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Protein chip antibody detection kit for avian infectious bronchitis virus and application thereof

A technology for bronchitis and antibody detection, applied in the field of immunology detection

Inactive Publication Date: 2018-02-23
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, based on iPDMS (initiator integrated poly(dimethysiloxane), iPDMS) as a protein chip membrane, a diagnostic method for IBV antibody detection has not been reported.

Method used

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  • Protein chip antibody detection kit for avian infectious bronchitis virus and application thereof
  • Protein chip antibody detection kit for avian infectious bronchitis virus and application thereof
  • Protein chip antibody detection kit for avian infectious bronchitis virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] [Example 1] Preparation of antigen

[0078] After the gene of IBV nsp5 was amplified by RT-PCR method, it was cloned into the prokaryotic expression vector pEGX-4T-1 and transformed into Escherichia coli; the recombinant bacteria carrying the IBV nsp5 gene were screened and expressed in large quantities under the induction of IPTG and fused with GST nsp5 recombinant protein; purified by GSTrap FF affinity chromatography column to obtain the purified IBV nsp5 recombinant protein, and the protein obtained after elution was sampled for SDS-PAGE analysis. The purified recombinant protein GST-nsp5 has a molecular weight of 60KD and high purity. Store at -20°C for later use. (Chicken infectious bronchitis virus nsp5ELISA antibody detection kit and its application. National invention patent application number: 201610239101.3, application publication number: CN 105785049 A, application publication date: 2016.07.20)

Embodiment 2

[0079] [Example 2] Preparation and screening of negative and positive serum

[0080] 1. Preparation of IBV nsp5 negative and positive serum

[0081] Four 3-month-old SPF chickens were randomly divided into two groups, two of which were immunized with the purified GST-nsp5 protein prepared in Example 1 to prepare positive serum; the other two were used as negative controls. The specific immunization process is as follows:

[0082] The purified GST-nsp5 protein was mixed with an equal volume of Freund's complete adjuvant, and after being fully emulsified, the first immunization was performed subcutaneously on the neck of chickens at a dose of 1 mg / bird. After that, every two weeks, take the same amount of GST-nsp5 protein as the first immunization and mix and emulsify it with an equal volume of Freund's incomplete adjuvant, then perform the second immunization and third immunization. 10 days after the third immunization, use nsp5-ELISA to measure the serum antibody level. Afte...

Embodiment 3

[0085] [Example 3] Optimization of IBV protein chip detection conditions

[0086] 1. The basic procedure of IBV protein chip reaction

[0087] (1) Preparation of protein chip:

[0088] After the concentration of the recombinant IBV nsp5 protein was determined by the BCA protein detection kit, it was stored at -20°C for future use, and protein microarray spotting was carried out in a 100,000-class clean room. Dissolve the recombinant IBV nsp5 protein in an amount of 1 mg / mL in 30% (v / v) acetonitrile aqueous solution, then dilute the antigen to the working concentration with spotting buffer, and spot on iPDMS conventional membrane or buffy coat. Prepare a 48-well protein chip. Vacuum-dried and packaged, stored at 4°C for later use;

[0089] The sample buffer solution is: 0.3M PB, 0.2% (v / v) glycerol, 0.01% (v / v) Triton and 1.5% (g / 100ml) mannitol.

[0090](2) The IBV nsp5 protein chip plate was taken out from 4°C and kept at room temperature, and rinsed with washing solution...

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Abstract

The invention discloses a protein chip antibody detection kit for an avian infectious bronchitis virus and application thereof. The kit comprises (1) an IBV nsp5 protein chip prepared from a recombinantly expressed IBV non-structural protein 5; (2) an IgY enzyme-labeled antibody solution diluted by using an antibody diluent; (3) a 20*TBST concentrated washing solution; (4) diluent serum; (5) a positive control sample, i.e., anti-IBV nsp5 chicken serum; (6) a negative control sample, i.e., negative SPF chicken serum; (7) chemiluminescent substrate liquid including chemiluminescent liquid A andluminescent substrate liquid B; and (8) a TMB color-developing solution. The IBV protein chip antibody detection kit provided by the invention uses the non-structural protein nsp5 as an antigen, has the characteristic of high coincidence rate with imported commercial kits and is substantially reduced in cost, faster in detection, higher in sensitivity and simple to operate.

Description

technical field [0001] The invention belongs to the field of immunological detection methods, and in particular relates to the development and application of a chicken infectious bronchitis virus protein chip antibody detection kit. Background technique [0002] Chicken infectious bronchitis (Infectious bronchitis, IB) is caused by infectious bronchitis virus (Infectious bronchitis Virus, IBV), chicken acute, highly contagious respiratory and reproductive tract infectious disease. Chicken infectious bronchitis virus has a variety of serotypes and multi-tissue tropism. Since the first report of respiratory type IB in 1931, subsequent reports of infectious bronchitis such as renal type, intestinal type, reproductive tract type, and glandular stomach type have been reported. More than 30 serotypes have been reported. In recent years, new serotypes and mutant strains are still emerging, and there is only partial or no cross-protection between the serotypes, which brings great di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/68
CPCG01N33/56983G01N33/68
Inventor 周继勇闫丽萍雷静胡建华施志玉
Owner NANJING AGRICULTURAL UNIVERSITY
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