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Development of analytical methods by capillary electophoresis for human blood isoenzymes.

开发人血液同工酶毛细管电泳分析方法。

基本信息

批准号：
07457617
负责人：
UJI Yoshinori
金额：
$ 0.77万
依托单位：
Kumamoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

Separation and quantitative estimation of the isoenzymes of lactate dehydrogenase (LD) is serum were accomplished with the Beckman P/ACE 2100 capillary electrophoresis (CE) system (Brea, CA,USA). A uncoated fused silica capillary column 50 cm long, 75 mum I.D.and substrate containing running buffer (51.6 mmol/L L-lactic acid, 8.26 mmol/L NAD in 25 mmol/L Tris (hydroxymethyl) aminoethane buffer, pH 8.7) were used. The resulting product NADH was detected at 340 nm. Injecting a sample (diluted 5 times by 25 mol/L Tris buffer, pH 8.7) by pressure 2 seconds. Separating the isoenzymes with 10kV applied for 5 min, turning off the voltage for 30 min of incubation at 24ﾟC and reapplying 10kV of 30 min. The results (LD1-LD5) obtained by the proposed CE method correlated well with those by REP (Helena Labs, TX.USA) gel electrophoresis system. Within-run precision CVs were excellent with 5 isoenzymes, respectively. Proposed LD isoenzymes analysis by CE system is sensitive, precise, easy for clinic … More al use. We demonstrated the separation of human serum proteins (SPE) by CE system. 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. Samples were diluted 11-fold in 20 mmol/L PBS (pH 7.0) before application to CE by pressure injection for 10 second. The CE column temperature is 24ﾟC,a voltage of 10 kV was applied 6.5 min separating the protein fractions, the peaks were detected at 200 nm. Human serum was fractionated into approximately 10 fractions by the proposed method. The results of analysis of 100 samples using the proposed CE system correlated well with by the cellulose acetate electrophoresis method (Olympus Optimal Co., Ltd., Tokyo, Japan). Beckman multi-channeled CE system Paragon CZE 2000 was evaluated for immunofixation electrophoresis by subtraction (IFE/s). Concordance studies between IFE/s and agarose gel imunofixation electrophoresis (IFE) showed a very good agreement on 30 monoclonal gammopathy patient samples. From these results, it is concluded that IFE/s is useful and suitable tool for routine clinical laboratory. Less

用Beckman P/ACE 2100毛细管电泳（CE）系统（Brea，CA，USA）对血清中乳酸脱氢酶（LD）同工酶进行分离和定量测定。使用未涂覆的熔融石英毛细管柱，50 cm长，75 μ m I.D.和含有运行缓冲液（51.6 mmol/L L-乳酸，8.26 mmol/L NAD，溶于25 mmol/L Tris（羟甲基）氨基乙烷缓冲液，pH 8.7）的底物。在340 nm处检测所得产物NADH。加压进样2秒（用25 mol/L Tris缓冲液（pH 8.7）稀释5倍）。用10 kV电压处理5 min，24 ℃下断电30 min，再加10 kV电压30 min，所得结果（LD_1-LD_5）与REP（Helena Labs，TX.USA）凝胶电泳系统的结果一致。5种同工酶的运行内精密度CV均极佳。建议用毛细管电泳系统分析LD同工酶，具有灵敏、准确、简便等优点， ...更多信息使用。我们用毛细管电泳系统分离了人血清蛋白质。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。样品在20 mmol/L PBS（pH 7.0）中稀释11倍，然后通过压力进样10秒应用于CE。毛细管电泳柱温为24 ℃，10 kV电压下分离6.5 min，在200 nm处检测峰。用该方法将人血清分成约10个组分。使用所提出的CE系统的100个样品的分析结果与通过乙酸纤维素电泳方法（Olympus Optimal Co.，有限公司、日本东京）。评价Beckman多通道CE系统Paragon CZE 2000的减影免疫固定电泳（IFE/s）。IFE/s和琼脂糖凝胶免疫固定电泳（IFE）之间的一致性研究表明，30个单克隆丙种球蛋白病患者样本非常好的协议。从这些结果中，可以得出结论，IFE/S是有用的和合适的工具，为常规的临床实验室。少