Generating Recombinant Monoclonal Antibodies From Mammalian Cell Cultures

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Isolation of Peripheral Blood Mononuclear Cells (PBMCs)

1. Centrifuge 25 ml of the participants' heparinized blood at 900 x g for 15 min, as soon as possible after the blood extraction. If there is less blood, scale down the reagents accordingly. Perform all the next steps in a hood.
2. Transfer the serum to a clean tube. It can be used in later steps for freezing PBMCs or in case of autoimmune patients, to test auto-antibodies.
3. Dilute the blood with 10 ml of Roswell Park Memorial Institute (RPMI) 1640 media and resuspend the cells by pipetting up and down.
4. Add 15 ml of a solution containing polysucrose and sodium diatrizoate (1.077 g/ml) to a new 50 ml tube.
5. Gently layer the blood on top of the solution in step 1.4 by bringing the two openings of the tubes close together until the two liquids come very slowly in contact, and then decant the PBMC suspension slowly on top of the 50 ml tube. This step is critical to have a good separation of the PBMCs.
6. Centrifuge the tube obtained in step 1.5 at 400 x g without brake at room temperature (RT) for 20 min.
7. After the centrifugation, recover with the pipet the white ring that appears in the middle part of the tube, which contains the PBMCs.
8. Wash the cells with 25 ml of RPMI 1640 media.
9. Centrifuge 300 x g at RT for 10 min.
10. Discard the supernatant and wash the cells with 10 ml of RPMI 1640. Centrifuge again as in step 1.9.
11. Count the PBMCs with a Neubauer chamber as previously reported.
12. Process the PBMCs as in section 2 or store them for later use as follows: 10 million PBMCs for freezing per Cryovial tube diluted in 1 ml of 10% dimethyl sulfoxide (DMSO) and 90% serum obtained in step 1.2. Freeze progressively and for long storage in liquid nitrogen.

2. Staining PBMCs for Sorting CD22+ and immunoglobulin G (IgG+) by Cell Cytometry

1. Plate the PBMCs in a 25 cm² cell culture flask with 6 ml of complete RPMI 1640 medium (supplemented with L-glutamine, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, 50 U/ml penicillin, 50 µg/ml streptomycin and 10% of fetal bovine serum) and let them recover O/N in the incubator at 37 °C at 5% carbon dioxide (CO₂).
2. Block sterile fluorescence-activated cell sorting (FACS) tubes with sterile 4% albumin phosphate buffered saline (PBS) solution overnight (O/N). Wash the tubes twice with PBS and fill them with 500 µl of complete RPMI 1640 medium. Use these tubes for recovering the cells after sorting.
3. Collect the PBMCs in a tube and centrifuge at 400 x g at RT for 5 min.
4. Resuspend the cells in labeling buffer (see steps 2.5 and 2.6) and count them. The labeling buffer is sterile 2% fetal bovine serum and 1 mM ethylenediaminetetraacetic acid (EDTA) in PBS.
5. Prepare 3 FACS tubes with 10⁵ cells each and resuspend them in 100 µl of labeling buffer. These tubes will serve for defining the gates of the sorting.
   1. In the first tube add 5 µl of anti CD22 PerCP antibody (recommended in manufacturer's datasheet), in the second tube 20 µl of anti IgG PE (recommended in manufacturer's datasheet), and nothing in the third tube. Incubate on ice and in dark during 30 min.
6. Resuspend 5 million cells in 200 µl of labeling buffer in a FACS tube and add 10 µl of anti CD22 PerCP and 40 µl of anti IgG PE. Incubate the cells on ice and dark during 30 min. If the sorting of a higher number of cells is desired, scale up all the reagents.
7. Centrifuge cells at 400 x g with a low brake for 5 min at RT.
8. Decant softly the supernatant.
9. Resuspend the cells in 500 µl of labeling buffer. Centrifuge cells at 400 x g with a low brake for 5 min at RT. Repeat steps 2.7 and 2.8
10. Decant softly the supernatant and resuspend the cells in 300 µl of RPMI complete media.
11. Pass the cells through a 0.45 µm filter. Cells are ready to sort.

