title: Immunoblot assays using recombinant antigens for the detection of Mycoplasma hyopneumoniae antibodies.
abstract: The 36kDa L-lactate dehydrogenase (LDH) and a 29kDa partial fragment of an ABC transporter ATP-binding protein analogue/multidrug resistance protein homologue (PR2) of Mycoplasma hyopneumoniae were tested for their potential as diagnostic antigens. Recombinant LDH was genetically engineered to contain six histidine residues at its C-terminal end, expressed in Escherichia coli and purified to a high degree using Ni(2+)-chelate affinity chromatography. A partial 262 amino acid segment representing the C-terminal end of the PR2 protein was cloned as a glutathione S-transferase (GST) fusion protein, expressed in E. coli and purified by urea extraction. Purified recombinant LDH-6xHis and PR2-GST were then reacted with pig sera in immunoblot assays. Our immunoblots showed that both proteins detected anti-M. hyopneumoniae antibodies in field and experimentally infected pig sera but not in any of 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 isolated in pigs but are not of particular concern.
authors.affiliation: Animal Health Biotechnology, Institute of Molecular Agrobiology, National University of Singapore, 1 Research Link, 117604, Singapore, Singapore.
authors.forename: S
authors.initials: S
authors.lastname: Subramaniam
authors.forename: J
authors.initials: J
authors.lastname: Frey
authors.forename: B
authors.initials: B
authors.lastname: Huang
authors.forename: S
authors.initials: S
authors.lastname: Djordjevic
authors.forename: J
authors.initials: J
authors.lastname: Kwang
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000276:immunology; D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood*; D000942:Antigens, Bacterial / Q000276:immunology; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D015151:Immunoblotting / Q000662:veterinary; D007770:L-Lactate Dehydrogenase / Q000276:immunology; D009174:Mycoplasma / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D009175:Mycoplasma Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D016133:Polymerase Chain Reaction / Q000662:veterinary; D011994:Recombinant Proteins / Q000276:immunology; D017422:Sequence Analysis, DNA; D013047:Specific Pathogen-Free Organisms; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis* / Q000382:microbiology
chemical_list: D018528:ATP-Binding Cassette Transporters; D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D017931:DNA Primers; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D007770:L-Lactate Dehydrogenase