An ELISA for five glycolipids from the cell wall of Mycobacterium tuberculosis: Tween 20 interference in the assay

Mycobacterium tuberculosis cell wall contains antigenic glycolipids: phenol -glycolipid (PGL), diacyltrehalose (DAT), triacyltrehalose (TAT), cord-fact or (CF), and sulpholipid-I (SL-I). In the last decade, the usefulness of th ese antigens for the serodiagnosis of tuberculosis has been evaluated mainl y using enzyme-linked immunosorbent assays (ELISA). Currently, there are no conclusive results about the utility of these glycolipidic antigens, becau se the results obtained by different groups are discrepant, In order to exp lain these discrepancies, we have investigated the methodological variation s in the ELISAs used previously. Specifically, we have studied the followin g: the coating solvent, the optimum amount of glycolipid coated per well, t he blocking agent, and the use of detergent (Tween 20) in the washing buffe r. The most significant. finding was that Tween 20 detaches PGL, DAT, TAT a nd SL-I from microtitre wells. However, Tween 20 does not remove CF from th e wells. In addition, we have found that the best solvent for coating is n- hexane, that the optimum antigen coating concentration is 1000 ng/well, and that BSA and gelatin are equally effective blocking agents. We can therefo re conclude that the use of Tween 20 as a detergent, and the lower antigen coating concentrations (100-200 ng/well), may well explain some of the disc repancies in previous studies. (C) 2001 Elsevier Science B.V. All rights re served.