Production of Monoclonal Antibodies Targeting Aminopeptidase N in the Porcine Intestinal Mucosal Epithelium

derived monoclonal antibodies are primarily used in diagnostic therapeutic immune reagents highlighting the need to produce stable and reliable antibodies in limited production prepared. The methods described here proved that recombinant antibody production could increase monoclonal antibody production efficiency and minimize labor and time associated costs. The methods are used to develop aminopeptidase and antibody-based antibody drug conjugates and other therapeutic products, which aids in clarifying the role of APN in the prevention and treatment of bacterial and viral infections.

Demonstrating the procedure will be Yan Li, a postgrad student from my laboratory. To begin, intraperitoneally inject 100 micrograms of APN protein into the selected adult female mice for a final antigen boost. After three 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 SP20 myeloma cells in a 25 square centimeter flask containing five 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 five to six 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 aspirate SP20 cells with a pipette from 8 to 10 bottles and suspend 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 SB20 cells at a ratio of 10:1 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 one milliliter of polyethylene glycol 1500 pre-warmed to 37 degrees Celsius dropwise to the loosened cell pellet over 45 seconds while gently rotating the bottom of the tube. Then slowly add one 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 degree 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 five days, add 100 microliters of fresh HAT medium to each well.

And again after five days of incubation, replace the medium with HAT medium. Use a microtiter plate coated with five micrograms per milliliter APN protein diluted in 0.05 molar PBS to analyze monoclonal antibodies in the hybridomas supernatant using ELISA assay. Grow the pET28a plus recombinant antibodies aminopeptidase NBL21 transformed bacteria in the presence of 0.4 millimolar beta-d-1-thiogalactopyranoside in an orbital shaker at 37 degrees Celsius for 10 hours.

Seed 100 microliters 0.5 times 10 to the fifth Chinese hamster ovary cells per well into a 96-well plate for incubation at 37 degrees Celsius and a 6%carbon dioxide atmosphere for 18 to 24 hours. When the cells reach 80 to 90%confluence, dilute the pIRES2-zs Green one recombinant antibodies aminopeptidase and plasmid with Opti-MEM to a final concentration of 0.1 micrograms per microliter. After five minutes, mix the 50 microliters of diluted pIRES2-zs Green one recombinant antibodies aminopeptidases and plasmid with one microliter of Lipofectamine 2000 and 49 microliters of Opti-MEM.

After 20 minutes of incubation, add 100 microliters of the mixture to each well of a 96-well plate containing CHO cells. At four to six hours post transfection, replace the medium with DMEM F12 supplemented with 10%FBS. After 48 hours of incubation, add 400 micrograms per microliter G418 to each well to select the stably transfected cells.

After 10 days of selection using DMEM F12 medium supplemented with 10%FBS and 400 micrograms per microliter G418, sort the cells with fluorescence-activated cell sorting. Serially dilute the harvested positive cells. Seed them at an average of 0.5 to 2 cells per well in a 96-well plate and culture in 37 degrees Celsius, 6%carbon dioxide incubator.

In representative microscopic analysis, all hybridomas cells appeared round, bright, and clear. The purified monoclonal antibodies possessing heavy 50 kilodalton and light 25 kilodalton chains were confirmed by SDS-PAGE and were found in the purified ascites. The titers of these anti-APN monoclonal antibodies in culture supernatants and ascites are shown here.

Mouse monoclonal antibody isotyping revealed that antibodies derived from clones 5B31, 5B36, 3C48, 5C51, and 6C56 possessed immunoglobulin G2B subclasses while APN 2A20 was an immunoglobulin G2A kappa type antibody and monoclonal antibody APN 3FD9, 3F10, and 10F3 belonged to immunoglobulin M type and processed kappa light chains. Most of these monoclonal antibodies showed AV values of over 50%indicating that they targeted different epitopes in the APN while the APN 5C51 antibody recognized antigenic epitopes like those recognized by APN 3C48, 5B31, and 6C56 monoclonal antibodies. The amplified APN 5B36 VHVL gene was ligated into a pET28a positive or pIRES2-zs Green one vector to construct the recombinant expression plasmids pET28a positive RABS APN and pIRES2-zs Green one RABS APN respectively.

The antibodies expressed by both pET28a plus recombinant antibodies aminopeptidase NBL21 and pIRES2-zs Green one recombinant antibodies aminopeptidase NCHO cells were purified and analyzed using ELISA and IFA assays. However, only the antibody expressed in the supernatant of pIRES2-zs Green one recombinant antibodies aminopeptidase NCHO cells recognize the APN protein. Binding of APN 5B36 monoclonal antibodies to APN proteins reached an equilibrium earlier than recombinant antibodies.

Purification of the monoclonal antibodies in the supernatant using protein A agarose is better than using saturated ammonia sulfate precipitation.