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A quantitative detection method for the titer of a swine fever neutralizing antibody in swine serum and a detection kit

A quantitative detection method and antibody titer technology, applied in measuring devices, instruments, scientific instruments, etc., can solve the problems of high professional skill requirements for operators, high cost of manpower and material resources, and restrictions on popularization and application, and achieve wide application value, The effect of low cost and broad market prospects

Inactive Publication Date: 2017-02-08
JINYUBAOLING BIO PHARMA CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Neutralizing rabbit experiments are routinely used in China, but some uncontrollable animal factors in the experiment, such as individual differences among animals, may cause changes in the results, and the manpower and material costs required for operation are high, and the professional skills of the operators are required High, not easy to operate in practical applications
However, the neutralization immunofluorescence test adopted in the world, due to the use of a virulent strain of classical swine fever virus as the test strain, its popularization and application is also limited due to factors such as its easy to disperse the virus and its difficulty in obtaining.

Method used

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  • A quantitative detection method for the titer of a swine fever neutralizing antibody in swine serum and a detection kit
  • A quantitative detection method for the titer of a swine fever neutralizing antibody in swine serum and a detection kit

Examples

Experimental program
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Effect test

Embodiment 1

[0052] Embodiment 1, preparation swine fever rabbitization attenuated cytotoxicity (neutralization poison) and TCID thereof 50 determination

[0053] 1. Virus cultivation and harvesting

[0054] Inoculate the monolayer of swine testis (Swine Testis, ST) cells (derived from ATCC) with 0.3% (mass volume ratio, W / V) of classical swine fever rabbit attenuated spleen gonorrhea (purchased from the Central Prison Institute), and use the maintenance solution MEM culture medium (purchased from Yixing Saier Biotechnology Co., Ltd.), newborn bovine serum (purchased from Inner Mongolia Jinyuankang Co., Ltd.) with a content of 1%-3% (V / V), harvested the cell culture after culturing at 37°C for 4 days, and recorded For 1 harvest, the 1 harvest culture was frozen and thawed three times and then inoculated on ST monolayer cells at 10% (V / V), cultured at 37°C for 4 days, and the cell culture was harvested, which was recorded as 2 harvests, and the 2 harvest cultures were After freezing and t...

Embodiment 2

[0059] Embodiment 2, the quantitative detection of swine fever neutralizing antibody titer in pig serum

[0060] The quantitative detection method of swine fever neutralizing antibody titer in pig serum of the present invention comprises the following steps:

[0061] 1) Inactivation of the pig serum to be tested: inactivate the aseptically collected pig serum to be tested in a 56°C water bath for 30 minutes (55-57°C water bath inactivation for 30-40 minutes is acceptable);

[0062] 2) Porcine serum dilution: Add 50 μL of MEM culture solution to each well of the cell culture plate, then add 50 μL of the porcine serum to be tested in the first well and mix, take out 50 μL with a micropipette, add to the second well, mix Take out 50 μL after homogenization, and then add it to the third well, and so on, until the last well (discard 50 μL of the mixed solution), the dilution ratio of pig serum is 1:2, 1:4, 1:8, 1:16 , 1:32, 1:64, set 2 (2-4) parallel wells for each different dilut...

Embodiment 3

[0081] Example 3, Quantitative detection kit for neutralizing antibody titer to classical swine fever in pig serum

[0082] The quantitative detection kit of the neutralizing antibody titer of classical swine fever in the swine serum of the present invention comprises the anti-CSFV E2 Monoclional Antibody (primary antibody) of classical swine fever monoclonal antibody and the goat anti-mouse IgG (secondary antibody) labeled with peroxidase ).

[0083] For the convenience of detection, the kit of the present invention also includes 0.01mol PBS with pH 7.4, peroxidase substrate chromogenic solution, 80% acetone fixative solution and the like.

[0084] For the method of using the kit of the present invention, refer to Example 2.

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Abstract

A quantitative detection method for the titer of a swine fever neutralizing antibody in swine serum and a detection kit are disclosed. The method includes neutralizing swine serum to be detected and having different degrees of dilution with swine fever viruses having a known amount, inoculating ST cells after neutralization, culturing for 2-5 days, and quantitatively detecting the titer (a neutralizing result) of the swine fever neutralizing antibody in the swine serum through an indirect immunoperoxidase process and a Karber process, thus indirectly acquiring the titer of the neutralizing antibody in the swine serum. The method and the kit are low in detection time, simple in operation, low in cost, accurate and specific in result and capable of truly reflecting the level of a neutralizing antibody in immunized swine flocks, can be used for formulation of a vaccine immunity procedure, vaccine quality identification, swine flock immune state monitoring, and the like and are wide in application.

Description

technical field [0001] The invention belongs to the technical field of biological detection, in particular to a quantitative detection method and a detection kit for the titer of swine fever neutralizing antibody in pig serum. Background technique [0002] Classical swine fever (CFS) is an acute, febrile and highly contagious infectious disease caused by classical swine fever virus (CSFV). . Swine fever virus is mainly transmitted through the respiratory tract and digestive tract, with rapid transmission, high morbidity and mortality, and has brought huge economic losses to the global pig industry. Swine fever is an important infectious disease that seriously endangers the global swine industry, and is one of the diseases that must be notified listed in the World Organization for Animal Health (OIE) disease catalog. In recent years, swine fever has occurred frequently in our country, mainly manifested as recessive virus infection and chronic infection, which has become a m...

Claims

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Application Information

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IPC IPC(8): G01N33/569
CPCG01N33/56983
Inventor 孔彩平刘国英范秀丽郝鹏纪燕温谢
Owner JINYUBAOLING BIO PHARMA CO LTD
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