Research of analytical methods for infection test by capillary electrophoresis.

我们开发并评估了毛细管电泳（CE）临床试验（蛋白质、同工酶和感染试验）的分析方法。在HBs抗原感染试验中，通过抗体的选择、运行缓冲液的选择、外加电压变化对样品分子结构的影响、分析个人计算机软件的开发等研究，建立了分析的最佳条件。实验条件为：用蒸馏水或HBs抗原标准品（日本东京Kyokuto制药公司）稀释50倍患者血清10 μl，乳胶带状抗HBs抗原单克隆抗体10 μl (Kyokuto)，抗HBs抗原单克隆抗体10 μl（日本大阪Wako纯化学公司）和FITC带状抗大鼠IgG单克隆抗体50 μl （Wako）混合。将混合物压入75μm X 20cm未涂覆的熔融毛细管柱中2秒（10nL）。CE （Paragon CZE 2000系统，Beckman Coluter Inc.Brea，…More USA）在100 mmol/l硼酸缓冲液（pH 10.04）中，24℃，10kV, 15 min， 480/520荧光检测HBs抗原活性。HBs抗原阴性样品未检测到抗原抗体反应产物峰，HBs抗原阳性样品约10 min出现抗原抗体反应产物峰。AXIM方法（ABBOTT， USA）的结果与该方法的峰面积具有良好的相关性（r=0.94）。标准HBs抗原与AXIM法结果的相关性极好（r=0.90），但在低活性方面差异不大。该方法重现性好（小于2%的CV）。各种抗凝血剂、白蛋白、葡萄糖、抗坏血酸和血红蛋白的正常浓度对结果无影响。结果表明，应用CE法测定HBs抗原简便、准确。此外，我们还在继续研究CE检测其他感染试验和脂质试验的方法。少

We have developed and evaluated the analytical method for clinical tests (proteins, isoenzymes and infection tests) by capillary electrophoresis (CE). In the infection test of HBs antigen, we established an analysis optimum condition passed through studies such as selection of antibody, selection of running buffer, change of the structure of sample molecule by change of applied voltage, development of analysis personal computer software. Assay conditions as follow : ten μl of 50 times diluted patient serum with distilled water or standard of HBs antigen (Kyokuto pharmaceutical Co., Tokyo, Japan), 10μL of latex banding anti-HBs antigen monoclonal antibody (Kyokuto), 10 μL of anti HBs antigen monoclonal antibody (Wako Pure Chemicals Co. Osaka Japan) and 50 μL of FITC banding anti rat IgG monoclonal antibody (Wako) were mixed. Mixture was introduced by pressure injection for 2 sec (10nL) to 75μm X 20cm uncoated fused capillary column. CE (Paragon CZE 2000 system, Beckman Coluter Inc.Brea, … More USA) was performed at 24℃, 10kV applied for 15 min in 100 mmol/l borate buffer (pH 10.04), HBs antigen activity was monitored by following fluorescence at 480/520. Peak of antigen-antibody reaction product was not detected for HBs antigen negative samples, peak of antigen-antibody reaction products were observed for HBs antigen positive samples approximately 10 min. Results of AXIM method (ABBOTT, USA) and the peak area of proposed method showed good correlation (r=0.94). Also, correlation of standard HBs antigen and results of AXIM method were excellent (r=0.90), however little differences were recognized in lower activity. The reproducibility for the proposed method were excellent (less than CV 2%). Normal concentration of various kinds of anticoagulants, albumin, glucose, ascorbic acid and hemoglobin had no effects on the results. We conclude that proposed HBs antigen assay by CE is easy and precise in clinical laboratory use. Furthermore, we are continue studying for the assay of other infection tests and the assay of lipids tests by CE. Less