|Williams, William - Paul|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 9/1/2005
Publication Date: 10/23/2005
Citation: Clements, M.J., Windham, G.L., Maragos, C.M., Williams, W.P., Brooks, T.D., Hawkins, L.K. 2005. Examination of error components associated with quantification of aflatoxin in ground corn grain with in-house CD-ELISA [abstract]. In: Proceedings of the 2005 Multicrop Aflatoxin/Fumonisin Elimination & Fungal Genomics Workshop, Raleigh, North Carolina. p. 130.
Technical Abstract: Genetic resistance is generally considered to be the most desirable means of minimizing aflatoxin accumulation in corn grain prior to harvest; however, variation associated with grain sampling and subsampling techniques, and quantitative analytical protocols greatly impedes accurate classification of genotypes as resistant or susceptible to aflatoxin accumulation in grain. Some widely accepted analytical protocols for aflatoxin quantification in grain include liquid chromatography (LC), thin-layer chromatography (TLC), immunoaffinity column assay (ICA), enzyme-linked immunosorbent assay (ELISA), and high-performance liquid chromatography (HPLC). Each of these protocols involves several components that may contribute substantial cost or variation to quantification of aflatoxin among replicated analyses of the same subsample. Our objective was to examine error components and inputs associated with grain sampling and an in-house competitive-direct ELISA (CD-ELISA) for quantification of aflatoxin in corn grain. Understanding of error components associated with the CD-ELISA will lead to management decisions that minimize cost and maximize data quality. The CD-ELISA was developed around a monoclonal antibody produced at the USDA-ARS Mycotoxin Research Unit, Peoria, IL. Aflatoxin concentration in corn grain determined with CD-ELISA correlated significantly (P<0.0001, r = 0.87) with aflatoxin concentration in grain determined with the widely accepted Vicam AflaTest. Preliminary analysis of error components revealed that greatest variation in aflatoxin concentration from sampling and laboratory assay was explained by 8-gram subsamples of ground grain (56.1%), followed by sub-subsamples of extract solution (24.1), and sub-sub-subsampling (19.8%). An increase in subsample number from 1 to 3 decreased LSR of entry means by approximately 30%, whereas increasing replicates of the assay or increasing sub-subsamples of extract solution had much less of an effect on reducing LSR of entry means. Increasing subsample size from 8 to 20, 50, 100, or 150 grams was not beneficial in reducing variation in aflatoxin concentration associated with subsampling of ground grain. Sensitivity of the assay when run with three subsamples and a 300-fold dilution of subsample extract is approximately greater than or equal to 4 ng aflatoxin per gram of ground corn. The CD-ELISA compares very favorably with the widely accepted Vicam AflaTest, therefore the assay may be used to differentiate corn genotypes that are susceptible or resistant to aflatoxin accumulation in grain.