Synthesis of hapten and conjugates of coumestrol and development of immunoassay.
3-O-Carboxymethylcoumestrol was prepared as the hapten for immunoassay by a partial alkylation of coumestrol with ethyl chloroacetate in acetone alkalized with potassium carbonate. 3-O-Ethoxycarbonylmethylcoumestrol was separated by column chromatography and finally was hydrolyzed with formic acid. 1H and 13C NMR data (APT, COSY, HMQC, and HMBC) revealed that the reaction was regioselective, as 3-O-ethoxycarboxymethylcoumestrol was the only monosubstituted derivative. The hapten was then conjugated to bovine serum albumin and used for immunization of rabbits. A radioimmunoassay (RIA) system was established based on the polyclonal antiserum and a 125I-labeled hapten-tyrosine methyl ester conjugate as the radioligand. Parameters of the RIA: sensitivity: 12 pg per tube, 50% intercept: 140 pg per tube, working range: 20-4000 pg per tube. The cross-reactivity of a panel isoflavonoid and lignan phytoestrogens was either negligible (e.g. formononetin 0.07%; biochanin A 0.06%) or not detectable at all. The major immunoreactive peak in HPLC fractions from an alfalfa extract had the same retention time as coumestrol standard and represented 94.8% of the signal. The remaining 5.2% of immunoreactivity was distributed between five minor peaks. We conclude that after the validation for particular matrices, the method will be a useful tool for analysis of coumestrol, especially in low volume and low concentration samples.
Lapcík
Oldrich
O
Department of Chemistry of Natural Compounds, Institute of Chemical Technology, Technická 5, 166 28 Praha 6, Czech Republic. oldrich.lapcik@vscht.cz
Stursa
Jan
J
Kleinová
Tereza
T
Vítková
Michaela
M
Dvoráková
Hana
H
Klejdus
Borivoj
B
Moravcová
Jitka
J
D000818:Animals; D000918:Antibody Specificity; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003375:Coumestrol / Q000031:analogs & derivatives / Q000032:analysis* / Q000138:chemical synthesis*; D006241:Haptens / Q000737:chemistry*; D007114:Immunization; D007074:Immunoglobulin G / Q000032:analysis; D007457:Iodine Radioisotopes; D007529:Isoflavones / Q000378:metabolism; D013058:Mass Spectrometry; D000455:Medicago sativa / Q000737:chemistry; D015394:Molecular Structure; D048789:Phytoestrogens; D028321:Plant Preparations / Q000378:metabolism; D011817:Rabbits; D011863:Radioimmunoassay / Q000379:methods*; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000276:immunology
D006241:Haptens; D007074:Immunoglobulin G; D007457:Iodine Radioisotopes; D007529:Isoflavones; D048789:Phytoestrogens; D028321:Plant Preparations; D012710:Serum Albumin, Bovine; D003375:Coumestrol