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Blocking test paper for swine fever antibody

A swine fever antibody and detection test strip technology, which is applied in the field of livestock disease antibody detection equipment, can solve the problems of inability to realize a large number of clinical samples, real-time detection, cumbersome operation, high cost, etc., and achieve easy large-scale promotion and application, broad market prospects, highly specific effect

Inactive Publication Date: 2019-07-26
HENAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Indirect ELISA has high sensitivity, is suitable for a large number of serological monitoring, can be standardized and the results are easy to analyze, but it cannot distinguish neutralizing and non-neutralizing antibodies, and cannot accurately evaluate the immune protection effect
The VN test is the most sensitive and specific method for detecting neutralizing antibodies. The level of neutralizing antibodies against swine fever is directly related to immune protection. However, this technical method is not only time-consuming, cumbersome, and expensive, but the virus must be cultured in a Class III biosafety laboratory. (P3) The operation, technology and safety measures are extremely demanding, and it is impossible to achieve a large number of real-time detection of clinical samples
Therefore, although the above detection methods can detect the level of swine fever antibodies, they all have complex test operations, time-consuming, specific professional skills and equipment, etc., are expensive, and are often limited to laboratories. It is difficult to popularize and promote at the grassroots level , not suitable for census of a large number of samples
In addition, there are large differences between the current widespread strains of classical swine fever virus and the commonly used vaccine strain HCLV in my country in terms of genetics, immune response and protective efficacy, and virulent infections of classical swine fever virus still occur in immunized animals from time to time. At the same time, due to the lack of serological markers and supporting differential diagnosis methods, its large-scale application in my country makes it difficult to distinguish attenuated immunity from wild virus infection through enzyme-linked immunosorbent assay (ELISA) and other antibody detection techniques, which is not conducive to the control of swine fever and purification

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  • Blocking test paper for swine fever antibody
  • Blocking test paper for swine fever antibody
  • Blocking test paper for swine fever antibody

Examples

Experimental program
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Effect test

preparation example Construction

[0055] (1) Preparation of swine fever virus immune antigen

[0056] Pig kidney cells (PK-15) were inoculated with Shimen strain, a standard virulent strain of classical swine fever virus, and the virus culture medium was collected for 48-60 hours, centrifuged at 3000 r / min at 4°C for 30 minutes at a low speed to remove impurities, and the virus culture medium was subjected to ultrafiltration with a 50kDa ultrafiltration membrane. Concentrate by filtration, carry out gel filtration chromatography purification to classical swine fever virus with Sepherose4 Fast Flow agarose column, measure half cell culture infection amount (TCID) of classical classical swine fever virus 50 ) up to 10 -7 Above, fluorescent quantitative PCR assay purified virus copy number up to 10 10 above.

[0057] (2) Preparation of Anti-CSFV Monoclonal Antibody

[0058] (2.1) Establishment of hybridoma cell lines

[0059] Mix the same amount of classical swine fever virus immune antigen with Freund's immu...

Embodiment 1

[0131] Example 1, monitoring the maternal antibody level of swine fever, collecting animal serum, taking 100 μL serum sample and adding 300 μL PBS or normal saline to dilute, preparing the serum solution to be tested, and using the swine fever antibody blocking test paper to detect according to the operation method (16) And the results are judged, and the maternal antibody level of swine fever is detected. There are two red bands (test line and quality control line) "||" on the test paper, and the color intensity of the test line T is equivalent to that of the blank control, which is negative for CSF antibody, indicating that the serum sample to be tested does not contain the maternal source of CSF Antibody; two red bands "||" appear, but the detection line T is significantly weaker than that of the blank control, which is weakly positive for CSF antibody, indicating that the serum sample to be tested contains a low level of maternal antibody to CSF; only one red band appears ...

Embodiment 2

[0132] Example 2: Evaluation of the immunization effect of the classical swine fever vaccine. 2 to 3 weeks after immunization with the inactivated classical swine fever vaccine, the serum of the immunized animal was collected, and 100 μL of the serum sample was added to 300 μL of PBS or normal saline for dilution or 1:2, 1:4, 1 : 8 ... Doubling dilution, prepare the serum solution to be tested, carry out detection and result judgment by (16) operating method with the swine fever antibody blocking detection test paper, detect the swine fever immune antibody level. Two red bands (test line and quality control line) "||" appear on the test paper, and the color intensity of the test line T is equivalent to that of the blank control, which is negative for swine fever antibody, indicating that the serum sample to be tested does not contain swine fever neutralizer Antibody; two red bands "||" appear, but the detection line T is significantly weaker than that of the blank control, whic...

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Abstract

The invention discloses a blocking test paper for swine fever antibody. The test paper is composed of a support plate, an antigen pad, a gold standard pad, a detection membrane and an absorbent pad, wherein the antigen pad absorbs the detection antigen of swine fever virus, the gold standard pad absorbs the colloidal gold labeled anti-swine fever virus monoclonal antibody mAb1, the detection membrane contains "|" blots of a detection line T and a quality control line C, the detection line T is the blot of an anti-swine fever monoclonal antibody mAb2 or an anti-swine fever polyclonal antibody pAb1, and the quality control line C is the blot of an anti-mouse IgG antibody pAb2 or a staphylococcus aureus SPA. The test paper provided by the invention realizes the rapid detection of neutralizingantibody of swine fever virus, can realize real-time monitoring of the maternal antibody and immune antibody level of the swine fever as well as the immune evaluation of the vaccine immune effect, and is simple in operation and applicable for everyone, which can well meet the needs of different levels of personnel, is easy to promote and apply in a wide range, and has broad market prospects and large economic and social benefits.

Description

technical field [0001] The invention relates to a detection device for livestock epidemic disease antibodies, in particular to a test paper for blocking detection of swine fever antibodies. Background technique [0002] Classical swine fever (CSF) is a highly contagious disease characterized by high fever, hemorrhage and high mortality caused by classical swine fever virus (CSFV). The World Organization for Animal Health (OIE) will It is included in the list of OIE epidemic diseases, and it is an animal infectious disease that must be declared, and it is listed as a first-class animal epidemic disease in my country. In my country, the hog cholera lapinized vaccine (HCLV) vaccine has been used to prevent and control the large-scale epidemic of CSF. However, in recent years, new changes have taken place in the prevalence and pathogenesis of CSF. Most of them are atypical, chronic and recessive infections. ", which is still one of the main infectious diseases that endanger the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/68G01N33/577G01N33/531C07K16/10C07K14/18C07K1/22C07K1/18C07K1/16C12N15/70C12N15/82
CPCC07K14/005C07K16/1081C12N15/70C12N15/8258C12N2710/12022C12N2800/22G01N33/531G01N33/56983G01N33/577G01N33/6854G01N2333/183
Inventor 王丽张改平郭军庆杨继飞张雨杭周人杰孙亚宁王瑞宁许倩茹杨艳艳李青梅柴书军
Owner HENAN ACAD OF AGRI SCI
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