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

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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