Development of an assay for quantification of linkage-specific O-acetylated sialoglycans on erythrocytes; its application in Indian visceral leishmaniasis

Abstract

We have developed a noninvasive approach for the quantification of linkage-specific 9-O-acetylated sialoglycans on mammalian erythrocytes using a lectin, Achatinin-H, whose lectinogenic epitope has previously been defined as 9-O-acetylated sialoglycoconjugates (9-O-AcSGs) α2→6 linked to subterminal GalNAc. Titration and checkerboard analysis were performed to optimize the assay using rabbit, rat and human erythrocytes that contain differing amounts of this glycotope. Assay specificity was established by decreased binding of erythrocytes to immobilised Achatinin-H when pre-incubated with excess lectin. The intra-assay coefficient of variation (CV) for rat and human erythrocytes was 8.6-9.2% and 11.1-13.0%, respectively. The inter-assay CV for rat and human erythrocytes was 9.9-10.1% and 15.2-16.6%, respectively. In previous studies, we have identified an enhanced presence of cell surface 9-O-AcSGs on the erythrocytes of patients with visceral leishmaniasis (VL) [Am. J. Trop. Med. Hyg. 58 (1998) 551]. Our assay when evaluated on erythrocytes from VL patients (n=30) showed a fourfold increase in lectin binding as compared to endemic controls. The mean±S.E.M. of the A₄₀₅ nm value was 1.14±0.04 vs. 0.23±0.03, respectively (p<0.0001). Following effective chemotherapy, a significant reduction of this glycotope on the erythrocytes of VL patients indicates that this assay has both a diagnostic and prognostic potential. Taken together, we conclude that this antigen-based assay is a specific and reproducible method for monitoring the disease status of VL patients and could be used in retrospective and prospective trials.