Basu Anupam, Maitra Saumen Kumar, Shrivastav Tulsidas G
Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, Munirka New Delhi 110067, India.
Anal Biochem. 2007 Jul 15;366(2):175-81. doi: 10.1016/j.ab.2007.04.015. Epub 2007 Apr 12.
The development of a simultaneous multianalyte immunoassay for the detection of progesterone and human chorionic gonadotropin (hCG) in serum is described. In this simultaneous multianalyte assay, two different enzymes, viz. horse radish peroxidase (HRP) and alkaline phosphatase (ALP), were used as markers. To the simultaneous immobilized progesterone and hCG antibody microwells, 50 microL of different concentrations of combined standards or serum samples was added in duplicate and then 100 microL of combined conjugate reagent, composed of 17-alpha-OH-P-ALP and hCG-biotin was added to all the wells and incubated for 1h at 37 degrees C. After incubation, the contents of the wells were decanted and washed thoroughly with running tap water. After washing, 100 microL alkaline phosphatase substrate along with streptavidin-horseradish peroxidase was added to all the wells and incubated for 0.5 h at 37 degrees C. After incubation, the developed color was measured at 405 nm. The absorbency at this stage provides the result for the progesterone assay. The contents of the wells were decanted and washed. In the next step, 100 microL of tetramethylbenzidene/H2O2 reagent was added to all the wells. After 15 min of incubation, 100 microL of 0.5 M H2SO4 was added to all the wells and the color was read at 450 nm. The absorbency at this stage provides the result for the hCG assay. Sensitivity of the progesterone and hCG assays were 0.118 ng/ml and 0.124 IU/ml respectively. Intra- and inter assay percentage coefficients of variation ranged from 1.8 to 7.1 and 9.1 to 11.5 for progesterone and from 2.1 to 10.4 and 7.2 to 11.3 for hCG. There was good correlation between the discrete and the simultaneous assays. For progesterone assay, R2 was 0.99 and for hCG R2 was also 0.99. The developed dual assay for progesterone and hCG may be useful for the diagnosis of abnormal pregnancies such as miscarriages and ectopic pregnancies.
本文描述了一种用于检测血清中孕酮和人绒毛膜促性腺激素（hCG）的同步多分析物免疫测定方法。在这种同步多分析物测定中，使用了两种不同的酶，即辣根过氧化物酶（HRP）和碱性磷酸酶（ALP）作为标记物。向同时固定有孕酮和hCG抗体的微孔板中，一式两份加入50微升不同浓度的混合标准品或血清样品，然后向所有孔中加入100微升由17-α-OH-P-ALP和hCG-生物素组成的混合共轭试剂，并在37℃下孵育1小时。孵育后，倒出孔中的内容物，并用自来水充分洗涤。洗涤后，向所有孔中加入100微升碱性磷酸酶底物以及链霉亲和素-辣根过氧化物酶，并在37℃下孵育0.5小时。孵育后，在405nm处测量显色。此阶段的吸光度提供孕酮测定结果。倒出孔中的内容物并洗涤。下一步，向所有孔中加入100微升四甲基联苯胺/H2O2试剂。孵育15分钟后，向所有孔中加入100微升0.5M H2SO4，并在450nm处读取颜色。此阶段的吸光度提供hCG测定结果。孕酮和hCG测定的灵敏度分别为0.118ng/ml和0.124IU/ml。孕酮测定的批内和批间变异系数百分比范围为1.8至7.1，hCG测定的批内和批间变异系数百分比范围为2.1至10.4和7.2至11.3。离散测定和同步测定之间具有良好的相关性。对于孕酮测定，R2为0.99，对于hCG，R2也为0.99。所开发的孕酮和hCG双重测定法可能有助于诊断异常妊娠，如流产和异位妊娠。
