Dutta Pratuyasha, Shrivastav Tulsidas G, Thakur Sonu Chand
The National Institute of Health and Family Welfare (NIHFW), New Delhi 110067, India; Jamia Millia Islamia (JMI), New Delhi 110025, India.
The National Institute of Health and Family Welfare (NIHFW), New Delhi 110067, India.
Anal Biochem. 2018 Oct 1;558:60-68. doi: 10.1016/j.ab.2018.07.010. Epub 2018 Jul 17.
In order to develop ELISA for medroxyprogesterone acetate, medroxyprogesterone acetate-3-carboxymethyloxime (MPA-3-CMO) was coupled to bovine serum albumin (BSA) for immunogen preparation and to horseradish peroxidase (HRP) for enzyme conjugate preparation by N-hydroxysuccinimide mediated carbodiimide reaction. The immunogen was used to raise the antiserum in New Zealand white rabbit. The immunoreactivity of MPA-3-CMO-BSA-antibody and MPA-3-CMO-HRP enzyme conjugate was checked by checkerboard assay. The MPA-3-CMO-HRP enzyme conjugate and MPA-3-CMO-BSA-antibody were used for further development, standardization and validation of the assay. Sensitivity, ED and affinity of the assay were found to be 0.114 ng/mL, 2.75 ng/mL and 9.9 × 10⁻⁸ L/mol respectively. The % cross-reaction of analogous steroids with MPA-3-CMO-BSA-antibody was less than 0.025%. The recovery of the exogenously spiked MPA serum pools were in the range of 96.83-105.47%. The intra- and inter-assay coefficients of variation was less than 7.02%. The correlation coefficient of the serum level of MPA measured by the developed assay with the commercially available kit was found to be 0.95 (n = 37). This developed ELISA was further validated by measuring serum level of MPA in rat after administering them different doses of MPA intramuscularly.
为开发醋酸甲羟孕酮的酶联免疫吸附测定法（ELISA），通过N-羟基琥珀酰亚胺介导的碳二亚胺反应，将醋酸甲羟孕酮-3-羧甲基肟（MPA-3-CMO）与牛血清白蛋白（BSA）偶联以制备免疫原，并与辣根过氧化物酶（HRP）偶联以制备酶结合物。用该免疫原在新西兰白兔中制备抗血清。通过棋盘滴定法检测MPA-3-CMO-BSA抗体和MPA-3-CMO-HRP酶结合物的免疫反应性。将MPA-3-CMO-HRP酶结合物和MPA-3-CMO-BSA抗体用于该测定法的进一步开发、标准化和验证。该测定法的灵敏度、检测限（ED）和亲和力分别为0.114 ng/mL、2.75 ng/mL和9.9×10⁻⁸ L/mol。类似甾体与MPA-3-CMO-BSA抗体的交叉反应率小于0.025%。外源性添加MPA的血清池的回收率在96.83 - 105.47%范围内。批内和批间变异系数小于7.02%。所开发的测定法测得的MPA血清水平与市售试剂盒测得的结果的相关系数为0.95（n = 37）。通过测定不同剂量MPA肌肉注射后大鼠血清中MPA的水平，对所开发的ELISA进行了进一步验证。
