ABSTRACT
An ion-sensitive field effect transistor (ISFET)-based immunosensor was developed to detect/quantitate beta-Bungarotoxin (beta-BuTx), a potent presynaptic neurotoxin from the venom of Bungarus multicinctus. A murine monoclonal antibody (mAb 15) specific to beta-BuTx was immobilized onto silicon nitride wafers after silanization and activation with glutaraldehyde. A chip based enzyme linked-immunosorbantassay (ELISA) was performed to ascertain antigen binding to the immobilized antibody. To develop an electrochemical immunosensing system for the detection/quantitation of beta-BuTx, an ISFET was used as a solid phase detector. MAb 15 was immobilized on the gate region of the ISFET. The antigen antibody reaction was monitored by the addition of urease conjugated rabbit anti-beta-BuTx antibodies. The sensor can detect toxin level as low as 15.6 ng/ml. The efficacy of the sensor for the determination of beta-BuTx from B. multicinctus venom was demonstrated in mouse model. Toxin concentration was highest at the site of injection (748.0+/-26 ng/ml) and moderate amount was found in the plasma (158.5+/-13 ng/ml).
Subject(s)
Biosensing Techniques/instrumentation , Bungarotoxins/analysis , Enzyme-Linked Immunosorbent Assay/instrumentation , Animals , Antibodies, Monoclonal/analysis , Antibodies, Monoclonal/immunology , Antigen-Antibody Complex/analysis , Antigen-Antibody Complex/immunology , Biosensing Techniques/methods , Bungarotoxins/blood , Bungarotoxins/immunology , Bungarus , Enzyme-Linked Immunosorbent Assay/methods , Equipment Design , Ions , Male , Mice , Rabbits , Reference Values , Reproducibility of Results , Sensitivity and Specificity , Subcutaneous Tissue/chemistry , Subcutaneous Tissue/immunology , Transistors, ElectronicABSTRACT
A highly sensitive avidin-biotin optical immunoassay (AB-OIA) has been developed for the detection of beta-bungarotoxin (beta-BuTx), a neurotoxin from the venom of Bungarus multicinctus, in whole blood, plasma, and urine. Affinity purified rabbit IgG anti-beta-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 beta-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 microl of biological fluid and can be performed without specialized equipment. The efficacy of the test for detection of beta-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 beta-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.
