Journal of Immunological Methods, 131 (1990) 77-82 77 Elsevier JIM 05621 Chromatographic enzyme immunoassay for T-2 toxin Beverly A. Warden *, Abdellah Sentissi * *, Markus Ehrat * * *, Douglas J. Cecchini * *, Kariman Alam and Roger W. Giese Department of Medicinal Chemistry in the College of Pharmacy and Allied Health Professions, and Barnett Institute of Chemical Analysis and Materials Science, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, U.S.A. (Received 5 January 1990, revised received 26 March 1990, accepted 26 March 1990) Both the active ester and maleimide moieties of the cross-linking reagent, N-[(v-maleimid0butyryl) oxy]succinimide (GMBS), were found to react with the primary amino groups on ribonuclease (RNase). This largely inactivated RNase towards a polymeric (but not monomeric) substrate. Citraconylating the RNase first, so that essentially only a single primary amino group remained to react with GMBS, overcame this problem. The subsequent maleimido-citraconyl-RNase was used to prepare a 1:1.1 M conjugate of anti-T-2 toxin Fab' and RNase (Fab'-RNase) in a 76% yield. The conjugate was used to detect as little as 0.1/~g of T-2 toxin based on the ability of T-2 toxin to specifically displace Fab'-RNase complexed to a T-2 agarose affinity gel. Key words: Enzyme immunoassay; Ribonuclease; T-2 toxin; Citraconylation; Chromatography Introduction T-2 toxin, the structure of which is shown be- low, belongs to the tricothecene family of fungal byproducts (Betina, 1984). This poisonous com- pound can be found on molds when they develop on stored grains such as wheat. Animals which then eat the grain tend to become sick. Thus there is an interest in developing convenient assays for this toxin that can be used in-the-field. Correspondence to: R.W. Giese, Department of Medicinal Chemistry in the College of Pharmacy and Allied Health Professions, and Barnett Institute of Chemical Analysis and Materials Science, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, U.S.A. * Present address: Florida International University, Miami, FL, U.S.A. ** Present address: State Laboratory Institute, Jamaica Plain, MA, U.S.A. *** Present address: Ciba Geigy, Basel, Switzerland. H ~ _H ^ H H [CHal2CHCH2C()z -~ CAH CH3CO2CH 2 3 ~'02CCH3 Starting with an antibody that recognizes T-2 toxin, we previously prepared an anti T-2 toxin Fab'-fluorescein conjugate and used it in a chro- matographic 'hit-and-run' fluoroimmunoassay to quantitate T-2 (Warden et al., 1987). In this assay, T-2 toxin was detected by its ability to elute Fab'-fluorescein complexed to a T-2 agarose affin- ity gel. As little as 1 ng of T-2 was detected in 11 rain, and the assay device was used repetitively to detect successive doses of T-2 with only periodic recharging with Fab'-fluorescein. Potentially the usefulness of a 'hit-and-run' im- munoassay can be broadened, especially for in- the-field applications, if this technique incorpo- 0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)