Meyer H H, Sauerwein H, Mutayoba B M
Institut für Physiologie der Süddeutschen Versuchs und Forschungsanstalt für Milchwirtschaft, Technische Universität München, Freising-Weihenstephan, F.R.G.
J Steroid Biochem. 1990 Feb;35(2):263-9. doi: 10.1016/0022-4731(90)90283-x.
A sensitive test system has been developed for estimation of estradiol-17 beta (E2) in bovine plasma. Plasma extracts are first purified by a selective immunoaffinity chromatography (IAC) using an antibody raised against estradiol-6-carboxymethyloxime-bovine serum albumin and immobilized to Sepharose. The eluate was analysed by a competitive enzyme immunoassay (EIA) on microtitration plates. For the assay the wells of microtitration plates were coated with affinity purified sheep IgG (antirabbit IgG) that binds the hormone specific antibody raised in rabbits against estradiol-17-hemisuccinate-bovine serum albumin. E2 is estimated by displacement of biocytinyl-E2, that was produced by ligation of estradiol-17 beta, D-glucuronic acid and biocytin. Bound biocytinyl-E2 is detected after binding of streptavidin-peroxidase and colour production by the enzyme. A very high amplification was possible with this technique and the absolute detection limit amounted to approximately 120 fg/well at 94% relative binding. By combination of IAC and EIA the following levels of E2 were found in bovine plasma: male or female calves less than 2.7 pg/ml, cycling cow 0.5-7 pg/ml, cow during the last month of pregnancy 9-310 pg/ml, mature bull 5-30 pg/ml. However, up to 1110 pg E2/ml were found in plasma of a calf after treatment with an illicit hormone preparation used for growth promotion; after 21 days levels declined to 6 pg/ml which is hardly different from controls. In conclusion, the IAC/EIA can be used for sensitive estimation of estradiol-17 beta in plasma from all type of cattle and for control of improper use of E2 after commitment of a threshold level.
已开发出一种灵敏的检测系统，用于测定牛血浆中的17β-雌二醇（E2）。血浆提取物首先通过选择性免疫亲和色谱法（IAC）进行纯化，该方法使用针对雌二醇-6-羧甲基肟-牛血清白蛋白产生的抗体，并固定在琼脂糖上。洗脱液通过微量滴定板上的竞争性酶免疫测定法（EIA）进行分析。对于该测定，微量滴定板的孔用亲和纯化的羊IgG（抗兔IgG）包被，该羊IgG可结合兔体内产生的针对17-半琥珀酸雌二醇-牛血清白蛋白的激素特异性抗体。E2通过生物素化E2的置换来估计，生物素化E2是由17β-雌二醇、D-葡萄糖醛酸和生物素连接产生的。结合链霉亲和素-过氧化物酶并通过酶产生颜色后，检测结合的生物素化E2。使用该技术可以实现非常高的放大倍数，在相对结合率为94%时，绝对检测限约为120 fg/孔。通过IAC和EIA相结合，在牛血浆中发现了以下E2水平：雄性或雌性犊牛低于2.7 pg/ml，发情母牛0.5 - 7 pg/ml，怀孕最后一个月的母牛9 - 310 pg/ml，成年公牛5 - 30 pg/ml。然而，在用用于促进生长的非法激素制剂处理后的一头犊牛血浆中发现高达1110 pg E2/ml；21天后水平降至6 pg/ml，与对照组几乎没有差异。总之，IAC/EIA可用于灵敏测定所有类型牛血浆中的17β-雌二醇，并用于在超过阈值水平后控制E2的不当使用。
