抗胰蛋白酶原激活肽单克隆抗体的制备与鉴定

Preparation and identification of monoclonal antibodies against trypsinogen activation peptides

目的制备抗胰蛋白酶原激活肽(TAP)单克隆抗体(TAP McAb),并进行初步鉴定.方法将人工合成的TAP与血蓝蛋白(KLH)交联作为免疫原,免疫Balb/c小鼠,经细胞融合,有限稀释法筛选出能稳定分泌TAP McAb的杂交瘤细胞株,并对单克隆抗体进行鉴定.结果细胞融合率85.03%,经4次克隆化后获得10株能稳定分泌TAP McAb的杂交瘤细胞株,经免疫球蛋白亚类鉴定,除1株为IgG1外,其余9株均为IgM类;染色体数目为92～101条;酶联免疫吸附测定(ELISA)检测杂交瘤细胞培养上清效价为1:20～1:160,腹水效价为1:3 200 ～1:25 600;特异性试验表明,抗TAP单克隆抗体与牛血清白蛋白(BSA)、人白蛋白、胰蛋白酶、淀粉酶均无交叉反应;中和抑制试验表明,培养上清及腹水中的抗体能明显被合成多肽TAP及为急性胰腺炎患者尿液所中和;对其中5株细胞株进行相对亲和力分析结果表明,2C3>8C6>6H7>6F7>8B10.结论成功制备了抗TAP单克隆抗体,为建立敏感、特异的TAP免疫检测方法奠定了基础.

Objective To prepare monoclonal antibody against trypsinogen activation peptides （TAP）, Methods The synthetical peptides of TAP linked by KLH was used as antigen. Balb/c mice were immunized with the joined antigen by intra-celiac and intra-venous injection. The TAP-induced spleen cells were fused with SP2/0 mydoma cell. The McAbs anti TAP were obtained by screening with indirect EI.ISA and 4 times clone, and were to be preliminary characterized. Results The rate of fusion was 85.03% . Ten hybridoma cell lines were developed which could secret McAb stably. The isotype of the ten McAb were all IgM,exccpt one （2C3）. The numbers of hybridoma chromosomes in ten hybridoma cell lines were between 92-101. Hybridoma cell culture supernatants titers were between 1 ： 20 to 1 ： 160, ascites titers were between 1 ： 3 200 to 1 ： 25 600. Relative affinity of five hybridoma cell lines were 2C3〉8C6〉6H7〉6F7〉8B10. Specificity analysis proved that all these McAbs reacted only with TAP and had no cross-reaction to Bovine serum （BSA）, human albumin, trypsinase and amylase. Neutralisation test showed that antibodies can be evidently neutralized by synthetical peptides of TAP and the urine of acute pancretitis（AP） valetudinarian. Conclusion The preparation of specific McAb to TAP has potential utility in the establishment of TAP determination for the early diagnosis of acute pancreatitis.