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HA (hemagglutinin) epitope-ELISA (enzyme-linked immunosorbent assay) kit for H5-subtype AIV (avian influenza virus) antibodies

A bird flu virus and kit technology, applied in the field of HA antigen epitope-ELISA kit, can solve the problems of consuming a lot of manpower and material resources, failing to meet production needs, increasing research and development costs, etc., to achieve efficient acquisition and epidemiological investigation and antibody monitoring for practical effects

Active Publication Date: 2012-08-22
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the HA gene contains many rare codons of Escherichia coli, when the full-length HA gene is expressed in prokaryotic cells, the densely distributed rare codons in the sequence will have a great impact on the expression yield of the recombinant protein, which cannot meet the production requirements. Need; moreover, these rare codons are widely distributed, and their transformation requires a lot of manpower and material resources, which greatly increases the cost of research and development

Method used

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  • HA (hemagglutinin) epitope-ELISA (enzyme-linked immunosorbent assay) kit for H5-subtype AIV (avian influenza virus) antibodies
  • HA (hemagglutinin) epitope-ELISA (enzyme-linked immunosorbent assay) kit for H5-subtype AIV (avian influenza virus) antibodies
  • HA (hemagglutinin) epitope-ELISA (enzyme-linked immunosorbent assay) kit for H5-subtype AIV (avian influenza virus) antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1, H5 subtype avian influenza virus infection serum antibody HA epitope-ELISA kit

[0045] The HA epitope-ELISA kit for detecting serum antibody infected by H5 subtype avian influenza virus of the present invention consists of HA epitope recombinant protein (coated antigen for short) of H5 subtype avian influenza virus, coating liquid, PBST The composition of buffer solution, blocking solution, sample diluent, TMB storage solution, TMB substrate dilution solution and stop solution, its preparation method or composition are as follows:

[0046] 1. Preparation and identification of HA epitope recombinant protein coated with antigen-H5 subtype avian influenza virus

[0047] 1. Cloning of HA gene of H5 subtype avian influenza virus

[0048] Total RNA was extracted from duck liver tissue infected with H5N1 avian influenza virus A / Duck / GX / 1 / 00 (H5N1) by conventional experimental methods such as virus isolation and hemagglutination inhibition test, and cDNA was obt...

Embodiment 2

[0080] Embodiment 2, the specific detection of the HA epitope-ELISA kit of serum antibody of H5 subtype avian influenza virus infection

[0081] Using the kit prepared in Example 1, adopting indirect enzyme-linked immunosorbent assay (indirect ELISA) to carry out H5 subtype poultry serum (serum to be tested) identified through conventional experimental methods such as hemagglutination inhibition test and agar diffusion test as follows Influenza virus detection: H5 subtype avian influenza positive serum (positive control), SPF chicken serum (negative control), H7 subtype avian influenza positive serum (serum 1 to be tested), H9 subtype avian influenza positive serum (to be tested) Serum 2), infectious rhinitis positive chicken serum (serum to be tested 3), Newcastle disease positive chicken serum (serum to be tested 4), egg drop syndrome positive chicken serum (serum to be tested 5), infectious bronchitis positive Chicken serum (serum to be tested 6), the specific steps are as ...

Embodiment 3

[0098] Example 3, Sensitivity Detection of the HA Epitope-ELISA Kit for Serum Antibodies Infected with H5 Subtype Avian Influenza Virus

[0099] One, the sensitivity of the coated antigen in the kit of the present invention

[0100] Dilute the coated antigen in the kit of the present invention to 20 μg / μL, 15 μg / μL, 10 μg / μL, 5 μg / μL, 2 μg / μL, 1 μg / μL, 0.5 μg / μL, 0.25 μg / μL, coat the ELISA plate according to the antigen coating method in step 2 in Example 2, and repeat 3 times for each dilution. The H5 subtype avian influenza chicken positive serum (positive control) and the SPF chicken serum (negative control) identified through the hemagglutination inhibition test method were diluted 100 times with the sample diluent respectively, and the coating was measured according to the steps 3-6 of Example 2 Antigen sensitivity. The results are shown in Table 4, and the values ​​in the table are the average values ​​of 3 repetitions.

[0101] Table 4. Sensitivity detection results ...

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Abstract

The invention discloses an HA (hemagglutinin) epitope-ELISA (enzyme-linked immunosorbent assay) kit for H5-subtype AIV (avian influenza virus) antibodies. The kit can be used for detecting or assisting in detecting antisera of H5-subtype avian influenza viruses, and the kit internally contains an independently-packaged protein shown in the following a) or b) as a detecting antigen: a) a protein consisting of amino acid sequences shown as a sequence 2 in a sequence table; and b) a protein which is formed by that amino acid sequences (shown as the sequence 2 in the sequence table) suffer from substitution and / or deletion and / or addition through one or more amino acid residues and can carry out specific binding with antisera of H5-subtype avian influenza viruses. Experiments show that the kit disclosed by the invention can specifically detect to-be-detected animal antisera containing H5-subtype avian influenza viruses, the sensitivity reaches 1:800, and the accuracy rate reaches more than 92%; the kit is suitable to be used for detecting antisera of H5-subtype avian influenza viruses from chickens, ducks, gooses, wild birds, pigs, cattle and persons and the like; the kit both can be applied to the early diagnosis of H5-subtype avian influenzas, and can be applied to antibody monitoring; and the kit is extremely practical in the aspects of epidemiological survey and antibody monitoring in the actual production.

Description

technical field [0001] The invention relates to an HA antigen epitope-ELISA kit for H5 subtype avian influenza virus antibody. Background technique [0002] Avian influenza virus (AIV) is a type A influenza virus that can cause severe acute infectious diseases in poultry. The avian influenza caused by it is listed as a category A animal infectious disease by the International Office of Epizootics (OIE). The virus is prone to mutation, and 16 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes have been discovered so far. Among them, H5 is a highly pathogenic subtype of AIV, which can cause mass mortality of poultry and cause huge economic losses to the poultry industry. Evidence shows that H5 subtype AIV can also be transmitted to humans and other mammals, such as cats, dogs, pigs and tigers, through various routes, leading to disease and even death. Therefore, rapid and accurate detection of H5 subtype avian influenza virus infection is of great significance for the inve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/543
Inventor 谢芝勋庞耀珊谢丽基谢志勤邓显文刘加波范晴
Owner GUANGXI VETERINARY RES INST
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