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CN-121991213-A - Double-antibody sandwich ELISA kit for detecting H5 subtype avian influenza virus M1 protein

CN121991213ACN 121991213 ACN121991213 ACN 121991213ACN-121991213-A

Abstract

The application relates to the technical field of protein detection, and particularly provides a double-antibody sandwich ELISA kit for detecting H5 subtype avian influenza virus M1 protein. The application adopts the inactivated H5 subtype avian influenza virus which is concentrated and purified by sucrose density gradient centrifugation as an immunogen to immunize a BALB/c mouse, utilizes the M1 protein of the H5 subtype avian influenza virus to screen and obtain a detection antibody, and prepares a capture antibody after immunizing SPF chickens. Based on the research and development, a double-antibody sandwich ELISA kit for detecting the H5 subtype avian influenza virus M1 protein is obtained. The technology can be used for quantitatively detecting H5 subtype avian influenza M1 protein, controlling the quality of avian influenza subunit products, ensuring the stable quality of products, and also can be used for detecting the expression characteristics of recombinant avian influenza baculovirus virus seed protein.

Inventors

  • PANG WENQIANG
  • HONG SUMEI
  • TIAN KEGONG
  • ZHANG XUKE

Assignees

  • 普莱柯生物工程股份有限公司
  • 普莱柯(南京)生物技术有限公司

Dates

Publication Date
20260508
Application Date
20250715

Claims (10)

  1. 1. An antibody specifically binding to H5 subtype avian influenza virus M1 protein is characterized in that sucrose density gradient centrifugation is adopted to concentrate and purify inactivated H5 subtype avian influenza virus as an immunogen, BALB/c mice are immunized, and H5 subtype avian influenza virus M1 protein is utilized to screen and obtain the antibody.
  2. 2. The antibody of claim 1, wherein the antibody comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID No.2 and a light chain variable region having an amino acid sequence as set forth in SEQ ID No. 4.
  3. 3. The antibody of claim 1, wherein the antibody is monoclonal antibody 2D1, the monoclonal antibody 2D1 heavy chain subclass is IgG1, and the light chain subclass is kappa.
  4. 4. A nucleic acid comprising sequences encoding the heavy chain variable region and the light chain variable region of the antibody of any one of claims 1 to 3; Alternatively, the nucleotide sequence encoding the heavy chain variable region is shown as SEQ.ID NO. 1; Alternatively, the nucleotide sequence encoding the light chain variable region is shown as SEQ ID NO. 3.
  5. 5. The H5 subtype avian influenza virus protein capture antibody is characterized in that sucrose density gradient centrifugation, concentration and purification are adopted to inactivate H5 subtype avian influenza virus as an immunogen, SPF chicken is immunized to prepare the capture antibody, and the capture antibody is a polyclonal antibody.
  6. 6. The capture antibody of claim 5, wherein the polyclonal antibody is H5 subtype avian influenza virus positive serum.
  7. 7. Use of an antibody according to any one of claims 1 to 3 or a capture antibody according to claim 5 or 6 for the preparation of a product for detecting the M1 protein of avian influenza virus subtype H5.
  8. 8. A double-antibody sandwich ELISA kit for detecting H5 subtype avian influenza virus M1 protein, characterized in that the kit comprises an ELISA plate coated with a capture antibody and a detection antibody, the detection antibody being an antibody according to any one of claims 1 to 3, the capture antibody being the capture antibody according to claim 5 or 6.
  9. 9. The kit of claim 8, further comprising HRP-labeled goat anti-mouse IgG, wash, diluent, substrate chromogenic solution, stop solution, standard, positive control, and negative control; optionally, the capture antibody is H5 subtype avian influenza virus positive serum; optionally, the coating dilution of the H5 subtype avian influenza virus positive serum is 1:500; Optionally, the dilution of the detection antibody is 1:3000; Alternatively, the HRP-labeled goat anti-mouse IgG was used at a dilution of 1:2000.
  10. 10. A method for detecting the M1 protein of the H5 subtype avian influenza virus, which is characterized in that a kit as claimed in claim 8 or 9 is used, a sample to be detected is added into an ELISA plate coated with a capture antibody for reaction at 37 ℃ for 60min, then a detection antibody is added, then goat anti-mouse IgG marked by HRP is added for reaction at 37 ℃ for 45min, and then the chromogenic detection is carried out, and the qualitative or quantitative detection is carried out on the sample to be detected.

