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ELISA kit for H9N2 influenza virus hemagglutinin protein

A technology of hemagglutinin protein and influenza virus is applied in the field of quantitative H9N2 influenza virus hemagglutinin protein double-antibody sandwich ELISA detection kit, which can solve the problems of difficult promotion, quantitative detection and poor specificity of influenza virus subtype detection. , to achieve the effects of high detection sensitivity and accuracy, convenient and easy inspection method, and low cost

Active Publication Date: 2015-07-01
北京义翘神州科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, H9N2 is still based on the quantitative PCR method based on nucleic acid detection. This method has high requirements on the environment, samples and instruments, requires professional operation, and is not easy to promote.
In addition, the existing H9N2 detection reagents based on the principle of ELISA have the disadvantage of being unable to carry out quantitative detection, and the detection specificity for different influenza virus subtypes is poor, so there is an urgent need for a quantitative detection reagent with simple operation, good sensitivity and high specificity

Method used

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  • ELISA kit for H9N2 influenza virus hemagglutinin protein
  • ELISA kit for H9N2 influenza virus hemagglutinin protein
  • ELISA kit for H9N2 influenza virus hemagglutinin protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1H9

[0015] Component preparation of embodiment 1H9N2 influenza hemagglutinin protein ELISA kit

[0016] 1. Preparation of mouse monoclonal antibody:

[0017] 1) Animal immunity

[0018] Balb / c mice were used as immunized animals, and the recombinant H9N2 influenza hemagglutinin protein (product number: 11229-V08H) produced by Sino Biological Technology Co., Ltd. protein. For the first immunization, the immunogen and the same amount of complete Freund's adjuvant were used to make an emulsifier, and the abdomen was injected subcutaneously at multiple points, and after an interval of 2 to 3 weeks, the same dose of the immunogen and the same amount of incomplete Freund's adjuvant were used to make an emulsifier , two times of booster immunization, and three times after the indirect ELISA method was used to measure the serum titer. After the serum titer reached 1:16000, the mouse with the highest titer was selected for a booster immunization once in the peritoneal cavity, and splenoc...

Embodiment 2H9

[0042] The formation of embodiment 2H9N2 influenza hemagglutinin protein ELISA kit

[0043] The assembled ELISA kit contains the following reagents:

[0044] a) mouse monoclonal coating antibody;

[0045] b) HRP-labeled rabbit polyclonal antibody;

[0046] c) H9N2 influenza hemagglutinin protein standard;

[0047] d) Coating buffer: pH9.6, 0.05mol / L carbonate buffer;

[0048] e) Blocking solution: Tris buffer containing 2% bovine serum albumin;

[0049] f) Sample diluent: phosphate buffer containing 0.1% bovine serum albumin;

[0050] g) Washing solution: phosphate buffer containing 0.1% Tween;

[0051] h) Substrate chromogenic solution: composed of chromogenic solution A and chromogenic solution B, chromogenic solution A is hydrogen peroxide or carbamide peroxide, and chromogenic solution B is tetramethylbiphenyl;

[0052] i) Stop solution: 2mol / L sulfuric acid

Embodiment 3H9

[0053] The preparation of embodiment 3H9N2 influenza hemagglutinin protein ELISA kit

[0054] 1. Use orthogonal experiments to explore the optimal antibody combination and working concentration of ELISA kits

[0055] According to the ultraviolet spectrophotometer method, the concentration of antibody and antigen was determined. Orthogonal test method was used to explore the best combination of antibodies and the best concentration of antibodies to use, dilute different anti-H9N2 influenza hemagglutinin protein monoclonal antibodies to concentrations of 4 μg / ml, 2 μg / ml, 1 μg / ml, recombinant hemagglutinin The protein concentration was diluted to 1000pg / ml, 100pg / ml, 0pg / ml, and the HRP-labeled rabbit polyclonal antibody was diluted to 4μg / ml, 2μg / ml, 1μg / ml, 0.5μg / ml. Considering the background of the blank well and the light absorption value of the positive test well, a mouse monoclonal antibody was selected as the coating antibody, and its optimal working concentration was c...

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Abstract

The invention discloses a double-antibody sandwich ELISA kit for H9N2 influenza A virus hemagglutinin protein. The kit comprises a solid phase vector coated with a monoclonal antibody, a rabbit polyclonal antibody labeled by horseradish peroxidase, an H9N2 hemagglutinin protein standard substance, a sample diluting liquid, a washing liquid, a substrate coloring liquid and a reaction terminating liquid. The kit is good in sensitivity, capable of performing quantitative detection for the H9N2 influenza virus hemagglutinin protein and specifically recognizing H9N2 influenza A viruses, and free of cross reactions with hemagglutinin protein of other main subtypes comprising H1N1, H2N2, H3N2, H5N1 and H7N7 of the influenza A viruses and hemagglutinin protein of influenza B viruses. The kit is simple to operate and capable of rapidly detecting a large number of samples simultaneously, can be used for supporting fundamental research of the H9N2 influenza viruses, and has important significance for performing epidemiologic study on influenza viruses.

Description

technical field [0001] The invention belongs to the field of immunology, and in particular relates to the preparation of H9N2 influenza virus hemagglutinin protein-specific monoclonal antibody and a quantitative H9N2 influenza virus hemagglutinin protein double-antibody sandwich ELISA detection kit. Background technique [0002] Avian influenza is a disease caused by avian influenza virus, and the clinical symptoms shown vary with the virus subtype. The 16 subtypes of hemagglutinin (H1~H16) and the 9 subtypes of neuraminidase (N1~N9) of influenza A virus that have been found so far can be isolated from birds, but not all of them can cause Avian Influenza. At present, the main avian influenza viruses that can infect humans are: H5N1, H9N2, H7N7, H7N2, H7N3 and H7N9 subtypes. [0003] H9N2 is an avian influenza virus that belongs to the genus Influenza A in the family Orthomyxoviridae. In 1966, HoMee isolated the first H9N2 subtype avian influenza virus from turkeys sufferi...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/535
CPCG01N33/531G01N33/56983G01N33/6803G01N2333/11
Inventor 谢良志罗春霞孙春昀张杰李东张延静李雁王加兰
Owner 北京义翘神州科技股份有限公司
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