Development of analytical methods by capillary electophoresis for human fluids.

我们演示了用Beckman P/ACE 2100毛细管电泳（CE）系统（Beckman Inc., Brea， CA，USA）分离人血清蛋白。采用20 cm无包被熔融石英毛细管柱（25mum， I.D.）和150 mmol/L硼酸盐缓冲液（pH 10.0）作为运行缓冲液。人血清样品在20 mmol/L PBS缓冲液（pH 7.0）中稀释11倍后，加压注射到CE柱上10秒（约10 nL）。色谱柱温度保持在23℃。施加10kV电压6.5 min，分离蛋白组分，在200 nm处检测到峰。用该方法将人血清样品分离成约10个馏分。我们用亲和柱分离血清蛋白的方法和抗体鉴定程序，证实了我们鉴定分离蛋白的有效性。这些蛋白峰分别为免疫球蛋白、补体C3、转移蛋白、α - 2巨球蛋白、接触球蛋白、α - 1抗胰蛋白酶、白蛋白、前白蛋白。此外，该方法对38个样品的分析结果与醋酸纤维素电泳法（Olympus Optical Co.Ltd, Tokyo, Japan）的结果具有良好的相关性。采用Beckman P/ACE 2100毛细管电泳法对人血清乳酸脱氢酶同工酶进行了分离和定量测定。采用50 cm长未包覆熔融石英柱，75 mmol/ d，底物含运行缓冲液[51.6 mmol/L L-乳酸，8.26 mmol/L NAD, 25 mmol/L Tris（羟甲基）氨基乙醇缓冲液，pH 8.7]。在340 nm处检测产物NADH。注射样品（血清样品用25 mmol/L Tris缓冲液（pH8.7）稀释5倍），加压注射2秒。在10kV电压下分离同工酶5 min，在24oc下关闭电压30 min（用于底物与同工酶之间的反应），然后重新施加10kV电压30 min。在这些条件下，LD5产生的NADH在20 min出现，LD-1峰在23.5 min出现，在LD-5和LD-1峰之间出现的其他同工酶的分离接近基线，另一个峰出现，称为样品冲击。所提出的CE方法获得的结果与REP （Helena Labs， T.X， USA）凝胶电泳的结果具有良好的相关性。综上所述，CE系统分析蛋白质和乳酸脱氢酶同工酶具有灵敏、准确、易于临床应用的特点。少

We have demonstrated the separation of human serum proteins with the Beckman P/ACE 2100 Capillary electrophoresis (CE) system (Beckman Inc., Brea, CA,USA). A 20 cm uncoated fused silica capillary column (25mum, I.D.) and 150 mmol/L borate buffer (pH 10.0) as the running buffer were used. Human serum samples were diluted 11-folds in 20 mmol/L PBS buffer (pH 7.0) before application to CE column by pressure injection for 10 second (approximately 10 nL). The CE column temperature was maintained at 23ﾟC.A voltage of 10kV was applied for 6.5 min separating the protein fractions and the peak were detected at 200 nm. Human serum sample was fractionated into approximately 10 fractions by the proposed method. We confirmed the validity of our identifying the proteins fractioned, employing method for separating serum proteins by affinity column and antibody identification procedure. These protein peaks were identified a immunoglobulin, complement C3, transferin, alpha 2-macroglobulin, haptoglobin, … More alpha 1-antitrypsin, albumin, prealbumin, respectively, according to increasing migration time with CE.Moreover, the results of analyzes of 38 samples using the proposed method correlated well with those obtained by the cellulose acetate electrophoresis method (Olympus Optical Co.Ltd, Tokyo, Japan). We also have developed separation and quantitative estimation of the isoenzymes of lactate dehydrogenase in human serum were accomplished with Beckman P/ACE 2100 capillary electrophoresis method. A uncoated fused silica column 50 cm long, 75 mum I.D.and substrate containing running buffer [51.6 mmol/L L-lactatic acid, 8.26 mmol/L NAD in 25 mmol/L Tris (hydroxymethyl) aminoethanol buffer, pH 8.7] were used. The resulting product NADH was detected at 340 nm. Injecting a sample (serum samples diluted 5 times by 25 mmol/L Tris buffer (pH8.7) by pressure injection for 2 seconds. Separating the isoenzymes with 10kV applied for 5 min, turning off the voltage for 30 min of incubation at 24ﾟC (for reaction between substrate and isoenzymes), and reapplying 10 kV 30 min. Under these conditions, the NADH generated by LD5 emerged at 20 min, the LD-1 peak 23.5 min with closed to baseline separation of the other isoenzymes which emerge between LD-5 to LD-1 peak, another peak appears, termed Sample Shock The results obtained by the proposed CE method correlated well those by REP (Helena Labs, T.X., USA) gel electrophoresis. In conclusion, proposed proteins analysis and lactate dehydrogenase isoenzymes analysis by CE system is sensitive, precise, easy for clinical use. Less