Abstract
Antigen Used for Immunization
The synthetic peptide sequence (Sema3Fc) was kindly provided by Professor Craig W. Vander Kooi (Department of Molecular and Cellular Biochemistry and Center for Structural Biology, University of Kentucky).(1)
Method of Immunization
Four BALB/c mice were subcutaneously injected with Sema3Fc to raise antibodies. Each mouse was immunized with 40 μg Sema3Fc in 0.4 mL phosphate buffered saline fully emulsified with 0.4 mL Freund's complete adjuvant. At days 14 and 28, each mouse was given 40 μg Sema3Fc in incomplete Freund's adjuvant. Tail vein blood was collected and the serum was subjected to indirect enzyme-linked immunosorbent assay (ELISA) to analyze antibody titers against Sema3Fc. The mouse with the highest titer was selected and intravenously injected with 20 μg Sema3Fc without Freund's adjuvant 3 days before cell fusion.
Parental Cell Line Used for Fusion
SP2/0 cell line and splenocytes from BALB/c mice were used for fusion.
Selection and Cloning Procedure
Cell colonies were selected and their supernatants were screened for antibody titers against Sema3Fc by indirect ELISA. The hybridoma cell colonies were cultured and recloned by limiting dilution method(2,3) in hypoxanthine, thymidine medium for 2 weeks.
Heavy and Light Chains of Immunoglobulin
Capture ELISA method indicated that Sema3F mAb was IgM isotype. The heavy chain is about 50 kDa and the light chain is about 25 kDa.
Specificity
To explore specificity and application of Sema3Fc mAb, we used the identified mAb as primary antibody in flow cytometry, immunofluorescence, and immunocytochemical staining. In all these assays, results showed that Sema3Fc mAb could specifically combine Sema3F compared with control. It demonstrated that our prepared antibody was not only applied into assays such as flow cytometry, immunofluorescence, and immunocytochemical staining, but also possessed high specificity.
Specific Antigen Identified
Semaphorin3Fc.