCG CKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTKPREEQINSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTI SKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK Light chain (SEQ ID NO:4): DVVMTQTPLSLPVSLGDQASISCRSSQSFVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLRISRVEAEDLGVYFCSQSAHIPPTFGGGTKLEIKRTVAAPSVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC The 27H4 sequence is as follows: Heavy chain (SEQ ID NO: 5): QVQLQQPGAELVKPGASVKLSCKTSGYIFTSYWMHWLKQRPGHGLEWIGEINPSNGRTNYNEKFKTKATLTVDKSSSTAYIQLSSLRSEDSAVYYCARDDYDGDWGQGTTLTVSSASTKGPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTKPREEQINSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK Light chain (SEQ ID NO: 6): DILMTQSPSSMSVSLGDTVSITCHASQGISSNIGWLQQKPGKSFKGLIYHGTNLEDGVPSRFSGSGSGADYSLTISSLEFEDFADYYCVHYAQFPYTFGGGTKLEIKRTVAAPSVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 1. Preparation of Ab19C7-0 and Ab27H4-0 Sequence synthesis and expression The monoclonal antibody sequences 19C7 and 27H4 were synthesized by Shanghai Sangon Biotech (Shanghai) Co., Ltd., and inserted into the PTT5 vector. Following the Thermo Fisher FreeStyle™ 293 Expression System User Manual, the vector containing the recombinant protein sequences was transfected into HEK293-F cells, and the cell culture supernatant was collected.

[0051] The above operations are known to those skilled in the art, and the above single-chain antibodies can also be obtained by other plasmid construction, cell transfection, and culture methods known in the art.

[0052] Separation and purification Collect the cell supernatant from HEK293-F cells and add it to a protein A affinity chromatography column equilibrated with 20 mM, pH 7.4 phosphate buffer at a linear flow rate of 200-400 cm / h to enrich the target protein. After loading, elute with 20 mM, pH 3.5 acetate buffer and collect the eluent.

[0053] Collect the avidin affinity chromatography elution fraction, concentrate it using a 30kD ultrafiltration tube, replace the buffer, and store the recombinant protein in 20mM phosphate buffer containing 100mM sodium chloride at pH 7.4.

[0054] The above operations are known to those skilled in the art, and other purification and separation methods known in the art can also be used to obtain the above recombinant protein.

[0055] To simulate the quality abnormalities of monoclonal antibodies due to production condition disturbances, the following measures were taken: HEK293 cells passaged for more than 30 generations were used for expression; the shaking incubator was stopped for 1 hour during expression; and the expression culture was continued until the cell viability was below 50%. These parameters represent poor conditions in routine production and can represent extreme fluctuations in production conditions during antibody production. Ab19C7-1, Ab19C7-2, Ab19C7-3 and Ab27H4-1, Ab27H4-2, Ab27H4-3 were prepared under the above conditions to simulate abnormal conditions.

[0056] Both the normal and abnormal antibodies mentioned above were detected by SDS-PAGE, and the results are as follows: Figure 1 As shown in the figure. The results indicate that the heavy and light chain bands of the antibodies are clear, their migration positions are correct, and their purity is not less than 90%. This demonstrates that conventional physicochemical detection methods cannot distinguish between antibodies produced under normal and abnormal production conditions.

[0057] Example 4: Detection of the performance of anti-H1N1 influenza virus NP protein monoclonal antibodies using different recombinant H3N2 NP proteins and recombinant H1N1 NP proteins. According to the test reagent preparation method disclosed in Chinese Patent CN120992935B, colloidal gold immunochromatographic test strips were prepared by pairing Ab19C7-0 and Ab27H4-0 with normal performance prepared in Example 3, as well as Ab19C7-1, Ab19C7-2, Ab19C7-3 and Ab27H4-1, Ab27H4-2, Ab27H4-3 prepared under abnormal conditions.

[0058] Using coating buffer, goat anti-mouse IgG polyclonal antibodies (Ab27H4-0, Ab27H4-1, Ab27H4-2, and Ab27H4-3) were diluted to 0.8 mg / mL and 0.4 mg / mL, respectively. Using a coating apparatus, each coating buffer was evenly spread onto a nitrocellulose membrane at a rate of 1 μL / cm, thus forming the control line C and the influenza A virus detection line T. The membranes were then dried at 55°C for 4 days.

