Yehudith Amir-Zaltsman

A Nonisotopic Enzyme-Based Immunoassay for Assessing Human Exposure to Genistein

Phytoestrogenic isoflavones that are abundant in soybeans may be an important group of natural products that could play a critical role in preventing several chronic human diseases. To facilitate studying the relationship of soya exposure and chronic diseases, we report a simple method for measuring an isoflavone, genistein, in human urine and plasma. The method is a competitive enzyme-linked immunoassay that utilizes a conjugate of horseradish peroxidase (HRP) and genistein as tracer and a monoclonal antibody to genistein (clone 10D8) generated through the 6-position of genistein. Genistein, in diluted hydrolyzed urine or plasma of subjects who ingested soy milk, competes with HRP-genistein conjugate for the binding sites of anti-genistein antibody on rabbit anti-mouse IgG-coated plates. After a one-hour incubation, the wall-bound genistein-HRP activity, after reaction with a chromogen, is measured colorimetrically at 450 nm and is inversely correlated with concentrations of genistein over the range of 0.1-32 ng/well. The sensitivity limit of the method is 0.5 ng of genistein per well or 0.5 ng per 10 microliters of urine and plasma. Urine and plasma levels of genistein measured by this immunoassay correlated well (R2 = 0.92 for urine and 0.77 for plasma) with those determined by chromatographic techniques. This method can be used to assess soya exposure in humans and could facilitate epidemiological studies of the relationship of soya diets and chronic diseases, including cancer.

ADP-ribosylation of microtubule proteins as catalyzed by cholera toxin.

Incubation of purified rat brain tubulin with cholera toxin and radiolabeled [32P] or [8‐3H]NAD results in the labeling of both alpha and beta subunits as revealed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). Treatment of these protein bands with snake venom phosphodiesterase resulted in quantitative release of labeled 5′‐AMP, respectively labeled with the corresponding isotope. Two‐dimensional separation by isoelectric focusing and SDS‐PAGE of labeled and native tubulin revealed that labeling occurs at least in four different isotubulins. The isoelectric point of the labeled isotubulins was slightly lower than that of native purified tubulin. This shift in mobility is probably due to additional negative charges involved with the incorporation of ADP‐ribosyl residues into the tubulin subunits. SDS‐PAGE of peptides derived from [32P]ADP‐ribosylated alpha and beta tubulin subunits by Staphylococcus aureus protease cleavage showed a peptide pattern identical with that of native tubulin. Microtubule‐associated proteins (MAP1 and MAP2) of high molecular weight were also shown to undergo ADP‐ribosylation. Incubation of permeated rat neuroblastoma cells in the presence of [32P]NAD and cholera toxin results in the labeling of only a few cell proteins of which tubulin is one of the major substrates.

The measurement of oestrone-3-glucuronide in urine by non-competitive idiometric assay

We report a novel non-competitive idiometric assay for the measurement of oestrone-3-glucuronide (EG) in diluted urine. The method is based on the use of two types of anti-idiotypic antibody, the beta-type and alpha-type, that recognize different epitopes within the hypervariable region of the primary anti-EG antibody (Ab1). The beta-type anti-idiotypic antibody is analyte sensitive and competes with the analyte for an epitope of the primary antibody at the binding site. On the other hand, the alpha-type is analyte insensitive, but does not bind the Ab1 in the presence of the beta-type due to epitope proximity. In the present format, reaction mixtures containing the europium labelled Ab1 are reacted sequentially with EG standards or diluted urine samples, with the beta-type anti-idiotypic antibody and biotinylated alpha-type anti-idiotypic antibody on immobilized streptavidin coated microtiter plates. After 1 h incubation, the fluorescence of europium is measured by a time-resolved fluorescence and is proportional to the concentration of EG over a range of 0-10 nmol/l. The method demonstrates good sensitivity, precision and comparability with an alternative competitive fluorescent immunoassay. The idiometric assay for EG may be applied for the monitoring of ovarian function in women and is suitable for dipstick technology.

