Avian influenza virus H9 subtype antibody enzyme-linked immunoassay kit

An enzyme-linked immunosorbent assay, avian influenza virus technology, applied in biological tests, measuring devices, material inspection products, etc., can solve the problems of human infection, high production cost, easy diffusion, etc., and achieve high sensitivity, improved efficiency, and repeated good effect

Active Publication Date: 2019-01-18
CHINA ANIMAL HUSBANDRY IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of these rely on purified viruses, the production cost is high, and there is a risk of infecting people and spreading easily. Therefore, it is urgent to develop a safe, efficient and convenient detection method for avian influenza antibodies

Method used

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  • Avian influenza virus H9 subtype antibody enzyme-linked immunoassay kit
  • Avian influenza virus H9 subtype antibody enzyme-linked immunoassay kit
  • Avian influenza virus H9 subtype antibody enzyme-linked immunoassay kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1, Preparation of Enzyme-linked Immunoassay Kit for Avian Influenza Virus H9 Subtype Antibody Coating Antigen

[0043] In this experiment, bioinformatics methods were used to accurately analyze the main epitopes of the HA protein of avian influenza virus H9 subtype, and suitable peptides were screened out. Three peptides were synthesized with an automatic peptide synthesizer, and the sequences were listed as the sequence table. As shown in Sequence 1, Sequence 2, and Sequence 3, the coated antigen with a purity of about 80% is prepared. The polypeptide synthesis method can be a conventional method, and the following method is used in the present invention to synthesize the three polypeptides of the present invention as the coating agent of the kit of the present invention.

[0044] The coated antigen of the present invention can be prepared using an Applied Biosystem automatic polypeptide synthesizer (model 433A). Using the Merrifield solid-phase synthesis meth...

Embodiment 2

[0073] Example 2, Preparation of Avian Influenza Virus H9 Subtype Antibody ELISA Kit

[0074] Avian influenza virus H9 subtype antibody ELISA kit includes:

[0075] (1) 96-well detachable polystyrene enzyme-linked reaction plate coated with H9 subtype HA antigen of avian influenza virus; 2×96 wells.

[0076] (2) Positive control serum: collect chicken serum after immunization with inactivated vaccine of avian influenza virus H9 subtype, and use it as the positive control serum of the kit (1 tube, 1.5ml / tube).

[0077] (3) Negative control serum: specific pathogen-free (SPF) chicken serum, used as the negative control serum of the kit (1 tube, 1.5ml / tube).

[0078] (4) Enzyme-labeled secondary antibody: prepared by diluting 1:30000 with horseradish peroxidase-labeled rabbit anti-chicken IgY (purchased from sigma company, product number A9046) as the stock solution, 2 bottles (12ml / bottle).

[0079] (5) Sample diluent: 0.01 M phosphate buffer solution with a pH value of 7.4 co...

Embodiment 3

[0086] Embodiment 3, sensitivity test of avian influenza virus H9 subtype antibody ELISA kit

[0087] 1. How to use the Avian Influenza Virus H9 Subtype Antibody ELISA Kit

[0088] 1. Equilibration: Take the kit out of the refrigerated environment, and put it at room temperature for 30 minutes for later use; mix the liquid reagents before use.

[0089] 2. Dosing: Dilute the concentrated washing solution 20 times with distilled water or deionized water to obtain washing buffer;

[0090] 3. Setting: 2 negative control wells and 2 positive control wells, and the rest are sample wells to be tested.

[0091] 4. Pre-dilution of the specimen to be tested: use the sample diluent to dilute the serum of the sample to be tested, the negative control serum, and the positive control serum at a ratio of 1:20.

[0092] 5. Adding samples: Add 100 μl of diluted samples to be tested in each well according to the preset setting. The time span of adding samples should be as short as possible. ...

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Abstract

The invention discloses an avian influenza virus H9 subtype antibody enzyme-linked immunoassay kit. The kit comprises an enzyme-labelled plate, a positive control serum, a negative control serum, an enzyme-labelled second antibody, a sample diluent, a sample diluent, a 20-fold concentrated washing solution, a substrate solution A, a substrate solution B and a stop solution, wherein the enzyme-labelled plate coats an avian influenza virus H9 subtype HA protein epitope polypeptide composition. The epitope polypeptide composition is any combination of one or more than two of polypeptide represented by a sequence 1 in a sequence list, polypeptide represented by a sequence 2 in the sequence list or polypeptide represented by a sequence 3 in the sequence list. The avian influenza virus H9 subtype antibody enzyme-linked immunoassay kit in the invention adopts an indirect method ELISA and uses a chemical synthetic antigen peptide to coat the enzyme-labelled plate; the use amount of antigen islow, and the sensitivity and the specificity are high; it can be effectively detected whether the avian influenza virus H9 subtype antibody exits. The kit in the invention has advantages of high sensitivity, good specificity, convenience for operations and good market aspect.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and more specifically, the invention relates to an ELISA detection kit for H9 subtype antibody of avian influenza virus. Background technique [0002] Avian influenza is a severe infectious disease of poultry caused by avian influenza virus (Avian influenza virus, AIV). The first outbreak occurred in Italy in 1878. At that time, the disease was called "fowl plague". Later, the disease was reported in many countries and regions, including the United States, Britain, Australia, Ireland, Belgium, the Netherlands, France, Russia, Canada, Israel, Hungary , Japan, China (including Hong Kong), etc. The H9N2 subtype avian influenza virus was first isolated from turkeys in North America in 1966, and began to appear in poultry in mainland China in 1994. [0003] Avian influenza virus is a type A influenza virus, belonging to the Orthomyxoviridae family Influenzavirus genus. It encodes 8 str...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/569G01N33/543
CPCG01N33/543G01N33/56983G01N33/6854
Inventor 肖进张蕾胡艳欣董春娜闫福栋王飞李静
Owner CHINA ANIMAL HUSBANDRY IND
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