Description

Double-antibody sandwich ELISA kit for detecting H5 subtype avian influenza virus M1 protein Technical Field The application relates to the technical field of protein detection, and particularly provides a double-antibody sandwich ELISA kit for detecting H5 subtype avian influenza virus M1 protein. Background Avian influenza (Avian influenza, AI) is an acute infectious disease caused by avian influenza virus (Avianinfluen virus, AIV) and can cause a variety of symptoms ranging from respiratory to severe systemic sepsis. AIV belongs to the genus influenza A in the family of orthomyxoviridae, consists of 8 single-stranded negative strand RNAs, and encodes 11 proteins. H5 subtype highly pathogenic avian influenza virus (Highly pathogenic avian influenza virus, HPAIV) can often cause complete death of infected poultry, causing outbreaks of epidemic situation. Meanwhile, H5 subtype HPAIV can cause morbidity and mortality of human beings, and forms a serious threat to human health. The M1 Protein (Matrix Protein 1) of the H5 subtype avian influenza virus is one of the most important and abundant structural proteins, playing a core role in the life cycle and particle structure of the virus. Mainly composed of an alpha-helix, with a flexible N-terminal and a more structured C-terminal domain. It can form dimers or even oligomers. Relative conservation among the different subtypes of influenza a virus (including the H5 subtype) makes it a target for diagnostic and classification studies. M1 itself is generally not the primary target antigen for influenza vaccines (inactivated, live attenuated or recombinant HA vaccines). However, it is a highly conserved source of T cell epitopes, has potential in inducing cellular immune responses, and is one of the subjects in the development of universal influenza vaccines (aimed at inducing cross-protective T cell immunity against multiple influenza strains). Up to now vaccination has been the most effective means of preventing and controlling avian influenza virus pandemics. The gene engineering subunit vaccine is one of the main directions of influenza vaccine production, the application of a baculovirus expression system is mature, the protein expressed by the system has no infectious activity and is relatively easy to purify, the operation of live viruses and the treatment of a large number of chick embryo residues can be avoided in the vaccine production process, the industrial development concept of no biological safety risk, environmental friendliness and easy control of product quality is met, and the development direction of the veterinary vaccine production process upgrading is achieved. In the development process of subunit vaccine, the M1 protein expressed by recombinant baculovirus needs to be quantified, and protein quantification cannot be performed by Western blot identification of the conventional method for detecting the protein. Therefore, a double-antibody sandwich ELISA detection kit for the H5 subtype avian influenza virus M1 protein needs to be developed. In view of this, the present application has been made. Disclosure of Invention The application aims to provide a double-antibody sandwich ELISA kit for detecting H5 subtype avian influenza virus M1 protein, so as to fill the blank of the existing quantitative detection method for H5 subtype avian influenza virus M1 protein. In order to achieve the above purpose, the present application adopts the following technical scheme: an antibody specifically binding to H5 subtype avian influenza virus M1 protein adopts sucrose density gradient centrifugation, concentration and purification of inactivated H5 subtype avian influenza virus as immunogen, BALB/c mice are immunized, and H5 subtype avian influenza virus M1 protein is utilized to screen and obtain the antibody. Further, the antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.2, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 4. Further, the antibody is monoclonal antibody 2D1, the heavy chain subclass of monoclonal antibody 2D1 is IgG1, and the light chain subclass is kappa. A nucleic acid comprising sequences encoding the heavy chain variable region and the light chain variable region of the above antibody; Alternatively, the nucleotide sequence encoding the heavy chain variable region is shown as SEQ.ID NO. 1; Alternatively, the nucleotide sequence encoding the light chain variable region is shown as SEQ ID NO. 3. An H5 subtype avian influenza virus protein capture antibody is prepared by taking an inactivated H5 subtype avian influenza virus which is concentrated and purified by sucrose density gradient centrifugation as an immunogen and immunizing SPF chickens, and the capture antibody is a polyclonal antibody. Further, the polyclonal antibody is H5 subtype avian influenza virus positive serum.