Tripathi V, Nara Seema, Chaube Shail K, Rangari Kiran, Saroha Ashish, Kariya Kiran P, Singh H, Shrivastav Tulsidas G
Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, New Delhi, India.
J Immunoassay Immunochem. 2008;29(2):117-27. doi: 10.1080/15321810801887599.
Using a homologous combination of immunogen and enzyme conjugate, a highly specific and sensitive Enzyme Linked Immunosorbent Assay (ELISA) was developed to measure 17-alpha-hydroxy-progesterone (17-alpha-OH-P) in human serum. The antiserum was raised against 17-alpha-hydroxy-progesterone-3-O-carboxymethyloxime bovine serum albumin (17-alpha-OH-P-3-O-CMO-BSA) in New Zealand white rabbits. The enzyme conjugate was prepared by labeling 17-alpha-hydroxy-progesterone-3-O-carboxymethyloxime with horseradish peroxidase (HRP). Checkerboard assay was performed to determine the working dilutions of antiserum and enzyme conjugate. Dose-response studies were carried out by incubating 25 microL enzyme conjugate along with 50 microL of standards on the primary antibody coated wells for 1 hour. The bound enzyme activity was measured colorimetrically using Tetramethyl benzidine/hydrogen peroxide (TMB/H2O2) as substrate. The enzyme substrate reaction was terminated with 100 microL of 0.5 M H2SO4 after 20 min and the intensity of the color was measured using Tecan ELISA reader at 450 nm. The assay was validated in terms of sensitivity, specificity, precision and recovery. The detection limit of the assay was 180 pg/mL. The assay was more specific as compared to most other reported immunoassays for 17-alpha-OH-P. Cross reaction with analogous C18, C19, and C21 steroids was less than 0.1% except for progesterone which showed 2.1% cross reaction. The intra- and inter-assay coefficients of variation ranges from 3.7-7.5% and 6.9-11.7%, respectively. The developed ELISA correlated well with established RIA, with a correlation coefficient of 0.9 (n=30).
采用免疫原和酶结合物的同源组合，开发了一种高特异性和高灵敏度的酶联免疫吸附测定法（ELISA），用于测定人血清中的17-α-羟基孕酮（17-α-OH-P）。在新西兰白兔中，用17-α-羟基孕酮-3-O-羧甲基肟牛血清白蛋白（17-α-OH-P-3-O-CMO-BSA）制备抗血清。通过用辣根过氧化物酶（HRP）标记17-α-羟基孕酮-3-O-羧甲基肟来制备酶结合物。进行棋盘滴定法以确定抗血清和酶结合物的工作稀释度。通过在包被有一抗的孔中孵育25微升酶结合物和50微升标准品1小时来进行剂量反应研究。使用四甲基联苯胺/过氧化氢（TMB/H2O2）作为底物，通过比色法测量结合的酶活性。20分钟后，用100微升0.5M H2SO4终止酶底物反应，并使用Tecan ELISA读数仪在450nm处测量颜色强度。该测定法在灵敏度、特异性、精密度和回收率方面进行了验证。该测定法的检测限为180pg/mL。与大多数其他报道的17-α-OH-P免疫测定法相比，该测定法更具特异性。与类似的C18、C19和C21类固醇的交叉反应小于0.1%，但孕酮的交叉反应为2.1%。批内和批间变异系数分别为3.7-7.5%和6.9-11.7%。所开发的ELISA与既定的放射免疫分析法（RIA）相关性良好，相关系数为0.9（n=30）。
