Generation of Murine Monoclonal Antibodies by Hybridoma Technology

Balb/c or NMRI mice (Mus musculus) from our breeding colony at the University of Potsdam (Potsdam, Germany) were used for the production of monoclonal antibodies. The animal work was conducted according to relevant national and international guidelines. The study was approved by the Brandenburg Ministry of Environment, Health, and Consumer Protection (reference number V3-2347-A16-4-2012).

1. Preparation of Immunoconjugates

1. For coupling the antigen to a carrier, use standard coupling protocols via amino-, carboxy, or sulfo-groups, such as by glutaraldehyde, N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC), or N-[y-maleimidobutyryloxy]succinimide ester (sulfo-GMBS).
2. For coupling (e.g., with glutaraldehyde)¹³, use the target protein or peptide and the carrier (ovalbumin, bovine serum albumin, or keyhole limpet hemocyanin) in a 1:1 ratio. Dilute the target protein or peptide in phosphate-buffered saline (PBS) and the carrier protein in 50 mM NaHCO₃ buffer. Adapt the volume and the concentration to the amount necessary for immunizations.
   1. Mix both solutions and add 1.25% glutaraldehyde. Mix and incubate the sample for 4 h at room temperature (RT). To select the hybridoma cells, make the same immunoconjugate, but with another carrier, in order to avoid the selection of unspecific carrier binders.
3. Dialyze the coupling sample by performing a fast dialysis with commercial coarse resin (e.g., Sephadex G25).
   1. Perforate the bottom of a 1.5-mL reaction vial with a hollow needle and place it in a 15-mL tube. Put glass wool in the bottom for sealing and cover it with 300 mg of coarse resin (to the calibration mark of 0.5 mL).
   2. Carefully, put 1 mL PBS dropwise into a 1.5-mL reaction tube and make sure that the glass wool is still sealing the perforation. Let the coarse resin swell and centrifuge the column at 500 x g for 2 min. One such column can be used to dialyze 200 µL of the coupling sample. For higher volumes, make more columns.
   3. Change the tube and drop the coupling solution onto the swelled coarse resin. Centrifuge again at 500 x g for 2 min. Remove the eluent from the tube and use it for immunization.

2. Immunization of Animals

1. Choose two or three 6- to 12-week-old Balb/c mice for immunization.
2. For one animal, prepare 150 – 200 µg of the immunoconjugate in 200 µL of PBS and mix it with 100 µL of complete Freund´s adjuvant (CFA). Mix the solution 3 times with a short and thin needle (0.6 mm diameter) on a 1-mL syringe. Inject the solution intraperitoneally into the mouse.
3. Give a booster injection 6 weeks later with the same amount of the immunoconjugate, but without CFA. Take blood 7 days later (out of the retroorbital sinus) and prepare the serum by incubating the blood for 3 – 4 h at room temperature (RT).
   1. Centrifuge the mixture at 3,000 x g for 5 min. Take the serum supernatant and transfer it into a new tube. Store it at -20 °C. Discard the pellet.
4. Analyze the sera via ELISA, as described in Step 3. For fusion, choose the mouse with the highest serum titer.
5. In preparation for cell fusion, boost the mouse with 150 – 200 µg of the immunoconjugate in 300 µL of PBS without adjuvant only 4 days before the fusion.

3. ELISA

1. Prepare an antigen solution with a concentration of 5 µg/mL in PBS and transfer 50 µL into each ELISA well of a 96-well plate.
2. Incubate the plate for 2 h at 37 °C or overnight at 4 °C in a humid chamber.
3. Wash the plate 3 times with tap water. Block the wells with 80 µL of PBS/5% neonatal calf serum (NCS) per well for 1 h at RT in a humid chamber. Prepare the serum dilutions in a 1:5 series starting with 1:50. For analysis of the cell culture, use the undiluted culture supernatant.
4. Wash the plate 3 times with tap water. Pipette 50 µL of the sample per well and incubate for 1 h at RT in a humid chamber. Prepare the secondary antibody solution in a 1:5,000 dilution of a goat anti-mouse IgG antibody conjugated to horseradish peroxidase (HRP).
5. Wash the plate 3 times with tap water and add 50 µL of the secondary antibody solution per well. Incubate for 45 min at RT in a humid chamber.
6. Prepare a fresh substrate solution. Make stock solutions of 0.1 M NaH₂PO₄ and 0.1% hydrogen peroxide. Take 5 parts NaH₂PO₄, 4 parts hydrogen peroxide, and 1 part tetramethylbenzidine for the fresh substrate solution.
   1. Wash the plate 10 times with tap water. Pipette 50 µL of fresh substrate solution into the wells and incubate for 5 – 10 min in the dark.
7. Stop the reaction with 50 µL 1 M H₂SO₄ per well. Using a plate reader, measure the optical density at 450 nm with a reference wavelength at 630 nm.

