Gillet D, Ezan E, Ducancel F, Gaillard C, Ardouin T, Istin M, Ménez A, Boulain J C, Grognet J M
Département d'Ingénierie et d'Etude des Protéines, (DIEP), Commissariat à l'Energie Atomique, Gif/Yvette, France.
Anal Chem. 1993 Jul 1;65(13):1779-84. doi: 10.1021/ac00061a023.
This paper describes a competitive enzyme immunoassay of rat prolactin (rPrl) using a recombinant conjugate as a colorimetric tracer. rPrl was inserted into the N-terminal end of Escherichia coli alkaline phosphatase (AP), using an expression vector which allows insertion of foreign DNA sequences between codons +6 and +7 of the phoA gene. The assay was performed in 96-well microtiter plates coated with a mouse monoclonal antibody raised against rabbit immunoglobulin G. Each component (recombinant tracer, rabbit antiserum against rPrl, and rPrl standard) was added in a volume of 50 microL. The sensitivity of the assay was sufficiently high to allow titration of rPrl in plasma. The detection threshold was 15 pg (0.3 ng/mL) and the B/B0 50% value was 150 pg (3 ng/mL). The intraassay coefficient of variation was less than 10% over a wide range of rPrl concentrations (2.9-50 ng/mL). The interassay coefficient of variation was less than 15% for rat plasma samples in the concentration range of 4-40 ng/mL. The good parallelism observed between the standard curve and sample dilution curves showed that the immunoreactivity in rat plasma behaves like standard rPrl. Together with recovery experiments, these results indicated that assay without extraction is possible. A single immunoreactive peak that comigrates with standard rPrl is observed after molecular sieve fractionation of plasma samples. The reliability of the assay was confirmed by good correlation with conventional radioimmunoassay (r = 0.996, slope 0.978).
本文描述了一种使用重组偶联物作为比色示踪剂的大鼠催乳素（rPrl）竞争性酶免疫测定法。利用一种表达载体将rPrl插入大肠杆菌碱性磷酸酶（AP）的N末端，该表达载体允许在phoA基因的密码子+6和+7之间插入外源DNA序列。该测定在包被有抗兔免疫球蛋白G的小鼠单克隆抗体的96孔微量滴定板中进行。每种成分（重组示踪剂、抗rPrl兔抗血清和rPrl标准品）的加入体积为50微升。该测定的灵敏度足够高，能够对血浆中的rPrl进行滴定。检测阈值为15皮克（0.3纳克/毫升），B/B0 50%值为150皮克（3纳克/毫升）。在较宽的rPrl浓度范围（2.9 - 50纳克/毫升）内，批内变异系数小于10%。对于浓度范围在4 - 40纳克/毫升的大鼠血浆样本，批间变异系数小于15%。标准曲线和样品稀释曲线之间观察到良好的平行性，表明大鼠血浆中的免疫反应性与标准rPrl相似。连同回收率实验，这些结果表明无需提取即可进行测定。血浆样本经分子筛分级分离后，观察到与标准rPrl共迁移的单一免疫反应峰。该测定的可靠性通过与传统放射免疫测定的良好相关性得到证实（r = 0.996，斜率0.9７8）。
