Abstract
A successful ELISA using monoclonal antibody (MAb) and one-step extraction method was established for aflatoxin (AF) B1 determination. Eight clones of stable hybridoma cells secreting IgG anti-AFB1 MAbs were prepared from spleen of BALB/c mice. The hybridoma cells were proliferated in culture and infused into pristane-induced BALB/c mice. Two weeks later, their ascites fluid were withdrawn and precipitated by 45% saturated ammonium sulfate. The IgG precipitate was dissolved in PBS and kept at -80°C for further test. The direct competitive ELISA (direct cELISA) system was developed for AFB1 determination in microplates by AFB1-horseradish peroxidase conjugated by carbodiimide method. The quality control was done. The minimum detectable limits of the direct cELISA with those monoclonal antibodies : AF 1, 2, 3, 4, 5, 6, 7 and 8 were 10, 20, 2, 2.5, 2, 5, 2 and 2.5 pg of standard AFB1 per assay, respectively. The specificity or cross reactivity to other aflatoxin derivatives was that MAbs were reactive with AFG1 as well as AFB1, weakly with aflatoxicol (AFL) I>AFL II>AFB2>AFG2>AFM1, respectively, except AF5 weakly reactive with AFG1. The result showed that MAb (AF5) was the best MAb for further use in the ELISA kit.
