Khosravi Javad, Krishna Radha G, Khaja Najmuddin, Bodani Umesh, Diamandi Anastasia
Diagnostic Systems Laboratories Inc., Toronto, Ontario, Canada.
Clin Biochem. 2004 May;37(5):370-6. doi: 10.1016/j.clinbiochem.2004.01.011.
Inhibin circulates in various molecular weight forms. Alpha (alpha)-subunit-directed total inhibin immunoassays, which detect all forms of alpha subunits plus the alpha/beta inhibin dimers, have been found valuable in the diagnosis and monitoring of ovarian cancer. Because of the dependency of the published methods on boiling sample pre-treatment with SDS and unavailability of a commercial assay, we developed an enzyme-linked immunosorbent assay (ELISA) for direct determination of total inhibin.
Method development involved a pair of well-characterized inhibin alpha subunit-directed antibodies and determination of the effects of various assay parameters. Selection of the optimized protocol was guided by the outcome of comparative sample analysis using previously reported boiling sample pre-treatment reagents and protocols.
We report development of a simplified ELISA for total inhibin. Method evaluation data demonstrated acceptable analytical performance characteristics with detection limit of 2 ng/l (recombinant inhibin-A), dynamic range of 12.5-500 ng/l, and intra- and inter-assay imprecision of 2.3-4.6% and 3.3-5.1% at total inhibin concentrations of approximately 60-400 ng/l, respectively. The mean (+/-SD) recovery from spiked serum samples averaged 109 +/- 14% and recovery in response to serial sample dilution was 99 +/- 10%. Serum values by the direct method (n = 40) correlated strongly with those obtained after sample pre-treatment by boiling with SDS (r = 0.97). As expected, the total inhibin immunoreactivity in human follicular fluid fractionated by HPLC gel filtration in multiple immunoreactive peaks (8-250 kDa). In serum samples from postmenopausal women with ovarian cancer, the assay detected significantly higher total inhibin levels than in samples from normal postmenopausal controls.
The development of a fast and simplified ELISA should facilitate wider investigations of pathophysiology and diagnostic potential of total inhibin measurement.
抑制素以多种分子量形式循环。α（α）亚基导向的总抑制素免疫测定法可检测所有形式的α亚基以及α/β抑制素二聚体，已被证明在卵巢癌的诊断和监测中具有重要价值。由于已发表的方法依赖于用SDS对样品进行煮沸预处理，且缺乏商业化检测方法，我们开发了一种酶联免疫吸附测定法（ELISA）用于直接测定总抑制素。
方法开发涉及一对特性明确的抑制素α亚基导向抗体，并确定各种检测参数的影响。通过使用先前报道的煮沸样品预处理试剂和方案进行比较样品分析的结果，指导优化方案的选择。
我们报告了一种用于总抑制素的简化ELISA的开发。方法评估数据显示出可接受的分析性能特征，检测限为2 ng/l（重组抑制素-A），动态范围为12.5 - 500 ng/l，在总抑制素浓度约为60 - 400 ng/l时，批内和批间不精密度分别为2.3 - 4.6%和3.3 - 5.1%。加标血清样品的平均（±标准差）回收率平均为109 ± 14%，连续样品稀释后的回收率为99 ± 10%。直接法测定的血清值（n = 40）与用SDS煮沸预处理后获得的值高度相关（r = 0.97）。正如预期的那样，通过高效液相色谱凝胶过滤分离的人卵泡液中的总抑制素免疫反应性呈多个免疫反应峰（8 - 250 kDa）。在患有卵巢癌的绝经后妇女的血清样本中，该检测方法检测到的总抑制素水平明显高于正常绝经后对照组的样本。
快速且简化的ELISA的开发应有助于更广泛地研究总抑制素测量的病理生理学和诊断潜力。
