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The production of monoclonal antibody by hybridoma fusion: Immortalization of sensitized B lymphocytes from immune mice.
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  Author Information
 
  Primary Author
  Jody D. Berry ( phagemid@hotmail.com )
 
  Affiliation
  University of Manitoba , Canada
 
  Co-Author(s)
  Franscesca Ranada 
 
 
  Protocol Information
 
  Protocol ID / PID
  PUB335
 
  Category
  Cell Biology
  Molecular Biology
 
  Title
  The production of monoclonal antibody by hybridoma fusion: Immortalization of sensitized B lymphocytes from immune mice.  
 
  Overview
  We outline a simple and contemporary protocol for the development of monoclonal antibodies using hybridoma fusion in immune mice (1). While the basic style of this fusion is similar to others (2,3), this protocol has several subtle but significant modifications. These include the use of: spleen perfusion rather than crushing to separate the spleen cells (which reduces the amount of contaminating fibroblast and lipoidal material) ; commercial Hybridoma CLoning Factors (rather than feeder layers); commercially prepared semi-solid HAT containing agarose, rather than limiting dilution. Elements of this protocol span several research institutions and many years of experience (4,5,6).  
 
  Material
  Sterile 10, 25, and 50 ml serological pipettes, Pipet-Aid, 15 and 50 ml centrifuge tubes (Falcon sterile), Tissue culture flasks (25 cm2, 75 cm2 and 125 cm2), indelible waterproof marker. Sterile 1 ml pipette tips for Gilson p1000 pipetteman.
370C waterbath and thermometer.
Humidified 370C, 5% CO2 tissue culture cabinet.
Class II Biological Safety Cabinet.
Inverted Microscope.
Benchtop centrifuge containing a 4 swinging bucket rotor, at room temperature.
Stopwatch or timer.
Multichannel pipettor and appropriate sterile tips, sterile disposable petri dishes. Sterile 96-well flat-bottomed cell culture plates.

Reagents:
1-3 x 10 8-9 immune spleen cells
1-6 x10 7-8 myeloma cells in log phase of growth
Complete Media No Sera (CMNS) for washing of the myeloma and spleen cells.
Hybridoma medium CM - HAT {Cell Mab (BD), 10% FBS (or HS) ; 5% Origen HCF (hybridoma cloning factor) containing 4mM L-glutamine and antibiotics} to be used for plating hybridomas after the fusion.

Hybridoma medium CM - HT (NO AMINOPTERIN) {Cell Mab (BD), 10% FBS 5% Origen HCF containing 4mM L-glutamine and antibiotics} to be used for fusion maintenance stored in the refrigerator at 4-60C. feeding fusions on days 4, 8, and 12, and subsequent passages.

Thawed inactivated and pre-filtered commercial Fetal Bovine serum (FBS) or Horse Serum (HS) stored in the refrigerator at 40C. Must be pretested for myeloma growth from single cells.

L-glutamine, 200mM, 100X solution stored at -200C freezer.
The L-gln is thawed and warmed until completely in solution. The L-gln is dispensed into media to supplement growth. L-gln is added to 2 mM for myelomas, and 4 mM for hybridoma media.

Penicillin, Streptomycin, Amphotericin (antibacterial-antifungal) is stored at -200C until needed and it is thawed and added to Cell Mab Media to 1%.

Cell Mab Media, Quantum Yield from BD is stored in the refrigerator at 40C in the dark. Myeloma growth media is Cell Mab Media with added L-gln to 2 mM and antibiotic/antimycotic solution to 1% and is called CMNS. Do not add antibiotics to media when growing myelomas for stock, only for pre-fusion growth. Indicate presence or absence of each reagent on the bottle label. Do not adjust the pH as it contains HEPES biological buffer already.

1 bottle of PEG 1500 in Hepes (Roche) (use fresh bottle that has never been opened)

8-Azaguanine is stored as the dried powder supplied by SIGMA at -700C until needed. Reconstitute 1 vial / 500 ml of media and add entire contents to 500 ml media (eg. 2 vials/ litre).

Myeloma Media is CM which has 10% FBS (or HS) and 8-Aza (1 X) stored in the refrigerator at 40c.

Clonal cell medium D (Stemcell, Vancouver) contains HAT and methyl cellulose for semi-solid direct cloning from the fusion. This comes in 90 ml bottles with a CoA and must be "melted at 37Oc in a waterbath in the morning of the day of the fusion. Loosen the cap and leave in CO2 incubator to sufficiently gas the medium D and bring the pH down.

Hybridoma supplements HT [hypoxanthine, thymidine] are to be used in medium for the section of hybridomas and maintenace of hybridomas through the cloning stages respectively.

Origen HCF can be obtained directly from Igen and is a cell supernatant produced from a macrophage-like cell-line. It can be thawed and aliqouted to 15 ml tubes at 5 ml per tube and stored frozen at -200C. Positive Hybridomas are fed HCF through the first subcloning and are gradually weaned. It is not necessary to continue to supplement unless you have a particularly difficult hybridoma clone. This and other additives have been shown to be more effective in promoting new hybridoma growth than conventional feeder layers.


 
 
  Procedure
  At least one week prior to expected fusion date thaw a fresh vial of myeloma cells. It is advisable to keep several flasks at different densities so that you can choose the best one on the day of the fusion. We generally try to use a flask that is actively dividing and at a cell density of 3-6x105 cells/ml. Do not let them overgrow or they will enter a decline phase.

Two to five days before the scheduled fusion give a final injection of ~5ug of antigen in PBS i.p. or intravenously in tail vein of the mouse (with high titer already determined).