3. Sorting of the B Cells CD22+ and IgG+

1. Use 3 control tubes to establish the viability of the cells. In the control with no staining, use the forward and size scatter plot to define the lymphocyte gating. The controls with the CD22 PerCP and anti-IgG PE serve to establish the gates, and the sorting gate. Perform following previously reported data.  
   NOTE: A gate is a set of value limits (boundaries) that serve to isolate a specific group of cytometric events, in our case cells, from a large set. In the case of a sorting gate the value limits allows the recovery of the cytometric events, cells, which are inside of the boundaries defined: CD22+ and IgG+.
2. As a collection tube in the sorting, use the blocked tubes with 500 µl of complete RPMI 1640 medium, obtained from step 2.2.
3. Proceed to sort double positive: CD22+ IgG+. NOTE the final number of cells in each condition. Plate sorted cells on top of feeder cells as soon as possible.

4. Irradiation of Feeder Cells

NOTE: Perform the preparation of the feeder cells between 1 – 3 days before sorting. At least 5,000 wi38 cells are needed per well in a 96 round well plate. Perform steps 4.1, 4.2 and 4.4 in a hood.

1. Cultivate the wi38 cells at 37 °C and 5% CO₂ in complete RPMI 1640 medium (same as for PBMCs) until the desired number of cell are reached.
2. Irradiate them at 50 Grays, following a protocol described before. Perform irradiation once the wi38 cells have been trypsinized and resuspended in complete RPMI medium, or with wi38 attached to the culture flask and trypsinized after the irradiation. Follow the method for trypsinization indicated by the supplier of wi38 cells. Irradiate the number of cells necessary to cover the wells needed for plating IgG+ B cells (1% of the total PBMC counted in step 1.11).
3. Plate 90 µl containing 5,000 irradiated wi38 cells in each well of the 96 round well plate. Leave the wells in the outside row of the plate empty (because they evaporate faster), and only use the 60 wells in the middle of the plate for plating cells. Fill the outer wells that are framing the plate with 200 µl of PBS.
4. Place them in an incubator at 37 °C at 5% CO₂, until needed.

