A kind of foot-and-mouth disease virus competition ELISA detection kit

The technology of a foot-and-mouth disease virus and a detection kit, which is applied in the field of immunoassay and corresponding biological substances, can solve problems such as economic loss, impact on the development of animal husbandry, and low fatality rate, and achieve reduced workload, effective and practical detection methods, and improved The effect of sensitivity

Active Publication Date: 2021-11-09
内蒙古必威安泰生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The fatality rate of foot-and-mouth disease is very low, but the virus can survive for a long time in the body, and the infected animals continue to detoxify, becoming an important source of infection, which will cause huge economic losses to animal husbandry production and import and export trade. The disease not only seriously affects the development of animal husbandry, but also serious threat to human health

Method used

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  • A kind of foot-and-mouth disease virus competition ELISA detection kit
  • A kind of foot-and-mouth disease virus competition ELISA detection kit
  • A kind of foot-and-mouth disease virus competition ELISA detection kit

Examples

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Effect test

Embodiment 1

[0025] The preparation of embodiment 1 monoclonal antibody

[0026] 1.1 Foot-and-mouth disease antigen (A / AKT-Ⅲ strain FMDV inactivated antigen) is kept in the laboratory.

[0027] 1.2 Preparation of immune splenocytes

[0028] Take the purified antigen and mix it well with an equal volume of adjuvant, inject it into the mouse body, each injection dose is 1ml, after the first immunization, second immunization, third immunization and booster immunization, measure the antibody titer in the mouse serum degree, will show sufficient antibody titer (1:10 8 ) (ELISA titer) mice were used as a source of immune splenocytes. The method for measuring antibody titer is ELISA detection method.

[0029] 1.3 Preparation of myeloma cells

[0030] Myeloma cells SP2 / 0 cells were preserved in the laboratory.

[0031] 1.4 Cell Fusion

[0032] 1.4.1 Cell Fusion

[0033] Spleen isolation was performed on mice with sufficient antibody titers in 1.2, and a spleen lymphocyte suspension was prep...

Embodiment 2

[0051] Example 2 Preparation of specific single domain antibody

[0052] A / AKT-Ⅲ strain FMDV inactivated antigen immunized Bactrian camels, isolated peripheral blood lymphocytes, extracted total RNA, synthesized cDNA by RT-PCR, and then used single-domain antibody-specific primers (upstream primer: CTTGGTCTCTGATGTGCAGCTGGTGGAGTC downstream primer: CTCGGATCCTCATGAGGAGACGGTGACCTGG) Three amplifications were performed to obtain the VHH gene. A phage display immune library of single domain antibody against type A foot-and-mouth disease virus was constructed. It was determined that the library capacity was 1.2×10 8 . After four rounds of panning, the sequence of type A FMDV single domain antibody was sequenced and named sdAb-A. After amplification, the target fragment was digested and ligated with pSMK vector to construct pSMK-sdAb-A prokaryotic expression vector, which was transformed into Escherichia coli BL21. Detected by SDS-PAGE, the recombinant sd Ab-A was expressed. Rec...

Embodiment 3 2

[0054] Example 3 Preparation and Labeling of Secondary Antibody

[0055] 3.1 Preparation of polyclonal antibodies

[0056] Select healthy and well-nourished mice as immunization objects. First, carry out basic immunization, inject the virus liquid into the mice and perform high-level immunization after recovery. Perform purification; immunize horses with purified serum, collect serum, and purify secondary antibody IgG.

[0057] 3.2 Labeling prepared horse anti-mouse IgG

[0058] 3.2.1 Prepare 1% chloroauric acid solution with 1g of chloroauric acid, store it in the dark at 4°C, and filter it with a 0.22um filter.

[0059] 3.2.2 Prepare 1% trisodium citrate solution with 1g of trisodium citrate and store at 4°C.

[0060] 3.2.3 Take 100ml of distilled water and heat it to 100°C, boil it, add 1ml of 1% chloroauric acid for about 1 minute, add 1ml of 1% trisodium citrate quickly, and time it for 3 minutes, and lower the temperature for 12 minutes. Cool at room temperature for ...

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Abstract

The invention relates to a competitive ELISA detection kit for foot-and-mouth disease virus antibody, the kit comprising: single domain antibody sdAb-A; A / AKT-III strain FMDV inactivated antigen; monoclonal antibody 9C10, blocking solution; gold-labeled secondary antibody ; and supporting media. By combining the FMD antigen with the specific single-domain antibody coated on the support medium, the monoclonal antibody 9C10 and the antibody in the serum to be tested compete with the antigen, and it is detected by gold-labeled horse anti-mouse IgG, when the sample is detected It only needs to add the sample to be tested and incubate with monoclonal antibody 9C10 for 1 hour, then add the gold-labeled secondary antibody for 10 minutes of color development, and the value can be obtained after washing. The operation is simple and the diagnosis is fast. The competitive ELISA detection kit of the invention has high detection sensitivity, and the coincidence rate with virus neutralization reaction is higher than other commercial detection kits.

Description

technical field [0001] The invention belongs to the field of immunoassay methods and corresponding biological substances, and in particular relates to a detection method and application of a foot-and-mouth disease virus antibody. Background technique [0002] Foot-and-mouth disease (FMD) is an acute, febrile, contagious animal disease that infects artiodactyla animals caused by foot-and-mouth disease virus (FMDV). FMD is a must-report infectious disease stipulated by the International Veterinary Bureau, and it is also recognized as a first-class infectious disease by China. The fatality rate of foot-and-mouth disease is very low, but the virus can survive for a long time in the body, and the infected animals continue to detoxify, becoming an important source of infection, which will cause huge economic losses to animal husbandry production and import and export trade. The disease not only seriously affects the development of animal husbandry, but also pose a great threat to...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577G01N33/569
Inventor 张涛徐娜刘月何召庆刘金萍张晓慧徐婉英张春辉孙聪
Owner 内蒙古必威安泰生物科技有限公司
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