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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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

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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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[0048] 5. Preparation of positive control and negative control

[0049] The standard positive serum (OD) obtained by immunization with recombinant chicken infectious bronchitis IBV-N protein 450nm ≥1.0), add 1000U / mL penicillin streptomycin, 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 penicillin streptomycin, aseptically filter, as a negative control in the indirect ELISA detection reagent for chicken infectious bronchitis antibody.

[0050] 6. Preparation of color developing liquid

[0051] Color developing solution A: Weigh 200mg of tetramethylbenzidine (TMB), dissolve it with 100mL absolute ethanol or DMSO, and dilute to 1000mL with double distilled water; Color developing 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 of 0.75% hydrogen peroxide urea, distille...

Example Embodiment

[0065] Examples:

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

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

[0068] 2) 10×Concentrated washing solution: 400mL (1∶10 dilution 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 with the sample diluent at 1:200, add 100 μL / well to the antibody detection plate, set a negative control and a positive control, and incubate at 37°C for 30 minutes;

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

[0079] 3. Add 100μL of enzyme con...

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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

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Claims

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Application Information

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Owner POULTRY INST SHANDONG ACADEMY OF AGRI SCI
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