German I, Kennedy R T
Department of Chemistry, University of Florida, Gainesville 32611-7200, USA.
Anal Chem. 2000 Nov 1;72(21):5365-72. doi: 10.1021/ac000549g.
Capillary reversed-phase liquid chromatography (RPLC) was coupled on-line to competitive capillary electrophoresis immunoassay (CEIA) to improve concentration sensitivity of the competitive CEIA and to provide a means for detecting multiple species that cross-react with antibody. A competitive CEIA for glucagon was used for demonstration of this technique. Five-microliter samples were injected onto a 4-cm-long by 50-micron-i.d. RPLC column. Sample was desorbed by gradient elution, mixed on-line with fluorescently labeled glucagon and anti-glucagon, incubated in a continuous-flow reaction capillary, and analyzed by capillary electrophoresis with flow-gated injection and laser-induced fluorescence detection. Electrophoretic analysis of the reactor stream was performed every 1.5 s, allowing nearly continuous monitoring of the RPLC separation. Preconcentration achieved by RPLC allowed improvement in the detection limit from 760 to 20 pM. Addition of the RPLC column also allowed multiple cross-reactive species to be differentiated by first separating them chromatographically and then detecting them with the immunoassay. The technique was used to measure glucagon secretion from single islets of Langerhans and to differentiate cross-reactive forms of glucagon with one assay.
毛细管反相液相色谱（RPLC）与竞争性毛细管电泳免疫分析（CEIA）在线联用，以提高竞争性CEIA的浓度灵敏度，并提供一种检测与抗体发生交叉反应的多种物质的方法。以胰高血糖素的竞争性CEIA为例演示了该技术。将5微升样品注入一根长4厘米、内径50微米的RPLC柱。通过梯度洗脱解吸样品，将其与荧光标记的胰高血糖素和抗胰高血糖素在线混合，在连续流动反应毛细管中孵育，然后通过带流动门控进样和激光诱导荧光检测的毛细管电泳进行分析。每隔1.5秒对反应流进行一次电泳分析，从而几乎可以连续监测RPLC分离过程。RPLC实现的预浓缩使检测限从760皮摩尔提高到20皮摩尔。添加RPLC柱还可以通过先进行色谱分离，然后用免疫分析进行检测，来区分多种交叉反应物质。该技术用于测量单个胰岛分泌的胰高血糖素，并通过一次分析区分胰高血糖素的交叉反应形式。
