Paramagnetic bead based enzyme electrochemiluminescence immunoassay for TNT

We have developed an electrochemiluminescence immunoassay system for TNT (2,4,6-trinitrotoluene) in which enzyme labelled antibodies bound to paramagnetic beads are concentrated on an electrode magnetically, and light emission is triggered electrochemically. Full details of the instrumentation used to carry out these immunoassays are described. The paramagnetic beads are coated with haptenylated dextrans prepared by substituting biotin and analogues of TNT into aminodextrans. Three methods for synthesizing aminodextrans are compared and a method for substituting haptens and biotin into aminodextrans is described. Antibodies to TNT were labelled with glucose oxidase, and used for immunoassays in which TNT and haptenylated dextran competed for a limited number of antibody binding sites. The haptenylated dextran was bound to streptavidin coated paramagnetic beads, and the antibodies bound to the dextran were separated from the sample by concentrating them on an electrode magnetically. Bound antibodies were quantified by integrating the light emitted when enzymatically generated H2O2 reacted with electrochemically-oxidized luminol. The time taken for the detection step was 80 s compared with 20 min for colorimetric ELISAs using the same reagents, and the limit of detection for TNT was 31 ppb (3×S.D. zero calibrator; n=8).