Development of ELISA and Serum Plate Agglutination for Detecting Antibodies of Mycoplasma gallisepticum using Strain of Thai Isolate

Abstract

The aim of the present study was to develop enzyme-linked immunosorbent assay (ELISA) and serum plate agglutination (SPA) for detecting antibodies of Mycoplasma gallisepticum (MG), using a Thai isolated field strain. MG isolation and identification was performed using conventional culture method, polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) and Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The growth characteristics of each strain were analyzed to determine the most appropriate conditions for the cultivation of MG. The results indicated that the Thai isolated field strain gave the highest growth rate of MG yield with a peak concentration of 1.4 X 108 cfu/ml at 36-48 hr post inoculation. The in-house agglutination antigen was evaluated for freedom of contamination and sterility, specificity and potency tests were conducted. All prepared antigens met all the quality criteria. The in-house serum plate agglutination antigen using a Thai isolated field strain gave 99% sensitivity and 100% specificity compared with a commercial agglutination antigen. The indirect ELISA test using a field strain was also developed with the purified antigen, and its potential for the detection of antibodies was compared with S6 and F strains. The in-house ELISA using the Thai isolated field strain provided 67% sensitivity and 95% relative specificity compared with the commercial ELISA test.