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Monoclonal Antibody Development Core

GENERAL FUSION PROTOCOL

Materials

P3X63Ag8.653 murine myeloma (maintained at < 1 x 106/ml)
50% w/v PEG 1500, warmed to 37° C

Medium

DMEM (4g/L glucose) supplemented with 20% fetal bovine serum, 4 mM L-glutamine, 1 mM sodium pyruvate, 50 U penicillin, 50 μg streptomycin and 50 μM 2-ME in the absence or presence of HAT or HT for selection and Hybridoma Cloning Factor (1% final).

Cell Preparation

  1. Tease spleen in ice cold serum-free medium (I-0). Pass cell suspension through a Falcon 70 micron cell filter and suspend in 50 ml of ice cold I-0. Centrifuge and wash cells three times at 4° C in I-0. Resuspend cells after the third wash in 10 ml I-0 and count viable cells. Keep cells on ice.

  2. Concurrently with the spleenocytes, centrifuge and wash myeloma cells three times, using I-0 and resuspend in I-0. Count viable cells.

  3. Add an appropriate number of myeloma cells to the entire volume of spleen cells according to the following ratios and centrifuge together.
SpeciesSpleenocytes: Myeloma
mouse - mouse5:1
hamster - mouse4:1

Fusion Protocol

Aspirate all supernatant and suspend pellet by tapping the end of the tube. Place tube in container of warm water (37°C). Gradually, over a period of 60 seconds, add 1 ml of 37° C 50% w/v PEG while tapping the side of the tube to achieve thorough mixing. Over the next 60 seconds continue to mix. Dilute the PEG/cell mixture slowly by adding dropwise 2 ml of I-0 over a 2 minute time span. Next use a 10 ml pipet and add 8 ml 37° C IMDM containing 10% FCS over a 4 minute period. Bring the total volume to 50 ml using I-10. Centrifuge at 4°C and resuspend in medium at the appropriate volume to bring the cells to the following concentrations:

TypeCell Concentration
mouse1.0 x 106 cells per ml
hamster0.6 x 106 cells per ml

Add 150 μl added per well. An additional 50 μl of a 4X HAT solution can be added at 16-24 hours after fusion.

Feeding Schedule

The day of the fusion is considered day 0. Fusion plates are examined at 24-48 hours for any abnormalities (i.e. bacterial contamination). On day 7, wells are inspected visually and then fed. One half of the volume in each well is aspirated using a sterile pasteur pipet. A new pipet is used for each plate. Wells are fed with 125 μl of I-20 + 2ME + HAT on days 7, 11 and thereafter as needed, i.e. Mon., Wed., Fri.

Cultures are examined visually at each feeding. Once a majority of wells appear 50% confluent for growth, supernatants are harvested for screening. Plates are fed at this time.

**Investigators should provide information on the serum titer of the immunized animal. If the titer is greater than 1:10,000, it may be important to feed the cultures at least 3-4 times prior to the screening. This will essentially "wash away" any antibody release from dying B cells and decrease background Ig levels.

Protocol References

Sheehan KCF, Lai KS, Dunn GP, Bruce AT, Diamond MS, Hentel JD, Dungo-Arthur C, Carrero JA, White JM, Hertzog PJ and Shreiber RD. Blocking monoclonal antibodies specific for mouse IFN-alpha/beta receptor subunit 1 (IFNAR1) from mice immunized by vivo hydrodynamic transfection. J Interferon Cytokine Res. 2006. 26:804-19.

Sheehan KCF. (2007) Production of Monoclonal Antibodies. In Making and using antibodies: a practical handbook. Eds GC Howard and MR Kaser. (73-94). Boca Raton, Fl. CRC Press.

Sheehan KC, Ruddle NH, Schreiber RD. Generation and characterization of hamster monoclonal antibodies that neutralize murine Tumor Necrosis Factors. J Immunol 142:3884-3893, 1989.

Sheehan KC, Calderon J, Schreiber RD. Generation and characterization of monoclonal antibodies specific for the human IFN gamma  receptor. J Immunol 140:4231-4237, 1988.

Sanchez-Madrid F, Szklut P, Springer TA. Stable hamster-mouse hybridomas producing IgG and IgM hamster monoclonal antibodies of defined specificity. J Immunol 130:309-312, 1983.

Kohler G, Milstein, C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495-497, 1975

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