Cossu G, Manca M, Pirastru M G, Bullita R, Bianchi Bosisio A, Righetti P G
J Chromatogr. 1984 Apr 13;307(1):103-10. doi: 10.1016/s0378-4347(00)84076-1.
A new isoelectric focusing technique for the separation and quantitation of glycosylated haemoglobin (HbA1c) is described. By using an equimolar mixture of two separators (0.2 M beta-alanine + 0.2 M 6-aminocaproic acid) a 2-pH unit Ampholine range (pH 6-8) is transformed in a shallow, 0.6-pH unit span (pH 6.7-7.3). This brings about an increment of resolution between HbA and HbA1c by a factor of about three, thus allowing proper densitometric evaluation of the trichloroacetic acid-fixed MetHb bands by conventional gel scanners. Excellent agreement is found among microchromatography, isoelectric focusing followed by densitometry in situ, and isoelectric focusing followed by band excision, elution and spectrophotometric determination. The present method also allows full resolution between HbA1c and fetal haemoglobins (F and Fac bands).
本文描述了一种用于分离和定量糖化血红蛋白（HbA1c）的新型等电聚焦技术。通过使用两种分离剂的等摩尔混合物（0.2Mβ-丙氨酸 + 0.2M 6-氨基己酸），一个2-pH单位的两性电解质范围（pH 6 - 8）被转化为一个浅的、0.6-pH单位的跨度（pH 6.7 - 7.3）。这使得HbA和HbA1c之间的分辨率提高了约三倍，从而允许使用传统凝胶扫描仪对三氯乙酸固定的高铁血红蛋白条带进行适当的光密度测定。在微量色谱法、原位光密度测定的等电聚焦法以及条带切除、洗脱和分光光度测定的等电聚焦法之间发现了极好的一致性。本方法还能实现HbA1c与胎儿血红蛋白（F和Fac条带）的完全分离。
