Immunoassay kit by using light initiated chemiluminescent assay for bovine mycoplasma antibody and detection method thereof

A light-activated chemiluminescence and Mycoplasma bovis technology, which is applied in the fields of chemiluminescence/bioluminescence, biological testing, fluorescence/phosphorescence, etc., can solve the problems of cumbersome operation and poor specificity, and achieve cumbersome operation, simple operation and high sensitivity. Effect

Inactive Publication Date: 2019-05-07
重庆市动物疫病预防控制中心 +1
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Problems solved by technology

[0004] Aiming at the deficiencies of the above-mentioned prior art, the object of the present invention is to provide a Mycoplasma bovis antibody photoactivated chemilu

Method used

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  • Immunoassay kit by using light initiated chemiluminescent assay for bovine mycoplasma antibody and detection method thereof
  • Immunoassay kit by using light initiated chemiluminescent assay for bovine mycoplasma antibody and detection method thereof
  • Immunoassay kit by using light initiated chemiluminescent assay for bovine mycoplasma antibody and detection method thereof

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Embodiment

[0036] 1. Antigen screening

[0037] The cell membrane surface protein of Mycoplasma bovis plays an important role in the process of pathogenicity and infection. The P30 gene was screened out by predicting the signal peptide and antigen epitope analysis of the cell membrane surface protein of Mycoplasma bovis, and the bioinformatics of the P30 gene and its encoded protein were further carried out. Scientific and immunological analysis proved that P30 is the surface membrane protein of Mycoplasma bovis, is a specific antigen of Mycoplasma bovis, has good immunogenicity, sequence conservation and strong stability. Therefore, the P30 protein can be used as the antigen in the photoactivated chemiluminescence immunoassay kit for the Mycoplasma bovis antibody.

[0038] 2. Prokaryotic expression and detection of Mycoplasma bovis membrane protein P30 protein

[0039] 2.1 Construction of pET28a(+)-P30 recombinant vector

[0040] According to the Mycoplasma bovis P30 gene sequence pub...

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Abstract

The invention discloses an immunoassay kit by using a light-initiated chemiluminescent assay for a bovine mycoplasma antibody and a detection method thereof. The kit comprises bovine mycoplasma antibody P30 protein monoclonal antibodies, goat anti-mouse IgG conjugated acceptor microbeads, His-tagged bovine mycoplasma antibody P30 protein antigens, and nickel chelated donor magnetic beads. The invention prepares outer membrane protein of bovine mycoplasma antibody P30 by using the genetic engineering technology, prepares bovine mycoplasma antibody P30 protein monoclonal antibodies by using thePEG1500 cell fusion technology, and the purified recombinant P30 protein is used as a product of interaction between the antigen and the prepared monoclonal antibody, competing with the monoclonal antibody by the antibody in the sample to bind and recombine the P30 protein, and establishing a method for detecting competitive bovine mycoplasma antibody AlphaLISA. According to the immunoassay kit byusing light initiated chemiluminescent assay for the bovine mycoplasma antibody and the detection method thereof, the detection method has the advantages of short detection time, good specificity, good sensitivity, no need for washing, simple operation and low detection cost.

Description

technical field [0001] The invention relates to the fields of veterinary medicine, immunology and biological products, in particular to a photo-activated chemiluminescence immunoassay kit for mycoplasma bovis antibody and a detection method thereof. Background technique [0002] Mycoplasma bovis (Mb) is the smallest and simplest prokaryon that can replicate autonomously. It can cause bovine pneumonia, mastitis, arthritis and other diseases, causing huge economic losses and seriously threatening the healthy development of my country's cattle industry. . At present, the commonly used detection methods for Mycoplasma bovis in veterinary laboratories include biological detection, molecular biological detection and immunological detection. Biological detection is mainly through the traditional isolation and identification of pathogens. Because mycoplasma is a facultative anaerobic organism, it has extremely strict nutritional requirements, and the culture period is long (it takes...

Claims

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Application Information

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IPC IPC(8): G01N21/76G01N21/64G01N33/68G01N33/577
Inventor 梁望旺吴胜昔曾政李俊萱李令臣鲁友铭侯力嘉蔺露陈忠琼徐缘李红
Owner 重庆市动物疫病预防控制中心
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