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Indirect ELISA kit for detecting various animal erysipelothrix rhusiopathiae antibody, preparation method, and application of indirect ELISA kit

A technology of Erysipelas erythematosus and kits, which are applied in biological testing, material inspection products and other directions, can solve the problems of inability to detect antibodies against Erysipelas erysipelas in various animals, low sensitivity and inappropriate high-throughput sample detection, etc. The high activity of the coated antigen and the effect of reducing the concentration

Inactive Publication Date: 2018-02-06
YUNNAN ANIMAL SCI & VETERINARY INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to overcome the defects that the above-mentioned existing Erysipelothrix rhusiopathiae agglutination test antibody detection method has low sensitivity and is not suitable for high-throughput sample detection and the defect that the existing ELISA kit cannot detect antibodies to Erysipelothrix rhusiopathiae of various animals, Provides an indirect ELISA kit capable of detecting antibodies to Erysipelothrix rhusiopathiae of pigs, cattle, sheep and rabbits. The method also has the advantages of sensitivity, rapidity and suitability for high-throughput sample detection

Method used

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  • Indirect ELISA kit for detecting various animal erysipelothrix rhusiopathiae antibody, preparation method, and application of indirect ELISA kit
  • Indirect ELISA kit for detecting various animal erysipelothrix rhusiopathiae antibody, preparation method, and application of indirect ELISA kit
  • Indirect ELISA kit for detecting various animal erysipelothrix rhusiopathiae antibody, preparation method, and application of indirect ELISA kit

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Embodiment 1

[0051] Example 1: Preparation method of an indirect ELISA kit for detecting antibodies to Erysipelothrix rhusiopathiae in various animals

[0052] The kit consists of enzyme label plate, serum dilution plate, coating antigen, coating buffer, positive control serum A, positive control serum B, negative control serum, HRP-SPA, skimmed milk powder, 100×TMB substrate solution, 10× Chromogenic diluent, 3% (v / v) H 2 o 2 Solution, stop solution, and 25× washing solution are assembled, and the specific methods of each component are as follows:

[0053] (1), preparation of coated antigen: screen the Erysipelothrix rhusiopathiae Yunnan isolate YN15075 with good antigenicity, inoculate it on a nutrient agar plate, cultivate it at 37° C. for 48 hours, elute the bacterium with sterilized physiological saline, and adjust its concentration to 2×10 8 CFU / mL, sonicate 100 times in an ice bath, 10 seconds each time, 10 seconds apart, ultrasonic power 100 watts, centrifuge the sonicated prote...

Embodiment 2

[0067] Embodiment 2: Application of this indirect ELISA kit to detect antibodies to Erysipelothrix rhusiopathiae of pigs, cattle, sheep and rabbits

[0068] The indirect ELISA kit can not only be used for qualitative detection of serum antibodies to Erysipelothrix rhusiopathiae of pigs, cattle, sheep and rabbits, but also can be used for semi-quantitative detection of serum antibodies. At the same time, it has the advantages of sensitivity, rapidity, high-throughput detection, economy and simplicity, and can be used for serum antibody detection and epidemiological investigation of Erysipelothrix rhusiopathiae. The specific steps of antibody ELISA detection are as follows:

[0069] (1) Antigen-coated ELISA plate: Take 12.5 μL of coated antigen and add it to 5 mL of coating buffer and mix well, add 100 μL of the above-mentioned diluted coated antigen to each well of the 1st to 4th column of the microtiter plate, 4 ℃ coated overnight;

[0070] Before proceeding to the next step...

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Abstract

The invention relates to erysipelothrix rhusiopathiae (a bacterial strain number: YN15075, and a bacterial strain preservation number: CGMCC No.14468) and an antibody indirect ELISA detection kit, a preparation method and application. The kit is prepared by assembling an elisa plate, a serologecal plate, a coating antigen, a coating buffer solution, positive control serum A, positive control serumB, negative control serum, horse radish peroxidase marked staphylococcus protein A (HRP-SPA), degreased milk powder, a 100*TMB substrate solution, 10*developing diluents, a 3 percent (v / v) H2O2 solution, a stop solution, and a 25*washing buffer solution. The kit can detect erysipelothrix rhusiopathiae antibodies of pigs, cattle, sheep and rabbits; a result judgment standard is as follows: on thepremise of ensuring the effectiveness of the test, the positive standard of the pig, cattle, sheep and rabbit erysipelothrix rhusiopathiae antibody is separately that S / P is greater than or equal to 0.30, S / P is greater than or equal to 0.28, S / P is greater than or equal to 0.26, and S / P is greater than or equal to 0.33 (S indicates a OD450nm value of the detected serum, and P indicates an averagevalue of the OD450nm value of the 2-pore positive control serum), otherwise, the result is determined as negative; the kit can be used for the erysipelothrix rhusiopathiae antibody detection and epidemiology investigation.

Description

technical field [0001] The patent of the present invention relates to Erysipelothrix rhusiopathiae (abbreviated as: E.rhusiopathiae, strain number: YN15075, strain preservation number: CGMCC No.14468) and its antibody indirect ELISA detection kit, preparation method and application . Especially an indirect ELISA kit for detecting antibodies to Erysipelothrix rhusiopathiae in pigs, cattle, sheep and rabbits. Background technique [0002] Erysipelothrix rhusiopathiae, also known as Erysipelothrix rhusiopathiae or Erysipelothrix suis, can infect many mammals such as pigs, cattle, sheep and rabbits, as well as poultry, fish and humans, and is a relatively harmful animal comorbid pathogens. Erysipelothrix rhusiopathiae infections usually result in massive acute mortality in animals and erysipelasoids in humans, and the disease has a worldwide distribution. In recent years, the morbidity and mortality of Erysipelothrix rhusiopathiae infection have increased significantly, prese...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/68
Inventor 李富祥李华春廖德芳宋建领杨仕标赵文华
Owner YUNNAN ANIMAL SCI & VETERINARY INST
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