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Isolation of Carbohydrate-Deficient Transferring (CDT) by Allo-A Sepharose 4B Affinity Chromatography
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摘要: 目的建立Allo-A Sepharose亲和层析技术分离血清中糖缺失转铁蛋白（CDT）和转铁蛋白（Tf）。方法Allo-A与的偶联反应在0.1mmol/L NaHCO3（含0.5mmol/L NaCl）内进行;Allo-A Sepharose 4B亲和层析技术用于分离血清CDT和Tf;ELISA用于定量CDT和Tf。结果Allo-A Sepharose 4B亲和层析图谱显示2个洗脱峰,CDT存在于第一洗脱峰,Tf存在于第二洗脱峰。ELISA测定结果显示,在5例正常血清中,CDT水平为4.534.76（CV=1.89.7%）,Tf水平为255.3524.9（CV=5.110.7%）;在5例患者血清中,CDT水平为3.044.20（CV=0.811.6%）,Tf水平为6.0534.73（CV=0.711.1%）。表明Allo-A Sepharose 4B亲和层析技术在分离CDT和Tf中,具有较理想的可重复性。结论Al...Abstract: Objective To establish Allo-A Sepharose 4B affinity chromatography for isolating CDT and Tf from serum.Methods Coupling reaction of Allo-A with CNBr-activated Sepharose 4B was conducted in 0.1mmol/L NaHCO3 containing 0.5mmol/L NaCl.Allo-A Sepharose 4B affinity chromatography was used to isolate CDT and Tf from serum.Tf-ELISA was used to determine the levels of the isolated CDT and Tf.Results Elution profiles showed two major peaks.CDT was found in the first elution peak and Tf in the second peak.In the sera from 5 healthy volunteers, the level of CDT ranged 4.534.76 (CV=1.89.7%) , and the level of Tf ranged 255.3524.9 (CV=5.110.7%) ;in the sera from 5 patients, the level of CDT ranged 3.044.20 (CV=0.811.6%) , and the level of Tf ranged 6.0534.73 (CV=0.711.1%) .These findings indicated that Allo-A Sepharose 4B affinity chromatography exhibited a good reproducibility when used in isolation of CDT and Tf.Conclusion Allo-A Sepharose 4B affinity chromatography is a acceptable method for isolation of CDT and Tf.Its sensitivity remains to be evaluated, conditioned by the availability of a panel of CDT reference sera.
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[1]Katsuko Y, Kazuo U, Tsuoneo S, et al.Carbohydrate Binding Spe-cificity of Immobilized Allmyrina dichotoma Lectin The journal of bio-logical chemistry[C].Vol.263, No.33 Laune of November 25Printed in U.S.A, 1988∶17482-17489. [2]赵永芳.生物化学技术原理及应用[M].第3版.北京:北京科学出版社, 2002∶124-134. [3]Kiyoshi Y, Kazuo U, Haruhide S, et al.Determination of carbohy-drate-deficient transferrin separated by lectin affinity chromatogra-phy for detecting chronic alcohol abuse[J].FEBS Letters, 1999, 458∶112-116. -
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