Development of analytical methods by capillary electophoresis for human fluids.

开发人体液体毛细管电泳分析方法。

• 批准号: 06672296
• 负责人: OKABE Hiroaki
• 金额: $ 1.34万
• 依托单位: Kumamoto University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06672296/
• 关键词: Capillary electrophoresis; Serum protein fractions; LD isoenzymes; Clinical test; 臨床検査医学

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

我们已经证明了用Beckman P/ACE 2100毛细管电泳（CE）系统（Beckman Inc.，Brea，CA，USA）。采用20 cm未涂层熔融石英毛细管柱（25 μ m，I.D.）以150 mmol/L硼酸盐缓冲液（pH 10.0）为运行缓冲液。将人血清样品在20 mmol/L PBS缓冲液（pH 7.0）中稀释11倍，然后通过压力进样10秒（约10 nL）上样至CE柱。将CE柱温保持在23 ℃。施加10 kV的电压6.5 min，分离蛋白质级分，并在200 nm处检测峰。人血清样品被分离成约10个馏分的建议方法。采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。这些蛋白质峰被鉴定为免疫球蛋白、补体C3、转铁蛋白、α 2-巨球蛋白、触珠蛋白， ...更多信息 α 1-抗胰蛋白酶，白蛋白，前白蛋白，分别根据增加迁移时间与CE。此外，38个样品的分析结果，使用所提出的方法与醋酸纤维素电泳法（奥林巴斯光学有限公司，东京，日本）获得的结果相关良好。用Beckman P/ACE 2100毛细管电泳法对人血清中乳酸脱氢酶同工酶进行了分离和定量测定。使用50 cm长、75 μ m内径的未涂覆熔融石英柱和含运行缓冲液[51.6 mmol/L L-乳酸、8.26 mmol/L NAD溶于25 mmol/L Tris（羟甲基）氨基乙醇缓冲液，pH 8.7]的底物。在340 nm处检测所得产物NADH。通过压力进样2秒，进样样品（用25 mmol/L Tris缓冲液（pH8.7）稀释5倍的血清样品）。用10 kV电压分离同工酶5 min，关闭电压，24 ℃孵育30 min（用于底物和同工酶之间的反应），并再次施加10 kV 30 min。在这些条件下，由LD 5产生的NADH在20 min出现，LD-1峰23.5分钟，在LD-5与LD-1峰之间出现的其他同工酶的基线分离接近，出现另一个峰，被称为样品冲击通过所提出的CE方法获得的结果与REP（HelenaLabs，T.X.，USA）凝胶电泳。结论：采用毛细管电泳系统进行蛋白质分析和乳酸脱氢酶同工酶分析，灵敏度高，准确度高，便于临床应用。少

项目成果

宇治義則、他: "キャピラリー電気泳動法臨床検査への応用" 九州臨床検査技師会精度管理セミナー資料集. 10. 10-12 (1994)

Yoshinori Uji 等：“毛细管电泳在临床检测中的应用”九州临床检验技师协会质量控制研讨会资料。10. 10-12 (1994)。

三浦雅一,他: "キャピラリー電気泳動法によるヒト血清蛋白の分離と同定" 臨床化学. Vol.23. 32-37 (1994)

Masakazu Miura 等人：“通过毛细管电泳分离和鉴定人血清蛋白”，《临床化学》，第 23 卷，32-37 (1994)。

宇治義則、他: "キャピラリー電気泳動法を用いたLDアイソザイム分析法の開発" 臨床病理. 42. 88-88 (1994)

Yoshinori Uji 等：“使用毛细管电泳的 LD 同工酶分析方法的开发”临床病理学 42. 88-88 (1994)。

宇治義則、他: "キャピラリー電気泳動を用いた臨床検査法の開発" 日本臨床化学会九州支部会雑誌. (印刷中). (1996)

Yoshinori Uji 等人：“使用毛细管电泳的临床测试方法的开发”日本临床化学学会九州分会杂志（1996 年出版）。

宇治義則、他: "キャピラリー電気泳動法を用いた臨床検査法の開発" 日本臨床化学会九州支部会雑誌. (1996印刷中).

Yoshinori Uji 等：“使用毛细管电泳的临床测试方法的开发”日本临床化学学会九州分会杂志（1996 年出版）。