A sensitive and validated immunomagnetic-bead based enzyme-linked immunosorbent assay for analyzing total T-2 (free and modified) toxins in shrimp tissues

Abstract

Accurate analyses of total T-2 (free and modified) in aquatic organisms including shrimp are important as the hazard caused by T-2 has been caught increasing attention. Therefore, acurate analysis of free T-2 especially of modified T-2 in shrimp tissues is important. A rapid, sensitive, and validated method for quantitative determination of free T-2 and modified T-2 toxin was developed using immunomagnetic-bead based enzyme-linked immunosorbent assay (IMB-ELISA). Super paramagnetic particles with a carboxyl group activated by an ester method coupled with envelope antigen 3- acetylneosolaniol- hemisuccinate - ovalbumin (3-Ac-NEOS-HS-OVA) was used to form immunomagnetic beads which could bind to T-2 skeletal structure antibodies. The conditions for magnetic bead coating of T-2 skeletal structure antibodies, and the concentrations of the polyclonal antibody and HRP-labeled goat anti-rabbit antibody were optimized. A good linear relationship with T-2 concentrations ranging from 5−75 ng/mL (R² =0.9965) was observed. The detection limit of different shrimp tissues of the IMB-ELISA ranged from 2.53 to 3.20 ng/mL. And the IC₅₀ was 63 ng/mL. The recovery varied from 86% to 99% with a standard deviation of 2.8−5.8%. The application of this method to study the distribution in tissues showed that the total T-2 concentration in hepatopancreas was 26.7 µg/kg > blood > head > muscle in the highest dose group of 12.2 mg/kg. Our research showed a combination of ELISA and immunomagnetic bead technology provide a new, convenient approach to significantly improve the accuracy and sensitivity of total T-2 measurement in shrimp tissues.