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Brucella outer membrane protein antibody detection ELISA (enzyme-linked immuno sorbent assay) kit

A technology for detection of outer membrane proteins and antibodies, applied in the biological field, can solve the problems of many false negative results, reduce test accuracy, false positives, etc., and achieve the effect of meeting the requirements of epidemic prevention

Inactive Publication Date: 2015-06-10
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Tiger red plate agglutination test and test tube agglutination test are easy to cause misjudgment due to many false positive and false negative results; dairy cow whole milk ring test results are easily affected by the internal environment of the animal body and cause misjudgment; complement fixation test is cumbersome to operate , time-consuming and less in practical applications
[0004] In recent years, ELISA has gradually become an important method for the detection of brucellosis due to its advantages of simple operation, convenient storage, high sensitivity and high specificity. Port inspection
In addition, intact bacteria are the most classic as the ELISA coating antigen, but because Brucella culture conditions are harsh, it is not easy to culture, and there are biological safety hazards
When ELISA is selected as a common method for detecting brucellosis at home and abroad, smooth lipopolysaccharide (sLPS) is mostly extracted from Brucella bovis S1119-3 or S99 strain as the antigen, which is easy to interact with other bacteria. Cross-reactivity, reducing the accuracy of the test

Method used

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  • Brucella outer membrane protein antibody detection ELISA (enzyme-linked immuno sorbent assay) kit
  • Brucella outer membrane protein antibody detection ELISA (enzyme-linked immuno sorbent assay) kit
  • Brucella outer membrane protein antibody detection ELISA (enzyme-linked immuno sorbent assay) kit

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Effect test

Embodiment 1

[0038] The Brucella outer membrane protein OMP28 that embodiment 1 Escherichia coli expresses is prepared

[0039] 1. Materials

[0040] 1.1 Strain and serum

[0041] Brucella bovis A544 strain was provided by the China Veterinary Drug Administration, the prokaryotic expression vector pET-28a (+), and Escherichia coli BL21 (DE3) were preserved by the Immunology Laboratory of Capital Medical University.

[0042] 1.2 Reagents

[0043] DNA polymerase, various restriction enzymes and T4 DNA ligase were purchased from NEB Company; Sephadex G25 and Superdex75 prepacked columns were purchased from GE Company; DNA gel recovery kit and plasmid extraction kit were purchased from Dalian Bao Biological Engineering Co., Ltd. .

[0044] 2. Primer Design

[0045] Referring to the gene sequence of Brucella bovis OMP28 (CP003176) provided by NCBI, primers were designed with Primer5.0 software and synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. The upstream prime...

Embodiment 2

[0057] The preparation of embodiment 2 antigen-coated microtiter plates

[0058] 1. Reagents

[0059] (1) Coating solution: 25mmol / L carbonate buffer solution, pH9.6. Na 2 CO 3 1.59g, NaHCO 3 2.93g,ddH 2 Add O to 1000mL, store at 4°C for later use;

[0060] (2) Washing solution storage solution (10*PBS): NaCl80g, KCl2g, NaCl 2 HPO 4 12H 2 O29g, KH 2 PO 4 2g,ddH 2 O was added to a constant volume of 1000mL;

[0061] (3) Washing solution (PBST): containing 0.05% Tween-20PBS, (pH7.4). Take 50mL10*PBS, 450mL ddH 2 O, 250uL Tween-20, stored at room temperature;

[0062] (4) Blocking solution: Weigh 5 g of skim milk, dissolve it in 100 mL of PBST washing solution, and store it at 4°C for later use.

[0063] 2. Coated

[0064] Under sterile conditions, dilute the purified recombinant protein OMP28 in the coating solution at 1 μg / ml, add 100 μL / well to a 96-well microtiter plate, and coat in a wet box at 37°C for 1 hour.

[0065] 3. Washing

[0066] Wash 4 times with...

Embodiment 3

[0071] Embodiment 3 Positive control serum, negative control serum preparation

[0072] 1. Reagents

[0073] Brucella standard positive serum and negative serum were purchased from China Veterinary Drug Administration, and thimerosal was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0074] 2. Positive Control Serum Production

[0075] Under sterile conditions, dilute the Brucella standard positive serum (1ml dry powder package) with 2ml of high-pressure distilled water, add 0.01% thimerosal for preservation, then subpackage and label it.

[0076] 3. Negative Control Serum Production

[0077] Under sterile conditions, dilute the Brucella standard negative serum (1ml dry powder package) with 2ml of high-pressure distilled water, add 0.01% thimerosal for preservation, then subpackage and label it.

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Abstract

The present invention relates to the field of biotechnology, in particular relates to a Brucella outer membrane protein antibody detection ELISA (enzyme-linked immuno sorbent assay) kit. The ELISA kit uses Brucella outer membrane protein OMP28 expressed by escherichia coli as antigen for package of an enzyme label plate, Brucella reference positive serum and negative serum are respectively used as a positive control and negative control, Goat Anti-Bovine IgG labeled with HRP (horseradish peroxidase) is used as an enzyme labeled antibody, and the ELISA kit is assembled by combination of a dilution liquid, a washing liquid, a substrate solution a, a substrate solution B and a termination liquid. The Brucella outer membrane protein antibody detection ELISA kit has the advantages of being safe, sensitive, specific, simple, fast and stable, can meet inspection and monitoring epidemic prevention requirements of Brucella antibody in cattles in China.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a Brucella outer membrane protein antibody detection ELISA kit. Background technique [0002] Brucellosis is a major zoonotic disease caused by bacteria of the genus Brucella, which seriously affects the productivity of livestock and the biosafety of animal products, and causes serious economic losses to the development of animal husbandry. According to the statistics of the world, the economic loss caused by brucellosis is nearly 3 billion US dollars every year. There are as many as one million cattle, sheep and pigs infected every year in my country, and the economic losses caused can reach more than one billion yuan. Based on estimates in Xinjiang and Gansu in my country, the annual direct economic loss caused by brucellosis is about 100 million yuan. According to statistics from the Ministry of Agriculture, in December 2011, there were 468 outbreaks of brucellosis in cattle and...

Claims

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Application Information

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IPC IPC(8): G01N33/68C12N15/70C12R1/01
CPCG01N33/56911G01N33/6854G01N2333/23
Inventor 韩雪清王慧煜林祥梅贾广乐张小娟
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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