Use of a Heterologous Bridge Coating Antigen for the Immunoassay of 3-Acetyldeoxynivalenol in Barley

Abstract

The study describes procedures that were used to develop a highly sensitive enzyme-linked immunosorbent assay (ELISA) for the quantitation of a trichothecene mycotoxin, 3-acetyldeoxynivalenol (3-AcDON), in barley. Polyclonal antibodies were produced in rabbits immunized with a conjugate of 3-AcDON and human serum albumin linked by an ester linkage (hemisuccinate bridge). High anti-3-AcDON titers were obtained after multiple immunizations. However, only a negligible degree of inhibition was obtained with 3-AcDON in a competitive ELISA when the coating conjugate contained the same ester linkage group (hemiglutarate bridge) as the immunogen. The use of a conjugate containing a heterologous ether linkage (O-methylcarboxyl bridge) compared to that of the immunogen yielded an inhibition curve for 3-AcDON that was highly sensitive (IC50 = 0.21 ng/ml) with essentially no interference from the bridging group. This conjugate was synthesized using iodoacetate and 1,1′-carbonyldiimidazole chemistries. The assay showed low cross-reactivity with other trichothecenes including several analogs of deoxynivalenol (DON) with the exception of acetylated DON. The ELISA developed on the basis of this new conjugate was able to detect low concentrations of 3-AcDON (16 ppb) in spiked barley without any cleanup treatments.

Production of a Sensitive Monoclonal Antibody to Sterigmatocystin and Its Application to ELISA of Wheat

“…It is not known, however, if this problem would arise as the previous authors did not verify their procedure using spiked grain samples. Such reactions have been reported before with polyclonal antibodies and are attributed to the nonspecific binding of antibody to the bridge between the hapten and the protein component of the immunogen (Nordblom et al, 1981;Wang et al, 1996).…”

Production and characterization of monoclonal antibodies against 3, 4, 15-triacetylnivalenol and 3,15-diacetyldeoxynivalenol

“…Wang and Chu (1991) used triacetyl-NIV-BSA conjugate as immunogen and obtained polyclonal antibodies which reacted with tetraacetyl-NIV, triacetyl-DON but not with DON, 4-ADON and NIV. Wang et al (1996) triacetylated DON (3,7,91.7%) and tetraacetylated NIV (3,4,7,15-TANIV, 24.5%), but they did not examine the reactivity against 3,15-DADON and 4,15-DANIV. The hydroxyl moiety at the C-7 position of NIV is known to be highly resistant to acylation, and harsh conditions are required to complete the reaction.…”