4. Preparation of Myeloma Cell Cultures

1. From now on, perform all work under sterile conditions.
2. Prepare a fresh culture medium for cell cultivation. Supplement 500 mL of RPMI1640 with 10% fetal calf serum (50 mL), 2 mM L-glutamine (5 mL), and 50 µM mercaptoethanol (5 mL). Substitute the glutamine every 7 days.
3. Thaw a vial of murine SP2/0 cells in warm water. Transfer the cell solution into 10 mL of fresh culture medium and centrifuge for 10 min at 200 x g. Discard the supernatant and resuspend the pellet in 1 mL of fresh culture medium.
4. Transfer the cells in a 25-cm² flask and incubate them at 37 °C and 5% CO₂ in an incubator. Expand the cells to two 75-m² flasks before fusion. Conserve aliquots for long-term storage, as described in Step 7.

5. Preparation of Feeder Cells

1. Sacrifice a 12-week-old NMRI mouse (according to national and institutional guidelines; e.g., by CO₂ inhalation). Remove the fur and clean the abdomen with 70% ethanol.
2. Inject 5 mL of ice cold RPMI medium into the abdomen, massage the abdomen with a tweezer, and aspirate the feeder cell suspension. Place the cells in an empty 50-mL tube. Perform this step once again. 7 to 10 mL of the feeder cell suspension should be retrieved.
3. Prepare a 100-mL feeder cell suspension by adding 90 mL of full cell culture medium (RPMI 1640, 10% fetal calf serum, 2 mM glutamine, and 50 µM mercaptoethanol). Mix it and transfer 100 µL/well into 10 x 96-well cell culture plates. Incubate the plates overnight at 37 °C and 5% CO₂ in an incubator.

6. Cell Fusion

1. Sacrifice the immunized mouse by cervical dislocation or CO₂ inhalation and excise the spleen¹⁴. Take additional blood from the heart and prepare the serum, as described in step 2.3, as a positive control in the ELISA.
2. Prepare a spleen cell suspension by pressing the spleen through a cell strainer. Fill up to 20 mL with full cell culture medium. Harvest the cells in 50-mL tubes by centrifugation (200 x g, 10 min, and 4 °C). Discard the supernatant.
3. Rinse the cell culture flask and harvest the myeloma cells by centrifugation (200 x g, 10 min, and 4 °C) and discard the supernatant.
4. Resuspend the cell pellets in 20 mL of balanced salt buffer (BSS) (125 mM NaCl, 5 mM KCl, 4 mM CaCl₂, 2.5 mM MgCl₂, and 5 mM Tris-HCl; pH 7.4) and centrifuge again. Repeat the washing steps three times.
5. For adjusting the splenocyte:myeloma cell ratio, count the cells after the second washing step by first resuspending the pellet in 1 mL of BSS buffer. Make a 1:10 dilution and transfer 10 µL to a cell counting chamber. Count the cells and determine the cell concentration in 1 mL.
6. Adjust the cell numbers in the BSS buffer to three-parts splenocytes and one-part myeloma cells in a 1-mL final volume. Take 200 µL of the cell suspension and mix it with 200 µL of PEG8000 (1 g in 4 mL of BSS buffer). Transfer the 400 µL into an electroporation cuvette with a diameter of 2 mm and set the pulse (600 – 650 V and 25 ms).
   1. After the pulse, incubate the cells at RT for 3 min in the cuvette. Remove the cells and put them dropwise into an Erlenmeyer flask with 100 mL of HAT Medium (RPMI 1640, 10% FCS, 2 mM glutamine, 50 µM mercaptoethanol, 100 µM hypoxanthine, 5.8 µM azaserine, and 16 µM thymidine).
   2. Repeat the fusion 4 times until the 1-mL batch is spent. Incubate the Erlenmeyer flask for 3 h at 37 °C and 5% CO₂ in an incubator.
7. Supplement the cell suspension again with hypoxanthine, azaserine, and thymidine before plating the cells on the feeder layer. Add 100 µL of the cell suspension per well to the feeder layer plates and incubate the plates at 37 °C and 5% CO₂ (in an incubator). The final concentration within the well should be 100 µM hypoxanthine, 5.8 µM azaserine, and 16 µM thymidine.
8. Perform a microscopic analysis (magnification: 10X objective lens, 10X eye-piece) for clonal growth in each well of the plate after 7 days in order to determine if the wells are mono- or polyclonal. Monoclonal cells are one cluster of cells that originate from one single cell. Polyclonal cells are multiple clusters of cells in one well. Change the complete media after 7 days and incubate again for 7 days in HAT medium.
9. Change the HAT medium and cultivate the cells in full cell culture medium. Use the supernatant to perform an ELISA, as described in Step 3. Positive hybridoma cells must be expanded in 24-well plates according to the tests for the production of specific antibodies. If the cells are polyclonal, a limited dilution has to be performed (described in step 8).

