Development of method for determination of AFB1, contaminater in corn seeds by strip-ELISA
1994
Limtrakul, P.L. | Sapyen, D. | Puttanont, P. (Chiang Mai Univ. (Thailand). Faculty of Medicine. Dept. of Biochemistry)
Because of frequent contamination of aflatoxin B1 (AFB1) in agricultural commodities such as peanuts, corn and animal feedstuffs, aflatoxin problems become a potential hazard to human and animal health. The degree of toxicity is determined by the level of aflatoxin fed. For monitoring AFB1 in cereals and food products, chemical methods such as thin layer chromatography (TLC), high performance liquid chromatography or mass spectrometry are not specific, but time consuming and need extensive clean up steps as well as expensive instruments. This paper will present home-made reagents for determining AFB1 artificially or naturally contaminated in corn seeds by competitive enzyme-linked immunosorbent assay (ELISA). The assay was directly performed from the methanol extract of corn samples. The ELISA method is sensitive, specific and inexpensive one which is convenient for a field test. Reproductibility, sensitivity and specificity of the method were evaluated. A microwell-strip ELISA using house reagents including polyclonal antibody specific to AFB1 and AFB1-conjugated horse radish peroxidase was evaluated by comparison with TLC and other commercially available AFB1 test kits. Up to 10 different corn samples can be prepared and analyzed within an hour. The range assay was limited between 1-80 ppb (ng/g corn seed). The assay can be both quantitative analysis of AFB1 by measuring with microwell reader and semiquantitative by measuring visually. The detection limit of the microwell-strip ELISA was as minimal as 1 pg/assay, shile the TLC was 10 pg/assay. Results of analysis of corn samples with each method evaluated with be reported. When the relationship between TLC and ELISA was investigated, results were found to have a good correlation coefficient (r=0.88). Chi-square analysis indicated that performance of the Afla 20-cup (USA), ID-block (USA) and RIDASCREEN (Germany) was not statistically different from that of the home-made ELISA. We are now developing the production of monoclonal antibodies specific for AFB1 and establishment of strip-ELISA using the antibody mainly for screening purposes only.
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