Lanz Christian, Marti Ulrich, Thormann Wolfgang
Department of Clinical Pharmacology, University of Berne, Murtenstrasse 35, CH-3010 Berne, Switzerland.
J Chromatogr A. 2003 Sep 26;1013(1-2):131-47. doi: 10.1016/s0021-9673(03)00566-1.
Capillary zone electrophoresis (CZE) with a dynamic double coating permits the simultaneous, individual, quantitative determination of transferrin (Tf) isoforms in human serum and thus carbohydrate-deficient transferrin (CDT), the most specific marker available today for the detection of chronic, excessive alcohol intake. CZE of serum Tf was carefully evaluated using the P/ACE MDQ with fused-silica capillaries of 50 microm I.D. and 60.2 cm total length, the CEofix CDT kit and the instrumental conditions recommended by the kit manufacturer. The precision performance assessed over a 20-day period according to the internationally accepted NCCLS EP5-A guidelines revealed the CZE assay as being highly reproducible with within-run and total precision being dependent on the Tf isoform level and RSD values ranging between 2.2 and 17.6%. Inter-day RSD values for asialo-Tf were noted to be between 9.8 and 11.5% and for disialo-Tf between 3.8 and 8.6%, whereas those for CDT levels of 0.87 and 4.31% of total Tf were determined to be 8.6 and 3.4%, respectively. The RSD values for trisialo-Tf, tetrasialo-Tf, pentasialo-Tf and hexasialo-Tf were found to be between 0.4 and 4.1%. Tf patterns are recognized and identified via detection times of Tf isoforms (intra-day and inter-day RSD values < 1.0% and < 1.7%, respectively), immunosubtraction of Tf and enzymatic sequential cleavage of sialic acid residues. Furthermore, heterozygous Tf BC and Tf CD variants are assigned via spiking with a known mixture of Tf isoforms (e.g. the serum of a healthy Tf C homozygote). Among the non-Tf peaks monitored, the CRP peak detected shortly before disialo-Tf was identified by immunosubtraction and peak magnitudes were found to correlate well with immunochemically determined CRP serum levels. The CZE assay with dynamic double coating could thereby be shown to be sensitive enough to determine elevated CRP levels in human serum. Furthermore, unusual peaks in the gamma-region were identified by customary serum protein CZE, immunosubtraction CZE and immunofixation.
采用动态双涂层的毛细管区带电泳（CZE）能够同时、单独且定量地测定人血清中的转铁蛋白（Tf）异构体，进而测定缺糖转铁蛋白（CDT），这是目前检测长期过量饮酒最具特异性的标志物。使用内径为50微米、总长60.2厘米的熔融石英毛细管的P/ACE MDQ、CEofix CDT试剂盒以及试剂盒制造商推荐的仪器条件，对血清Tf的CZE进行了仔细评估。根据国际认可的NCCLS EP5 - A指南，在20天内评估的精密度性能表明，CZE测定具有高度可重复性，批内和总精密度取决于Tf异构体水平，相对标准偏差（RSD）值在2.2%至17.6%之间。去唾液酸Tf的日间RSD值在9.8%至11.5%之间，双唾液酸Tf的日间RSD值在3.8%至8.6%之间，而CDT水平分别为总Tf的0.87%和4.31%时，其日间RSD值分别为8.6%和3.4%。发现三唾液酸Tf、四唾液酸Tf、五唾液酸Tf和六唾液酸Tf的RSD值在0.4%至4.1%之间。通过Tf异构体的检测时间（批内和日间RSD值分别<1.0%和<1.7%）、Tf的免疫扣除以及唾液酸残基的酶促顺序裂解来识别和鉴定Tf模式。此外，通过加入已知的Tf异构体混合物（如健康Tf C纯合子的血清）来确定杂合Tf BC和Tf CD变体。在所监测的非Tf峰中，通过免疫扣除鉴定出在双唾液酸Tf之前不久检测到的CRP峰，并且发现峰大小与免疫化学测定的CRP血清水平密切相关。由此表明，采用动态双涂层的CZE测定足够灵敏，能够测定人血清中升高的CRP水平。此外，通过常规血清蛋白CZE、免疫扣除CZE和免疫固定鉴定出γ区域中的异常峰。
