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Mycoplasma synoviae antigen protein LP85, ELISA detection method of corresponding antibody and kit

An antigen protein, detection method technology, applied in chemical instruments and methods, botanical equipment and methods, biochemical equipment and methods, etc. good, repeatable results

Active Publication Date: 2021-03-23
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The serum plate agglutination (SA) method is quick and easy to operate, and it is often used for the general screening of mycoplasma infection in chicken flocks, but it is prone to cross-reactions and high false positives; the MS antibody detection kit of IDEXX company has stable and reliable results, but is expensive

Method used

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  • Mycoplasma synoviae antigen protein LP85, ELISA detection method of corresponding antibody and kit
  • Mycoplasma synoviae antigen protein LP85, ELISA detection method of corresponding antibody and kit
  • Mycoplasma synoviae antigen protein LP85, ELISA detection method of corresponding antibody and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Identification, cloning and expression of the antigenic protein LP85 (MS LP85) of Mycoplasma gallisovii.

[0065] 1.1 Screening of MS immunogenic proteins

[0066] 1.1.1 MS membrane protein extraction

[0067] The MS WVU1853 culture in the logarithmic growth phase was transferred to the mycoplasma liquid medium at a ratio of 5%, cultured statically at 37° C. for 30 h, and the bacteria were collected by centrifugation at 12000 g. After washing twice with PBS, resuspend in PBS with 1% bacterial volume, ultrasonicate for 20 min (power 40%, work for 5 s, rest for 5 s) to obtain MS whole bacterial protein. The Mycoplasma Membrane Protein Extraction Kit (Shanghai Beibo) was used to extract the membrane protein from MS whole bacterial protein according to the method in the manual, and the protein concentration was determined with the BCA kit, and stored at -80°C after aliquoting.

[0068] 1.1.2 Western blot analysis of MS immunogenic proteins and mass spectrometry analysis ...

Embodiment 2

[0116] This example illustrates the ELISA detection method for establishing the Mycoplasma gallisovialum antibody by using the Mycoplasma gallisovialis antigenic protein LP85 screened and identified in Example 1, and illustrates the corresponding ELISA kit and usage method.

[0117] According to embodiment 1, the ELISA detection method of Mycoplasma gallisovum antibody can be set up as follows:

[0118] 2.1 Determination of optimal reaction conditions

[0119] 2.2.1 Determination of optimal antigen coating concentration and optimal serum dilution factor

[0120] Dilute the rMS LP85 protein antigen with the aforementioned carbonate coating buffer to 10 μg / ml, 7.5 μg / ml, 5.0 μg / ml, 2.5 μg / ml and 1.25 μg / ml, and coat them in an amount of 100 μl / well ELISA plate; MS standard positive serum and SPF chicken negative serum were diluted 1:200 and 1:500 times with the aforementioned antibody diluent, respectively, as positive serum control and negative serum control; the enzyme-labele...

Embodiment 3

[0159] This example is to carry out specificity test, sensitivity test and repeatability test on the ELISA detection method and corresponding kit provided in Example 2.

[0160] 3.1 Specificity test

[0161] The indirect ELISA detection method established with the above optimized conditions was respectively effective for MS clinical isolates JS1, SD1, SH1 and HB1 positive sera, Mycoplasma gallisepticum MG R low strain positive serum, Mycoplasma Iowa MI 695 strain positive serum, Escherichia coli EC O1, O2 and O78 trivalent positive serum, pullorum salmonella typhi SPG positive serum, Newcastle disease virus NDV positive serum, infectious bronchitis virus IBV positive Serum and chicken infectious bursal virus IBDV positive serum were tested, and MS standard positive serum and SPF chicken serum (MS negative serum) were set as controls at the same time, and OD was determined 450 Value, the commercial Mycoplasma synovialum antibody detection kit (IDEXX) was used as method control...

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Abstract

The invention provides a mycoplasma synoviae antigen protein LP85, an established ELISA method for detecting a corresponding antibody of mycoplasma synoviae, an ELISA kit prepared according to the method and a corresponding using method. Based on the mycoplasma synoviae antigen protein LP85, especially a mycoplasma synoviae recombinant expression protein LP85, the ELISA detection method of the corresponding antibody and the ELISA kit prepared according to the method have the characteristics of better specificity and sensitivity and good detection repeatability compared with an existing detection form.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an ELISA detection method and a kit for the mycoplasma gallisovialum antigenic protein LP85 and corresponding antibodies. Background technique [0002] Mycoplasma synoviae (Mycoplasma synoviae, MS, or Mycoplasma synovialum) is one of the important poultry pathogens, can infect chickens and turkeys, cause joint synovial bursitis and tenosynovitis, can also cause air sacs, and in severe cases cause systemic In recent years, there have been more and more reports that MS infection in laying hens leads to decreased egg production, egg quality and hatchability (3, 4, 5). [0003] MS is endemic worldwide, and epidemiological surveillance in several countries has shown a high prevalence of MS in commercial chicken flocks (6,7). The latest serological survey shows that from 2010 to 2015, chicken serum samples from many provinces and regions in my country, including Guangxi, Guang...

Claims

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Application Information

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IPC IPC(8): C07K14/30C12N15/31C12N15/70G01N33/569
CPCC07K14/30C12N15/70G01N33/56933C12N2800/22G01N2333/30G01N2469/20Y02A50/30
Inventor 于圣青祁晶晶李浩然丁铲王宇王少辉田明星李涛
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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