Studies on establishment of methods for determination of growth hormone, insulin and thyroxine levels in serum of bovine by means of enzyme immunosorbentassay system

为建立适合兽医临床用药的牛血清促生长激素、胰岛素和甲状腺素浓度的测定方法，对从脑下垂体、胰脏和甲状腺前叶分离纯化生长激素的方法进行了改进，建立了亲和素-生物素-过氧化物酶免疫分析法(ABC)测定牛血清中生长激素(BGH)、胰岛素和甲状腺素(T_4)的分析条件。BGH、胰岛素和T_4是从分离的组织中用众所周知的方法分离和纯化的。我们通过高效液相色谱和凝胶过滤层析对其进行了进一步纯化。经SDS-PAGE和等电聚焦电泳法鉴定，这些蛋白质组分均为纯化的单一组分。此外，通过免疫机器人的方法，这些抗体在与每个纯化的抗体的反应中都显示出一条单一的条带。经分析，这些蛋白质组分分别为BGH、胰岛素和T_4。用提取的激素、纯化的抗体、ABC和碱性磷酸酶(ALP)研究了EIA的基本反应条件。不能制备每条标准曲线，呈现稳定的重现性。用EIA法和间接免疫法测定了健康牛和不同病牛血清中BGH、胰岛素和T_4的浓度。虽然EIA的数据显示出比RIA略高的水平，但这两个测试之间的相关性相对较大。因此，为建立临床EIA方法，有必要对抗体固相偶联和血清缓冲液处理方法进行改进。

In order to establish some methods for the determination of concentration of growth hormaone, insulin and thyroxine in the serum of bovine that be able to apply veterinary clinical medication in the bedside, modifications on the separation and purification methods of these hormones from anterior lobe of pituitary, ppancreas and thyroid gland and analytical conditions of enzyme immunosorbentassay (EIA) using avidin-biotin-peroxidase complexes (ABC) for the determination of bobine growth hormone (bGH), insulin and thyroxine (T_4) in serum of bovine were studied.1. bGH,insulin and T_4 was separated and purified from avobed mertioned tissues by well-known methods. We purified these further by HPLC and gel filtration chromatography. These protein fractions were recognized to be each a purified single fraction by SDS-PAGE and isoelectric focusing electriphoresis. Furthermore, those showed each a single band in the reaction with each purified antibodies by means of immunobotting. These protein fractions were isolated to be each of bGH,insulin and T_4 by their analytical techniques.2. The basical reactional conditions of EIA were studied using each extracted hormones, purified antibodies and ABC and alkaline phosphatase(ALP). We could t prepare the each standard curves, which showed stabilized reappearance.3. Concentrations of bGH,insulin and T_4 in serum from healthy and various patiant cattle were determined by means of two methods of EIA and dadioimmunoassay. Relatively significant correlation between these two tests was found, though data of EIA has shown a slightly high level more that RIA.4. Accordingly, it is considered necessary to modify about conjugation of antibody to solid phase and treatment of serum with some buffered solution for establishment of clinical EIA method.