title: Use of biotinylated 17beta-estradiol in enzyme-immunoassay development: spacer length and chemical structure of the bridge are the main determinants in simultaneous streptavidin-antibody binding.
abstract: 17beta-estradiol (E2) concentrations are in the low pg/ml range in plasma. To develop a sensitive enzyme immunoassay (EIA) for E2-determination a highly specific antibody raised against a 6-carboxymethyl (CMO)-E2-bovine serum albumine conjugate was used. Based on 6-CMO-E2 and 6-amino-E2, four biotinylated tracers with two different spacer lengths between E2 and biotin were synthesized using biotinylation reagents in one step reactions. All amino-based tracers were unsuitable for assay development because the antibody binding was too weak compared to the analyte E2. For 6-CMO-based tracers the simultaneous binding of the tracer to the antibody and streptavidin seems to be the determining step in the procedure depending on incubation temperature and spacer lengths. While a short spacer of 9 carbon atoms was susceptible to room temperature, a longer spacer of 16 carbon atoms showed nearly the same results for incubation at 4 degrees C or at room temperature. The absolute detection limit of this system was 0.63 pg/well. For sample clean-up, porcine plasma was solvent-extracted and depending on the initial plasma volume further purified by solvent partition. Determination of reproducibility resulted in intraassay coefficients of variation of 13% and 5.3% for samples with E2-levels of 15 pg/ml and 236 pg/ml, respectively. Measurement of E2-spiked blood plasma revealed recoveries of 83% up to 100% for E2 concentrations between 50 pg/ml and 1000 pg/ml. Only for the lowest concentration (20 pg/ml) a recovery of 58% was observed. Correlation of the EIA with an established radio immunoassay resulted in r=0.991 using the same antibody.
authors.affiliation: Institute of Animal Husbandry and Animal Breeding, Section of Animal Husbandry and Regulation Physiology, University of Hohenheim, Garbenstr. 17, 70599 Stuttgart, Germany. m-lacorn@uni-hohenheim.de
authors.forename: Markus
authors.initials: M
authors.lastname: Lacorn
authors.forename: Kerstin
authors.initials: K
authors.lastname: Fleischer
authors.forename: Stephanie
authors.initials: S
authors.lastname: Willig
authors.forename: Sabine
authors.initials: S
authors.lastname: Gremmel
authors.forename: Hans
authors.initials: H
authors.lastname: Steinhart
authors.forename: Rolf
authors.initials: R
authors.lastname: Claus
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology*; D019921:Biotinylation; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004958:Estradiol / Q000031:analogs & derivatives* / Q000097:blood* / Q000737:chemistry* / Q000276:immunology; D004966:Estrogens, Conjugated (USP) / Q000276:immunology; D011817:Rabbits; D012680:Sensitivity and Specificity; D019809:Streptavidin / Q000276:immunology*; D013552:Swine
chemical_list: D000906:Antibodies; D004966:Estrogens, Conjugated (USP); C002912:estradiol-6-(O-carboxymethyl)oxime; D004958:Estradiol; D019809:Streptavidin