Heather A. Lee

A nonisotopic estrogen receptor-based assay to detect estrogenic compounds

We have used the ligand binding domain of the recombinant human estrogen receptor (hER) to develop a nonisotopic assay for detection of estrogenic compounds. The assay is based on competition of the estrogenic ligand with 17β-estradiol for binding to the receptor, which leaves 17β-estradiol free to bind to an anti–17β-estradiol antibody. Unbound anti–17β-estradiol antibody then binds to immobilized 17β-estradiol–protein conjugate (to which hER is unable to bind for steric reasons), and is detected by an enzyme-labeled anti-rabbit IgG antibody. We used the assay to detect estrogenic compounds (mainly members of the flavonoid group of plant polyphenols) in a variety of commonly consumed plant foods.

An enzyme‐linked immunosorbent assay for deoxynivalenol in wheat, utilizing novel hapten derivatization procedures

Polyclonal anti‐deoxynivalenol (vomitoxin) antisera was produced in rabbits. The immunogen was synthesized by using acetyl esterase to deacylate 3‐acetyldeoxynivalenol hemiglutarate and coupling the product to bovine serum albumin using the mixed anhydride reaction. The antiserum was very specific for deoxynivalenol in a microtitration plate ELISA that had a limit of detection of 90 pg per well. A simple, rapid extraction procedure was employed to enable the ELISA to be used for analysis of the deoxynivalenol in wheat.

Production and characterization of polyclonal and monoclonal antibodies against aflatoxin B1 oxime-BSA in an enzyme-linked immunosorbent assay.

From a single aflatoxin B1 oxime — bovine serum albumin conjugate, polyclonal and monoclonal antibody preparations were produced. The four rabbit polyclonal antisera were specific for aflatoxin Bi in a microtitration plate enzyme — linked immunosorbent assay. The monoclonal antibodies showed a wide range of differing specificities, recognizing, for example, aflatoxins B1, B2, G1 and G2; B1 and B2; B1 and G1; and G1 alone. No antibody preparations reacted with aflatoxin M1. The significance of these results to the strategy of anti-aflatoxin antibody production for use in quantitative enzyme immunoassays is discussed.

Rapid enzyme‐linked immunosorbent assays for the detection of salmonella enteritidis in eggs

Rapid antibody‐based assays have been developed for the detection of Salmonella enteritidis in eggs. The assays consist of an initial non‐selective enrichment step in a chemically defined medium followed by either an indirect competitive enzyme‐linked immunosorbent assay or an antibody‐capture enzyme‐linked immunosorbent assay. The limits of detection are 6 × 105 cells and 5×104 cells/ml respectively, and both assays are highly reproducible. A positive result is obtained from an egg inoculated with 10 S. enteritidis cells in 18 h using either assay. The potential for further improvements is discussed.

Immunoprobes for polychlorinated dibenzodioxins: Synthesis of immunogen and characterization of antibodies

We have synthesized a novel dioxin immunogen, 2‐adipamide‐7, 8‐dichlorodibenzodioxin‐BSA, with a view to generating antibodies suitable for use in an immunoaffinity column. The specificity of antisera raised in rabbits to this conjugate was assessed in a competitive enzyme‐linked immunosorbent assay which was sensitive to 1 ng 2,3,7,8‐tetrachlorodibenzodioxin well‐1. Cross‐reactivity studies indicated that the antibodies were more sensitive to this congener than to the more highly chlorinated structures which are considered less toxic. 3,3’,4,4,‐Tetrachlorobiphenyl was unable—at any concentration—to displace antibody from the solid phase. We consider such specificity would be eminently suitable for immunoaffinity columns in the clean‐up and concentration of samples prior to dioxin determination by gas chromatography/mass spectrometry.