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PRV (pesudorabies virus) gB monoclonal antibody and application thereof

A monoclonal antibody and protein technology, applied in the field of immunobiology, can solve the problems of long operation time and high price

Pending Publication Date: 2017-12-12
HENAN CENT FOR ANIMAL DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the prior art, serological diagnostic methods for PRV antibodies, such as agar diffusion test, indirect ELISA, latex agglutination test, hemagglutination inhibition test and microneutralization test, etc., require too high purity of the antigen, so the clinical testing process cannot be avoided The non-specific reaction often occurs in the test, which leads to the determination of false positive results, so it is necessary to further improve it; although there are blocking ELISA methods and reagents based on monoclonal antibody detection of PRV gB in foreign countries, they are expensive and take a long time to operate. It is difficult to adapt to the needs of the current epidemic detection at the grassroots level

Method used

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  • PRV (pesudorabies virus) gB monoclonal antibody and application thereof
  • PRV (pesudorabies virus) gB monoclonal antibody and application thereof
  • PRV (pesudorabies virus) gB monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] This example briefly introduces the preparation and identification process of the monoclonal antibody against PRVgB as follows.

[0084] 1. Preparation of porcine pseudorabies virus (immune antigen)

[0085] The preparation of porcine pseudorabies virus (antigen) is mainly the expansion culture, concentration and purification of the virus. The relevant steps are briefly introduced as follows.

[0086] 1. Subculture the self-isolated porcine pseudorabies virus variant strain HeNZK-2014 on PK-15 cells;

[0087] According to the Reed-Muench method, taking the cytopathic effect (CPE, cytopathic effect) of PK-15 cells as the result, the preliminary research results of the strain showed that its TCID 50 up to 10 -9.77 / 0.1mL;

[0088] After the expanded culture, the culture medium containing the virus was repeatedly frozen and thawed 3 times, and then the culture medium was inactivated with 0.1% formaldehyde solution at 37°C for 18 hours (that is, the virus was inactivated...

Embodiment 2

[0221] Using the monoclonal antibody prepared in Example 1 (taking the monoclonal antibody secreted by 6E9H7 cell line as an example) can be used to prepare a typical one-step competitive ELISA kit for detecting porcine pseudorabies virus gB antibody, which includes an ELISA detection plate , enzyme-labeled anti-pseudorabies virus monoclonal antibody, TMB chromogenic solution, stop solution, etc., are briefly introduced as follows.

[0222] One-step competitive ELISA kit for detecting porcine pseudorabies virus gB antibody, including: 1 piece of 96-well ELISA detection plate, 1 bottle of enzyme-labeled anti-pseudorabies virus monoclonal antibody (10mL / bottle), 2 tubes of negative and positive controls ( 1.5mL / tube), 1 bottle of 20-fold concentrated washing solution (30mL / bottle), 1 bottle of TMB chromogenic solution and 1 bottle of stop solution (10mL / bottle);

[0223] The 96-well ELISA detection plate is an antigen-coated plate, which is prepared according to the following st...

Embodiment 3

[0247] In order to verify the application effect of the kit provided in Example 2, the sensitivity, specificity, accuracy, repeatability and other tests were carried out. The relevant tests are briefly introduced as follows.

[0248] sensitivity test

[0249] Divide 70 unimmunized healthy pigs into 7 groups, monitor the PRV gB antibody of pigs in different periods after immunization with porcine pseudorabies vaccine, and use this kit to detect the collected serum samples. The test results are shown in the table below.

[0250] Sensitivity Verification:

[0251] .

[0252] It can be seen from the data in the above table that when most of the sera were tested positive after 14 days of immunization, all samples were tested positive after 21 days, indicating that the sensitivity of the kit is better.

[0253] specificity test

[0254] Standard positive serum samples of classical swine fever, porcine blue ear disease, porcine circovirus disease, porcine parvovirus disease, and...

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Abstract

The invention belongs to the technical field of immunobiology and particularly relates to application for a patent of a PRV (pesudorabies virus) gB monoclonal antibody and application thereof. The antibody is obtained by screening PRV variant strains HeNZK-2014 and PRV gB proteins with hybridoma cells. The hybridoma cells capable of stably secreting the PRV gB protein monoclonal antibody is obtained through screening, so that the gB protein monoclonal antibody with high potency is obtained, a finished kit is prepared from the antibody, and a matched one-step competitive ELISA detection method is established. Through a series of verification for specificity, sensitivity and repeatability, the result indicates that according to the PRV gB antibody provided in the application and the established one-step competitive ELISA detection method, a comparatively accurate and comparatively stable detection result is achieved, the result can be better used for detection and evaluation of the PRV gB antibody, and the foundation is laid for preventing and controlling a PRV.

Description

technical field [0001] The invention belongs to the technical field of immunobiology, and specifically relates to a patent application for a porcine pseudorabies virus (PRV) gB monoclonal antibody and its application. Background technique [0002] Porcine pseudorabies (PR) is a highly contagious and acute infectious disease caused by porcine herpesvirus type 1 (Pseudorabies virus, PRV) with fever, miscarriage, and stillbirth as the main clinical symptoms. After pigs are infected with PRV, they show different symptoms due to different age groups and different strain virulence. The main symptoms of pregnant sows are abortion, weak piglets, stillbirth and mummified fetuses; newborn piglets often show acute lethal process. , typical neurological symptoms, paralysis, and a mortality rate of nearly 100%; most adult pigs are recessively infected. PRV has been widely prevalent in the world and has brought huge economic losses to the pig industry. It is one of the major infectious d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08G01N33/577G01N33/569G01N33/543
CPCC07K16/085G01N33/543G01N33/56994G01N2469/10
Inventor 闫若潜班付国吴志明马震原王淑娟谢彩华王华俊王东方赵雪丽曹伟伟方先珍赵明军刘梅芬赵国然张淼洁孙淑芳于辉杨海波李秀梅陈兴安
Owner HENAN CENT FOR ANIMAL DISEASE CONTROL & PREVENTION
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