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Indirect enzyme-linked immunosorbent assay adsorption detection method for deer tuberculosis natural infection and BGG immunization

An enzyme-linked immunosorbent adsorption and natural infection technology, applied in the direction of measuring devices, biological testing, material inspection products, etc.

Inactive Publication Date: 2015-04-29
杜锐 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

For the differential diagnosis of deer tuberculosis natural infection and BCG immunity, to overcome the lack of effective methods to distinguish natural infection and BCG immunity

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  • Indirect enzyme-linked immunosorbent assay adsorption detection method for deer tuberculosis natural infection and BGG immunization

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Embodiment Construction

[0032] 1. The purified protein GST-Rv3872-74-75, which was expressed and purified and tested for reactogenicity, was used as an antigen, and Staphylococcus protein A (SPA)-horseradish peroxidase (HRP) produced by Shanghai Biological Products was used for preparation Enzyme-labeled antibody, autopsy, and bacteriological examination were all positive tuberculosis deer serum, and allergy, autopsy, and bacteriological examination were negative deer serum.

[0033] 2. Preparation of rabbit anti-deer IgG

[0034] Take healthy deer serum and saturate it with 50% (NH4) 2 SO 4 After salting out once, then saturated with 37.5% (NH4) 2 SO 4 Salt out three times, extract deer serum IgG: fully dialyze to no NH 4+ , SO 4 2+ . The balance was dialyzed against 0.01M pH8.0 phosphate buffer and passed through a DEAE-32 column. Elute with 0.01M pH8.0 phosphate buffer, collect the protein fraction, and determine the protein content; identify its purity by polyacrylamide disc electrophore...

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Abstract

The invention discloses an indirect enzyme-linked immunosorbent assay adsorption detection method for deer tuberculosis natural infection and BGG immunization. The biomaterials selected by the detection method comprise purified protein GST-Rv3872-74-75, BSA, IgG-HRP, standard positive blood serum, standard negative blood serum and to-be-detected blood serum; the detection method comprises the steps of enveloping antigen, closing, adding the blood serum, adding the enzyme combination IgG-HRP, adding substrate, stopping reaction, conducting detection through an enzyme-linked immunosorbent assay detector. The method has the positive results that indirect ELISA diagnosis antigen of deer tuberculosis natural infection and BGG immunization is further screened on the basis that second antibody of the IgG-HRP is fulfilled, and adopts the purified protein GST-Rv3872-74-75 as optimal antigen and establishes optimal test steps and conditions; the differential diagnosis method is high in specificity and sensitivity, simple, convenient, fast, and low in cost.

Description

technical field [0001] The present invention belongs to the field of veterinary medicine. In particular, it relates to an indirect enzyme-linked immunosorbent assay. Background technique [0002] Epidemiological surveys show that Deer tuberculosis has become the most serious infectious disease that endangers the deer industry in my country. Studies have shown that Bacillus Calmette-Guérin (BCG) is an effective agent for preventing tuberculosis in deer. However, with the application of BCG in deer herds, a serious problem arises, that is, it is impossible to distinguish BCG-immunized deer from wild virus-infected deer with current diagnostic methods. This is a major problem that has plagued the deer industry for a long time. The emergence of this problem seriously interferes with the diagnosis and control of deer tuberculosis, limits the application of BCG in deer herds, and makes the epidemic of deer tuberculosis more and more serious. Currently, there are no diagnostic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543
CPCG01N33/53G01N33/543
Inventor 杜锐王文玉曾范利时坤宗颖李健明刘东旭
Owner 杜锐