Generation of an Anti-idiotypic Antibody as a Surrogate Ligand for Sulfamethazine in Immunoassay Procedures

We report the generation of an anti-idiotypic antibody, clone 12E12, against antisulfamethazine (SMZ) as a surrogate ligand for SMZ in immunoassay procedures. This has been achieved by using the primary monoclonal anti-SMZ antibody as an immunogen in hybridoma technology followed by screening of the resulting hybridromas for binding to the SMZ site of the primary anti-SMZ antibody. Characterization of a strong anti-idiotypic antibody of the betatype, clone 12E12, capable of mimicking SMZ, permitted the development of immunoassay formats for SMZ based on the idiotypic and anti-idiotypic approach and the direct measurement of SMZ content in diluted pig urine. The availability of the strong betatypic anti-idiotypic antibody acting as a surrogate ligand for SMZ enables antibodies to be labeled instead of ligands and offers interesting possibilities for the development of competitive type non-isotopic immunoassays for SMZ.

Labeling of specific proteins in rat ovarian plasma membranes with [γ-32P]GTP

Guanosine 5’triphosphate (GTP) has been implicated in a number of biological processes associated with protein synthesis [ 11, microtubular function [2,3] and in the control exerted by a large number of hormones [4]. Our interest in plasma membrane-associated reactions that specifically utilize GTP has been stimulated by the recognition that this nucleotide is directly involved in the control of adenylate cyclase activity [4] and by the demonstration of specific binding sites for GTP in plasma membranes of hepatocytes and adipocytes [.5]. A hormone-sensitive GTPase in turkey erythrocytes [6] and a GTP-binding protein associated with pigeon erythrocyte adenylate cyclase have been described [7]. We undertook a search for GTP-dependent phosphorylation in ovarian membranes since in vitro desensitization of LH-sensitive adenylate cyclase has been postulated to involve phosphorylation reactions [8,9]. This desensitization process in the rat ovary is dependent on low concentrations of GTP even in the presence of ATP [ 10-121. Similar findings have been made also in normal rat kidney cells [ 131. Here we report evidence that [T-“~P] GTP preferentially labels two proteins in rat ovary and parotid membranes [ 141 that differ structurally from the proteins that are substrates for ADP-ribosylation by cholera toxin [15-l 71 and which are thought to be involved in the regulation of adenylate cyclase by GTP.

Characterization of a prolactin-daunomycin ligand as a probe for drug targeting.

Conjugates of ovine prolactin and daunomycin were prepared for use as affinity-labelled drug carriers in cancer cells carrying the prolactin receptor. The binding affinity of the conjugates to prolactin receptors in rat liver membrane preparations and in viable granulosa cells derived from estradiol- and pregnant mare serum gonadotropin (PMSG)-treated immature female rats was less than an order of magnitude lower than prolactin. The toxicity of the conjugate in cultured granulosa cells was dependent upon the concentration of the daunomycin present in the culture. The cytotoxic effect of the ligand was abolished by the addition of free prolactin or NH4Cl to the granulosa cell cultures. These conjugates may be useful probes in drug targeting against hormone-sensitive cancer.

The Interaction of the LH/hCG Receptor with Adenylate Cyclase in the Rat Ovary

The response of the gonads to luteinizing hormone (LH) and the related human chorionic gonadotropin (hCG) appears to depend on stimulation of the membrane-bound enzyme, adenylate cyclase (AC), (Lamprecht et al., 1973; Koch et al., 1974; Lindner et al., 1974; Marsh, 1976; Birnbaumer et al., 1976; Catt and Dufau, 1976; HunzickerDunn and Birnbaumer, 1976; Mintz et al., 1978; Bramley and Ryan, 1978a). A wide variety of hormones share this dependence of their action on cyclase mediation. The mechanism by which adenylate cyclase responds to hormones has therefore attracted intensive investigation in recent years. Some of these studies made use of membrane preparations or cells from rat liver (Pohl et al., 1971; Rodbell et al., 1971; Birnbaumer, 1973; Londos et al., 1974; Salomon et al., 1974; Rodbell et al., 1975), in which the enzyme is sensitive to glucagon; from fat tissue (Rodbell et al., 1970; Harwood et al., 1973; Rodbell, 1975), in which the enzyme is sensitive to several hormones such as epinephrine, glucagon, adrenocorticotropic hormone (ACTH), secretin, and LH; or from gonadal tissues (Birnbaumer et al.,1976; Mintz et al., 1978; Harwood et al., 1978; Bramley and Ryan, 1978b; Dufau et al.,1978; Catt et al., 1979), in which the enzyme is sensitive to gonadotropins.