[0059] Ab19C7-0, Ab19C7-1, Ab19C7-2, and Ab19C7-3 were conjugated onto latex microspheres and sprayed onto the conjugate pads. The dosage was 0.8 μg of influenza A 19C7 antibody per centimeter of conjugate pad. Afterward, the microspheres were placed in a 37°C drying oven for 2.5 hours, and finally assembled into a colloidal gold immunochromatographic test strip.

[0060] The four recombinant proteins (Ib-H3N2, Sol-H3N2, Ib-H1N1, and Sol-H1N1) prepared in Examples 1 and 2 were diluted to 2.5 μg / mL using sample extraction buffer. These diluted solutions were then used as samples for detection. The colorimetric results of the detection line (T line) were interpreted after 15 minutes. The results are shown in Table 1. Table 1. Detection results of different recombinant NP proteins against normal / abnormal antibody test strips.

[0061] The colorimetric reaction of recombinant H2N3 NP protein on normal / abnormal antibody test strips is as follows: Figure 2 , Figure 3 As shown, S: sample well (sample loading area); C: control line; T: detection line.

[0062] "Strong positive" means the T line is darker, "medium positive" means the T line is moderately bright, "weak positive" means the T line is lighter, and "negative" means the T line is not bright.

[0063] The results showed that only the inclusion body-folded recombinant H3N2 NP protein (Ib-H3N2) exhibited a significant weak positive result on the abnormal antibody test strips, while the other three control proteins showed strong positive results on all test strips. This result indicates that the inclusion body-folded recombinant H3N2 NP protein provided by this invention can sensitively respond to performance differences in antibodies produced under different abnormal production conditions or perturbations in production parameters, and its colorimetric gradient has a discriminative ability that is visible to the naked eye. Further analysis of the data in Table 1 shows that this antigen exhibits a stable dose-response relationship and good batch-to-batch differentiation efficacy in different batches of test strips. This characteristic makes it not only a primary screening tool for antibody batch-to-batch quality (for rapid assessment of antibody consistency), but also has the potential to become a standard antigen for performance verification of H1N1 influenza test kits—subsequently, a semi-quantitative standard curve corresponding to the T-line gray value can be established to achieve rapid semi-quantitative assessment of antibody titer.

[0064] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A quality control method for antibodies against influenza A virus NP protein, characterized in that, Includes the following steps: S1. Provide a quality control antigen, wherein the quality control antigen is a recombinant influenza A virus H3N2 subtype NP protein prepared by inclusion body refolding process; S2. Contact the monoclonal antibody against the NP protein of influenza A virus to be tested with the quality control antigen, and detect the binding activity between the antibody to be tested and the quality control antigen. The monoclonal antibody against the NP protein of influenza A virus to be tested is Ab19C7 and Ab27H4. S3. Based on the detected binding activity, determine the performance of the antibody to be tested; if the binding activity detection result is weakly positive or negative, then determine that the antibody to be tested is unqualified or has defects. If the test result is strongly positive, the antibody to be tested is deemed to be of qualified performance.

2. The quality control method for anti-influenza A virus NP protein antibody according to claim 1, characterized in that, The amino acid sequence of the recombinant influenza A virus H3N2 subtype NP protein is shown in SEQ ID NO:

1.

3. The quality control method for anti-influenza A virus NP protein antibody according to claim 1, characterized in that, The inclusion body refolding process includes: separating the recombinant H3N2 subtype NP protein from the host cell in the form of inclusion bodies, dissolving it in a buffer solution containing a denaturing agent, and refolding it by gradually reducing the concentration of the denaturing agent.

4. The quality control method for anti-influenza A virus NP protein antibody according to claim 3, characterized in that, The host cell is Escherichia coli; the denaturing agent is urea; and the refolding is performed using gradient dialysis.

5. The quality control method for anti-influenza A virus NP protein antibody according to claim 1, characterized in that, The detection of binding activity in step S2 is performed on an immunochromatographic platform.

6. The quality control method for anti-influenza A virus NP protein antibody according to claim 5, characterized in that, The immunochromatographic platform is a colloidal gold immunochromatographic test strip.

7. The quality control method for anti-influenza A virus NP protein antibody according to claim 6, characterized in that, In step S3, a strong positive result is defined as a darker test line (T line) on the test strip, while a weak positive result is defined as a lighter color on the test line (T line).

8. The use of a recombinant influenza A virus H3N2 subtype NP protein in the preparation of a quality control standard for evaluating batch-to-batch consistency of anti-influenza A virus NP protein antibodies, characterized in that, The amino acid sequence of the recombinant influenza A virus H3N2 subtype NP protein is shown in SEQ ID NO:1, and it was prepared by inclusion body refolding process.

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