Fractionation, characterization and diagnostic potential of circulating leprosy antigens isolated from sera of lepromatous leprosy patients.

Circulating antigen was isolated from lepromatous sera by ammonium sulfate precipitation. The protein fraction between 36% and 75% ammonium sulfate was reactive with leprosy sera. Further fractionation of Ultrogel AcA 34 gel filtration column gave four protein fractions, CLA1, CLA2, CLA3 and CLA4. CLA1 and CLA2 showed antigenic activity. On SDS-PAGE analysis and elution of the protein fractions, CLA1-7, CLA2-1 and CLA2-7 were found to be reactive with leprosy sera. On evaluating the diagnostic utility of these fractions, CLA1-7 could detect IgG antibodies in 80% of the lepromatous (LL) and in 40% of the tuberculoid (TT) serum samples. Fraction CLA2-1 reacted with IgM antibodies in 80% of the LL and TT patients; fraction CLA2-7 reacted with IgM antibodies in 70% of the LL and TT sera. Biochemical characterization indicated that CLA1-7 was a glycoprotein while CLA2-1 and CLA2-7 were lipoproteins in nature. When tested by an inhibition ELISA, fraction CLA2-7 inhibited the binding of anticeramide antibodies to a ceramide-coated plate while thin-layer chromatography of fractions CLA2-1 and CLA2-7 showed a spot with an Rf value similar to that of standard ceramide. This study thus shows for the first time the presence of ceramide in circulating leprosy antigen.