Abstract
Antigen Used for Immunization
Faraz-Institute for Cancer Research (ICR) whole cell from pancreatic cancer was used as antigen.
Method of Immunization
Whole cell immunization was done by intraperitoneal injections.
Parental Cell Line Used for Fusion
Mouse myeloma SP2/0 cells (National Cell Bank of Iran, Pasteur Institute of Iran, Tehran) were used for fusion.
Selection and Cloning Procedure
Spleenocytes and myeloma cells were mixed at a ratio of 5:1, and the mixture was washed twice with prewarmed Roswell Park Memorial Institute medium (RPMI)-1640. Fusion was then performed using prewarmed polyethylene glycol 1500. The resulting hybridoma cells were dispensed at 2 × 105 cells per well in 96-well plates and cultured in RPMI medium containing 20% fetal bovine serum, 1% penicillin/streptomycin, 1% sodium pyrophosphate, and 1% Non-Essential Amino Acids solution 100X and supplemented with hypoxanthine-aminopterin-thymidine medium supplement. Fourteen days after fusion, the reactivity of the culture supernatants from wells containing hybrids was identified by enzyme-linked immunosorbent assay. Finally, positive hybridomas were cloned by the limiting dilution process using conventional methods. Then, positive clones were subcloned and rescreened by ELISA and immunocytochemistry.
Heavy and Light Chains of Immunoglobulin
Heavy chain: IgM
Light chain: kappa
Specificity
Immunofluorescence staining
Different cell lines containing pancreatic cell lines, breast cell lines, and mesenchymal stem cells were grown on slides, and after fixation and blocking, they were incubated with 1C11 clone culture supernatant as a primary monoclonal antibody. Finally, cells were incubated with secondary fluorescent FITC-conjugated antibody. 1C11 mAb reactivity and target localization of antigens were observed using a fluorescent microscope ni-80 (Nikon, Japan).
Western blotting
For determination of 1C11 mAb specificity and its target molecular weights, Western blotting was performed. For this purpose, 30 μg of cell lysate was run on a 12% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis gel and electrophoretically transferred to polyvinylidene fluoride membrane (PVDF) membranes by a semidry blotter. The membranes were blocked and then incubated with mouse ascites at different concentrations as primary antibody (overnight at 4°C) and horseradish peroxidase-conjugated antimouse antibody as secondary antibody. After washing with tris buffer saline plus 1% tween 20, the membrane was developed by treatment with enhanced chemiluminescence substrate and the resulting chemifluorescence signals recorded on X-ray film.
Two dimensional immunoblotting
Faraz-ICR cell lysates were separated in two dimensions. For the first dimension, 400 μg protein lysate was loaded onto immobilized pH gradient (IPG) gel strips (pH 3–10 NL; 18 cm, GE Healthcare) by gel rehydration for 18 hours at 50 V and then isoelectric focusing (IEF) was performed on an IPGphor IEF unit system (Bio-Rad, Hercules, CA) for a total of 55,000 Vh. For the second dimension, strips were run on 12% SDS polyacrylamide gel at a constant current of 30 mA. Protein spots were observed by a modified Coomassie Brilliant Blue staining method. For 2D immunobloting, the 2D gel was transferred across the PVDF membrane by a semidry blotter (BioRad) for 1.5 h followed by a routine Western blot procedure for detecting 1C11 clone target in the Faraz-ICR cell lysate. In this process, 1C11 clone ascites fluid was used as a primary antibody. Immunoreactive protein spots were manually cut from 2D gel stained with Coomassie and sent for MALDI-TOF/TOF MS analysis to the United Kingdom (Department of Biology, The Proteomics and Analytical Biochemistry Labs, University of York, United Kingdom).
Specific Antigen Identified
Human mortalin (heat shock protein 70).