Preparation of Monoclonal Antibody against Extracellular Domain of TNF-related Apoptosis-induced Ligand

Objective To prepare the monoclonal antibody(McAb)against amino acids 114-281 at extracellular domain of TNF-related apoptosis-induced ligand (TRAIL), i.e. sTRAIL. Methods BALB / c mice were immunized with recombinant sTRAIL, and their splenic cells were fused with SP2 / 0 cells, and positive clones were screened by capture ELISA to prepare McAb in ascites. The prepared McAb was determined for titer by indirect ELISA and analyzed for subclass and specificity. Meanwhile, the chromosome of hybridoma cells and recognition site of McAb were analyzed. The culture supernatants of hybridoma cells 3 months after culture in vitro and 6 months after storage in frozen were determined for McAb titers respectively. Results All the McAbs secreted by 3 hybridoma cell strains were IgG1, and their titers in ascites were higher than 10-6. The number of chromosome in hybridoma cells was 94～98. All the McAbs recognized the linear epitopes on sTRAIL molecules. The McAb titers in culture supernatants of hybridoma cells showed no significant change after culture in vitro for 3 months or storage in frozen for 6 months. Conclusion Three hybridoma cells stably secreting McAbs against sTRAIL were successfully established, and McAbs were prepared, which laid a foundation of qualitative and quantitative assays as well as expression of TRAIL.