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Higher concentration and less cytotoxicity

Q: We’ve been using Mab-ZAP to test our primary mouse monoclonal antibody in cytotoxicity assay. Relative to the Primary mAb-Mab-ZAP complex, the Mab-ZAP alone has quite a bit of activity (40-60% growth inhibition) at the recommended concentrations used (45 or 100 ng/well) or even half that dose. Even though we get a dose response, the higher concentrations of primary antibody give me less cytotoxic activity than the lower concentrations. Can you give your thoughts on this matter?

A: The effect you are seeing is something that is actually typical and indicates that you are using the material correctly. Described in Kohls et al., unbound primary antibody will compete with primary antibody bound to a secondary conjugate may reduce cytotoxicity through competitive inhibition of the primary antibody-Mab-ZAP complex. This is especially noticeable with our Fab-ZAP line of secondary conjugates.

We still recommend that our customers try a 10 nM dose as a starting point, but always recommend adjusting the concentration to better accommodate their experiments. As a reference, our data sheets show a cytotoxicity curve with a starting concentration of 10 nM and an ending concentration of 1 fM.

Related Product: Mab-ZAP (Cat. #IT-04)

References

  1. Kohls MD et al. Mab-ZAP: A tool for evaluating antibody efficacy for use in an immunotoxin. BioTechniques 28(1):162-165, 2000.