Competitive photometric and visual ELISA for aflatoxin B1 based on the inhibition of the oxidation of ABTS

This work reports on a sensitive colorimetric immunoassay for aflatoxin B 1 (AFB 1 ) in foodstuff. The reagent L-ascorbic acid 2-phosphate (AAP) is added to the system, and alkaline phosphatase (ALP) hydrolyzes AAP under formation of ascorbic acid and phosphate. The ascorbic acid produced reduces chloroauric acid to form zero-valent gold (in the form of nanoparticles). Hence, Au(III) is no longer available to oxidize 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) to form a green product. Rather, the solution remains colorless. By using ALP-labelled monoclonal anti-AFB 1 antibody, a competitive enzyme-label immunoassay was developed for AFB 1 in a microplate coated with the AFB 1 -BSA conjugate. Under optimal conditions, the absorbance of the solution at 415 nm increases linearly with increasing AFB 1 concentration in range from 10 pg·mL −1 to 100 ng·mL −1 (while the color gradually turns to green), and the detection limit is 7.8 pg·mL −1 . The precision of the method (expressed as RSD) is ±9.7%. The accuracy was validated by analyzing both naturally contaminated and spiked food samples, and the results matched the results obtained by ELISA very well. Schematic of a colorimetric aflatoxin B 1 immunoassay. Antibody is labeled with alkaline phosphatase which catalyzes the hydrolysis of ascorbic acid 2-phosphate (AAP) to form ascorbic acid which reduces chloroauric acid to form gold nanoparticles. Hence, Au(III) is no longer available to oxidize 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) to form a green product.