1. Spin down myelomas and wash with 30 ml serum free media (CMNS has glutamine). Use tabletop centrifuge at 850 rpm for 12 minutes. Perform viable cell count with trypan blue exclusion principle, and wash cells with 30 ml of RPMI-CMNS. Spin down as above, resuspend in CMNS and disperse. Leave at 37C until spleens are retrieved.
Test aminopterin sensitivity. Keep 1 million myeloma cells for control plate and transfer into a 15ml conical. To do so, add 15 ml of HAT media to the million myeloma cells and plate out 2 drops/well on a 96 well plate.

2. Remove spleen from mouse in the biohazard facility. Euthanise the mice and submerge it in 70% ETOH. Let the mouse air dry on its right side on a paper towel. Remove spleen using sterile instruments and carefully put into labeled 10 ml of RPMI-CM with antibiotics and 20% FCS for transport back to the lab. Dispose of mouse and leave facility.

3. Place spleen into sterile petri dishes. Add 10 ml of RP-I-CMNS and perfuse the cells out of the spleen. Poke the spleen 8-10 times with an 18 ga needle (hold with sterile forceps). Use a 21 ga on a 3 ml syringe to draw up some RPMI. Inject the RPMI slowly into the spleen about 50-100 times until nearly all the cells are washed out. Discard the spleens into the biohazards bag.

4. Collect and transfer the spleen cells to a new 50 ml conical tube. Rinse out the dish 2X with 10 ml of RPMI-CMNS and pool with the first 10 ml (the use of perfusion removes the production of large debris seen with grinding, and obviates the need to let the debris settle). Spin down at 900 rpm for 12 minutes. Discard the supernatant to bleach container. Wash the cells with another 30 ml RPMI-CMNS. Remove a small sample and count the viable cell/ml and spin again as above. Combine the cells at a ratio of 5:1 (spleen cells: myeloma cells) and never 1X10 myeloma cells.

5. Wash both the myeloma and spleen cells 2 more times with 30 ml of RPMI-CMNS. Spin at 800 rpm for 12 minutes.

6. Remove supernatant and resuspend cells in 5 ml of RPMI-CMNS and pool together. Fill volume to 30 ml and spin down as before.

7. Aspirate all fluid into bleach vessel. Break up pellet by gently tapping on the flow hood surface. Add 1 ml of BMB REG1500 (prewarmed to 37C) dropwise with 1 cc needle over 1 minute. Swirl and tap the conical gently while adding the PEG to resuspend the cells.

8. Add 1 ml of RPMI-CMNS to the PEG cells gently over 1 minute while swirling (to dilute the PEG).

9. Add 8 ml RPMI-CMNS over 2 minutes to slowly dilute out the PEG.

10. Incubate the cells in the 37C waterbath for 10 minutes. Centrifuge the cells at 700 rpm for 10 minutes (the membranes are still very weak).

11. Aspirate all fluid, and add 5 ml of RPMI-CM-10% FCS WITHOUT RESUSPENDING THE CELLS! The cells will disperse adequately by simply adding the media at this point.

12. Incubate @ 37C another 10 minutes.

(12.5 Meanwhile put aside 1 ml of Clonacell medium D for myeloma testing. )

13. Gently dilute cells in 5 ml of Complete media and transfer into 95 ml of Clonacell Medium D (HAT) media (with 5 ml of HCF) and plate out 10 ml per small petri plate.

14. Dilute about 1000 P3X63 Ag8.653 myeloma cells into 1 ml of mediu D and transfer into a single well of a 24 well plate. This is the myeloma/HAT control. P

15. Place plates in incubator two plates inside of a large petri plate, with an additional petri plate full of dH20 without a lid for humidity. Leave for 10-18 days under 5% CO2 overlay at 37 degrees.

15. Pick clones from semisolid agarose into 96 well plates containing 150-200 ul of CM- HT. Screen sups 4 days later in ELISA. Move positive clones up to 24 well plates.

16. Heavy growth will require changing of the media at day 8 (+/- 150 ml). Should see macroscopic colonies at this time.
At this time can decrease the HCF to 0.5% (gradually- 2%, then 1%, then 0.5%) in the cloning plates.

17. Isotype via supernatants and grow up for ascites/ large flask production and further freeze down.  
 
  Troubleshooting
 
I strongly reccommend to use Southern Biotech Goat anti-Mouse Ig (H+L)HRP Chains for screening supernatants. We have screened and developed Mabs to various antigens and have surprisingly found out that using some secondary reagents from several other major companies can result in weak or no reactivity.



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

2) Lane, R.D. A short duration polyethylene glycol fusion technique for increasing production of monoclonal antibody-secreting hybridomas. 1985. J. Immunol. Meth. 81:223-228.

3) Harlow, E. and D. Lane. Antibodies: A laboratory manual.Cold Spring Harbour Laboratory Press. 1988.

4) Kubitz, D. The Scripps Research Institute. La Jolla. Personal Communication.

5) Zhong, G., Berry, J.D., and Choukri, S. (1996) Mapping epitopes of Chlamydia trachomatis neutralizing monoclonal antibodies using phage random peptide libraries. J. Indust. Microbiol. Biotech. 19, 71-76.

6) Berry, J.D. , Licea, A., Popkov, M., Cortez, X., Fuller, R., Elia, M., Kerwin, L., and C.F. Barbas III. (2003) Rapid monoclonal antibody generation via dendritic cell targeting in vivo. Hybridoma and Hybridomics 22 (1), 23-31.  
 
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