Prakash B S, Mondal Mohan, Anandlaxmi N
Division of Dairy Cattle Physiology, National Dairy Research Institute, Karnal, Haryana, India.
J Immunoassay Immunochem. 2003;24(4):409-20. doi: 10.1081/IAS-120025778.
A simple and highly sensitive enzymeimmunoassay (EIA) for GH determination in buffalo plasma on microtitreplates using biotin-streptavidin amplification system and the second antibody coating was developed. Biotin was coupled to GH and used to bridge between streptavidin-peroxidase and immobilized antiserum in competitive assay. The EIA was carried out directly in 100 microL buffalo plasma. The GH standards ranging from 0.05 ng/well/100 microL to 12.8 ng/well/100 microL were prepared in hormone free plasma collected from an aged (> 15 years) senile buffalo. The sensitivity of the EIA procedure was 50 pg/well GH. which corresponded to 0.50 ng/mL plasma; the 50% relative binding sensitivity was seen at 800 pg/well/100 microL. Plasma volumes for the EIA, viz., 25, 50, and 100 microL did not influence the shape of standard curve, even though a slight drop in the OD450 was seen with higher plasma volumes. For the biological validation of the assay, 12 Murrah buffalo calves were used. Six of these were administered synthetic bovine growth hormone-releasing factor (10 microg/100 kg body weight, i.v., and the remaining six animals were administered sterile normal saline and kept as controls. Jugular blood samples were collected at -60, -45, -30, -15, -10, -5, 5, 10, 15, and 30 min and, thereafter, at an interval of 15 min using an indwelling jugular catheter, beginning 1 h prior to GRF injection up to 8 h post treatment. In all animals, a peak of GH was recorded within 5 to 20 min of GRF administration, which confirms the biological validation of the EIA. To confirm homogeneity of buffalo GH with bovine GH, a parallelism test was conducted between the buffer standard curve of bovine GH and GH measured from serial dilution of buffalo plasma containing a high level of endogenous growth hormone.
建立了一种简单且高度灵敏的酶免疫分析（EIA）方法，用于在微量滴定板上测定水牛血浆中的生长激素（GH），该方法采用生物素-链霉亲和素放大系统和第二抗体包被。生物素与GH偶联，并在竞争性分析中用于连接链霉亲和素-过氧化物酶和固定化抗血清。EIA直接在100微升水牛血浆中进行。在从一头年龄大于15岁的老年水牛采集的无激素血浆中制备了浓度范围为0.05纳克/孔/100微升至12.8纳克/孔/100微升的GH标准品。该EIA方法的灵敏度为50皮克/孔GH，相当于0.50纳克/毫升血浆；50%相对结合灵敏度出现在800皮克/孔/100微升。EIA所用的血浆体积，即25、50和100微升，虽随着血浆体积增加OD450略有下降，但不影响标准曲线的形状。为进行该分析方法的生物学验证，使用了12头摩拉水牛犊牛。其中6头静脉注射合成牛生长激素释放因子（10微克/100千克体重），其余6头注射无菌生理盐水作为对照。在注射生长激素释放因子前1小时至治疗后8小时，使用颈静脉留置导管每隔15分钟在-60、-45、-30、-15、-10、-5、5、10、15和30分钟采集颈静脉血样。在所有动物中，注射生长激素释放因子后5至20分钟内记录到GH峰值，这证实了EIA的生物学验证。为确认水牛GH与牛GH的同源性，在牛GH缓冲液标准曲线与从含有高水平内源性生长激素的水牛血浆系列稀释液中测得的GH之间进行了平行性试验。
