| Identification and characterization of both black and white sesame seed proteins were investigated using biochemical and IgE-binding techniques. Extracts were made from both non-defatted and defatted seeds using both phosphate-buffered saline (PBS; pH 7.4) and deionized water (D-H2O). Extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by IgE-immunoblotting, and radioallergosorbent assay (RAST) with sera from sesame seed-allergic subjects. The SDS-PAGE results revealed differences in the extractable proteins obtained from each extract. Sesame seed extracts contained several proteins (13–26) with molecular weights in the range of 6.5–151 kD. Immunoblotting studies demonstrated the presence of several IgE-binding proteins with estimated molecular weights in the range of 6.5 to 87 kD, and IgE-binding patterns and RAST results differed between extracts for each sesame seed-allergic subject.; Polyclonal antibodies against the mixture of one part black and three parts of white sesame seeds were produced in goats, sheep, rabbits, and chickens. Antibodies were purified from the animal antisera and egg yolk. The immune response of each animal was monitored using an indirect enzyme-linked immunosorbent assay (ELISA). The sheep, goats, and chickens generated good immune responses to the sesame seed immunogen, while rabbits gave a weaker immune response to the immunogen.; A sandwich ELISA was developed using the goat serum and chicken egg yolk antibodies for quantification of sesame seed residues in foods with a detection limit of 2 ppm sesame seed flour. Goat IgG and chicken egg yolk IgY antibodies were used as capture and detector antibodies, respectively. Binding was visualized by addition of commercial goat anti-chicken IgY-alkaline phosphatase conjugate and subsequent substrate addition. Validation of the assay included testing pasta containing different known amounts of sesame seed flour (0-100 ppm), positive and negative samples, and cross-reactivity assessment. Minimal cross-reactivity was observed with allspice and kidney beans. A limited retail survey and positive samples demonstrated that the assay produced statistically consistent results (P > 0.05). |
