Abstract
Antigen Used for Immunization
Glutathione S-transferase-spermidine/spermine-N1-acetyltransferase (GST-SSAT).
Method of Immunization
Subcutaneous injection.
Parental Cell Line Used for Fusion
Sp2/0.
Selection and Cloning Procedure
After fusion for 4 days, the medium was changed and 7 days later ELISA was used for screening. Strong positive clones were subcloned by limited dilution.
Heavy and Light Chains of Immunoglobulin
Three are IgG1 and one is IgG2b, light chains are all kappa chains.
Specificity
To ensure high specificity, we took the following strategies. First, GST-SSAT was used to inject mice and SSAT was used to screen the positive clone. The injection and screening protein were all of high purity. Second, we used siRNA on SSAT and compared the SSAT level in knockdown and normal cells. Results showed that the monoclonal antibody specifically recognized SSAT.
Specific Antigen Identified
Before overexpression, the SSAT cDNA was sequenced. The sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) confirmed that the protein size was right.