Abstract
Antigen Used for Immunization
Recombinant human leptin (Biomyx Technology, San Diego, CA).
Method of Immunization
BALB/c mice were immunized three times at an interval of 1 week with leptin-KLH. The immunization was performed using IMMACCEL (PickCell Laboratories, Amsterdam, The Netherlands). Complete Freund's adjuvant was first used (10 μg KLH-leptin); incomplete Freund's adjuvant was used for the second (20 μg KLH-leptin), and third (40 μg KLH-leptin) immunizations. Three days before the cell fusion, 20 μg of KLH-leptin (without any adjuvant) was injected intravenously.
Parental Cell Line Used for Fusion
Mouse myeloma SP2/0 cells.
Selection and Cloning Procedure
Mouse myeloma SP2/0 cells were mixed with the splenocytes of the immunized mouse at a ratio of 1:10. The cell mixture was washed twice with RPMI. Pre-warmed (37°C) 50% polyethylene glycol 1500 (PEG, Sigma) was added to the cell pellet slowly with continuous mixing. Cells were then centrifuged at 22°C for 5 min at 500 g. HAT selection medium (Sigma) was then added to the wells and hybridomas were allowed to grow.
Heavy and Light Chains of Immunoglobulin
Mouse immunoglobulin G (IgG).
Specificity
Western blot, immunocytochemistry, and flow cytometry experiments were used to characterize the anti-leptin antibody. The antibody had a high affinity (around 1.13 × 10−9 M) for its antigen. The saturation of the antibody with leptin (20 moles leptin per 1 mole antibody) in Western blot analysis proved that the antibody had specific binding to its antigen. Immunocytochemistry and flow cytometry on JEG-3 (human placental choriocarcinoma cells) revealed that the anti-leptin antibody recognized intracellular leptin.
Specific Antigen Identified
Human leptin.