蟾蜍血清梭曼水解酶的酶学性质及其单克隆抗体的制备

Study on the characters and preparation of monoclonal antibodies of toad serum Somanase

蟾蜍血清梭曼水解酶 ( TSS)已被纯化。采用微量联苯胺法对该酶的酶学性质进行研究表明 ,该酶有两个最适 p H,分别是 p H7和 p H1 0 ;最适作用温度为 30℃ ;以梭曼为底物 ,在 p H7.73,1 /1 5mol/L PB,30℃条件下测定该酶的米氏常数为 5 .864mmol/L,最大反应速度为 692 μmol/L。Mg2 + 、Ca2 + 对 TSS有激活作用 ,而 Ni2 + 、Cu2 + 、Fe2 + 、Na+ 、K+ 对酶活性无明显影响。用 TSS免疫Bal b/c小鼠 ,取其脾细胞在 PEG40 0 0作用下与 P3x63- Ag8.65 3小鼠骨髓瘤细胞进行融合 ,获得 8株能分泌抗 TSS单克隆抗体的杂交瘤细胞株 :AC1 1 ,AE8,DA3,DE5 ,DG3,EB4,EC4,EH4。用酶联免疫吸附试验对其分泌的抗体滴度进行检测 ,其腹水滴度最高可达 2× 1 0 7,对其中 4株杂交瘤分泌的抗体相对亲合力进行测定 ,它们的大小顺序为 DA3>EH4>DG3>EC4;对其中 6株杂交瘤分泌的抗体进行免疫球蛋白类型鉴定表明 ,6株单抗均为 Ig G1类。上述 8株杂交瘤在体外连续培养 6个月 。

Toad serum Samanase(TSS) was purified from toad serum. The enzyme activity was optimal at pH7,pH10, and 30℃. The K m and V max values with Soman were 5.864 mmol/L and 692 μmol/L respectively. The enzyme was markedly activated by Mg 2+ and Ca 2+ ,but inhibited by over dose of the substrate. McAbs were prepared by fusing P3x63 Ag8.653 myeloma cells with spleen cells of Bal b/c mice immunized with the antigen off TSS. The antibodies inhybridoma culture supernatant and mouse ascites were detected by ELISA, and positive hybridomas were coloned by the thchnique of limiting dilutions. 8 McAbs: AC11, AE8,DA3,DE5,DG3,EB4,EC4 and EH4 against TSS were produced and characterized. The highest titer of the McAbs in ascites was 2×10 7. The Ig subclasses of AC11, AE8,DA3,DG3,EC4,EH4 are all IgG1. The relative affinity constants are DA3>EH4>DG3>EC4. After cultur of 6 months, the 8 hybridoma cell lines can steadily produced high titer McAbs.