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Antibody detection kit for infectious bovine rihinotracheitis virus and application thereof

A rhinotracheitis virus and antibody detection technology, which can be applied to measurement devices, instruments, scientific instruments, etc., can solve the problems of cross-contamination of experimental waste, complicated and complicated operations, and difficult to carry out, and achieve obvious technical improvement, high detection sensitivity, and consumption. short time effect

Active Publication Date: 2019-02-22
北京纳百生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The industry standard of the Ministry of Agriculture "NY / T 575-2002 Diagnostic Techniques for Bovine Infectious Rhinotracheitis" lists three IBR detection methods: virus isolation and identification, trace serum neutralization test, enzyme-linked immunoassay method, the first two methods The operation is cumbersome and complicated, time-consuming, requires many reagents and equipment, and is difficult to carry out in grassroots pastures; while the enzyme-linked immunoassay takes nearly 4 hours as a whole, and requires the preparation of various solutions and preparation components. For grassroots veterinary laboratories words, it is also difficult to develop
[0004] Furthermore, the enzyme-linked immunosorbent assay described in the standard is an indirect enzyme-linked immunosorbent assay with rabbit polyclonal antibody, which uses virus antigens to coat the enzyme-linked immunoassay plate, which has a certain risk of poisoning. Improper handling of experimental waste may also lead to crossover. Pollution

Method used

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  • Antibody detection kit for infectious bovine rihinotracheitis virus and application thereof
  • Antibody detection kit for infectious bovine rihinotracheitis virus and application thereof
  • Antibody detection kit for infectious bovine rihinotracheitis virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: Preparation of recombinant gD protein

[0035] (1) Preparation and purification of recombinant gD antigen

[0036] According to the gene information of bovine infectious rhinotracheitis virus in Genbank NC 001847, DNAStar was used to analyze the signal peptide, antigenicity and hydrophilicity of the amino acid sequence of gD protein, remove the signal peptide sequence, screen the antigenic region, and finally intercept Two pieces of protein sequence were combined in tandem to synthesize the gD gene sequence as SEQ ID No.2 sequence, and a pair of primers were designed, the upstream primer sequence was SEQ ID No.3 sequence, and the downstream sequence was SEQ ID No.4 sequence.

[0037] Using the synthetic gene as a template, carry out PCR reaction. The PCR reaction system is 50 μL. Add the following reagents to a sterile 0.2mL EP tube: template DNA 0.5μL, 10XEx TaqTN Buffer (Mg 2+ free) 5μL, dNTP Mixture (2.5mM each) 2μL, MgCl 2 (25mM) 4μL, upstream pr...

Embodiment 2

[0042] Example 2: Preparation and purification of gD protein-specific monoclonal antibody

[0043]After the prepared recombinant gD antigen was emulsified with an equal amount of Freund's adjuvant in an amount of 50 μg, several Balb / C mice were immunized by subcutaneous injection at multiple points. The adjuvant for the first immunization was complete Freund's adjuvant, and the adjuvant for subsequent immunization was incomplete Freund's adjuvant. The immunization interval was 2 weeks, and the serum titer was determined by direct ELISA after 3 immunizations. Screen the mice with high serum titer and low cross-reactivity, and perform cell fusion according to conventional methods, and screen hybridoma cell lines secreting specific monoclonal antibodies.

[0044] Screening antigens are inactivated bovine infectious rhinotracheitis virus, bovine viral diarrhea virus, bovine foot-and-mouth disease virus, etc., and recombinant gD protein antigens, and use negative serum as a refere...

Embodiment 3

[0047] Example 3: Identification of IBR-specific monoclonal antibodies

[0048] The prepared monoclonal antibodies were identified by using a commercial IgG subtype identification test kit. The results showed that 5A1-1A11 and 7C10-1B1 were IgG2a and IgG2b subtypes, respectively. The specific operations were as follows:

[0049] 1) Dilute the prepared purified monoclonal antibody with PBS solution at 1:10000, 1:15000, 1:20000, 1:25000, 1:30000, 1:35000 and 1:40000.

[0050] 2) Take the gD protein-coated plate (0.1 µg / ml), wash the plate once with 300 μl / well of 1× washing solution, and discard the washing solution. Add the corresponding diluted monoclonal antibody, 50 μl per well, make 5 replicate wells for each monoclonal antibody, shake and mix for 1 minute. After incubating at 37°C for 30 minutes, take out the reaction plate, discard the reaction solution, add 300 μl of 1× washing solution to each well, wash 3 times, and spin dry.

[0051] 3) Dilute IgG1, IgG2b and IgG3 1...

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Abstract

The invention provides an antibody detection kit for an infectious bovine rihinotracheitis virus. The detection kit is composed of a recombinant gD antigen coated elisa plate, negative control, positive control, a horse radish peroxidase marked IBR specific monoclonal antibody, a sample diluting solution, a washing solution, a primer solution and a terminating solution. By means of the kit, whether a cattle is infected with infectious bovine rihinotracheitis or not can be detected within a relatively short time, and a corresponding management policy is made. For a complex situation of currentanimal disease prevention and control, the kit has a very wide market prospect and can play a great role in basic level detection and government regulation.

Description

technical field [0001] The invention belongs to the technical field of rapid detection of animal epidemic diseases, and relates to a bovine infectious rhinotracheitis virus antibody detection kit and application thereof. Background technique [0002] Infectious bovine rhinotracheitis (IBR for short) is a viral infectious disease caused by bovine herpesvirus type 1 (BHV-1) infecting domestic cattle and wild cattle, and is listed as B by the World Organization for Animal Health. The Ministry of Agriculture of my country lists it as a second-class animal disease. The disease is widely distributed around the world and has a low mortality rate. Many infected cattle pass through with subclinical symptoms, often resulting in more serious respiratory diseases and mixed infections due to bacterial secondary infection, so it is very important for early diagnosis and detection of IBR infection. [0003] The industry standard of the Ministry of Agriculture "NY / T 575-2002 Diagnostic Tec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/531
CPCG01N33/531G01N33/56983
Inventor 杨春江于在江赵荣茂曹倩倩李月莫勋孙海霞王军陈曼利
Owner 北京纳百生物科技有限公司
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