Enzyme-linked immunosorbent assay employing monoclonal antibody specific for deoxynivalenol (vomitoxin) and several analogs

Abstract

A monoclonal antibody was prepared against deoxynivalenol (DON, vomitoxin), a trichothecene mycotoxin occurring in grain contaminated with Gibberella zeae (anamorph = Fusarium graminearum), and incorporated into competitive direct and indirect enzyme-linked immunosorbent assays (ELISAs). DON antibodies were secreted by hybridomas derived from mice inoculated with DON conjugated to bovine serum albumin. Conjugation of DON to carrier proteins was facilitated by conversion of DON to 3-O-hemisuccinyl-DON after protection of two of the three available hydroxyls with a cyclic boronate ester. DON was detectable at 10-250 ng/assay (0.2-5.0 µg/mL) for direct ELISA and 10-150 ng/assay (0.2-2.0 µg/raL) for indirect ELISA. The monoclonal antibody cross-reacts with 3-acetyl-DON, 3-Ohemisuccinyl-DON, DON, 12,13-deepoxy-DON, nivalenol, and fusarenone X (in order of decreasing affinity) but has low affinity for 15-acetyl-DON and T-2 toxin. Deoxynivalenol (DON, vomitoxin) is one of the sesquiterpene mycotoxins classified as 12,13-epoxytrichothecenes (Mirocha et al, 1977). It occurs naturally in infected corn (Hart et al., 1982;Mirocha et al., 1976), small grains (Hart and Braselton, 1983; Neish and Cohen, require little sample preparation, are rapid and relatively simple to execute, yet are sensitive. In addition, enzyme immunoassays lack the health hazards associated with radioimmunoassays. Monoclonal antibodies have important advantages over polyclonal antibodies. They may be extremely specific, even if the immunizing antigen was not pure, and offer a theoretically unlimited supply of homo-

Rapid detection of nivalenol and deoxynivalenol in wheat using surface plasmon resonance immunoassay

“…Enzyme-linked immunosorbent assay (ELISA) [11], lateral-flow immunoassay [12], and radioimmunoassay [13] are all effective methods for mycotoxin detection. In addition to these immunoassays, surface plasmon resonance (SPR) has recently been applied to mycotoxin analysis [14].…”

Development of an immunochromatographic strip test for the rapid detection of deoxynivalenol in wheat and maize

“…Some researchers found that proper orientation of DON on the carrier protein was critical to antibody production. Since DON cannot be directly conjugated to carrier proteins, chemical groups capable of covalent linkage to protein must be introduced onto the molecule (Casale et al, 1988). In this paper, DON-CBSA conjugate was used as the immunogen.…”

“…But they are thought to be unsuitable for measuring many samples within a short time. In addition, some researchers have established enzyme-linked immunosorbent assay (ELISA) for DON (Casale, Pestka, & Hart, 1988;Sinha, Savard, & Lau, 1995), and it is also unsuitable for on-site detection.…”

Heterologous screening of hybridomas for the development of broad-specific monoclonal antibodies against deoxynivalenol and its analogues

“…So far, all the previously reported DON-MAbs were selected by homologous screening and most of them displayed much higher affinity either to 3-acetyl-DON (3-ADON) (Baumgartner et al, 2010;Casale et al, 1988;Maragos and McCormick, 2000;Tangni et al, 2010) or to 15-acetyl-DON (15-ADON) (Dos Santos et al, 2011;Maragos et al, 2011;Nicol et al, 1993;Sinha et al, 1995;Xu et al, 2010), the two derivatives that often co-occur with DON. Thus, the relevant immunoassays could only be used to screen for DON/3-ADON or DON/15-ADON, not three of them simultaneously.…”