摘要
以重组人红细胞ＮＡＤＨ－细胞色素ｂ５还原酶为抗原免疫ＢＡＬＢ／ｃ小鼠；通过常规方法将小鼠脾细胞与Ｓｐ２／０骨髓瘤细胞融合，经ＥＬＩＳＡ筛选及有限稀释法克隆化，选出６株阳性克隆。其中两株分泌ＩｇＧ，其余均分泌ＩｇＭ。免疫印迹分析表明，两株ＩｇＧ单克隆抗体对人红细胞ＮＡＤＨ－细胞色素ｂ５还原酶有较高的特异性和亲和力。这一研究为开展对人红细胞ＮＡＤＨ－细胞色素ｂ５还原酶的研究和遗传性高铁血红蛋白血症的免疫诊断奠定了基础。
Human erythrocyte NADH-cytochrome b5 reductase,or Cyt b5R,is one of the redox enzymes playing important roles in vivo.Deficiency of this enzyme will lead to hereditary methemoglobinemia.We immunized BALB/c mouse with recombinant Cyt b5R. Hybridoma cells were prepared through conventional cell fusion of Sp2/0 myeloma cells with splenocytes of the immunized BALB/c mouse.Six clones of ELISA-positive hybridoma cells were picked out by ELISA-based screening and limiting dilution,with two clones secreting IgG isotypes and the rest secreting IgM.In immunoblotting,the two IgG monoclonal antibodies showed high specificity and affinity to Cyt b5R.These monoclonal antibodies provide a useful tool for the investigation of Cyt b5R and for the immunodiagnosis of hereditary methemoglobinemia.
基金
总后卫生部"八五"重点资助
关键词
红细胞
细胞色素酶
还原酶
辅酸I
单克隆抗体
NADH-cytochrome b5 reductase
antibody,monoclonal
hereditary methemoglobinemia
ELISA
immunoblotting
