| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1999: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
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,
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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
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