基于磁珠微阵列的多重血型检测技术

Multiplex blood phenotype assay based on bead-microarray

目的建立并评价基于磁珠微阵列技术的血型表型的鉴定方法。方法用Tris-HCl（0.01 mol/L,p H 7.4）裂解浓缩红细胞作为待检样本,将探针抗体包被于经醛基修饰的三维基片上,将待测样本与抗体进行反应,最后用C1q包被的磁珠识别抗原抗体复合物,在磁针的磁力吸附下将未结合的磁珠清除。显微摄影每个微阵列检测点,通过Photoshop图像分析判断信号强度。结果针对108个检测样本的5种抗原（A、B、D、M、N）进行分析,以含有40%甘油的PBS做为反应介质能有效避免咖啡环的形成,方法敏感性为86%~93%,特异性为98%~100%。通过C1q包被的磁珠识别抗原抗体复合物能在10~20 min完成检测,所需样本10μL。结论本方法有较高的敏感性与特异性,同一个芯片上可同时检测5种血型抗原,节约样本,且不受样本损伤的影响,为血型鉴定提供了一种新的手段。

Objective To develop and evaluate a new blood phenotype assay based on bead-microarray technique. Method The concentrated red blood cells were collected from the tested subjects and lysed by Tris-HCl buffer. The 3D-aldehyde-polymer modified slide was used to solidify the blood group antibody probes. After sampling onto the testing spot of the slide for the reaction with antibody,C1q-coupled magnetic beads were added to recognize the antibody-antigen complex. The unbounded beads were removed by attraction of magnetic pin. Each testing spot of the array was photomicrographed and the mean gray value as the signal intensity was analyzed by photoshop image software. Results Five phenotypes of blood group antigens（ A,B,D,M,N） from 108 subjects were determined.For avoiding coffee-ring formation,PBS-glycerinum was used as reaction medium instead of water or normal saline. The sensitivity of the method varied from 86% to 93% and the specificity varied from 98% to 100%. A volume of 10 μL sample was sufficient for the test and the assay could be completed within 10 to 20 minutes. Conclusion The developed method in this study displayed high sensitivity and specificity. On the same chip 5 kinds of blood group antigen could be simultaneously detected by using small amount of sample. The damage of sample would not affect the results. The bead-microarray may provide a new means for identification of blood phenotype.