Development of analytical methods by capillary electophoresis for human fluids.

我们已经使用Beckman P/ACE 2100毛细管电泳仪(Beckman Inc.，Brea，CA，USA)演示了人血清蛋白的分离。20厘米未涂层熔融石英毛细管柱(25微米，内径)运行缓冲液为150 mm o l/L硼酸盐缓冲液(pH 10.0)。人血清样品在20 mmol/L PBS缓冲液(pH 7.0)中稀释11倍后，用加压进样方式进入毛细管电泳柱10秒(约10nL)。毛细管电泳柱温度保持在23ﾟC，用10KV电压分离蛋白质组分6.5min，在200 nm处检测到峰。用所提出的方法将人血清样本分成大约10个组分。采用亲和层析和抗体鉴定相结合的方法对分离的血清蛋白进行鉴定，验证了所提分离蛋白的有效性。这些蛋白质峰被鉴定为免疫球蛋白、补体C3、转铁蛋白、α2-巨球蛋白、结合珠蛋白、…随着毛细管电泳法迁移时间的延长，α1-抗胰蛋白酶、白蛋白、前白蛋白的含量分别增加。此外，对38个样品的分析结果与日本东京奥林巴斯光学公司的醋酸纤维素电泳法的分析结果具有很好的相关性。用Beckman P/ACE2100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。采用未涂层熔融硅胶柱，柱长50 cm，内径75um，底物内含运行缓冲液[51.6mmo1/L L-乳酸，8.26mmo1/L，25 mmo1/L三(羟甲基)氨基乙醇缓冲液，pH 8.7]。在340 nm处检测到产物NADH。加压进样(血清样品用25 mmo1/L Tris Buffer(pH 8.7)稀释5倍，进样2秒。分离同工酶，10KV作用5min，24ﾟC关断电压30min(底物与同工酶反应)，10KV重复作用30min。在此条件下，LD5产生的NADH在20min出现，LD-1峰在23.5min出现，其他同工酶在LD-5和LD-1峰之间出现接近基线的分离，出现另一个峰，称为样品休克。综上所述，所提出的蛋白质分析和乳酸脱氢酶同工酶分析系统是灵敏、准确、易于临床使用的。较少

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