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Mycoplasma ovipneumoniae antibody indirect ELISA detection kit

A technology of Mycoplasma pneumoniae and detection kit, which is applied in biological tests, measuring devices, immunoassays, etc., can solve problems such as cross-reaction of whole bacterial antigen coating, unsuitable for serological diagnosis technology, etc.

Inactive Publication Date: 2020-05-08
SOUTHWEST UNIVERSITY FOR NATIONALITIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no commercialized ELISA antibody detection kit for Mycoplasma ovis pneumoniae. Although there is an ELISA method using whole bacterial antigens in China, due to the presence of Mycoplasma conjunctiva and other mycoplasma close to Mycoplasma pneumoniae in sheep, the whole Bacterial antigen coating will have obvious cross-reaction, so it is not suitable for the establishment of serological diagnostic technology

Method used

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  • Mycoplasma ovipneumoniae antibody indirect ELISA detection kit
  • Mycoplasma ovipneumoniae antibody indirect ELISA detection kit
  • Mycoplasma ovipneumoniae antibody indirect ELISA detection kit

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1 Preparation of Mycoplasma pneumoniae Antibody Indirect ELISA Antigen and Detection of Immunogenicity

[0030] 1. Materials

[0031] (1) Strains and plasmids: positive serum of Mycoplasma ovis pneumoniae and negative serum of Mycoplasma ovis pneumoniae, positive serum of Pasteurella, Staphylococcus aureus, Mannella haemolyticus and Mycoplasma mycoplasma goat subspecies serum, positive serum of Mycoplasma hyopneumoniae, Serum of Mycoplasma filamentous subspecies were all existing materials, which were stored at -20°C by the Veterinary Laboratory of Southwest University for Nationalities; Escherichia coli and Salmonella factor serum were purchased from Sichuan Institute of Biological Products; clinical isolate of Mycoplasma pneumoniae SC01 was obtained from this Separated and stored in the laboratory; the prokaryotic expression vector pET-32a(+) is the product of Novagen; the competent cells DH5ɑ and Rosetta (DE3) are the products of TaKaRa;

[0032] (2) Main re...

Embodiment 2

[0061] Example 2 Establishment of Indirect ELISA Method for Antibody of Ovine Mycoplasma Pneumoniae and Condition Optimization

[0062] ① Determination of the optimal working concentration of antigen and antibody

[0063] The optimum working concentration of antigen and antibody was determined by Founder titration. According to the prepared recombinant protein concentration, the antigen was diluted to 4 μg / ml, 2 μg / ml, 1 μg / ml, 0.5 μg / ml, 0.25 μg / ml in sequence using coating buffer, and each dilution was repeated three times. Use the serum diluent to dilute the negative and positive serum of Mycoplasma pneumoniae in sequence 1:100 times, 1:200 times, 1:400 times and 1:800 times, and seal with 2% BSA. The rest of the steps follow the method of 2.3.1, read Take the results, calculate the P / N value, and select the antigen concentration when the average positive OD450 is around 1 and the P / N is the largest as the optimal concentration of the antigen. The serum dilution correspon...

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Abstract

The invention relates to the technical field of veterinary biological product detection, in particular to a mycoplasma ovipneumoniae antibody indirect ELISA detection kit. Wherein the coating antigenis mycoplasma ovipneumoniae adhesin gene P113 recombinant protein rP113 (C); the primer sequence of the primer is as follows: P113 (c) F: 5 to CGCGGATCCGAAGGTGCTCAAGACCAAGGTA-3, and P113 (c) R: 5 to CCGCTCCGTTGTTGTTGTTGAGGTGGTGTATCAGGT-3. The invention provides the indirect ELSIA detection kit for the mycoplasma ovipneumoniae antibody, which is established by taking purified P113 recombinant protein rP113 (C) as a coating antigen, and a powerful tool is provided for epidemiological investigation, disease diagnosis and vaccine immune effect evaluation.

Description

technical field [0001] The invention relates to the technical field of detection of veterinary biological products, in particular to an indirect ELISA detection kit for antibodies to Mycoplasma pneumoniae. Background technique [0002] Mycoplasma ovis is the main pathogen that causes atypical pneumonia in sheep and goats. At the same time, the susceptibility of sheep to Pasteurella multocida, Mannella haemolytica and parainfluenza virus also increases significantly after being infected with Mycoplasma ovis. At present, the pathogen is distributed all over the world, causing significant economic losses to the sheep and goat breeding industry, and also seriously threatening the health of wild small ruminants. In my country, the infection of Mycoplasma pneumoniae in sheep is also very common and the harm is serious. [0003] Accurate diagnosis and epidemiological investigation of Mycoplasma pneumoniae infection in sheep are the key links to take comprehensive control measures....

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/569G01N33/543
CPCG01N33/6854G01N33/56933G01N33/543G01N2469/20
Inventor 杨发龙王远微刀筱芳
Owner SOUTHWEST UNIVERSITY FOR NATIONALITIES
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