A Hybridoma Technology for Producing Monoclonal Antibodies against a Metallopeptidase

Take a mouse immunized with a metallopeptidase. Harvest the spleen and obtain a cell suspension.

Filter the suspension through a mesh and centrifuge, removing tissue debris.

The isolated cells include metallopeptidase-specific B cells, producing antibodies against metallopeptidase epitopes.

Add immortal myeloma cells lacking the HGPRT enzyme, preventing the salvaging of nucleobases for nucleotide synthesis. They survive via de novo nucleotide synthesis.

Centrifuge to obtain a pellet and discard the supernatant.

Add PEG and incubate, inducing myeloma-B cell fusion to form HGPRT-positive immortal hybridomas.

Add HAT medium comprising hypoxanthine, aminopterin, and thymidine. Transfer into wells containing a peritoneal macrophage feeder layer, promoting hybridoma growth.

Aminopterin blocks de novo nucleotide synthesis, causing unfused myeloma cells to die. Unfused B cells die due to their short lifespan.

The HGPRT-positive hybridomas salvage the nucleotide precursors hypoxanthine and thymidine, proliferate, and secrete metallopeptidase-specific monoclonal antibodies.

Harvest the supernatant to confirm antibody production via downstream assays.

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.