This protocol is significant because it can be used to interrogate the immune repertoires of human donors to identify rare, antigen-specific B-cells. This technique allows us to obtain natively-paired heavy and light chains, which can be rapidly cloned, and then directly tested against the antigen or antigens of interest. Demonstrating the procedure will be Mike Morton, a technician from our laboratory.
At least one hour after thawing and recovery, collect the donor PBMC by centrifugation. And re-suspend the cells in 50 milliliters of ice cold MES Buffer for counting. Pellet the cells by another centrifugation, and isolate the CD22-positive B-cells by positive selection with CD22 microbeads according to standard protocols.
Collect the bead isolated CD22-positive cells by centrifugation and resuspend the cells at four times 10 to the seventh cells per milliliter of Vax buffer concentration. Add an IgG CD19 CD27 antibody cocktail to the cells according to the manufacturer's dilution recommendations with gentle mixing, and aliquot 10 to the seventh cells into a 1.5 milliliter microcentrifuge tube for the negative control. Add a dual-labeled biotin steptavidin tetramers to the negative control tube at a final concentration of 36 nanomolar each with a PE and APC, multiplied by the number of peptides in the sort tube.
Bring the final volume in each tube 2.5 milliliters with fresh Vax buffer and add the peptide tetramers to the tube sort at 36 nanomolar each PE and APC. After centrifuging, bring the cells to two times 10 to the 6th per 100 microliters of TBS concentration for a 30 to 60 minute incubation, in the dark, at four degrees Celsius, with rotation. At the end of the incubation, wash the cells two times in fresh TBS buffer per wash before straining the samples into individual five milliliter filter-cap tubes.
Stain the samples with a 0.3 micromolar final concentration of DAPI as a marker of cell membrane integrity. And run the entire negative control on the cell sorter to set the flow cytometry gates for isolating the appropriate cell populations for single cell sorting. Plot the antigen PE versus antigen APC and set the R8 gate as an antigen PE-positive, and APC-positive quadrant with a low number of events in the double positive gate.
Then, sort the single CD19-positive CD27-positive antigen PE-positive, antigen APC-positive cells into individual wells of a Master Mix prepared 96 well plate. At the end of the sort, cover the plate with aluminum tape pads, and centrifuge the cells for one minute at 400 times g before minus 80 degree storage. To perform a reverse transcriptase reaction, thaw the sorted B-cells on ice for two minutes before spinning the plate for two minutes at 3300 times g to pull the well contents to the bottom of the plate before opening.
After treating the cells with an appropriate enzyme Master Mix, add 2.5 microliters of complimentary DNA as a template for each PCR reaction, and add primers to a final concentration of 1 micromolar to each reaction. Then, add a 2X concentration of 10X polymerase buffer to each well to bring the final volume to 25 microliters per reaction. And run the heavy and light chain PCR reactions each for 50 cycles.
At the end of the reactions, run the samples on 1%agarose gel to visualize the positive amplification hits. Isolate both the paired heavy and light chain reactions from the same cell via gel extraction. Determine the DNA fragment concentration by the optical density to 60 for accurate ligation mix calculations.
Combine the recovered heavy and light chain fragments with a linker fragment and the expression vector backbone using a four fragment ligation reaction according to the manufacturer's instructions. And transform the ligation reactions into chemically competent bacteria according to the manufacturer's instructions. Once plated onto antibiotic plates, add four milliliters of growth medium with antibiotics to the remaining transformation culture for incubation overnight at 37 degrees Celsius at 250 rotations per minute.
The next morning, prepare a miniprep DNA from the overnight ligation mix cultures according to the manufacturer's instructions and determine the resulting plasmid DNA concentration. To confirm the antigen specificity of the isolated antibodies, transfect 10 micrograms of the miniprep DNA with cationic lipid base reagent in a 10 milliliter suspension of 293 cells. Incubate the cell culture three to four days at 37 degrees Celsius and 8%carbon dioxide and 125 rotations per minute.
At the end of the incubation, centrifuge the culture for 10 minutes at 1000 times g, and recover the clarified medium. Measure the IgG concentration in the supernatant via affinity to protein A and test each antibody in the supernatant at a 25 microgram per milliliter concentration by ELISA against the individual peptides used for the sort according to standard ELISA protocols. Then, confirm the screen positive hits with an additional ELISA using dilutions of IgG supernatant to generate a concentration curve starting at 20 microliters per milliliter and plotting against the optical density for each antigen showing reactivity to the initial screen ELISA.
To isolate antigen specific antibodies from human donors, a series of cytometry gates can be devised to isolate the target memory B-cells. The lymphocytes are isolated based on their cell size and granularity, using forward and side scatter. Following the exclusion of doublets and dead cells, phenotypic markers allow the segregation of IgG-positive CD19-positive B-cells and CD27-positive memory B-cells.
The first readout of the single cell cloning is a confirmation of amplification of the respective heavy and light variable chains. Here, an example of a very efficient amplification from 24 single cells with a 42%paired recovery after PCR is shown. Following IgG cloning, the IgG expression vector is transpected into human embryonic kidney 293 cells.
Recovered antibodies are screened against the original panel of Tau peptides scored for reactivity by ELISA. Additional confirmation is then completed using a concentration curve of the same recombinant antibody samples against the peptides identified in the initial screen. For each positive hit, an individual plasmid clone can be isolated from the transformed pool and reconfirmed by the same ELISA method.
The goal is the amplify sequences from single cells, so the most important thing to remember is that any contamination will be amplified during the PCR portion of this procedure. This procedure yields intact human recombinant antibodies, which allows for the purification and functional characterization of as much antibody as you wish to generate.
