4.1.5. Quantification of AFB1 in Feed Samples

The extract was prepared and analyzed for AFB1 using AFB1-ELISA low matrix kit (Helica Biosystems Inc.®) according to manufacturer's instructions but with modification. Briefly, 100 µL of the extract, appropriately diluted in phosphate buffered saline containing tween 20 (PBS-T) was loaded on polystyrene ELISA micro-plate with immobilized mouse anti-AFB1 monoclonal antibodies, incubated at ambient temperature for 30 min, contents decanted and plate washed with PBS-T (this was prepared by dissolving contents of PBS-T packet in 1 L distilled water) to remove non-specific reactants, 100 µL of AFB1-horseradish peroxidase conjugate added and incubated again for 30 min, contents decanted and washed with PBS-T to remove non-specific reactants. Substrate-chromogen reagent (100 µL) was added and incubated at ambient temperature for 10 min before stopping the reaction with 100 µL of acid solution and the optical density (ODs), which is inversely proportional to aflatoxin concentration measured at 450 nm on a multi-channel micro-plate reader (BDSL Immunoskan Plus, Finland) inter-faced to a personal computer (Dell®, Precision 470, Cherrywood, Ireland) and the OD s recorded using Eiaquik program (© M.C Eisler, 1995). By setting the "zero" standard as 100% binding (B0), percent binding for each standard and sample as a percent of the zero binding (% B/B0) was calculated. Average absorbance values for each standard and sample extract dilution (B) and that of reagent blank (B0) were used to calculate percentage inhibition (B/B0%) for each standard and sample dilution, and for construction of 4-parameter calibration curve (log of standard concentration versus logit of B/B0%) using programmed template spreadsheets (Microsoft Excel Office 1997–2003). The calibration curve was used to calculate AFB1 concentrations of unknown samples. For the conventional method (Groups VI and VIII), 80% acetonitrile (ACN) extract to PBS-T ratio was 100:900, *v/v*., while for the novel method (Groups II and V) this was modified to 100:650, *v/v* (Table 2).