5. Plating Sorted PBMCs, Epstein-Barr Virus (EBV) Infection and Growing

1. Dilute the sorted PBMCs, to plate 50 cells in 50 µl of RPMI 1640 complete medium per well in the 96 round well plate (previously plated with wi38 irradiated cells). Perform steps in a hood equipped for EBV work. 
   NOTE: the infection of 50 cells per well is optimized and increases the likehood to produce a monoclonal antibody, however, it is recommended to verify this by polymerase chain reaction (PCR) as described before.
2. Add 60 µl of EBV supernatant (containing 3 – 4 x 10⁸ viral copies/ml) to each well in the 96 round well plate. CAUTION should be taken during EBV work.
3. Add 1 µg/ml of CpG (ODN2006) per well.
4. Leave the cells in an incubator at 37 °C at 5% CO₂.
5. Visually monitor clone growing under the microscope after one week.
   NOTE: Some examples of B cell growth can be seen below in the results section. Notice that growth rates of the clones may vary, and extra time might be necessary to start seeing some cell mass growing in the middle of the well.
6. After the 1^st two weeks monitor clone growing under the light microscope and proceed to the first media exchange. Slowly pipet 90 µl of medium from the top part of the well and collect in a clean 96-well plate. (B cells lie in the bottom of the well, so there is no risk to aspirate them). 
   1. Add to each well 100 µl of complete RPMI 1640 media supplemented with 1 µg/ml of CpG (ODN2006) and 50 U/ml of IL2. If clones are growing, analyze this first media exchange by enzyme-linked immuno sorbent assay (ELISA) as in step 6.
7. Monitor clone growing and change media after the 3^rd and the 4^th week of growing again by taking 90 µl of media and adding 100 µl of complete RPMI 1640 media supplemented with 1 µg/ml of CpG (ODN2006) and 50 U/ml of IL2. Use the collected supernatant to monitor IgG production by ELISA as in step 6.
   NOTE: After one month clone growing should be evident by observing aggregates of round cells that usually lie in the middle of the round bottom well.
8. At this step, change media in the same way that the previous weeks but only with complete RPMI 1640 media. A good indicator to know the right moment to change media is the media color, if it is becoming yellowish cells need a media exchange.
9. When 96-well plates are containing big clones (approximately 5 x 10⁵ cells), transfer them to a flat well of a 24-well plate. Add 300 µl of complete RPMI 1640 media to the new well. Resuspend the 96-well growing clone, by pipetting up and down several times, and transfer cells to the new well, distributing homogeneously. Add new media when needed.
   1. When wells of the 24-well plate are full of cells (approximately 5 x 10⁶ cells), transfer to a flat well of a 6-well plate. Add 2 ml of complete RPMI 1640 media to the new well. Resuspend the cells in the 24-well plate by pipetting up and down several times, and distribute them homogeneously in the new well.
10. Add media when needed. If cells are not to be maintained in culture, pellet the cells at 400 x g for 5 min at RT. Store the supernatants for ELISA testing. Wash the pellet of cells once with 1 x PBS, centrifuge again at 400 x g for 5 min, and stored dry at -80 °C until RNA extraction.
    1. When the cells in the 6-well plate become confluent (approximately 20 x 10⁶ cells), transfer to a 60 mm culture plate. Add 4 ml of complete RPMI 1640 media to the new plate. Resuspend the cells in 6-well plate and transfer them as done is steps 5.9 and 5.10.
11. Add new media when needed. At this step, freeze cells at 10 – 30 million/ml in 90% fetal calf serum and 10% DMSO. If larger amounts of cells are wanted, continue expansion of the clones in bigger surface plates.

6. ELISA for IgG Antibody Detection

1. Dispense 50 µl/well in an ELISA plate of goat F(ab)₂ anti-human Fc antibody diluted 1 200 in coating buffer. Coating buffer contains 50 mM sodium carbonate (Na₂CO₃) pH 9.6.
2. Seal the plates with a plastic sticker, and incubate 1 hr at 37 °C. Alternatively, incubate at 4 °C O/N.
3. Wash each well 6 times with 200 µl of washing buffer. Washing buffer contains 0.05% Tween-20 in 1 x PBS. Do not touch or scratch the well surface where the antibody has bound.
4. Block with blocking buffer, 100 µl/well. Blocking buffer contains 4% of non-fat dry milk in PBS. Seal the plate and incubate 1 hr at 37 °C.
5. Do not wash. Discard blocking buffer and slap the plate 3 times upside down on a paper towel to remove residual liquid.
6. Incubate clones' supernatants and standards.
   1. For a first test, dilute clone supernatants obtained in step 5.6 or 5.7 1/3 in RPMI. For the standards dilute with RPMI medium human IgG solution to get: 1,000 ng/ml, 500 ng/ml, 250 ng/ml, 125 ng/ml, 62.5 ng/ml, 31.25 ng/ml, 15.6 ng/ml and blank. Use 50 µl/well and incubate at 37 °C for 60 min. Analyze further dilutions of the clone' supernatant to test the antibody affinity, such as 1/10 or 1/100.
7. Wash as done in step 6.3.
8. Use 50 µl/well of goat F (ab)2 anti-human IgG Fc peroxidase conjugated diluted 1:20,000 in the incubation buffer. Incubation buffer contains 1% bovine serum albumin (BSA) and 0.02% Tween 20 in 1x PBS. Incubate at 37 °C for 60 min.
9. Wash as done in step 6.3.
10. Add 100 µl/well substrate solution containing 0.1% 3,3',5,5'-Tetramethylbenzidine (TMB). Incubate 10 min until a stable blue color forms in the wells. Be careful; do not wait too long!
11. Stop the reaction by adding 50 µl of 2 M sulfuric acid (H₂SO₄). Measure the absorption at 450 nm, within 30 min after stopping the reaction.
    NOTE: All the clones' supernatants with 3 standard deviations over the blank will be considered positive for IgG human antibodies. For confirmation of the positive clones this ELISA should be repeated at least three times in different supernatants of the same clone. Also to discard the possibility of unspecific cross reactivity is recommended to assure that there is no signal when supernatants are incubated in uncoated but blocked wells. At this step, a detection assay to screen for the antigenic specificity of the antibodies of interest can be performed prior to proceed to section 7.

