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Monoclonal antibody for identifying N6 subtype avian influenza virus neuraminidase protein and application of monoclonal antibody

A neuraminidase protein, avian influenza virus technology, applied in the direction of anti-virus immunoglobulin, anti-enzyme immunoglobulin, antibody, etc., can solve the problems of difficult to popularize and use, long experimental period, etc., and achieve good development prospects, High sensitivity and good repeatability

Active Publication Date: 2020-05-08
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the NA subtype detection method recommended by WHO is the neuraminidase inhibition test, but the test period of this method is long, and the experimental reagent sodium arsenite is a highly toxic reagent, so it is difficult to promote the use of this method in clinical practice

Method used

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  • Monoclonal antibody for identifying N6 subtype avian influenza virus neuraminidase protein and application of monoclonal antibody
  • Monoclonal antibody for identifying N6 subtype avian influenza virus neuraminidase protein and application of monoclonal antibody
  • Monoclonal antibody for identifying N6 subtype avian influenza virus neuraminidase protein and application of monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1N6

[0038] Preparation and Identification of Example 1N6 Subtype Avian Influenza Virus Neuraminidase Protein Monoclonal Antibody

[0039] 1.1 Preparation of immunogen and animal immunization

[0040] Three N6 subtype avian influenza viruses in different evolutionary branches were selected (CK / HuN / S3003 / 2009(H6N6), referred to as HuN3003; DK / HuB / SP143 / 2014(H5N6), referred to as HuB143; DK / HuN / SE226 / 2017 (H5N6), referred to as HuN226) as an immunogen, wherein the amino acid homology between HuN226 and HuN3003 is 95%, the amino acid homology between HuN3003 and HuB143 is 94.1%, and the amino acid homology between HuN226 and HuB143 is 91.7%. Firstly, the viruses HuN226, HuN3003 and HuB143 were diluted 10 times, and inoculated with 9-11-day-old SPF chicken embryos, placed in an incubator at 37°C for 48 hours, collected allantoic fluid, and added β- Propanolactone inactivates the virus. After the chicken embryo is inoculated, it is tested whether the virus inactivation is sufficient, ...

Embodiment 2

[0067] Example 2 Establishment of N6 Subtype Avian Influenza Virus Neuraminidase Protein Antibody Blocking ELISA Detection Method

[0068] 2.1 N6 subtype avian influenza virus neuraminidase protein antibody blocking ELISA operation method

[0069] (1) Set the titer of HA to 2 8 The inactivated antigen A / chicken / Hunan / S3003 / 2009 (H6N6) was diluted 1:80 with coating solution (50mmol / L carbonate buffer (pH 9.6)) and then coated with a microtiter plate, 100μL per well , overnight at 4°C.

[0070] (2) Discard the supernatant, wash the microplate with PBST solution (50mmol / L pH 7.2 phosphate buffer containing 0.05% Tween-20), 200μl per hole, wash 3 times and add 5% w / w The PBST solution of skim milk was used to block, 200 μL per well, and incubated at 37 ° C for 1 h.

[0071] (3) Discard the supernatant, wash the microtiter plate with PBST solution, 200 μl per well, wash 3 times, add the serum to be tested, negative serum and positive serum, 100 μL per well, and incubate at 37°C ...

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Abstract

The invention discloses a monoclonal antibody for identifying a N6 subtype avian influenza virus neuraminidase protein and an application of the monoclonal antibody. The monoclonal antibody is secreted by a hybridoma cell strain 3H7 with a preservation number of CGMCC No.18900. Experiments show that the monoclonal antibody 3H7 obtained by the invention can specifically bind to N6 subtype avian influenza viruses and can bind to 15 strains of N6 subtype avian influenza viruses with different evolutionary branches, thereby indicating that the 3H7 has a good broad spectrum; an ELISA detection kitfor N6 subtype avian influenza virus neuraminidase protein antibody blocking and a detection method are established by using the better broad spectrum and specificity of the monoclonal antibody; and experiments show that the method has high sensitivity and good reproducibility, can be used for detection of the antibody level of the N6 subtype avian influenza virus neuraminidase protein in a clinical serum sample, and has good development prospects.

Description

technical field [0001] The invention relates to a hybridoma cell strain capable of secreting a monoclonal antibody to N6 subtype avian influenza virus neuraminidase protein, as well as the monoclonal antibody secreted by the hybridoma cell strain and its application. The invention belongs to the field of biotechnology. Background technique [0002] Avian influenza (AI) is an acute infectious disease of poultry caused by influenza A virus of the Orthomyxoviridae family, and is listed as a severe infectious disease of type A by the World Organization for Animal Health. Avian influenza virus (AIV) is a single-stranded negative-segmented RNA virus with numerous subtypes, which can be divided into 16 according to the antigenic differences of the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). In addition, H17N10 and H18N11 subtype influenza viruses were isolated from bats. [0003] NA is a type II glycoprotein encoded by fragment 6. The NA protein forms a heter...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/10C07K16/40G01N33/577G01N33/569A61K39/42A61P31/16C12R1/91
CPCA61K2039/505A61P31/16C07K16/1018C07K16/40C07K2317/33C07K2317/35C07K2317/76G01N33/56983G01N33/577
Inventor 邓国华崔鹏飞陈化兰施建忠
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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