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Indirect ELISA (Enzyme-Linked Immuno Sorbent Assay) diagnostic kit of mycoplasma gallisepticum

A technology of Mycoplasma gallisepticum and a diagnostic kit, which is applied in biological testing, material testing products, measuring devices, etc., can solve the problems of low specificity, low sensitivity, lack of test results, etc.

Inactive Publication Date: 2014-01-15
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide an antimicrobial method according to the problems of the existing whole-bacteria diagnostic antigens, such as messy antigen sites, low specificity, easy cross-reaction with other mycoplasma, low sensitivity, and lack of accuracy in detection results. Mycoplasma gallisepticum indirect ELISA diagnostic kit, the kit has the characteristics of species specificity and immunogenicity, high detection rate and strong specificity

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Mycoplasma gallisepticum indirect ELISA antibody detection kit

[0028] The composition is as follows: ELISA plate coated with recombinant fusion protein, antibody diluent is 10mMPBST, pH7.4 containing 0.5% bovine serum albumin, elution buffer is 10mMPBST containing 0.1% Tween 20, pH7.4, enzyme The secondary antibody is horseradish peroxidase-labeled goat anti-chicken IgG, the substrate chromogenic solution is 0.01% tetramethylbenzidine, and the stop solution is 2M sulfuric acid solution.

Embodiment 2

[0029] Example 2 Preparation of Mycoplasma gallisepticum Indirect ELISA Antibody Detection Kit

[0030] Expression and purification of recombinant fusion protein

[0031]The fusion protein gene PvpA was inserted downstream of the plasmid PET-41αT7 promoter, and the host bacteria for expression was Escherichia coli BL21 (DE3), and the recombinant plasmid PET-PvpA was transformed into Escherichia coli BL21 (DE3) and high-copy recombinants were screened to construct Recombinant Escherichia coli BL21 (DE3) engineering bacteria. The above-mentioned recombinant Escherichia coli BL21 (DE3) engineered bacteria were induced to express, and the medium used was LB liquid medium containing 25 μg / ml kanamycin. The induction expression conditions were: 28°C, the final concentration of IPTG was 1.0mmol / L, and the induction time was 5h. The obtained bacterial solution was centrifuged at 4°C, 9000r / min for 15min, the supernatant was discarded, the above bacterial pellet was re-selected wit...

Embodiment 3

[0038] Example 3 The use method of Mycoplasma gallisepticum indirect ELISA antibody detection kit

[0039] 1. Put the antigen-coated ELISA plate and various solutions in the kit at room temperature and return to temperature

[0040] 2. Adding samples: dilute the serum to be tested with antibody diluent by a certain factor, add 100 μl to each well, and use the standard negative and positive serum as a control, place the microtiter plate at room temperature or 37°C for 1 hour, then shake dry, add to each well Wash 3-5 times with 300μl elution buffer.

[0041] 3. Add enzyme-labeled secondary antibody: add 100 μl of diluted enzyme-labeled secondary antibody to each well, place the enzyme-labeled plate at room temperature or 37°C for 1 hour, then spin dry, add 300 μl elution buffer to each well and wash 3 to 5 times .

[0042] 4. Add chromogenic solution: Add 100 μl of TMB chromogenic solution to each well, and keep the microplate plate away from light for 10 minutes at room te...

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Abstract

The invention discloses an indirect ELISA (Enzyme-Linked Immuno Sorbent Assay) diagnostic kit of mycoplasma gallisepticum, which comprises an ELISA plate coated by species specificity proteins PvpA, an elution buffer solution, an antibody diluting solution, an ELISA secondary antibody, a substrate color developing solution and a stop solution. The kit disclosed by the invention selects the species specificity proteins PvpA, covers the high-frequency variation regions: DR-1 and DR-2 regions of the PvpA proteins, has high specificity and immunogenicity, improves the specificity and sensitivity of the detection result, lowers the production cost and is suitable for popularization and use in grassroots veterinarian mechanisms.

Description

technical field [0001] The invention relates to the technical field of kit preparation, in particular to a Mycoplasma gallisepticum indirect ELISA diagnostic kit. Background technique [0002] The current antibody detection methods mainly include agar gel immunodiffusion test (AGID), convective immunoelectrophoresis test (CIET), hemagglutination inhibition test (HI), immunofluorescent antigen test (IFAT), and enzyme-linked immunosorbent assay (ELISA). Among them, ELISA is currently the most researched and fastest-growing technology. It has been widely used in clinical medicine, biochemistry, analytical chemistry, food analysis and other fields, but its application in animal husbandry has not been widely used in China. The theoretical basis of ELISA is the immobilization of antigens or antibodies and the enzymatic labeling of antigens or antibodies. [0003] Agar gel immunodiffusion experiments and convection immunoelectrophoresis experiments are prone to cross-reactions, an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/535
Inventor 蒋红霞周云雷曾振灵刘雅红陈杖榴沈祥广
Owner SOUTH CHINA AGRI UNIV
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