抗PEA受体结合区单克隆抗体的制备及鉴定

目的探讨绿脓杆菌外毒素A(PEA)中和性单克隆抗体在预防和治疗绿脓杆菌感染中的免疫保护作用.方法以重组绿脓杆菌外毒素A受体结合区蛋白免疫Balb/c小鼠,将免疫鼠脾细胞与SP2/0骨髓瘤细胞融合,用ELISA方法筛选细胞培养上清,采用有限稀释法对阳性孔进行克隆,3次克隆后获得4株能稳定分泌抗PEA单克隆抗体的杂交瘤细胞株,分别命名为1C4,1D4,2E12和3C6.结果细胞培养上清ELISA效价为4000～8000,腹水ELISA效价可达25600～51200,其中3C6抗体在细胞上的中和试验显示具有较高的中和效价,其亲和常数为8.93×108.结论获得的单克隆抗体可在抗烧伤绿脓杆菌感染中发挥重要作用.

Preparation and Identification of Monoclonal Antibodies Against PEA Receptor Binding Subuni Protein

Objective： To study the immunoprotection of neutralizing monoclonal antibody against Pseduomonas - aeruginosa exotoxin A （PEA） so as to prevent and to treat the infection of Pseduomonas - aeruginosa bacillus. Method： Balb/c mice were immunized with the recombinant protein of the receptor binding domain of pseudomonas exotoxin A （PEA） . The splenic cells from the immunize mice were fused with SP2/0 myeloma cells. The cells were screened and the clear supernatant was cultivated in ELISA. The positive hybridoma was cloned in limited dilution method so as to subclone the positive clones. After three cycles of subcloning, four McAbs against the PEA were obtained, designated asl CA, 11M, 2E12 and 3C6. Results： The ELISA titers of culture supematant and ascites were 4000 -8000 and 25600 -51200 respectively. The 3C6 McAb had higher neutralization titers, the affinity constant was 8.93 ×108M - 1. Conclusion： The monoclonal antibody obtained in the study may bring the important effect on resisting Pseduomonas - aeruginosa infection.