7. Cryoconservation of Hybridomas

1. Cryoconserve positive mono- and polyclonal hybridomas for long-term storage. Harvest the cells from a 24-well plate with a confluence of > 60% by centrifugation (200 x g, 10 min, and 4 °C).
2. Resuspend the pellet in 500 µL of full cell culture media and transfer it into a cryotube. Add 500 µL of freeze medium (RPMI 1640, 20% FCS, and 15% dimethylsulfoxide), mix it, and put it in in a Styrofoam box or a special freezing box. Store at -80 °C for 3 days. After 3 days, the cryotube can be transferred to liquid nitrogen for long-term storage.

8. Limiting Dilution of Polyclonal Hybridomas

1. Make a feeder layer culture, as described in Step 5. Harvest positive polyclonal cells, as described in Step 7.1. Count the cells, as described in Step 6.5, and adjust the cell number to 10 cells/mL, with a final volume of 10 mL for a 96-well plate. Add 100 µL/well onto the feeder layer.
2. Incubate for 7 days at 37 °C and analyze the clonal growth microscopically (magnification: 10X objective lens, 10X eye-piece). Make an ELISA of the culture supernatant, as described in Step 3, and identify the appropriate monoclonal hybridomas for mass culture.

9. Mass Culture and Purification of Monoclonal Antibodies

1. Transfer the monoclonal hybridomas with the appropriate ELISA signals to 25 cm² or 75 cm² flasks and collect up to 500 mL of the culture supernatant. Test the culture weekly for specific antibody production and conserve 3 – 5 aliquots per hybridoma for long-term storage, as described in step 7.
2. Centrifuge the culture supernatant at 4,900 x g and 4 °C for 15 min and filter it through a 0.45-µm filter. Mix the supernatant with binding buffer (4 M NaCl and 2 M glycine NaOH, pH 8.9) in a 3:1 ratio.
3. Prepare a protein A column by washing it with washing buffer (dilute the binding buffer 1:3 in dH₂O). Pass the supernatant from Step 9.2 over the column and collect the flow-through. Elute the bound antibody with 0.1 M citrate, pH 3.5 and neutralize the pH immediately with 500 µL of Tris-HCl, pH 9.0.
4. Characterize the eluted antibody by SDS-PAGE, Western Blot, and ELISA, and validate it for the final application.
   1. For the SDS PAGE, take 3 – 5 µg of purified antibody solution per lane (a 20-µL sample), add 5 µL of 4x SDS sample loading buffer (8% SDS, 20% 2-mercaptoethanol, 40% glycerin, and 0.015% bromophenol, reduced), and heat the probes at 95 °C for 5 min. As a control, use the same sample with a purified antibody of the same isotype but irrelevant specificity.
   2. Load the samples on a 12.5% SDS gel, place 5 µL of an unstained protein ladder in an additional lane, and separate them by electrophoresis. Stain the gel with Coomassie Brilliant Blue and document the results. A basic SDS protocol was established by Laemmli in 1970¹⁵.