Bovine brucella indirect ELISA antibody detection kit

A technology for the detection of Brucella bovis and antibodies, applied in measuring devices, color/spectral characteristic measurements, instruments, etc., can solve the problems of missed detection, high proportion of kits, etc., to control the reaction background, shorten the time, and reduce non-specific The effect of heterosexuality

Inactive Publication Date: 2015-11-18
CHINA INST OF VETERINARY DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

That is to say, the production of Virb8 antibody has the specificity of infecting the host and Brucella species, and the cattle that can detect the Virb8 protein antibody are likely t

Method used

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  • Bovine brucella indirect ELISA antibody detection kit
  • Bovine brucella indirect ELISA antibody detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0108] ——Comparison of sensitivity and specificity of LPS extraction from different species of Brucella

[0109] 1. LPS purification

[0110] B. suis S2, B. melitensis 16M, and B. bovis A99 were inoculated with 20 kelp flats in TSA medium, respectively, and cultured at 37°C for 72 h. The cultured bacteria were washed with 0.5% carbolic acid physiological saline and centrifuged. Take 10 g of the wet weight of the bacterial body, and carry out LPS extraction and purification according to the "LPS extraction and purification method" in step 2 of the present invention.

[0111] 2. Preparation of LPS antigen-coated plates

[0112] After quantifying the purified LPS, it was diluted to 10 μg / ml with 0.05M carbonate buffer at pH 9.6, added to the ELISA plate, 100 μl per well, and coated for 16 hours at 2-8°C. The cells were washed once with 1× washing solution, 200 μl of blocking solution (0.01M PBS containing 3% gelatin, pH 7.4) was added to each well, and the cells were blocked a...

Embodiment 2

[0116] ——Research on optimization of kit components

[0117] 1. Selection of coating solution

[0118] Select citrate buffer solution (pH5.0), phosphate buffer solution (pH7.2), Tris-HCL buffer solution (pH8.5) and carbonate buffer solution (pH9.6) as coating solution respectively for coating, The concentration of the coated antigen was 10 μg / ml. After blocking with the blocking solution, the one with the highest OD value of the positive serum reaction was used as the coating solution with the best antigen adsorption effect. Results By comparing the four coating solutions, it was shown that the carbonate buffer solution with pH 9.6 had the best coating effect as the coating solution (see the appendix for the results). figure 2 ).

[0119] 2. Selection of blocking solution

[0120] PBS containing 3% gelatin, PBS containing 10% skimmed milk powder, PBS containing 10% horse serum and PBS containing 3% BSA were used as blocking agents for blocking respectively, and the blockin...

Embodiment 3

[0127] ——Preparation of positive control serum for Brucella bovis indirect ELISA antibody detection kit

[0128] The brucella tiger red plate agglutination test, in vitro agglutination test and complement fixation test were used to detect the clinically healthy adult cows around 18 months of age, and screen brucellosis antibody-negative cows. To screened negative bovine Brucella vaccine strain A19 inactivated antigen with 8 × 10 10CFU / bovine neck subcutaneous immunization, double-dose booster immunization 1 time after 3 months. Blood was collected 2 weeks after booster immunization, and the agglutination titer was about 1:1300 by test tube agglutination test. Phlebotomy was performed, serum was separated, and filter sterilized through a 0.45 μm filter. Then, the brucellosis-positive serum was diluted with the qualified negative control serum to a test tube agglutination titer of 1:320, added with proclin300 to a final concentration of 0.05%, aseptically packaged, and stored ...

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Abstract

The invention relates to a bovine brucella indirect ELISA antibody detection kit. The kit uses the lipopolysaccharide (LPS) of brucella suis S2 acclimated and induced by the application institution in the 1960s as the coating antigen, and improves the sensitivity of detection. The study improves the LPS purification and purity determination method, and endows the developed kit with good specificity. The kit not only can effectively eliminate the interference of conventional Gram negative bacteria to Brucella, but also can get rid of the technical defect that general brucellosis indirect ELISA (enzyme-linked immunosorbent assay) kits cannot distinguish enterocolitis Yersinia O9 and Escherichia coli O157 interference, and improves the specificity of detection. Through improvement and optimization of the main reagent formula, the reaction background of the kit is effectively controlled, and also the time required by detection is shortened.

Description

technical field [0001] The invention relates to a method for diagnosing brucellosis in animals - an indirect ELISA antibody detection kit for Brucella bovis, which belongs to the technical field of biological product detection. technical background [0002] Brucellosis (brucellosis) is a zoonotic disease characterized by miscarriage and fever caused by Brucella or Brucella, which seriously threatens the life and health of humans and various animals . This disease not only has serious harm to the reproduction and production performance of animals, but more importantly, it is often difficult to cure people infected with Brucella, thus causing serious public health problems. Therefore, in countries where Brucella is endemic, elimination of brucellosis has been one of the most important goals of public health programs. [0003] Brucellosis has existed in the world for a long time. Humans' understanding of the disease has gradually deepened and the level of diagnosis has been c...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N21/31
CPCG01N33/56911G01N21/31
Inventor 丁家波王芳冯宇朱良全王楠毛开荣
Owner CHINA INST OF VETERINARY DRUG CONTROL
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