A Hybridoma Technology for Producing Monoclonal Antibodies against a Metallopeptidase

To begin, intraperitoneally inject 100 micrograms of APM protein into the selected adult female mice for a final antigen boost. After 3 days of injection, collect the spleens from mice, and wash with DMEM twice to remove blood and fat cells.

Filter the spleen cell suspension using a 200-mesh copper grid to remove tissue debris, and harvest spleen cells using centrifugation to remove the spleen membrane. Seed the mouse SP2/0 myeloma cells in a 25-square centimeter flask containing 5 milliliters of DMEM supplemented with 6% fetal bovine serum, and culture the cells at 37 degrees Celsius and 6% carbon dioxide atmosphere to maintain cell viability.

After 5 to 6 days of culture, the cells should reach 80% to 90% confluence post-resuscitation and appear round, bright, and clear under the microscope. One day before hybridization, collect macrophages from the peritoneal cavities of the mice.

Seed peritoneal macrophages at a density of 0.1 to 0.2 times 10 to the fifth per milliliter in 96-well plates, each containing 100 microliters of HAT medium, and incubate them overnight. For hybridization, gently aspirated SP2/0 cells with a pipette from 8 to 10 bottles and resuspend them in 10 milliliters of serum-free DMEM medium.

Wash the cells with fresh DMEM and centrifuge them twice, then resuspend them in 10 milliliters of DMEM. Mix the quantified spleen cells with SP2/0 cells at a ratio of 10-to-1 and transfer them into 50-milliliter tubes.

After centrifugation, discard the supernatant and collect the pellets at the bottom of the tubes. Tap the tube to loosen the pellets before hybridization. Using a dropper, add 1 milliliter of polyethylene glycol 1,500 pre-warmed to 37 degrees Celsius drop-wise to the loosened cell pellet over 45 seconds while gently rotating the bottom of the tube.

Then, slowly add 1 milliliter of DMEM pre-warmed to 37 degrees Celsius to the mixture for 90 seconds, followed by another 30 milliliters of fresh DMEM, and place the fusion tube into a 37 degrees Celsius water bath for 30 minutes.

After incubation, harvest the cells. Resuspend the HAT medium and culture in a 96-well plate inoculated with peritoneal macrophages. After 5 days, add 100 microliters of fresh HAT medium to each well. And again, after 5 days of incubation, replace the medium with HT medium. Use a microtiter plate coated with 5 micrograms per milliliter APN protein diluted in 0.05 molar PBS to analyze monoclonal antibodies in the hybridoma supernatant using an ELISA assay.