Cam M C, McNeill J H
Division of Pharmacology and Toxicology, University of British Columbia, Vancouver, Canada.
J Pharmacol Toxicol Methods. 1996 Apr;35(2):111-9. doi: 10.1016/1056-8719(96)00021-4.
There has been a notable lack of consistency in plasma insulin values reported in the literature. In an attempt to find a more reproducible and sensitive insulin radioimmunoassay (RIA) method for the measurement of plasma insulin, the charcoal adsorption method for separation of free from antibody-bound antigen was investigated. The applicability of this method towards the measurement of plasma insulin, however, has been known to be severely limited because of interference by plasma proteins with charcoal binding. Indeed, the assay resulted in negative fasting insulin values and unacceptable interassay variability, which was not improved by adding charcoal-extracted plasma (CEP) to the standard curve. In this paper, we demonstrate a process by which relatively small volumes (25 microL) of plasma from control, diabetic, and fasted rats can be assayed reproducibly with charcoal in the final separation step. It was observed that the assay of insulin-free plasma, in the form of CEP, could represent a distinct "zero insulin" level specific for plasma samples, in place of the zero insulin standard (insulin-free standard buffer) routinely used. Normalizing plasma samples against CEP resulted in lower intra-assay and interassay variability (coefficient of variation < 10%) and enhanced sensitivity to 7 microU/mL. Recovery was tested by adding 25 microL of known insulin standards to plasma samples in which insulin levels had been determined previously. Whereas the recovery of total insulin in the presence of plasma was low (38%-89%), recovery of total insulin from two standards was > 95%. Following correction, recovery of total insulin in 25-microL plasma was improved to > 90%. Hence, this approach provides a simplified way of correcting for plasma effects and greatly enhances the feasibility of using the charcoal-separation technique for the measurement of plasma insulin.
文献报道的血浆胰岛素值明显缺乏一致性。为了找到一种更具可重复性和敏感性的胰岛素放射免疫测定（RIA）方法来测量血浆胰岛素，研究了用活性炭吸附法分离游离抗原与抗体结合抗原。然而，由于血浆蛋白对活性炭结合的干扰，已知该方法在测量血浆胰岛素方面的适用性受到严重限制。实际上，该测定法得出的空腹胰岛素值为阴性，且批间变异度不可接受，即使在标准曲线中加入活性炭提取血浆（CEP）也未得到改善。在本文中，我们展示了一个过程，通过该过程，在最终分离步骤中使用活性炭可以对来自对照、糖尿病和禁食大鼠的相对少量（25微升）血浆进行可重复测定。据观察，以CEP形式存在的无胰岛素血浆测定可以代表血浆样品特有的明显“零胰岛素”水平，取代常规使用的零胰岛素标准品（无胰岛素标准缓冲液）。以CEP对血浆样品进行标准化处理可降低批内和批间变异度（变异系数<10%），并将灵敏度提高到7微单位/毫升。通过向先前已测定胰岛素水平的血浆样品中加入25微升已知胰岛素标准品来测试回收率。虽然在有血浆存在的情况下总胰岛素的回收率较低（38%-89%），但两种标准品的总胰岛素回收率>95%。校正后，25微升血浆中总胰岛素的回收率提高到>90%。因此，这种方法提供了一种校正血浆效应的简化方法，并大大提高了使用活性炭分离技术测量血浆胰岛素的可行性。
