Tao L, Kennedy R T
Department of Chemistry, University of Florida, Gainesville 32611-7200, USA.
Anal Chem. 1996 Nov 15;68(22):3899-906. doi: 10.1021/ac960560+.
An on-line competitive immunoassay for insulin has been developed and applied to monitoring insulin concentration in a flowing stream. In the assay, solutions of fluorescein-labeled insulin (FITC-insulin), monoclonal anti-insulin, and sample containing insulin are pumped into a cross where they begin to mix. The mixture flows through a fused silica reactor capillary to a flow-gated interface. During transfer to the interface, insulin and FITC-insulin compete to form a complex with the antibody. At the interface, a plug of the mixture is injected into a separation capillary, where the bound and free FITC-insulin are separated and detected by capillary electrophoresis with laser-induced fluorescence detection. The amount of bound FITC-insulin, amount of free FITC-insulin, or bound/free ratio can be used to quantify insulin concentration. Typical relative standard deviations of bound over free ratio are 5%. The detection limit of the immunoassay in the on-line mode is < 0.3 nM. Each separation requires as little as 3 s, and over 1600 consecutive assays can be acquired with no need to rinse the separation capillary. Thus, the system can be used to monitor insulin in a flowing stream for flow injection analysis or for sensor-like monitoring. Dilution and zone broadening during transfer of sample to the interface limit the response time of the on-line system to about 25 s. As a demonstration of the on-line immunoassay, the insulin content of single islets of Langerhans was determined by flow injection analysis.
一种用于胰岛素的在线竞争性免疫测定法已被开发出来，并应用于监测流动样品中的胰岛素浓度。在该测定法中，将荧光素标记的胰岛素（FITC - 胰岛素）溶液、抗胰岛素单克隆抗体以及含有胰岛素的样品泵入一个交叉点，在那里它们开始混合。混合物通过熔融石英反应毛细管流至流动门控接口。在转移至接口的过程中，胰岛素和FITC - 胰岛素竞争与抗体形成复合物。在接口处，将一小段混合物注入分离毛细管，在其中结合态和游离态的FITC - 胰岛素通过激光诱导荧光检测的毛细管电泳进行分离和检测。结合态FITC - 胰岛素的量、游离态FITC - 胰岛素的量或结合/游离比可用于定量胰岛素浓度。结合态与游离态比值的典型相对标准偏差为5%。在线模式下免疫测定法的检测限<0.3 nM。每次分离只需3秒，并且无需冲洗分离毛细管即可连续进行1600多次测定。因此，该系统可用于监测流动样品中的胰岛素，以进行流动注射分析或类似传感器的监测。样品转移至接口过程中的稀释和区带展宽将在线系统的响应时间限制在约25秒。作为在线免疫测定法的一个实例，通过流动注射分析测定了单个胰岛的胰岛素含量。
