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Blocking ELISA kit for detecting neutralizing antibody of infectious bovine rhinotracheitis virus and application of blocking ELISA kit

A rhinotracheitis virus and infectious technology, which is applied in the field of neutralizing antibody detection, can solve the problems of irreplaceable neutralization test and inability to detect IBRV neutralizing antibody, etc., and achieves good consistency, good specificity and good repeatability Effect

Active Publication Date: 2021-06-18
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing ELISA kits can detect IBRV antibodies, but cannot detect IBRV neutralizing antibodies, so they cannot replace neutralization tests

Method used

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  • Blocking ELISA kit for detecting neutralizing antibody of infectious bovine rhinotracheitis virus and application of blocking ELISA kit
  • Blocking ELISA kit for detecting neutralizing antibody of infectious bovine rhinotracheitis virus and application of blocking ELISA kit
  • Blocking ELISA kit for detecting neutralizing antibody of infectious bovine rhinotracheitis virus and application of blocking ELISA kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1. Preparation of recombinant gD protein and anti-IBRV monoclonal antibody

[0055] 1 Preparation of recombinant gD protein

[0056] 1.1 Gene and primer design

[0057] Download the IBRV gD gene sequence from the GenBank database (gene sequence accession number: MH751901.1), use the online analysis website (http: / / www.cbs.dtu.dk / services / TMHMM / ) to analyze and predict the transmembrane region of the sequence , designed a truncated gD gene sequence (SEQ ID NO: 1), which encodes a truncated gD protein (SEQ ID NO: 2). In order to promote the protein purification after secreting and expressing the truncated gD protein in insect cells, it is necessary to artificially introduce a 6×His sequence at the C-terminus of the protein. The optimized gD gene sequence (SEQ ID NO: 3) was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. Use Oligo7.0 software to design specific primers (Gd-F / Gd-R) for the truncated gD gene, and introduce restriction enzyme sites Nde...

Embodiment 2

[0141] Example 2. Establishment of the blocking ELISA method for detecting antibodies against bovine infectious rhinotracheitis virus

[0142] 1. Horseradish peroxidase-conjugated mAb

[0143]Mab 3E8 was labeled with horseradish peroxidase (HRP) by the sodium periodate method.

[0144] (1) Weigh 5 mg of HRP (SIGMA, SRE0082) and dissolve it in 1000 μL of double distilled water, and continue to add 500 μL of newly prepared 0.1M NaIO 4 , mix well, and incubate at 4°C in the dark for 30min.

[0145] (2) Add 500 μL of freshly prepared 0.2M ethylene glycol (SIGMA, 324558) solution, mix well, and incubate at 4°C in the dark for 30 minutes.

[0146] (3) Add 5 mg of purified monoclonal antibody 3E8 to the above solution, mix well and put it into a pre-treated dialysis bag (Beijing Suolaibao, MW14000), and put it in 0.1M carbonate buffer solution with pH9.6 overnight During dialysis, the fluid was changed three times during the dialysis period.

[0147] (4) Pour the dialyzed liquid ...

Embodiment 3

[0227] Example 3. Preparation of an ELISA kit for detecting antibodies against bovine infectious rhinotracheitis virus

[0228] Kit assembly: recombinant gD protein (recombinant gD protein prepared in Example 1), enzyme-labeled plate, blocking solution (chicken serum), enzyme-labeled monoclonal antibody (HRP-labeled anti-IBRV monoclonal antibody prepared in Example 2), secreted the The preservation number of the hybridoma cells of the monoclonal antibody is CGMCC No. 21015), TMB chromogenic solution, stop solution (2M H 2 SO 4 ) were aseptically packaged separately, all were put into the kit, and the kit label was affixed on the casing of the kit. Recombinant gD protein is pre-coated on the microtiter plate, or packaged separately from the microtiter plate, and coated by the user before use.

[0229] The method of using the kit is as follows:

[0230] (1) Coating: Use 0.1M pH8.5 Tris-HCL buffer to prepare recombinant gD protein solution with a concentration of 2.5-5 μg / mL, ...

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Abstract

The invention relates to detection of a neutralizing antibody of an infectious bovine rhinotracheitis virus, in particular to a blocking ELISA kit for detecting the neutralizing antibody of the infectious bovine rhinotracheitis virus and application of the blocking ELISA kit. The invention provides a monoclonal antibody with neutralizing activity for resisting infectious bovine rhinotracheitis virus. The monoclonal antibody is secreted by hybridoma cells with the preservation number of CGMCC No.21015. The invention further provides a kit for detecting the neutralizing antibody of the infectious bovine rhinotracheitis virus. The kit comprises the monoclonal antibody. Based on the monoclonal antibody and truncated gD protein designed in the invention, a blocking ELISA method for detecting a neutralizing antibody of the infectious bovine rhinotracheitis virus is established, and the blocking ELISA method has the advantages of strong specificity, high sensitivity and good repeatability.

Description

technical field [0001] The invention relates to the detection of neutralizing antibodies to bovine infectious rhinotracheitis virus, in particular to a blocking ELISA kit for detecting neutralizing antibodies to bovine infectious rhinotracheitis virus and its application. Background technique [0002] Infectious Bovine Rhinotracheitis (IBR) is a febrile, acute and highly latent infectious disease caused by Infectious Bovine Rhinotracheitis Virus (IBRV). Since Miller first reported the disease in the United States in 1995, IBR has shown a global epidemic trend, which has caused huge economic losses to the world cattle industry. The main danger of IBRV is that after the virus invades the host, it can latently infect the ganglion of the host, causing the sick cow to be infected for life, which brings a huge challenge to the prevention and control of the disease. In view of its harmfulness, the World Organization for Animal Health (OIE) classifies it as a Class B infectious dis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08C12N5/20G01N33/543G01N33/569
CPCC07K16/085G01N33/543G01N33/56983G01N2333/06
Inventor 刘文晓李永清洪家兵江波许健程晶郭楠楠王晓玥
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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