Lacorn Markus, Fleischer Kerstin, Willig Stephanie, Gremmel Sabine, Steinhart Hans, Claus Rolf
Institute of Animal Husbandry and Animal Breeding, Section of Animal Husbandry and Regulation Physiology, University of Hohenheim, Garbenstr. 17, 70599 Stuttgart, Germany.
J Immunol Methods. 2005 Feb;297(1-2):225-36. doi: 10.1016/j.jim.2004.12.011. Epub 2005 Jan 28.
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.
血浆中17β-雌二醇（E2）浓度处于低皮克/毫升范围。为开发一种用于测定E2的灵敏酶免疫分析（EIA）方法，使用了针对6-羧甲基（CMO）-E2-牛血清白蛋白偶联物产生的高度特异性抗体。基于6-CMO-E2和6-氨基-E2，使用生物素化试剂通过一步反应合成了四种在E2与生物素之间具有两种不同间隔长度的生物素化示踪剂。所有基于氨基的示踪剂均不适用于分析方法的开发，因为与分析物E2相比，抗体结合力太弱。对于基于6-CMO的示踪剂，示踪剂与抗体和链霉亲和素的同时结合似乎是该过程中的决定性步骤，这取决于孵育温度和间隔长度。虽然9个碳原子的短间隔在室温下敏感，但16个碳原子的较长间隔在4℃或室温下孵育显示出几乎相同的结果。该系统的绝对检测限为0.63皮克/孔。对于样品净化，猪血浆经溶剂萃取，并根据初始血浆体积通过溶剂分配进一步纯化。重现性测定结果显示，E2水平为15皮克/毫升和236皮克/毫升的样品的批内变异系数分别为13%和5.3%。对添加E2的血浆进行测定，结果显示，E2浓度在50皮克/毫升至1000皮克/毫升之间时，回收率为83%至100%。仅在最低浓度（20皮克/毫升）时观察到回收率为58%。使用相同抗体，该EIA与既定放射免疫分析的相关性为r = 0.991。
