Immunoblot assays using recombinant antigens for the detection of Mycoplasma hyopneumoniae antibodies

The 36 kDa L-lactate dehydrogenase (LDH) and a 29 kDa partial fragment of a n ABC transporter ATP-binding protein analogue/multidrug resistance protein homologue (PR2) of Mycoplasma hyopneumoniae were tested for their potentia l as diagnostic antigens. Recombinant LDH was genetically engineered to con tain six histidine residues at its C-terminal end, expressed in Escherichia coli and purified to a high degree using Ni2+-chelate affinity chromatogra phy. A partial 262 amino acid segment representing the C-terminal end of th e PR2 protein was cloned as a glutathione S-transferase (GST) fusion protei n, expressed in E. coli and purified by urea extraction. Purified recombina nt LDH-6xHis and PR2-GST were then reacted with pig sera in immunoblot assa ys. Our immunoblots showed that both proteins detected anti-M. hyopneumonia e antibodies in field and experimentally infected pig sera but not in any o f the SPF control sera. The two proteins were specific for M. hyopneumoniae as they did not react with sera of pigs infected with the closely related Mycoplasma flocculare and Mycoplasma hyorhinis which are frequently isolate d in pigs but are not of particular concern. (C) 2000 Elsevier Science B.V. All rights reserved.