Evaluation of a rapid serological test for leprosy classification using human serum albumin as the antigen carrier.

The presence of anti-BSA antibodies may interfere in serological tests, as ELISA or immunochromatographic assays. BSA is frequently used as a blocking agent or as "inert" carrier of antigens, such as the NT-P-BSA, the semi-synthetic trisaccharide analogue of the PGL-I (phenolic glycolipid-I) antigen from the cell wall of the Mycobacterium leprae. PGL-I was prepared and linked to human serum albumin based in the hypothesis that replacing BSA by a human protein carrier would enhance the performance of leprosy serological tests. A total of 1162 serum samples were tested by ELISA and by the ML Flow rapid test using NT-P-BSA or NT-P-HSA antigens. When grouping leprosy patients as paucibacillary (PB) or multibacillary (MB) according to the Ridley & Jopling classification, ML Flow BSA and ML Flow HSA tests correctly allocated 70.9% and 68.6% of patients in the PB group, and 87% and 81% of patients in the MB group, respectively. Concordant results were found in 82.0% (953/1162) (kappa value=0.637; sd=0.023) of samples between ML Flow tests and 85.7% (996/1162) (kappa value=0.703; sd=0.021) between ELISA tests. ML Flow results were statistically similar and the same was true for ELISA tests using HSA or BSA. However, we noticed a tendency to decreased capacity to detect MB patients and an increased positivity among PB patients, HHC, TB patients and healthy controls by the HSA carrier in both ML Flow and ELISA. The PGL-I serology performed by the ML Flow test with BSA or HSA as antigen carriers can be a useful, friendly auxiliary tool to identify patients with higher bacterial load.

Chemical synthesis and seroreactivity of O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-O-(2,3-di-O-methyl- alpha-L-rhamnopyranosyl)-(1----9)-oxynonanoyl-bovine serum albumin--the leprosy-specific, natural disaccharide-octyl-neoglycoprotein.

The outer disaccharide segment, namely, O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-2,3-di-O-methyl-alpha -L-rhamnopyranose, of the trisaccharide-containing, leprosy-specific, phenolic glycolipid I has been synthesized as the 8-(methoxycarbonyl)octyl glycoside in high yield and absolute stereospecificity by a series of modified Koenigs-Knorr and Helferich reactions. A particular feature of the synthetic pathway involves methylation of the 2-hydroxyl group of the rhamnose moiety under neutral conditions, after first preparing the 8-(methoxycarbonyl)octyl glycoside as the alpha anomer via the 1,2-orthoacetate, and thus precluding the possible formation of an anomeric mixture. The 8-(methoxycarbonyl)octyl O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-2,3-di-O-methyl-alpha -L-rhamnopyranoside was converted into the crystalline hydrazide, and this was coupled to bovine serum albumin (BSA), via intermediate acyl-azide formation, to produce the corresponding neoglycoprotein, O-(3,6-di-O-methyl-beta-D-glucopyranosyl)- (1----4)-O-(2,3-di-O-methyl-alpha-L-rhamnopyranosyl)- (1----9)-oxynonanoyl-BSA, the so-called natural disaccharide-octyl-BSA. Extensive serological testing of this product against sera from leprosy patients and control populations, and comparison with the native glycolipid and previously synthesized neoglycoproteins, have shown that it is unparalleled in terms of sensitivity and specificity, and highly suited to replace the native glycolipid for the serodiagnosis of worldwide lepromatous leprosy.