Filtration-based tyramide amplification technique—a new simple approach for rapid detection of aflatoxin B₁

Abstract

The catalyzed reporter deposition (CARD) method of signal amplification, also called “tyramide signal amplification”, has been used in immunoassays not only to increase sensitivity but also to reduce assay time. The current approach to tyramide amplification in immunoassays involves slow incubation with agitation. In this paper we describe new filtration-based tyramide amplification and substrate visualization techniques. Compared with the standard method, this new approach greatly enhances spot intensities in membrane immunoassay and reduces biotinylated tyramide (B-T) and substrate consumption approximately fiftyfold, without loss of specificity. An improved test device and a cost-effective method for preparation of membranes for Super-CARD amplification have also been developed. The techniques have been used for rapid detection of aflatoxin B₁ (AFB₁) in a variety of foodstuffs with a detection limit of 12.5 μg kg⁻¹. The assay procedure involves sequential addition of standards or sample, AFB₁–horseradish peroxidase (HRP) conjugate, B-T, avidin–HRP, and substrate solution over anti-AFB₁ antibody-spotted zones of the membrane surface. The method saves time, improves reproducibility, eliminates many washing steps and avoids manipulation of the membranes between the different steps, while maintaining the sensitivity of the standard method. Average recoveries from different non-infected food samples spiked with AFB₁ at concentrations from 25 to 100 mg kg⁻¹ were between 95 and 105%. AFB₁ results obtained on different days for Aspergillus parasiticus infection of corn and groundnut samples correlated well with estimates obtained by HPLC.