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Develop antibodies? Accurately determine antibody isotypes.


Whether you are a core facility that makes monoclonal antibodies every day or a scientist that is trying to generate a monoclonal antibody for the first time, the process can be long and arduous.  Because we screen thousands of clones each year, we developed an easy and accurate assay for isotyping monoclonal antibodies during hybridoma development and we would like to share this assay with you.

One of the needs an antibody developer has is the ability to identify, with confidence, heavy and light chain types during various steps of antibody development.  The assay and techniques we outline below will show you how to improve your own accuracy and confidence while developing monoclonal antibodies!

The Isotyping ELISA Technique

In this example, we fused a mouse spleen and then expanded 12 clones (arranged horizontally) that were positive against a target protein into a 24-well plate.  After growing the clones for few days, the clones were retested against the target and their isotypes checked using the Mouse Ig Isotyping ELISA RSG® Kit (IgG1, IgG2a, IgG2b, IgG3, IgA, IgM, κ chain, λ chain).

Initial Isotype Results

isotyping results first screening resized 600

(In the figure above, the color intensity correlates with the signal intensity)

What does this data mean and where to go next?

At this stage, the hybridoma cells most likely are not monoclonal and the antibodies secreted into the supernatant are mixed with antibodies produced from spleen cells before those cells were killed off under selection pressure.  This is why it is not surprising to see multiple isotypes and a strong IgM signal.

For this example:

  • We are not interested in IgM producing hybridomas so hybridomas 1 and 11 are eliminated.  
  • Clone 4 is promising as it has a nearly clean IgG1 kappa isotype and could be monoclonal already.
  • Clones 2, 3, 5, 6, 7, 8, 10 and 12 are showing multiple isotypes. It is necessary to subclone these hybridomas to either confirm their monoclonality or isolate clonal ones.

We decide to move forward with clones 4, 6 and 8 and subclone them at a relatively high dilution. For each clone, we visually select 12 monoclonal subclones from the 96 well plates, and expand them in 24 well plates and test their isotypes and reactivity against the target protein.

Subcloning Isotype Results

Clone 4

clone 4 isotype results resized 600

Clone 4 was fairly clean prior to subcloning and all the subclones from clone 4 recognize the target protein and have very clean IgG1 kappa isotypes.

Clone 6

clone 6 isotype results resized 600


Clone 6 started with a mixed isotype of IgG2b, IgM and kappa.  After subcloning, there is one subclone that is IgM kappa, which does not recognize the target, and 11 clean IgG2b kappa subclones that do recognize the target of interest.

Clone 8

clone 8 isotype result resized 600

Clone 8 started with a mix of IgG1, IgM, kappa and lambda. All the subclones are either IgG1 kappa, IgG1 lambda or a mix of IgG1 kappa/lambda. Interestingly, only the subclones that have a kappa isotype are recognizing the target. The original well from the initial screening had at least a mix of 2 antibodies: an IgG1 kappa that recognizes the target and an IgG1 lambda that does not recognize the target

The subclones 3, 6, 7 and 10 are clean IgG1 kappa.

In these 3 examples, the hybridomas need to be subcloned another time at low dilution and screened again to assure or confirm their monoclonality before deciding upon the clone to make your antibody.

Final Thoughts

If you are working with hybridomas, we hope you try utilizing the Isotyping ELISA kit assay outlined above and experience isotyping with confidence!

Let us know of your antibody development success using this kit and technique by commenting on this blog!


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