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Protocols

Cloning method

The objective of cloning the cells producing the antibody of interest is to ensure that the desired hybridoma cell line produced is obtained from a single fused cell.  After a fusion, many different hybrid cells will be present in a single well resulting in the growth of multiple colonies in each well. Your specific antibody-secreting colony is therefore likely to be mixed with other cells that are either non-secreting or which are producing an antibody of undesired specificity.

Figure – Outline showing the various major stages of the cloning methodology.
The purpose of the cloning technique is to grow a clone of cells from an isolated single cell and is outlined above. The method that we use for cloning is that of limiting dilutions. This means you dilute the cells to a concentration calculated so that each well of a 96 well plate will contain one, two or no cells after plating out. Once the cells have begun growing to form a clone, you can view each well under the microscope and discount any well with more than one clone or no clones at all.

Method

Using the cloning method to test the quality of your FCS/FBS batch

It is extremely important to use good quality FCS/FBS when trying to produce or grow hybridoma cells.  To test the quality of a potential new batch of FCS/FBS it is recommended that you clone a hybridoma cell line, using the method above, in the FCS/FBS you are currently using and in the new serum test batches.  You would expect to find that the new batch had a cloning efficiency similar to or slightly higher than your original batch.  If you have no previous batch for comparison, you should select a batch that has a cloning efficiency of at least 20% and which results in subsequent healthy cell growth.