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目的评价3种芒果致敏原提取方法,分析与血清特异性IgE结合的芒果蛋白成分,为临床检测sIgE提供最佳抗原制备方法。方法分别用三氯乙酸沉淀法、三氯乙酸/丙酮沉淀法、裂解液提取法粗提芒果蛋白,并通过SDS-PAGE和二维电泳技术分析不同提取方法提取的芒果蛋白组分,用免疫印迹和二维免疫印迹的方法鉴定粗提液中与患者特异性IgE结合的蛋白成分。结果 3种方法提取的蛋白组分不同,单例病人血清与3种不同方法提取的蛋白结合的强度和所识别的蛋白组分存在一定差异,三氯乙酸沉淀法和三氯乙酸/丙酮法提取的蛋白反应条带主要是30、40、44、47和90 kD,而裂解液提取的蛋白反应条带主要是23、32、40、46、73、90 kD。结论 3种提取方法各有特点,三氯乙酸沉淀法提取的芒果蛋白粗提液致敏原组分较完整,重复性好,操作简便,能够满足检测芒果sIgE所需已知抗原的要求。
Objective To evaluate the extraction method of three kinds of mango allergens and to analyze the components of mango proteins that bind to serum specific IgE, so as to provide the best method for the preparation of sIgE. Methods The mango proteins were extracted by trichloroacetic acid precipitation method, trichloroacetic acid / acetone precipitation method and lysate extraction method respectively. The components of mango proteins extracted by different extraction methods were analyzed by SDS-PAGE and two-dimensional electrophoresis. And two-dimensional immunoblotting method to identify protein components in crude extract that bind to patient-specific IgE. Results The protein components extracted by the three methods were different. The strength of the protein extracted by the three different methods was different from that of the protein extracted by the three different methods. Trichloroacetic acid precipitation and trichloroacetic acid / acetone extraction Of the protein reaction bands are mainly 30, 40, 44, 47 and 90 kD, while the protein bands extracted by the lysate are mainly 23, 32, 40, 46, 73, 90 kD. Conclusion The three extraction methods have their own characteristics. The components extracted from the crude extract of mango protein by trichloroacetic acid precipitation are more complete, reproducible and easy to operate, which can meet the requirement of detecting the known antigen of mango sIgE.