Direct competitive ELISA based on a monoclonal antibody for detection of aflatoxin B₁. Stabilization of ELISA kit components and application to grain samples

A direct competitive enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody has been developed and optimized for detection of aflatoxin B₁ (AFB₁), and an ELISA kit has been designed. This immunoassay was highly specific, sensitive, rapid, simple, and suitable for aflatoxin monitoring. AFB₁ concentrations determinable by ELISA ranged from 0.1 to 10 μg L-¹. The IC₅₀ value was 0.62 μg L-¹. Recovery from spiked rice samples averaged between 94 and 113%. The effect of different reagents on the stability of HRP-AFB₁ conjugate solution was studied. The performance of a stabilized enzyme tracer in ELISA was determined and compared with that of a freshly prepared control solution of HRP-AFB₁ conjugate. The results showed that stabilizing media containing 0.02% BSA, 0.1% Kathon CG, and 0.05 mol L-¹ calcium chloride in 0.05 mol L-¹ Tris-HCl buffer (pH 7.2) maintained the activity of HRP-AFB₁ at a dilution of 1:1000 for a period of at least 12 months at room temperature whereas the reference conjugate solution without the additives lost its activity within a few days. Several additives were tested for their stabilizing effect on a monoclonal antibody (MAb) immobilized on the surface of polystyrene microtitre plates. It was shown that immobilized MAb, treated with post-coating solutions containing PVA, BSA, and combinations of these substances with trehalose, retained its activity for at least 4 months at 4°C, whereas the untreated MAb-coated plate lost its activity within 2 days.