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        The production of monoclonal antibody by hybridoma

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        2597

         

          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
        37°C 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 37°C) 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 37°C 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 @ 37°C 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.

        Keywords: monoclonal antibody ; hybridoma ; fusion



         
         
          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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