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Indirect ELISA kit for detecting avian infectious bronchitis virus antibody

A bronchitis and chicken infectious technology, applied in the biological field, can solve the problems of complex fluorescent antibody technology and neutralization test operation, agar diffusion test sensitivity, poor stability, low sensitivity of colloidal gold technology, etc., and achieve good antigenicity. , easy operation, low cost effect

Inactive Publication Date: 2011-06-15
POULTRY INST SHANDONG ACADEMY OF AGRI SCI
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the sensitivity and stability of agar diffusion test are poor; the sensitivity of colloidal gold technology is not high; IBV has no hemagglutination before untreated, and the preparation process of this hemagglutination inhibitory antigen is complicated; the fluorescent antibody technology and neutralization test operation are complicated , time-consuming and laborious, not easy to make a quick diagnosis

Method used

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  • Indirect ELISA kit for detecting avian infectious bronchitis virus antibody
  • Indirect ELISA kit for detecting avian infectious bronchitis virus antibody
  • Indirect ELISA kit for detecting avian infectious bronchitis virus antibody

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preparation example Construction

[0048] 5. Preparation of Positive and Negative Controls

[0049] The standard positive serum (OD) obtained by immunizing with chicken infectious bronchitis IBV-N recombinant protein 450nm ≥1.0), add the penicillin streptomycin of 1000U / mL, sterile filter, as the positive control in the chicken infectious bronchial antibody indirect ELISA detection reagent; The SPF chicken standard negative serum (OD 450nm ≤0.200), add 1000U / mL of penicillin streptomycin, sterile filter, as the negative control in the chicken infectious bronchitis antibody indirect ELISA detection reagent.

[0050] 6. Preparation of chromogenic solution

[0051] Chromogenic solution A: weigh 200mg of tetramethylbenzidine (TMB), dissolve it with 100mL of absolute ethanol or DMSO, and dilute to 1000mL with double distilled water; Chromogenic solution B: weigh 21g of citric acid (C 6 h 8 o 7 ·H 2 O), 28.2g anhydrous disodium hydrogen phosphate (Na 2 HPO 4 ), 6.4mL 0.75% urea hydrogen peroxide, distilled wat...

Embodiment

[0066] 1. Chicken infectious bronchitis antibody ELISA detection kit, including the following components:

[0067] 1) ELISA strips (96 wells): 5 pieces

[0068] 2) 10× concentrated washing solution: 400mL (diluted 1:10 before use)

[0069] 3) Sample diluent: 200mL

[0070] 4) Enzyme conjugate working solution (rabbit anti-chicken enzyme-labeled secondary antibody): 50mL

[0071] 5) Chromogenic solution A: 50mL

[0072] 6) Chromogenic solution B: 50mL

[0073] 7) Stop solution: 60mL

[0074] 8) Positive control (+): 2mL

[0075] 9) Negative control (-): 2mL

[0076] 2. Operation steps:

[0077] 1. Dilute the serum to be tested 1:200 with the sample diluent, add 100 μL / well to the antibody detection plate, set a negative control and a positive control at the same time, and incubate at 37°C for 30 minutes;

[0078] 2. Discard the liquid in the reaction well, add 300 μL of washing solution to each well, wash 5 times with an interval of 1 min between each time, and pat dry;...

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Abstract

The invention discloses an indirect enzyme-linked immuno sorbent assay (ELISA) kit for detecting avian infectious bronchitis virus (IBV) antibody. The kit contains an ELISA plate enveloped by IBV-N recombinant protein serving as antigen. The IBV-N recombinant protein is obtained by the following method: designing a pair of specific primers according to an IBV-N gene sequence; amplifying the N gene of IBV by using a reverse transcription polymerase chain reaction (RT-PCR) method, directionally inserting the N gene into a pET-32a(+) expression vector, and screening to obtain a positive recombinant expression plasmid of pET-32a(+)-IBV-N; and transferring the plasmid to a BL21 competent cell, and performing isopropyl thiogalactoside (ITPG) induction expression to obtain the IBV-N recombinant protein. The kit is low in cost, easy, convenient and quick to operate, and particularly suitable for detecting batch samples, and greatly improves the efficiency of serodiagnosis of the avian infectious bronchitis.

Description

technical field [0001] The invention relates to biotechnology, in particular to an indirect ELISA kit for detecting IBV antibodies, which is specially used for rapid detection of chicken infectious bronchitis virus antibodies. Background technique [0002] Chicken infectious bronchitis is an acute highly contagious infectious disease caused by infectious bronchitis virus (IBV). IBV mainly infects the respiratory tract, digestive tract, reproductive system and kidney of chicks, and IBV has different degrees of harm to chickens of different ages. IBV often causes mixed infections (such as Newcastle disease, chronic respiratory disease, etc.), and can cause secondary bacterial diseases such as colibacillosis, salmonellosis, etc., thereby aggravating the harm to chickens. The loss caused by the decline in the quality of poultry products caused by the disease is usually greater than the loss caused by death. The domestic chicken industry causes direct economic losses of hundreds...

Claims

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Application Information

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IPC IPC(8): C12N15/09G01N33/569G01N33/535
Inventor 亓丽红黄兵宋敏训艾武王莉莉刘涛秦卓明
Owner POULTRY INST SHANDONG ACADEMY OF AGRI SCI
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