Preparation and Testing of Antigens for the Demonstration of Mycoplasma Antibodies in Pigs by a Solid-phase Radioimmunoassay

Hampl J., J. Tomanek, L. Rodak, F. Kuksa and M. Sedlacek: Preparation and Testing of Antigens for the Demo7JStration of Mycoplasma Antibodies in Pigs by a Solid-Phase Radioimmunoassay. Acta vet. Brno. 50, 1981: 67-72. Three types of antigen, namely corpuscular, disaggregated with sodium dodecyl sulphate and disaggregated by sonication, were prepared from Mycoplasma hyopneumoniae and Mycoplasma hyorhinis strains and used for the demonstration of antibody by a solid-phase radioimmunoassay in the sera of pigs infected experimentally with these two mycoplasma strains. The best suited from the practical point of view was the antigen prepared by ultrasonic disaggregation, diluted 1: 50 and incubated in polystyrene microtubes for 16 hours. The test serum and 125I-Iabelled antibody were incubated for 3 hours; prolongation of this period had no effect on the results. The radioactivity of the administered 1'5I-Iabelled antibody ranged from 10000 to 80000 counts per minute (cpm); its level had no significant effect on antibody titres demonstrated in the sera. Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, enzootic pneumonia, corpuscular antigen, disaggregated antigen, 125 I-labelled antibody to swine IgG. Radioimmunoassay (RIA) is being used increasingly in the diagnosis of farm animal diseases. It is sensitive and reliable, allows an objective evaluation of the results and is economic in terms of cost where large numbers of samples are to be processed. One 'of the main problems in pig herds under intensive husbandry is respiratory disease conditions. A considerable role in its development is played by mycoplasmas, particularly Mycoplasma hyorhinis and Mycoplasma hyopneumoniae, which have been isolated from the lungs of pigs with enzootic pneumonia. Their isolation presents great difficulties because it requires special media and prolonged cultivation. The demonstration of antibodies in pigs exhibiting no clinical and pathological signs of enzootic pneumonia may be an indication of their subclinical disease and allows early culling of such possible sources of infection. The present study was designed to test three differently prepared mycoplasma antigens as to their suitability for solid-phase RIA and other conditions of the assay. Preliminary results were presented previously (Hampl et al. 1978). Recently Taylor (1979) described demonstration by solid-phase RIA of immunoglobulin class-specific antibody in mice after infection with Mycoplasma pulmonis. Materials and Methods Mycoplasma hyorhinis and M. hyopneumoniae strains used for the preparation of antigens were grown in a liquid medium according to GoB et al. (1975) for 48 and 120 hrs, respectively. The medium was then centrifuged at 10 000 rpm for 30 minutes and the sediment was washed three times with phosphate buffered saline (PBS), pH 7.2, resuspended in PBS to give a volume 100 times smaller than the original culture medium volume and used for the preparation of antigens.