CHARACTERIZATION OF ENTEROTOXIGENIC BACTEROIDES-FRAGILIS BY A TOXIN-SPECIFIC ENZYME-LINKED-IMMUNOSORBENT-ASSAY

Within the past decade, certain strains of Bacteroides fragilis have b een associated with diarrhea in humans and cytotoxic activity on certa in colon carcinoma cell lines. An enzyme-linked immunosorbent assay (E LISA) for detecting the enterotoxin of B. fragilis in cultures and sto ols was developed by using high-titer monospecific goat and rabbit ant itoxins in an indirect format. The lower limit of detection for purifi ed toxin was approximately 0.05 mu g/ml; the linear range was from 0.0 5 to 10 mu g/ml. Using the ELISA to screen cultures of toxigenic and n ontoxigenic strains of B. fragilis, we observed 100% correlation with 16 known toxigenic strains which had various cytotoxic activities on H T-29 cells. In addition, we found 6 of 62 previously untested strains also to be positive in both assays. Stability studies revealed that al though the cytotoxic activities of crude and purified toxin preparatio ns incubated at elevated temperatures were rapidly lost, the ELISA res ponses were not significantly reduced. Sodium dodecyl sulfate(SDS)-pol yacrylamide gel electrophoresis and SDS capillary electrophoresis show ed that the purified toxin autodigested to several stable peptides. St udies on partially purified membranes from the toxigenic strains revea led the presence of several membrane-associated components which were noncytotoxic hut strongly immunoreactive in the ELISA. preliminary stu dies with spiked feces indicated that the ELISA may be useful for scre ening not only cultures for the enterotoxigenic B. fragilis but also s tool specimens. Ongoing studies are focusing on determining the nature of the toxin's apparent proteolytic capabilities and investigating th e feasibility of using the ELISA on stool specimens from healthy and d iarrheic humans.