Cattle and sheep brucellosis indirect enzyme-linked immunosorbent assay antibody detection kit and preparation method thereof

An enzyme-linked immunosorbent assay, Brucella bovis and sheep technology, applied in measuring devices, instruments, scientific instruments, etc., can solve the problems of low specificity and low sensitivity, and achieve good repeatability and specificity. The effect of good performance and good detection effect

Inactive Publication Date: 2014-01-29
INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the existing problems of low sensitivity and low specificity in the detection of cattle and sheep brucellosis by other antigens, the present invention provides an indirect ELISA antibody detection kit for cattle an...

Method used

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  • Cattle and sheep brucellosis indirect enzyme-linked immunosorbent assay antibody detection kit and preparation method thereof
  • Cattle and sheep brucellosis indirect enzyme-linked immunosorbent assay antibody detection kit and preparation method thereof
  • Cattle and sheep brucellosis indirect enzyme-linked immunosorbent assay antibody detection kit and preparation method thereof

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preparation example Construction

[0091] The preparation method of cattle and sheep brucellosis indirect enzyme-linked immunosorbent assay antibody detection kit of the present invention, the conditions and steps of the method are as follows:

[0092] 1. Antigen-coated microtiter plate

[0093] (1) Coating: Using the recombinant Brucella BCSP31 protein induced and expressed in Escherichia coli BL21 as an antigen, dilute the purified lyophilized antigen with antigen diluent to obtain an antigen diluent with a concentration of 119 μg / mL, add to each well 100 μL antigen diluent, coated with microtiter plate, overnight at 4°C,

[0094] (2) Washing: Discard the antigen solution, add 300 μL of washing buffer to each well for washing, repeat the washing three times, each time for 3-5 minutes,

[0095] (3) Blocking: Place the microplate plate on filter paper and pat dry until there is no water, add 100 μL of blocking solution to each well, and incubate at 37°C for 1-2 hours.

[0096] (4) Washing: Discard the blockin...

Embodiment 1

[0115] Embodiment 1 specificity test

[0116] Utilizing the determined ELISA reaction conditions, the indirect enzyme-linked immunosorbent assay antibody detection kit for cattle and sheep brucellosis was assembled, and the recombinant BCSP31 protein antigen was used to coat the microtiter plate, Escherichia coli positive serum, Salmonella positive serum, Pasteurella The rod-positive serum bacteria and streptococcus-positive serum were detected and analyzed by ELISA, and the bovine brucellosis-positive serum and negative serum were used as controls. The results are shown in Table 1: OD of Escherichia coli-positive serum, Salmonella-positive serum, Pasteurella-positive serum bacteria, and Streptococcus-positive serum 450nm Values ​​are all less than 0.366, therefore, these several bacterial positive sera are all negative under this ELISA reaction system, and the OD of bovine brucellosis positive serum 450nm The value is greater than 0.433, which indicates that the specificity ...

Embodiment 2

[0119] Embodiment 2 sensitivity test

[0120] Utilizing the determined ELISA reaction conditions, an indirect enzyme-linked immunosorbent assay antibody detection kit for cattle and sheep brucellosis was assembled, and the optimal coating concentration of the recombinant BCSP31 protein antigen was used to coat the enzyme plate, and the bovine brucellosis Mycosis positive serum was diluted according to the eight dilutions of 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, 1:2560, and other conditions were based on the best response Conditions for ELISA detection test. The results are shown in Table 2: when the positive serum dilution of the recombinant BCSP31 protein antigen was 1:640, the OD 450nm value is critical.

[0121] Table 2

[0122]

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Abstract

The invention relates to a cattle and sheep brucellosis indirect enzyme-linked immunosorbent assay antibody detection kit and a preparation method thereof, belongs to the field of detection of pathogens of zoonotic infections and aims at solving the problems of low sensitivity, poor specificity and the like in detection of Brucella by adopting other antigens. The kit is assembled by taking a recombinant Brucella BCSP31 protein with expression induced by SUMO as an expression vector in Escherichia coli to serve as an antigen-coated ELISA (enzyme-linked immunosorbent assay) plate, adding serum to be detected and rabbit anti-bovine HRP-IgG or rabbit anti-goat HRP-IgG and adding a washing buffer solution, a blocking solution, a TMB (3,3',5,5'-Tetramethylbenzidine) primer solution and a stop solution for matching. The prokaryotic expression vector, namely the SUMO, is utilized to express the Brucella BCSP31 protein in an efficient and soluble manner, the kit is used for establishing a Brucella ELISA detection method, the detection has better repeatability and specificity, and cattle and sheep Brucella can be fast and efficiently detected by utilizing the method.

Description

technical field [0001] The invention relates to the technical field of detection of zoonotic infectious disease pathogens, in particular to an indirect enzyme-linked immunosorbent assay antibody detection kit for cattle and sheep brucellosis and a preparation method thereof. Background technique [0002] The Brucella cell surface protein 31ku (BCSP31) gene was cloned, sequenced and expressed in vitro in 1988. It encodes a soluble cell surface protein with an immunogenicity of 31ku. Except for Brucella ovis epididymis, The expression of BCSP31 protein could be detected in the other 6 species of Brucella. The BCSP31 gene of Brucella has a homology of 99.3% in 8 Brucella strains from different genera. It is a Brucella species-specific gene and is highly conserved. [0003] At present, some scholars have expressed BCSP31 protein by eukaryotic expression method, and used its eukaryotic expression product to immunize mice to study its ability to resist Brucella infection in mice;...

Claims

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Application Information

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IPC IPC(8): G01N33/569
CPCG01N33/56911G01N33/6893G01N2333/23
Inventor 杨艳玲郭利杨福合程世鹏李光玉陈立志李春义温永俊
Owner INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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