Use of an Artificial Antigen Containing the 3,6-di-O-methyl- -D-glucopyranosyl Epitope for the Serodiagnosis of Leprosy

Abstract

The coupling of synthetic 3,6-di-O-methyl-β-d-glucopyranosyl-(1→4)-2,3-di-O-methyl-α-l-rhamnopyranose, the hapten determinant of phenolic glycolipid I from Mycobacterium leprae, to bovine serum albumin (BSA) by reductive amination produced the antigen ε-N-l-[1-deoxy-2,3-di-O-methyl-4-O-(3′,6′-di-O-methyl-β-d-glucopyranosyl)rhamnitol]-lysyl-BSA, which proved highly sensitive in ELISA and showed good concordance with the native glycolipid in analysis of serum samples from 223 leprosy patients. Conjugates prepared from 6-O-methyl-β-d-glucopyranosyl- or β-d-glucopyranosyl-containing disaccharides were inactive and those containing noncyclic 3,6-di-O-methyl-glucitol showed little activity. Thus 3,6-di-O-methyl-β-d-glucopyranose in its cyclic hemiacetal form is necessary for binding anti-glycolipid IgM from leprosy patients. Analysis of serum samples from healthy subjects showed a false-positive rate of 2.4% (four of 169) against the glycolipid and 3.6% (six of 169) against the glycoconjugate. Comparable figures for samples of sera of tuberculosis patients were 3.0% (two of 66) and 9.0% (six of 66), respectively. Alternative synthesizing strategies may diminish this cross-reactivity. The prospects of a fully synthetic specific antigen for the worldwide serodiagnosis of leprosy look promising.