This method can help answer key questions in the bispecific antibody field, such as how to produce a bispecific antibody in bacteria and determine the function of the purified antibody. The main advantage of this technique is that GPC3-S-Fab can be easily produced and purified from E.coli. To prepare the periplasmic fraction, first centrifuge the cell suspension at 4, 000 g for 30 minutes at four degrees Celsius.
Then, expel the medium, and weigh the cells. After centrifugation, dissolve the cell pellet in four milliliters of ice-cold sucrose solution for each gram of cells. Incubate the cell suspension on ice for 15 minutes.
After 15 minutes, centrifuge the cell suspension at 8, 500 g for 20 minutes at four degrees Celsius. Then, pipette out the supernatant containing the sucrose fraction, and store it on ice. Resuspend the cell pellet in ice-cold mixture of five-millimolar magnesium chloride and one-millimolar PMSF.
Then, add 40 microliters of 15 milligrams per milliliter of the lysosome stock to the cell suspension. After adding the lysosome stock, leave the cell suspension on ice for 30 minutes. After the incubation period is over, combine the sucrose and the periplasmic fractions.
Then, centrifuge the sucrose-periplasmic fraction at 30, 000 g for 30 minutes at four degrees Celsius. After centrifugation is over, transfer the supernatant in a 50-milliliter conical tube on ice. Next, mix and resuspend one milliliter of nickel-NTA agarose with the supernatant to obtain a homogeneous suspension, then one milliliter of nickel-NTA agarose to a fresh 15-milliliter conical tube.
To the agarose, add 10 column volumes of the equilibration buffer. Then, centrifuge the agarose at 400 g for five minutes. Post-centrifugation, carefully remove the supernatant, and add the nickel-NTA agarose to the sucrose-periplasmic fraction.
Then, rock the sucrose-periplasmic fraction-containing tube at four degrees Celsius for two hours. Again, centrifuge the mixture. Then, carefully remove the supernatant to a fresh ice-cold tube since this is the unbound fraction.
After the supernatant is transferred, add the nickel-NTA agarose in the gravity column. Then, add three column volumes of elution buffer to the column. Next, collect the eluate as elution fractions one, two, three, and four, respectively.
Dilute and seed 5, 000 cells in 100 microliters in each well of a 96-well plate. Incubate the plate at 37 degrees Celsius for six to eight hours for attachment. To prepare the PBMCs, first dilute the freshly prepared blood with an equal volume of phosphate-buffered saline in a 50-milliliter conical tube.
Pipette 15 milliliters of lymphocyte separation medium in a fresh 50-milliliter conical tube. Next, carefully transfer the diluted blood along the walls of the tube to the lymphocyte separation medium. Centrifuge the cells at 400 g for 35 minutes at room temperature with the brakes off.
Post-centrifugation, carefully draw the upper plasma layer out. Then, pipette the white blood layer containing the PBMCs. Dilute these PBMCs obtained with double the volume of phosphate-buffered saline with 2%fetal bovine serum.
Then, centrifuge the PBMC suspension. Wash the cell pellet with phosphate-buffered saline containing 2%fetal bovine serum, and again centrifuge. After centrifugation, count the cell number.
To prepare the natural killer cells, reconstitute the PBMCs in phosphate-buffered saline with 2%fetal bovine saline. Then, transfer the mixture in a polystyrene round-bottom tube. Add 50 microliters per milliliter of natural killer cell enrichment cocktail to the PBMC mixture.
Mix the contents, and leave the tube for 10 minutes. Next, resuspend and mix the magnetic particles present in the enrichment cocktail by pipetting multiple times, such that the particles are uniformly suspended. Then, add 100 microliters per milliliter of magnetic particles to the cell suspension.
Mix the cell suspension magnetic particles, and incubate at room temperature for five minutes. After five minutes, add 2.5 milliliters of phosphate-buffered saline with 2%fetal bovine serum to the cell suspension. Gently pipette two to three times to mix the cells in the tube.
Place the tube on the magnet, and let the magnetic beads settle down for 2.5 minutes at room temperature. Then, in one stroke of action, invert the tube, and pour the enriched cell suspension in a new tube. Again, count the number of cells.
Once the counting is over, dilute the natural killer cells in the culture medium. Then, add 100 microliters of the cell suspension to the tumor cells plated in a 96-well plate. To set up the cytotoxicity assays, add 10 microliters of varying concentrations of GPC3-S-Fab antibody to the 96-well plate.
Then, incubate the plate at 37 degrees Celsius for 72 hours. After the designated incubation time, aspirate the medium. Then, add 100 microliters of DMEM medium containing 10 microliters of CCK reagent in each well of the 96-well plate.
Again, incubate the plate at 37 degrees Celsius. Post-incubation, read the plate at 450 nanometers every consecutive hour until four hours after the addition of the CCK8 reagent. The GPC3-S-Fab antibody is purified using a two-step affinity purification.
Here, the two panels show that the antibody is purified using the nickel-NTA agarose and the IgG-CH1 affinity purification columns, respectively. Next, the binding affinity of the GPC3-S-Fab antibody is evaluated using GPC3-positive and negative cancer cell lines. The GPC3-S-Fab binds to all the GPC3-positive cells, such as Hep3B, Huh7, Hep2G, and CHO/GPC3.
Interestingly, no or minimal binding is observed when GPC3-negative cells, such as MHCC-97H and CHO, are used. The cytotoxicity of the GPC3-S-Fab is also studied in the presence of the tumor cells incubated with fresh natural killer cells. The antibody GPC3-S-Fab elicits strong cytotoxicity against HepG2, Hep3B, and Huh7 seven cells in a dose-dependent manner in the presence of the natural killer cells.
However, no such cytotoxicity is observed in the absence of the natural killer cells on the MHCC-97H cells. Once mastered, this technique can be done in six hours, if it is performed properly. While attempting this procedure, it's important to remember to keep the steps on ice.
Following this procedure, other methods like xenograft modeling can be performed in order to answer additional questions, like in tumor effect in vivo. After its development, this technique paved the way for researchers in the field of bispecific antibody to explore antitumor effects in vitro. After watching this video, you should have a good understanding of how to produce a functional bispecific antibody targeting the tumor cells, especially the isolation of the NK cells.
