Isolation of Mycoplasma pulmonis membranes and identification of surface antigens.

Labeling of Mycoplasma pulmonis cells by iodination with the Bolton-Hunter reagent was shown to efficiently label membrane-associated proteins without significant loss of viability. Labeled proteins proven to be surface exposed by differential proteolytic digestion were analyzed by autoradiography of two-dimensional polyacrylamide gel electrophoresis (PAGE), and seven labeled major polypeptides were identified. To identify all the membrane-associated antigens, pure membranes were isolated by using the surface-labeled proteins as markers. Analysis of the isolated membranes by autoradiography of both sodium dodecyl sulfate-PAGE and the two-dimensional PAGE indicated that the labeled surface proteins were retained in the pure membranes; by immunoblotting with sera from naturally infected animals, these surface proteins were shown to be the predominant antigens recognized by the host during natural infection.