7. RNA Isolation and First Strand complementary DNA (cDNA) Synthesis of the Producing IgG Clones

1. As soon as the production of IgG is confirmed by ELISA, extract the RNA of the clone.
2. For extracting RNA from cells growing in steps 5.7 or 5.9, use the superscript III cells direct cDNA synthesis kit, which allows obtaining DNA from 10,000 cells to one cell.
3. For extracting RNA from larger amounts of cells, or from the pellet stored in step 5.11 use the high pure RNA isolation kit. Other systems of RNA extraction can also be suitable at this step. Follow the instructions of the manufacturer.
4. For cell numbers between 1 x 10⁶ and 1 x 10⁴ use the reverse transcription system, following the instruction of the manufacturer. Other systems for first strand cDNA synthesis can also be used.

8. 1^st and 2^nd PCR for Amplification of the Heavy and Light Chains of the IgG-producing B-cell Clones

1. With the cDNA of the cell clones, obtained in step 5.6 and 5.7 and positive in the IgG ELISA in step 6, perform PCR with the primers listed in Table 1.
2. Set up independent PCRs for each IgG heavy, kappa and lambda chain. Make a stock solution with forward and reverse primers in equal concentrations. Add them to the reaction at 0.4 µM final concentration.
3. Use 1 µl of the cDNA synthesis to perform the 1^ST PCR. Here, perform 20 µl reactions using Takara Taq manufacturer instructions. Alternatively, use other polymerases.
4. Place the 1^st PCR under the following cycle conditions: 94 °C for 5 min; (50 cycles of: 94 °C for 30 sec; 58 °C for 30 sec, and 72 °C for 45 sec; 72 °C for 7 min; let it cool down at 4 °C.Include a blank in the reaction, to detect possible contaminations.
5. Prepare 1x TBE (89 mM Tris-borate and 2 mM EDTA). In a volume of 100 ml of 1x TBE add 1 g of agarose (1% agarose gel). Heat the solution in the microwave for approximately 1 min, until the agarose gets dissolved. Then add 4 µl of 10 mg/ml ethidium bromide (or equivalent DNA dye) and mix.
   1. Pour the content in a tray and wait until gets polymerized. Run 5 µl of the PCR mix in the gel to visualize the bands. Heavy chain fragments should be around 400 – 550 bp, while light chain should be around 300 – 400 bp. If the bands are not detected, proceed likewise to 2^nd PCR.
6. Use 1 µl of the first PCR mix to perform the 2^nd PCR. Use the same reagents that in step 8.2 but using the suitable mix of primers (see Table 1). For the 2^nd PCR use the following conditions: 94 °C for 5 min; (50 cycles of: 94 °C for 30 sec; 56.5 °C for 30 sec and 72 °C for 55 sec); 72 °C for 7 min; let it cool down at 4 °C. Include a blank in the reaction, to detect possible PCR contaminations.
7. Prepare an agarose gel as described in 8.5. Run the gel to visualize the PCR product as in 8.5.1 Use the amplifications which contain the right size fragments for cloning in step 9 and produce the antibodies in step 10.
8. Sequence the DNA, by using 10 µl reaction containing: 100 ng of the 2^nd PCR, 0.2 µM of the primer or primer mix, 1 µl of the terminator and 1 µl of the buffer. Perform the sequencing reaction under these conditions: (25 cycles of: 96 °C for 10 sec; 50 °C for 5 sec; and to 60 °C for 4 min); 60 °C for 7 min; let it cool down at 4 °C.
9. Purify the sequencing reactions with microspin G50 columns following manufacturer's instructions. Evaluate the sequences of the ligation in an automatic sequence analyzer as described before. Electropherograms should be clean and correspond to a single immunoglobulin sequence.         
   NOTE: If the PCR products show unclear or mixed immunoglobulin sequences, please consider to clone them using the TopoTA system, to obtain isolated sequences and then proceed to step 9.

