Abstract
Antigen Used for Immunization
A synthetic peptide derived from the last 50 amino acids of the extracellular domain of sortilin protein was selected and conjugated to keyhole limpet hemocyanin and used to immunize mice.
Method of Immunization
Female BALB/c mice were used for immunization with peptide-KLH conjugate. The first immunization was performed using Freund's complete adjuvant. Incomplete Freund's adjuvant was used for the second, third, fourth, and fifth immunizations. Finally, 3 days before the cell fusion, KLH-peptide (without any adjuvant) was injected intravenously. Mice with higher titers of specific antibody were selected for fusion.
Parental Cell Line Used for Fusion
Mouse myeloma SP2/0-Ag14 cells.
Selection and Cloning Procedure
The mouse spleen cells were obtained 3 days after the last booster immunization and fused with SP2/0-Ag14 cells at a ratio of 5:1 in 50% (W/V) polyethylene glycol 1500 (PEG 1500) (Sigma, St. Louis, MO). The cell fusion method was accomplished according to the technique described by Köhler and Milstein.(1) The hybridoma cells were cultured in 96-well plates containing feeder cells with RPMI-1640, 20% FBS, and hypoxanthine-aminopterin-thymidine (HAT, Sigma). Hybridoma cells producing anti-sortilin antibodies were screened using indirect ELISA by coating immunizing sortilin peptide according to the method described previously.(2) The positive clones were subcloned four times using limiting dilution until the final clone was obtained.
Heavy and Light Chains of Immunoglobulin
Mouse IgG1/κ.
Specificity
The anti-sortilin monoclonal antibody (MAb), clone 2D8, was purified from supernatant of final hybridoma clone using peptide affinity chromatography column. Reactivity of antibody with the immunizing peptide was assessed in ELISA. Furthermore, application of 2D8 in Western blot analysis was proved by specific detection of sortilin protein in a number of ovarian carcinoma tissues, within which the sortilin overexpression was reported previously.(3) Indeed, cell surface staining of some ovarian carcinoma cell lines expressing sortilin on their surfaces, including Caov-4, A2780S, SKOV-3, and 2008/C13.R, demonstrated that 2D8 could react with an extracellular epitope of sortilin protein. MAb 2D8 did not react with NIH/3T3-vector cells (negative control) that confirmed its specificity for sortilin in flow cytometry.
Specific Antigen Identified
Sortilin in human tissues and cell lines.