Michael R. A. Morgan

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.

Mycotoxin immunoassays (with special reference to elisas)

Abstract Immunoassays are now in routine use for analysis of mycotoxins. This paper presents background information on antibody production and on immunoassay formats, particularly the enzyme-linked immunosorbent assay (ELISA). Applications of immunoassay technology to determination of aflatoxins, trichothecenes, ochratoxin A and others are reviewed and their performance discussed.

Immunochemical identification of LMW subunits of glutenin associated with bread-making quality of wheat flours

A murine monoclonal antibody (IFRN 0067), one of a library developed against prolamin fractions fromTriticum aestivum, has been characterised using a combination of immunoassay and immunoblotting techniques. The antibody was specific for two glutenin polypeptides which appeared by 2-dimensional electrophoresis to belong to the B group of LMW subunits. From results of antibody-binding studies with material extracted from genetic stocks, it was deduced that the target polypeptides were encoded on the short arm of chromosome 1D. The antibody was used in an immunoassay of bread wheats with a range of anticipated baking scores and for flours of known baking performance. Significant correlations were found between immunoassay and test-bake results. Indeed, correlation of IFRN 0067 binding with loaf volume was equal or better than that provided by alveograph parameters. The results provide evidence that LMW subunits contribute to the bread-making properties of wheat glutenin, as identified by the use of immunological techniques. The use of particular monoclonal antibodies, such as IFRN 0067, in the further development of simple, rapid diagnostic tests for flour quality predictions is discussed.

Solanidine is Present in Sera of Healthy Individuals and in Amounts Dependent on their Dietary Potato Consumption

1 Solanidine, a steroidal alkaloid, and its glycosides have been reported to have caused poisoning in man and animals. These alkaloids are normally present in small amounts in potatoes. Measurement of solanidine in body fluid would be expected to establish the real incidence of acute toxicity and help to resolve the question of any chronic toxicity including teratogenicity. 2 We report the detection of solanidine in the serum of 57 normal healthy volunteer subjects in whom it measured 4.0-56.3 nmol/l (1.6-22.5 ng/ml) before the midday meal. There was a significant correlation between serum solanidine concentration and normal dietary intake of potato by the individual concerned. 3 When two subjects abstained from potato and its products serum solanidine fell markedly and became minimal after the second week onwards.

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.

Development of an ELISA for sulfachlorpyridazine and investigation of matrix effects from different sample extraction procedures

The development of an enzyme-linked immunosorbent assay (ELISA) for the detection of residues of sulfachlorpyridazine (SCP) is described for the first time. The assay is highly specific for SCP, is simple to perform and has a lower detection limit of 0.65ng/ml in assay buffer. The potential application of the assay to detect residues of SCP at the 0.1mg/kg level in eggs, milk, beef, lamb, pork, chicken, turkey, porcine kidney, porcine liver and pig feedstuffs is discussed with regard to the effects of sample extracts on the standard curves. The antibody exhibits a rare stability in assay buffers containing up to 30% methanol. It is concluded that the ELISA for SCP has the appropriate characteristics for development into a robust method for the detection of this sulphonamide in agri-food materials.

Studies on the detrimental effects of bivalent binding in a microtitration plate ELISA and possible remedies

During the development of a microtitration plate enzyme-linked immunosorbent assay (ELISA) for 3-acetyldeoxynivalenol, using an immobilised hapten:protein conjugate, problems were encountered in obtaining an inhibition curve with hapten. Investigations into this phenomenon were conducted. Univalent Fab fragments were prepared and their binding compared with untreated antiserum on plates coated with different immobilised coating concentrations and with conjugate of a lower hapten:protein ratio. The studies showed that standard curves obtained with Fab fragments using the coating conjugate of high hapten ratio were more sensitive and showed a greater degree of inhibition. However with the conjugate of lower hapten ratio the untreated antiserum gave the more sensitive curve although inhibition was incomplete. From these results it is concluded that the antiserum probably contains two populations of antibodies, one of which binds bivalently to the plates at all concentrations of immobilised hapten.

Determination of solanidine in human plasma by radioimmunoassay

A radioimmunoassay for solanidine, the major hydrolysis product of potato glycoalkaloids, has been established and validated for application to human plasma. All 34 samples of human plasma tested contained solanidine, with a mean of 1.27 +/- 0.97 ng/ml (3.18 +/- 2.43 nmol/litre).

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.

Characterization of a panel of monoclonal anti-gliadin antibodies.

A panel of monoclonal antibodies has been raised against a total gliadin fraction prepared from the hard bread wheat cultivar Avalon. The antibodies have been characterized by enzyme-linked immunosorhent assay and immunoblotting techniques for their interactions with a range of cereal prolamins, purified gliadins and short synthetic peptides. The antibodies showed broad, intermediate and narrow specificities. The immunological approach offers the protein chemist an additional means of studying the structures and interrelationships of cereal proteins.