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.

Detection of S-100 protein and anticeramide antibodies in leprosy patients by ELISA.

The status of assay for S-100 antigen protein and anticeramide antibodies in serum in understanding nerve damage in different forms of leprosy were evaluated by the enzyme immunoassay. Based on the clinical and smear examination, patients were classified as indeterminate (Ind), tuberculoid (TT), borderline tuberculoid (BT), borderline lepromatous (BL) and lepromatous (LL). Antibody levels against ceramide were observed in sera of leprosy patients with 37.5% of Ind, 28% of TT, 66% BT, 78% BL and 62% LL patients positive as against 8% endemic normal sera. The mean OD ranged from 0.141 to 0.275 in different groups of leprosy. In contrast, S-100 was detected in 71.4% Ind, 88.8% TT, 76.4% BT, 100% BL and 95.8% LL, while 5% of ENL samples were positive for S-100 antigen. Mean S-100 levels in these different categories of patients were significantly higher Ind--0.45 ng/ml, TT--0.32 ng/ml, BT--0.23 ng/ml, BL--0.23 ng/ml, LL--0.19 ng/ml as compared to that of normal 0.07 ng/ml. In general S-100 seems to be a more sensitive and reliable marker than anticeramide antibodies for nerve damage. Five out of 7 indeterminate cases show increased levels of S-100, showing an extent of nerve damage similar to that of TT and could be a useful marker for assessing nerve damage in indeterminate patients for better management.

Detection of S-100 antigen and anti ceramide antibody in sera of leprosy patients with and without reaction.

Levels of anticeramide antibodies and S-100 antigen in leprosy patients with and without reaction are compared in this study. The increase in levels of IgM anti ceramide antibody in the tuberculoid group of patients with reaction, when compared to those without reaction, is significant (P < 0.05). Similarly, significant increase (P < 0.01) was observed in the borderline group with reaction. No significant change in anti ceramide antibody level was observed in the lepromatous group of patients with and without reaction. Mean levels of S-100 were slightly lower in all three groups of patients with reaction, but the differences were not statistically significant.

Penicillinase ELISA for detection of tubercular antigen in tuberculosis.

Stick sandwich enzyme linked immunosorbent assay (ELISA) using rabbit anti PPD-RT 23 immunoglobulins and enzyme penicillinase has been explored for detection of tubercular antigen in sera and CSF samples of pulmonary tuberculosis and tubercular meningitis (TBM) respectively. The analysis of sera showed 73.3% of pulmonary tuberculosis cases, 16.2% of healthy controls and 44.4% of Hansen's disease positive for tubercular antigen. The accuracies of positive and negative predictive values were 69% and 82% respectively. The analysis of CSF samples showed the presence of tubercular antigen in 76.4% of TBM, 16.6% of pyogenic meningitis cases, 19.4% of neurological diseases other than meningitis and 16.1% non-neurological disease controls. The accuracies of positive and negative predictive values were 48% and 94% respectively. Hence this simple test using economical and indigenous reagents can be applied for the diagnosis of pulmonary and extra-pulmonary tuberculosis.