Improved GM1-enzyme-linked immunosorbent assay for detection of Escherichia coli heat-labile enterotoxin

Abstract

A GM1-ELISA [ganglioside-enzyme-linked immunosorbent assay] that employed anti-cholera toxin to make it comparable in sensitivity to the Y-1 adrenal [mouse] cell assay was modified. When 5 media commonly used for LT production were compared, Mundell''s Casamino Acids medium was significantly superior. Lincomycin (45 .mu.g/ml) added to E. coli cultures significantly increased net optical densities in the GM1-ELISA, a direct measure of the amount of LT. Treatment of broth cultures or bacterial cell pellets with polmyxin B or extension of culture time to 48 h also significantly increased net optical density by allowing enhanced release of periplasmic LT. A major innovation involved the direct culture of E. coli strains in GM1-coated wells of microtiter plates followed by ELISA. This direct culture method GM1-ELISA (DCM-GM1-ELISA) saved assay time, materials and reagents. The net optical densities that result from this assay allow the test to be read visually without a spectrophotometer. Three independent observers read plates with E. coli tested by DCM-GM1-ELISA. Thirty-four of 35 adrenal cell-positive strains (97% sensitivity) and 30 of 30 LT-negative control E. coli strains (100% specificity) were identified by all 3 observers reading coded plates. The DCM-GM1-ELISA provides a simple, practical and efficient assay for LT for less sophisticated laboratories.