Duhau L, Grassi J, Grouselle D, Enjalbert A, Grognet J M
Département de Biologie, CEN/Saclay, Gif sur Yvette, France.
J Immunoassay. 1991;12(2):233-50. doi: 10.1080/01971529108055069.
Pure acetylcholinesterase (EC 3.1.1.7) from Electrophorus electricus has been covalently coupled to rat prolactin using the heterobifunctional reagent: N-succinimidyl-4 (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC). This conjugate was used as a tracer in a competitive enzyme immunoassay using a rabbit antiserum, raised against rat prolactin, as first antibody. The assay was performed in 96-well microtiter plates coated with a mouse monoclonal anti-rabbit immunoglobulin antibody. This second antibody solid phase ensured separation of bound and free moieties of the tracer during the specific immunoreaction. The total reaction volume was 150 microliters. Each component (tracer, antiserum and standard) was added in a volume of 50 microliters. The sensitivity of the assay was good since calculation indicated a detection threshold of 25 pg (0.5 ng/ml) and a B/Bo 50% value of 220 pg (4.4 ng/ml). Intra-assay variation was better than 10% over a wide range (135 to 2500 pg) with an optimum of 4% at 300 pg. The inter-assay coefficient of variation was less than 15% for rat plasma samples in the concentration range of 8 to 1000 ng/ml. The good parallelism observed between the standard curve and sample dilution curves, and recovery experiments, indicated that direct assay is possible. This was confirmed by 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).
来自电鳗的纯乙酰胆碱酯酶（EC 3.1.1.7）已使用异双功能试剂：N-琥珀酰亚胺基-4（N-马来酰亚胺甲基）环己烷-1-羧酸盐（SMCC）与大鼠催乳素共价偶联。这种偶联物在竞争性酶免疫测定中用作示踪剂，使用针对大鼠催乳素产生的兔抗血清作为一抗。该测定在涂有小鼠单克隆抗兔免疫球蛋白抗体的96孔微量滴定板中进行。这种第二抗体固相确保了在特异性免疫反应过程中示踪剂结合部分和游离部分的分离。总反应体积为150微升。每种成分（示踪剂、抗血清和标准品）以50微升的体积加入。该测定的灵敏度良好，因为计算表明检测阈值为25 pg（0.5 ng/ml），B/Bo 50%值为220 pg（4.4 ng/ml）。在较宽范围（135至2500 pg）内，批内变异优于10%，在300 pg时最佳为4%。对于浓度范围为8至1000 ng/ml的大鼠血浆样品，批间变异系数小于15%。在标准曲线和样品稀释曲线之间观察到的良好平行性以及回收实验表明可以进行直接测定。血浆样品的分子筛分级分离证实了这一点。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）。
