Development of an indirect ELISA for detecting humoral immunodominant proteins of Mycoplasma hyopneumoniae which can discriminate between inactivated bacterin-induced hyperimmune sera and convalescent sera

BACKGROUND: Mycoplasma hyopneumoniae (M. hyopneumoniae) is the primary pathogen of porcine enzootic pneumonia, which has been associated with economic losses due to reduced daily weight gain and feed efficiency. Although it has a small genome and no more than 1000 genes, M. hyopneumoniae can be cultured in cell free media. However, some proteins were not expressed or were only expressed in negligible amounts under culture conditions. Nevertheless, some of these proteins can be expressed at a high level and induce a strong and rapid immune response after M. hyopneumoniae infection. The unexpressed or less expressed proteins may play critical roles in pathogenesis and/or immune response. In order to find the differentially expressed proteins of M. hyopneumoniae between culture condition and infected animals, we established an indirect ELISA for the detection of humoral immunodominant proteins which can discriminate between inactivated bacterin-induced hyperimmune sera and convalescent sera by using Mhp366 protein which did not react with sera from bacterin-immunized pigs, but revealed a strong immunoreaction with porcine convalescent sera. RESULTS: The checkerboard titration method was done by using porcine convalescent sera as positive sera and inactivated bacterin-induced hyperimmune sera as negative sera. The bacterial lysates of fusion proteins and free GST protein without dilution were the optimal coating antigens. The optimal blocking buffer was PBS with 10% FBS and 2.5% skimmed milk. In the checkboard ELISAs, when the sera were diluted at 1:500 and the HRP-labeled rabbit anti-pig IgG were diluted at 1:20000, most positive result was obtained for the assay. CONCLUSIONS: This established indirect ELISA can be used as a tool for the detection of humoral immunodominant proteins of M. hyopneumoniae which can discriminate between inactivated bacterin-induced hyperimmune sera and convalescent sera.