Cloning of hybridomas

Centrifuge cells from at least two wells of a 24-well plate that contain confluent monolayers of hybridoma cells. 

Count the cells and then dilute to give 50 hybridoma cells in 20 ml of medium containing feeder cells. 

Examine the plates seven to ten days later and screen those wells containing only single colonies. 

Pick cells from positive wells into 24-well plates, expand, and freeze in liquid 

Long-term storage of cells in liquid nitrogen 

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Monoclonal antibody, an antibody produced by a single clone of cells (specifically, a single clone of hybridoma cells) and therefore a single pure homogeneous type of antibody that binds to the same target. 

Parks, D. R., Bryan, V M., Oi, V. I, and Herzenberg, L. A (1979) Antigen-specific identification and cloning of hybridomas with a fluorescent-activated cell sorter (FACS) Proc Natl Acad Sci USA 76, 1962-1966... 

The standard protocol used today for the production of hybridomas is somewhat different from that employed by Kohler and Milstein in 1975. The major modifications (proposed by De St Groth and Scheideger (1980)) involve the use of polyethylene glycol (PEG) as a fusion agent, the use of myeloma cells that do not produce antibodies, the cloning of hybridomas using limitant dilution, and cultivation on plastic plates. 

Five steps are important in the production of hybridomas to be used for the secretion of mAbs of a determined specificity (Nelson et al., 2000) (1) immunization of mice, (2) fusion and selection of secreting hybridomas (3) cloning of hybridomas, (4) definition of the isotype of mAbs obtained, and (5) further development (Figure 17.3). 

Monoclonal Antibodies from Murine Hybridomas. Murine hybridomas are hybrid cells formed by the fusion of a mouse cancer cell with lymphocytes committed to the secretion of antibodies. The lymphocytes are obtained from the spleens of mice immunized with antigens of interest, e.g. extracellular glycoproteins from a fungal plant pathogen, and under normal circumstances would produce antibodies, but would be unable to divide. Fusion with cancer cells yields hybrids that continue to divide, as well as secrete antibodies. Thus, from a population of lymphocytes from the spleen of an immunized mouse it is possible to generate numerous clones of hybridomas and each clone will yield a single antibody, i.e. monoclonal antibodies. 

Jefferis, R., Glycosylation of recombinant antibody therapeutics. BiotechnoL Prog., 21, 11, 2005. Marder, et al.. Selective cloning of hybridoma cells for enhanced immunoglobulin production using flow cytometric cell sorting and automated laser nephelometry. Cytometry, 11, 498, 1990. 

Mouse antibodies can also be displayed on phage, and a series of primers which will pick up almost all mouse V genes has been described [38]. These are likely to be very useful for the cloning of hybridoma V regions and for making antibody libraries from immunized mice. 

Feeder cells for fusion cultures and cloning of hybridomas (essential for fusions using rat myelomas) rat fibroblast cell lines derived from the xiphisternae of various strains are suspended in DMEM and irradiated with about 30 Gy (3000 rad) of X- or gamma rays, then frozen in 95% FCS, 5% DMSO, and stored in liquid nitrogen as aliquots of 5 X 10 cells. Use one aliquot for each 200 ml of HAT or HT medium. Alternatively, use thymocytes from spleen donors. 

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