Indirect immunoperoxidase localization of aflatoxin B1 in rat liver.

Aflatoxin B2a (AFB2a) antibody was used as a histochemical probe in the indirect immunoperoxidase localization of aflatoxin B1 (AFB1) bound to rat liver. The efficacy of the indirect method was initially demonstrated by detecting AFB1 covalently bound to DNA in an enzyme-linked immunosorbent assay. AFB1-modified DNA was attached to a polystyrene microtissue culture plate (solid phase) and then subjected to sequential incubation with AFB2a antiserum followed by goat anti-rabbit peroxidase conjugate. Assays for bound peroxidase revealed that the AFB2a antiserum could be diluted 200,000-fold and still yield a signal-to-noise ratio of 10 when compared to an unmodified DNA control. When the same indirect immunoperoxidase protocol was applied to the light-microscopic localization of AFB1 in liver sections of rats treated in vivo with the mycotoxin, bound toxin could be identified in excellent detail in tissues fixed with periodate-lysine-paraformaldehyde and embedded in glycol methacrylate, but was detectable with only poor resolution in unfixed cryostat sections. Peroxidase-positive reactions in hepatocytes typically exhibited strong nuclear and relatively lighter cytoplasmic staining. Greater concentrations of peroxidase-positive hepatocytes were detected in the periportal area than in the area of the central vein, suggesting a circulatory pattern for AFB1 binding in the liver.