Enzyme immunoassay for aflatoxin B-1-lysine adduct and its validation

A simple procedure was developed for in vitro synthesis and characterizatio n of aflatoxin B-1-lysine adduct using aflatoxin B-1, N-alpha -acetyl lysin e and m-chloroperbenzoic acid (MCPBA). At a molar ratio of 1:16 (aflatoxin B-1:N-alpha -cetyl lysine), the recovery of adduct was 62%. Analysis of the adduct by thin-layer chromatography showed a single spot (R-f = 0). Absorp tion spectra of the adduct showed 2 peaks at 275 and 335 nm. Liquid chromat ographic (LC) analysis of the AFB(1)-lysine adduct showed a relative retent ion time of 2.1 min. Using the same epoxidation procedure, BSA-AFB(1) adduc t and ovalbumin-AFB(1) adduct were synthesized for production of antibodies and as coating antigen, respectively. Control rat serum, spiked with AFB(1 )-lysine adduct and subjected to LC analysis showed a retention time of 2.1 min, which is similar to that of AFB(1)-lysine reference standard, synthes ized. Further, enzymatically hydrolyzed, control rat serum spiked with BSA- AFB(1) adduct showed 2 peaks with retention times of 2.1 and 2.7 min. Based on the LC analysis, recovery of BSA-AFB(1) in terms of AFB(1)-lysine adduc ts was 67 +/- 5%. The major peak (2.1 min) accounted for 72% of the adduct; the second minor peak (2.7 min) accounted for 28% of the total AFB(1)-lysi ne adducts formed. Stability studies on the AFB(1)-lysine adduct synthesize d, indicated that it was stable for 1 month. Antibody capture assay showed an absorbance of 0.9 to 1.0 at a dilution of 1:50 000 when ovalbumin-AFB(1) was used as a coating antigen. Indirect competitive ELISA showed 50% displ acement (IC50) of the antibodies at a concentration of 13 ng AFB(1)-lysine, whereas the IC50 for AFB(1) was 7 ng. The recovery of AFB(1)-lysine adduct spiked to control rat serum followed by enzymatic hydrolysis and immunoana lysis (indirect ELISA) was 93 +/- 6%. The enzyme immunoassay was validated by a rodent model, in which the animals were exposed to aflatoxin B-1 (20 m ug AFB(1)/kg body mass/day). The level of AFB(1)-lysine adduct in the rat s erum was 27.3 +/- 4.37 mug/mg albumin.