分泌抗过氧化氢酶单克隆抗体的研制及特性鉴定

Establishment of Monoclonal Antibodies against Catalase

用从微球菌中提取纯化的过氧化氢酶为免疫原，以硝酸纤维膜皮下埋植法免疫ＢＡＬＢ／ｃ小鼠，取其脾细胞和ＳＰ２／０骨髓瘤细胞融合，获得１１株能稳定分泌抗体的杂交瘤细胞系。其中１Ａ１０、３Ｃ５、４Ｅ１２和４Ｆ６四株经连续传代三个月、冻存１０个月后复苏，仍能稳定分泌抗体．腹水效价在１０－４～１０－６之间．Ｉｇ类型鉴定，１Ａ１０与４Ｅ１２为ＩｇＧ２ａ，４Ｆ６为ＩｇＧ３亚类，３Ｃ５为ＩｇＭ类．染色体数介于８２～１１０之间．将４株ＭｃＡｂ与溶菌酶、纤维结合蛋白等１１种蛋白及多肽物质做交叉反应试验，均无明显交叉反应．以固相抗原竞争ＥＬＩＳＡ试验和ＥＬＩＳＡ相加试验作位点分析，两法一致表明ＭｃＡｂ４Ｆ６与另３株ＭｃＡｂ１Ａ１０、３Ｃ５和４Ｅ１２所针对的抗原位点有差别。４株ＭｃＡｂ相对亲和力测定结果依次为：４Ｅ１２＞１Ａ１０＞３Ｃ５＞４Ｆ６．用亲和层析柱纯化的４Ｆ６ＭｃＡｂ做过氧化氢酶催化活性试验，发现能激活过氧化氢酶对底物索曼的活性，抑制过氧化氢酶对底物过氧化氢的分解．

The catalase purified from the micrococcus lysodeiktcus was used to immunize BALB/c mice with the method of nitrocellulose subcutaneous implanting. Splenocytes from mice were fused with Sp2/0 myeloma. Eleven stable hybridoma clones were obtained after two to three clonizations. IA10, 3C5, 4E12 and 4F6 could stably secrete monoclonal antibodies after being subcultured for 3 months and preserved in liquid nitrogen for 10 months. Ascitic titres were between 10-4 and 10-6. Identification of Ig subclass: IA10 and 4E12 were IgG2a; 4F6 and 3C3 were IgG3 and IgM,respectively.Chromosomal numbers in the cells were between 82 and 101. The cross-reaction of the four McAbs with lysozyme, fibronectin and so on was not detected. The reaction spot analysis had been performed with the ELISA additivity test and solid ELISA competitive inhibition test,which demonstrated that the difference of the antigen reaction spot existed between McAb 4F6 and the other three McAbs (lA10, 3C5 and 4E12).The relative affinities of the four McAbs were as follows: 4E12 >1A10 >3C5 >4F6. It was found with the catalytic activity tests that McAb 4F6 which was purified with the protein G chromatography could activate catalase for the soman and inhibit the decomposition of H,O, as substrate of the catalase.