等电聚焦电泳/免疫固定和考马氏蓝染色技术检测缺糖转铁蛋白

Isoelectric focusing/immunofixation/Comassie blue staining for detection of carbohydrate-deficient transferrin

目的 建立等电聚焦电泳／免疫固定（ＩＥＦ／ＩＦ）和考马氏蓝染色技术检测缺糖转铁蛋白（ＣＤＴ）方法。方法选择３２例酒精性肝病患者，血清铁饱和后，进行水平板式超薄层等电聚焦电泳，免疫固定和考马氏蓝染色后，蛋白条带进行等电点分析。结果 酒精性肝病组ＣＤＴ阳性率为８７．５％。ＣＤＴ诊断酒精性肝病的灵敏度为８７．５％，特异度为６９．３９％。结论ＩＥＦ／ＩＦ结合考马氏蓝染色技术是一种操作方便、经济、灵敏度高、分辨率高、适用于进一步大样本检测血清ＣＤＴ的方法。

Objective To establish the detect method of Carbohydrate - deficient Transferrin. Methods According to the-diagnosis criteria for alcoholic liver disease established by Chinese Medical Association in NanJing 1995,32 alcoholic liverdisease patients were selected. Each serum sample was iron saturated with l0mmol/L Fe solution giving a 1:4 find dilutionat 37d. Different molecular forms of transferrin were identified by isoelectric focusing on polyacrylamide gel, pI5- 7. Prepared samples were applied after prefocusing of the gel for 500Vh (l800V,20mA,15W),the samples were applied to the gel for 1 hour (1800V,2OmA,15W),and isoelectric separation was done by 1800V,20mA,15W and 10°C .After isoelectric focusing, draw a diagram of ph - cm according to the ph value of gel from anode to cathode. Immunofixation was employed to identify transferrin. The gel were incubated at 37°C for 2 hours with anti - human transferrin serum. The unprecipitated protein were washed with 10% NaCl, 0.9% NaCl and distilled water. Comassie blue staining were used to show the protein zone. Carbohydrate - deficient Transferrin were defines as had transferrin band at pI≥ 5.7. Results The positive ratio of Carbohydrate - deficient Transferrin was to be raised in alcoholic liver disease (87.5%) . Conclusion Carbohydrate - deficient transferrin can be detected by isoelectric focusing / immunofixation / Comassie blue stailning.