Achieving Monoclonality in Hybridoma Cultures

In this tech tip you will learn about subcloning techniques, including limiting dilution assay in liquid media and semi-solid cloning, for establishing monoclonality in hybridoma cultures

The ability of monoclonal antibodies (mAbs) to bind to a specific epitope on a target antigen makes them attractive candidates for a variety of applications, including cell phenotyping, diagnostics, and therapeutics. As an early step in monoclonal antibody development, hybridomas are generated by fusing antibody-secreting B cells with myeloma cells. To ensure that the hybridomas generated only secrete monoclonal antibodies of appropriate specificity, a monoclonal cell line is generated by a procedure called subcloning. Typically the following subcloning methods are used for achieving monoclonality in hybridoma cultures:

  • Subcloning using limiting dilution assay: Conventionally, hybridoma cultures have been subcloned using limiting dilution assay (LDA) in liquid selection media. This method is also compatible with modern liquid handling platforms. In this technique, a liquid medium such as ClonaCell™-HY Medium E or ClonaCell™-HY Liquid HAT Selection Medium is used to dilute the hybridoma cells and plate at a very low dilution, typically 0.5 or 1 cell/well, in a 96-well or 384-well plate. Monoclonality can be confirmed by viewing under a microscope or by digital imaging to verify that only a single cell is seeded into each well. LDA plates should be cultured for 7 - 10 days to expand surviving monoclonal hybridomas prior to screening.
  • Subcloning using semi-solid media: Hybridoma cultures can also be subcloned using semi-solid media. ClonaCell™-HY Medium D , a methylcellulose-based semi-solid medium containing selective agents hypoxanthine, aminopterin, and thymidine (HAT), allows for discrete colony growth required for subcloning. Hybridomas can be diluted in ClonaCell™-HY Medium D and plated at low density on a 100 mm dish. To ensure discrete colonies, plating should be done at several dilutions, e.g. 50, 100, 250, 500, and 1000 cells per dish. The subcloned hybridoma should be cultured in the semi-solid medium for 10 - 14 days until discrete colonies are visible. The colonies can then be picked and expanded in ClonaCell™-HY Medium E or ClonaCell™-HY AOF Expansion & Cloning Medium for screening.

Viable monoclonal hybridoma colonies from either LDA or semi-solid subcloning should be verified for antibody expression using a screening method such as ELISA, flow cytometry, or Western blot. Positively expressing monoclonal hybridoma cell lines can be expanded and then cryopreserved for safekeeping using a cryopreservation medium, such as CryoStor® CS10 .

Products for Subcloning Hybridoma Cultures

Catalog #
ClonaCell™-HY Hybridoma Kit
ClonaCell™-HY Medium D
ClonaCell™-HY Medium E
ClonaCell™-HY Liquid HAT Selection Medium

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