Abstract
Antigen Used for Immunization
His-SpiC prokaryotic expression, soluble recombinant protein.
Method of Immunization
Following a previously published protocol, (14,15) 6-week-old female BALB/c mice were injected subcutaneously with 100 μg of purified rHis-SpiC protein mixed with an equal volume of Freund's complete adjuvant (Sigma-Aldrich, St. Louis, MO). Booster injections of the same antigen mixed with an equal volume of Freund's incomplete adjuvant (Sigma-Aldrich) were given twice subcutaneously at 2-week intervals, and a final intravenous injection was given exactly 3 days before harvesting splenocytes.
Parental Cell Line Used for Fusion
The splenocytes were harvested and fused with SP2/0 myeloma cells following the lymphocyte hybridoma technique.
Selection and Cloning Procedure
Hybridoma cells that secreted MAbs directed against SpiC protein were subcloned by serial dilution. The titers of the hybridoma culture supernatants were determined with an indirect ELISA based on purified rGST-SpiC protein, and the results were used to evaluate the hybridoma cells. In this assay, a polyclonal antibody (serum from BALB/c mice immunized with rHis-SpiC) was used as the positive control, and the supernatant of SP2/0 myeloma cells was used as the negative control.
Heavy and Light Chains of Immunoglobulin
Specificity
To confirm whether all the MAbs recognized both the rHis-SpiC and rGST- SpiC proteins, the purified MAbs were tested in an immunoblotting assay. Both the expected 20 kDa and 40 kDa bands were detected in this assay. The results confirmed that all seven MAbs bound to both the rHis-SpiC and rGST-SpiC proteins and did not react with the His and GST proteins from E. coli BL21(DE3)(pET) and E. coli BL21(pGEX-6p-l), respectively.
Specific Antigen Identified
GST-SpiC, prokaryotic expression, recombinant protein.