Escherichia coli heat-stable enterotoxin (STa)-biotin conjugates for the titration of STa antisera by an enzyme-linked immunosorbent assay.
The development of a new approach to the diagnosis of infectious diarrhoea, caused by Escherichia coli heat-stable enterotoxin (ST), was preceded by a preliminary study. The purpose of the latter was to establish whether three preparations of ST produced by a human isolate of enterotoxigenic E. coli (STa), obtained at different steps of the purification procedure (involving Amberlite XAD2 resin chromatography (P3), a gel filtration chromatography on a Biogel P4 (P2) or a disc-gel electrophoresis (P1)), could be employed to titrate antisera to STa using an ST-biotin enzyme-linked immunosorbent assay (ELISA). The solid-phase STa was obtained by first coupling the toxin to biotinyl-N-hydroxysuccinimide and then binding this conjugate to avidin adsorbed to flat-bottomed polystyrene microtitre plates. Using these reagents, the assay conditions were examined. Checkerboard tests determined optimal biotin-P3, P2 or P1 toxin conjugate concentrations to be used as the immunosorbent for P3, P2 and P1 antiserum titration. The immunosorbent prepared with STa purified only on Amberlite XAD2 resin was unable to differentiate significantly between P3, P2 or P1 antisera. Immunosorbent prepared with P2 or P1 detected widely differing titres between the three antisera and gave more sensitive results. Only small but questionable differences were observed between P2 and P1 toxin preparations.
Germani
Y
Y
Laboratoire des Bactéries Entéropathogènes, Institut Pasteur de Nouvelle-Calédonie, Nouméa.
deRocquigny
H
H
Guesdon
J L
JL
D000818:Animals; D001360:Avidin; D001427:Bacterial Toxins / Q000302:isolation & purification; D001710:Biotin; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003967:Diarrhea / Q000382:microbiology; D004589:Electrophoresis, Disc; D004768:Enterotoxins / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004926:Escherichia coli / Q000302:isolation & purification; D004927:Escherichia coli Infections / Q000175:diagnosis; D007164:Immunosorbents; D000922:Immunotoxins; D051379:Mice
D001427:Bacterial Toxins; D004768:Enterotoxins; D007164:Immunosorbents; D000922:Immunotoxins; D001360:Avidin; D001710:Biotin