9. Cloning and Sequencing of the Heavy and Light Chains of the Producing IgG clones

1. Prepare 1 µg of DNA of vectors pFUSE2ss-CLIg-hk, pFUSE2ss-CLIg-hl2 and pFUSEss-CHIg-hG1.
2. Digest these vectors with the appropriate restriction enzymes. Use EcoRI and BsiWI for pFUSE2ss-CLIg-hk, EcoRI and AvrII for pFUSE2ss-CLIg-hl2, and EcoRI and NheI for pFUSEss-CHIg-hG1. Use 3 units of each restriction enzyme for 1 µg of DNA of vector.
   1. Digest with one enzyme and purify the digested product with a PCR Purification Kit. Digest with the second enzyme and purify with a Gel Extraction Kit (according to manufacturer's protocol). Cut the fragment of interest from the agarose gel. Prepare agarose gel as in step 8.5.
3. Digest the 2^nd PCR products of the producing IgG clone: EcoRI and BsiWI for kappa chain amplification, EcoRI and AvrII for lambda chain amplification, and EcoRI and NheI for heavy chain amplification. Digest with one enzyme and purify the digested product with a PCR Purification Kit. Digest with the second enzyme and purify the digested product with a PCR Purification Kit. Use same units of enzymes and DNA amount as in step 9.2.
4. Ligate the PCR fragments inside of the vectors with T4 DNA ligase 16 °C O/N following manufacturer's recommendations. The ratio used should be 1 : 2 (vector : insert).
5. Use 1 µl of the ligation to transform DH5α bacteria. Follow DH5α manufacturer' conditions for transformation. Spread bacteria in blasticidin or zeocin plates, depending on the type of vector used. Incubate the plates O/N at 37 °C.
6. Grow single colonies, and extract DNA with a miniprep kit, following manufacturer' instructions. Use for single colony grow and DNA extraction the manufacturer's, recommendations from the DNA miniprep kit. Analyze them by digestion with the enzymes used for preparing vectors and inserts as in steps 9.2 and 9.3.
7. Sequence the DNA, by using 100 ng of the vector as done in steps 8.8 and 8.9.

10. Production of Antibodies in HEK cells

1. Grow HEK cells in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum, 1% L-glucose, 1% penicillin/streptavidin (DMEM+) to a confluency of 75% in a 150 mm cell culture plate. Perform in a hood step 10.1 to 10.7.
2. Before transfection, exchange the medium to medium as before but without fetal calf serum.
3. For each heavy, light chain transfection, prepare a transfection mix with 2.5 ml of the DMEM (without serum), 9 µg of the vector with the heavy chain, 6 µg of the vector with the light chain, and 100 µl of polyethylenimine (PEI) solution (from a 1 µg/µl stock). Incubate at RT for 15 min.
4. Gently add 2.5 ml of the transfection mix prepared in step 10.3 to the HEK cells in the plate. Rock the plate to distribute homogeneously.
5. Incubate the cells in the incubator at 37 °C with 5% CO₂ for 24 hr.
6. Change the culture medium to medium without fetal calf serum.
7. Collect the medium from the plates 4 days later.        
   NOTE: The antibodies in the media can be used to characterize their reactivity in vitro and in vivo.

Table 1. Primers used.