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目的构建一种高通量蛋白芯片检测人血清中血红蛋白氧载体类兴奋剂的方法。方法用自制SEC微柱快速地将人血清中血红蛋白聚合体(氧载体)和血红蛋白单体(另外加入的内标)分离,逐滴收集洗脱液作为探针,点样结合在蛋白质基片上,与其相对应的荧光标记的多克隆抗体进行杂交,扫描芯片荧光强度检测人血红蛋白氧载体。结果自制SEC微柱能够将戊二醛聚合的人血红蛋白聚合体和单体有效分离,在蛋白芯片的检测中能较好地检测出人血清中1.5%的聚合体人血红蛋白氧载体。结论以SEC技术和蛋白芯片相结合是一种高通量、灵敏、重现性好的检测人血清中的以人血红蛋白为基质氧载体类兴奋剂新方法。
Objective To construct a high-throughput protein chip for detection of hemoglobin oxygen carrier stimulants in human serum. Methods Separated hemoglobin aggregates (oxygen carrier) and hemoglobin monomer (internal standard added separately) were separated by SEC microcolumn in human body. The eluate was collected as a probe by drop, and then spotted on the protein substrate. Hybridization with its corresponding fluorescent labeled polyclonal antibody, scanning the chip fluorescence intensity detection of human hemoglobin oxygen carrier. Results The self-made SEC microcolumn was able to separate glutaraldehyde-polymerized human hemoglobin aggregates and monomers effectively and detect 1.5% polymeric human hemoglobin oxygen carrier in human serum in protein chip detection. Conclusion The combination of SEC and protein chip is a high-throughput, sensitive and reproducible method for the detection of human hemoglobin-based oxygen carrier stimulants in human serum.