论文部分内容阅读
目的提取红细胞RhD膜蛋白并将其固化至乳胶微球以获得固相化RhD蛋白靶分子。方法采集3份志愿者全血标本,分别使用Yared MA法提取浓缩红细胞、试剂盒法提取浓缩红细胞及血影细胞膜蛋白的方法提取标本红细胞RhD膜蛋白,通过蛋白质定量检测及免疫印迹试验评价不同方法的提取效果。将不同方法提取出的RhD蛋白固化至乳胶微球,并通过流式细胞仪分析其抗原活性。结果 RhD膜蛋白浓度(mg/m L)分别为(n=3):0.24±0.04(Yared MA法提取浓缩红细胞)、4.82±0.09(试剂盒法提取浓缩红细胞)和4.31±0.39(试剂盒法提取血影细胞)与浓缩红细胞上的RhD膜蛋白浓度(mg/m L)。免疫印迹试验:Yared MA法从浓缩红细胞提取的RhD蛋白仅有1条RhD特异性条带;试剂盒法从浓缩红细胞提取的RhD蛋白无特异性条带,从血影细胞提取的RhD蛋白有2条RhD特异性条带。流式分析(n=3):Yared MA法提取的浓缩红细胞样品与试剂盒法提取的血影细胞样品、浓缩红细胞样品的荧光强度分别为:98.00±12.53、92.67±10.69、50.00±13.89。结论 Yared MA法提取出的RhD膜蛋白固化于乳胶微球可获得RhD蛋白纯度及抗原性较佳的固相靶分子。
Objective To extract RhD membrane protein and immobilize it on latex microspheres to obtain immobilized RhD protein target molecules. Methods Blood samples were collected from 3 volunteers. Yared MA method was used to extract the concentrated erythrocytes, and the kit was used to extract the membrane proteins of concentrated erythrocytes and ghost cells. RhD membrane proteins of the samples were extracted and quantified by Western blotting and Western blotting The extraction effect. RhD proteins extracted by different methods were immobilized to latex microspheres and their antigenic activity was analyzed by flow cytometry. Results RhD membrane protein concentration (mg / m L) was 0.24 ± 0.04 (Yared MA extraction of concentrated erythrocytes), 4.82 ± 0.09 (kit concentration of concentrated red blood cells) and 4.31 ± 0.39 Extract ghost cells) and RhD membrane protein concentration (mg / m L) on concentrated erythrocytes. Western Blotting Test: The RhD protein extracted from the concentrated erythrocytes by the Yared MA method has only one RhD-specific band; the RhD protein extracted from the concentrated erythrocytes by the kit method has no specific band, and the RhD protein extracted from the ghost cells has 2 RhD-specific bands. Flow analysis (n = 3): The fluorescence intensity of the concentrated erythrocyte samples extracted by Yared MA method and the reagent kit method were 98.00 ± 12.53,92.67 ± 10.69,50.00 ± 13.89, respectively. Conclusion The RhD membrane protein extracted by Yared MA method can be immobilized on latex microspheres to obtain solid phase target molecules with better purity and antigenicity of RhD protein.