A new avidin–biotin optical immunoassay for the detection of beta-bungarotoxin and application in diagnosis of experimental snake envenomation

Abstract

A highly sensitive avidin–biotin optical immunoassay (AB-OIA) has been developed for the detection of beta-bungarotoxin (β-BuTx), a neurotoxin from the venom of Bungarus multicinctus, in whole blood, plasma, and urine. Affinity purified rabbit IgG anti-β-BuTx antibody was immobilized on an optically active silicon surface (SILIAS™ wafer). The test sample was incubated and the antigen–antibody reaction was monitored by the addition of a biotinylated monoclonal antibody (mAb 15) specific to the toxin, avidin–horseradish peroxidase (HRP) and tetramethylbenzidine substrate. The silicon assay surface technology enables us to directly visualize a physical change in the optical thickness of the antibody thin film. The change in thickness is due to the specific capture of the toxin on the surface and when the substrate is added, the binding event is amplified, which then alters the reflected light path and a change in colour is visualized. The assay could detect β-BuTx levels as low as 16 pg/ml in sample buffer and 100 pg/ml in whole blood or plasma. The AB-OIA is simple, requires only 40 μl of biological fluid and can be performed without specialized equipment. The efficacy of the test for detection of β-BuTx in blood or plasma obtained from mice during experimental envenomation with B. multicinctus venom was demonstrated. The AB-OIA was also used to quantitate the postmortem level of β-BuTx in various organs such as brain, liver, and kidney, as well as the tissue at the site of injection. Development of a simple, rapid snake toxin detection kit based on AB-OIA technique potentially applicable in the clinics as well as in the field is discussed.