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目的:制备可同时识别血红蛋白A2(HbA2)与血红蛋白A(HbA)的单克隆抗体(mAb),即与血红蛋白的δ链与β链结合,而不与γ链结合。方法:以原核表达的重组Hbδ链免疫小鼠,常规融合制备杂交瘤细胞,以离子交换层析分离的天然HbA2与HbA为抗原同时进行筛选;所获mAb用间接ELISA、基于变性与非变性聚丙烯凝胶电泳的Western blot、等离子表面共振及流式细胞术进行鉴定。结果:所获mAb2C9以高亲和力与纯化的天然HbA2与HbA结合,经等离子表面共振测定KA值分别为2.25×1011、2.16×1010;该mAb不与胎儿血红蛋白(HbF)、血红蛋白α链及重组血红蛋白ζ(zeta)链结合。结论:获得1株可结合HbA2与HbA的mAb2C9,提示其识别血红蛋白δ链与β链的共同表位。该抗体将成为研究与诊断血红蛋白病的有效工具。
OBJECTIVE: To prepare a monoclonal antibody (mAb) that recognizes both hemoglobin A2 (HbA2) and hemoglobin A (HbA), ie binds to the δ and β chains of hemoglobin but not to the γ chain. METHODS: Mice were immunized with the recombinant Hbδ chain expressed in prokaryotic cells, and the hybridoma cells were prepared by conventional fusion. Native HbA2 and HbA were separated by ion exchange chromatography and screened simultaneously. The obtained mAbs were purified by indirect ELISA on the basis of denatured and non-denatured poly Western blot analysis of propylene gel electrophoresis, plasma surface resonance and flow cytometry were identified. Results: The obtained mAb2C9 with high affinity and purified native HbA2 and HbA binding by plasma surface resonance KA were 2.25 × 1011, 2.16 × 1010; the mAb does not with fetal hemoglobin (HbF), hemoglobin α chain and recombinant hemoglobin ζ (zeta) chains. Conclusion: Obtain a mAb2C9 that can bind HbA2 and HbA, suggesting that it recognizes the common epitope of hemoglobin δ chain and β chain. This antibody will be an effective tool for the study and diagnosis of hemoglobinopathies.