A novel broad-specific noncompetitive immunoassay and its application in the determination of total aflatoxins.
Broad-specific immunoassays are an emerging technology that allows simultaneous determination of a class of closely related small molecular weight analytes. The current approaches are mainly based on the competitive format and involve complicated or expensive processes hindering their wide application. Herein we describe a simple approach for performing broad-specific noncompetitive immunoassays for the determination of total aflatoxins (AFB(1)+AFB(2)+AFG(1)+AFG(2)) using a highly specific polyclonal antibody against AFB(1). The method is based on blocking the free sites of the capture antibodies by an AFB(1)-protein conjugate followed by the replacement of antibody-bound AFs by an enzyme-labeled AFB(1). The rates of displacement of weakly bound AFB(1) congeners by the AFB(1)-HRP conjugate are faster than that of AFB(1). Consequently, the measured signals from cross-reactants are higher and almost linearly correlated to the AFB(1) concentration. Different proteins such as casein, ovalbumin and BSA were utilized for the preparation of AFB(1) conjugates and their binding efficiencies were investigated. The limit of detection of AFs by the present assay was 5 pg/well (0.1 microgL(-1)). Spiked and contaminated corn samples were analyzed without sample cleanup. The matrix interferences were eliminated by diluting the sample 10-fold with the assay buffer. The values obtained for the infected samples correlated well (R(2)=0.99) with estimates by the ELISA kit. This approach can be applied to any analyte for the development of noncompetitive immunoassay in broad-specific or specific format provided the corresponding anti-hapten antibody has negligible cross-reactivity with structurally related compounds.
Acharya
Debopam
D
Drug Development, Diagnostics and Biotechnology Division, Indian Institute of Chemical Biology, Council of Scientific and Industrial Research, 4, Raja S.C. Mullick Road, Jadavpur, Calcutta 700032, India.
Dhar
Tarun K
TK
D016604:Aflatoxin B1 / Q000032:analysis / Q000276:immunology; D000348:Aflatoxins / Q000032:analysis* / Q000276:immunology; D000906:Antibodies; D002364:Caseins; D005506:Food Contamination; D006801:Humans; D007118:Immunoassay / Q000379:methods; D007202:Indicators and Reagents; D010047:Ovalbumin; D012710:Serum Albumin, Bovine; D003313:Zea mays
D000348:Aflatoxins; D000906:Antibodies; D002364:Caseins; D007202:Indicators and Reagents; C027955:aflatoxin G1; D012710:Serum Albumin, Bovine; C029753:aflatoxin B2; D010047:Ovalbumin; D016604:Aflatoxin B1