Isolation of Carbohydrate-Deficient Transferring(CDT) by Allo-A Sepharose 4B Affinity Chromatography

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 4.53~4.76(CV=1.8~9.7%),and the level of Tf ranged 255.3~524.9(CV=5.1~10.7%);in the sera from 5 patients,the level of CDT ranged 3.04~4.20(CV=0.8~11.6%),and the level of Tf ranged 6.05~34.73(CV=0.7~11.1%).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.