Enzyme immunoassay for aflatoxin B1-lysine adduct and its validation.

A simple procedure was developed for in vitro synthesis and characterization of aflatoxin B1-lysine adduct using aflatoxin B1, N-alpha-acetyl lysine and m-chloroperbenzoic acid (MCPBA). At a molar ratio of 1:16 (aflatoxin B1:N-alpha-cetyl lysine), the recovery of adduct was 62%. Analysis of the adduct by thin-layer chromatography showed a single spot (Rf = 0). Absorption spectra of the adduct showed 2 peaks at 275 and 335 nm. Liquid chromatographic (LC) analysis of the AFB1-lysine adduct showed a relative retention time of 2.1 min. Using the same epoxidation procedure, BSA-AFB1 adduct and ovalbumin-AFB1 adduct were synthesized for production of antibodies and as coating antigen, respectively. Control rat serum, spiked with AFB1-lysine adduct and subjected to LC analysis showed a retention time of 2.1 min, which is similar to that of AFB1-lysine reference standard, synthesized. Further, enzymatically hydrolyzed, control rat serum spiked with BSA-AFB1 adduct showed 2 peaks with retention times of 2.1 and 2.7 min. Based on the LC analysis, recovery of BSA-AFB1 in terms of AFB1-lysine adducts 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 AFB1-lysine adducts formed. Stability studies on the AFB1-lysine adduct synthesized, 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-AFB1 was used as a coating antigen. Indirect competitive ELISA showed 50% displacement (IC50) of the antibodies at a concentration of 13 ng AFB1-lysine, whereas the IC50 for AFB1 was 7 ng. The recovery of AFB1-lysine adduct spiked to control rat serum followed by enzymatic hydrolysis and immunoanalysis (indirect ELISA) was 93 +/- 6%. The enzyme immunoassay was validated by a rodent model, in which the animals were exposed to aflatoxin B1 (20 microg AFB1/kg body mass/day). The level of AFB1-lysine adduct in the rat serum was 27.3 +/- 4.37 microg/mg albumin.

Camel kidney ferritin: isolation and partial characterization.

Camel kidney ferritin was isolated from a tissue homogenate by thermal denaturation, ammonium sulphate fractionation, Sephacryl S-300 gel filtration and DEAE-blue gel affinity chromatography. The yield and the iron and neutral carbohydrate contents were 0.012 mg/g wet tissue, 4.0% and 2.7%, respectively. The phosphate:iron ratio was 0.13, twofold lower than that reported for camel liver ferritin. Native gel electrophoresis revealed the presence of a monomeric ferritin. SDS gel electrophoresis and immunoblotting showed two types of subunits, heavy and light, contrary to the extensive heterogeneity observed in camel liver ferritin. In general, the tissue ferritins shared a similar amino acid composition. However, a twofold lower glycine and an eightfold higher arginine content were recorded for camel kidney ferritin. In addition, kidney ferritin had a relatively high content of glutamic acid. Cross-reactivity studies by Ouchterlony double diffusion and noncompetitive indirect ELISA revealed a distinct cross-reactivity between buffalo ferritin antiserum and camel liver ferritin, but camel liver ferritin showed only weak cross-reactivity.

Purification and characterization of liver ferritins from different animal species.

The ferritins were purified from liver homogenates of buffalo, camel, cattle, sheep and shark by thermal denaturation, ammonium sulphate fractionation, Sephacryl S-300 gel filtration and DEAE-blue gel affinity chromatography. The yield and iron content of affinity-purified liver ferritins ranged from 0.008 to 0.052 mg/g and 3.17% to 11.4% respectively. As they are glycoproteins, the ferritins contained variable amounts of neutral carbohydrates. Except for shark ferritin, the ferritins all exhibited immunological cross-reactivity with anti-buffalo liver ferritin and anti-horse spleen ferritin by immunodiffusion and immunoelectrophoresis. Gel electrophoresis, gel filtration and ultracentrifugal analysis indicated the presence of a monomeric ferritin in all cases. SDS-gel electrophoresis of shark ferritin gave a protein band of 18 kDa. Ovine, buffalo and bovine ferritin comprised two protein subunits, the H (20 and 21 kDa) and the L types (18 and 19 kDa). Oligomeric ferritin subunits with molecular weights of 27, 37 and 55 kDa were also found for bovine and buffalo ferritin. SDS-PAGE of camel ferritin revealed a complex pattern with four prominent bands of 61, 51, 44 and 39 kDa. Two fast-migrating components of 15 and 16 kDa were also found in the purified liver ferritins, including reference preparations. The PO4(3-)/Fe ratios of purified shark (0.10) and bovine ferritin (0.12) were similar to that of standard equine spleen ferritin (0.11). However, the ratio was higher in ovine (0.17), camel (0.22) and bovine (0.26) ferritins. The amino acid compositions, molecular weights and sedimentation coefficients of the different liver ferritins were similar.