Antibodies with Specialized Specificity

In any polyclonal purification setting typically the antigen is immobilized on an affinity matrix (aka a column), relevant antibodies are bound to this during an incubation with the serum, the column is washed to remove low-binding or non-relevant entities, and finally the specific antibodies are eluted from the column with a low pH buffer.  The defining characteristic of how the process is different for more highly specific needs is the introduction of a negative selection step.  With this, whatever reactivity is not desired can be removed, then a final positive purification can be performed on the resulting ‘cleaned-up’ material that maximizes the required specificity.

Several ways negative selection can be utilized are:

• Non-modified peptide for modification specific antibody (e.g., phospho-specific)
• Spanning peptide for a cleavage site specific antibody
• Wild-type peptide for a mutation site specific antibody
• Closely related paralog peptide for cross-reactivity in same protein family
• Ortholog peptide for unwanted species reactivity for same protein

Our standard practice with all polyclonal projects of this type is to perform 1x negative selection then proceed to positive selection.  Most of the time this is sufficient to remove the unwanted reactivity, but there are situations where this can persist (such as when the ELISA shows particularly high non-specific signal).  It is possible to perform multiple successive negative selections to correct for this at an additional cost.

We have highlighted our polyclonal process for the development of these types of reagents, but it is of course possible to make them as monoclonals as well.  The drawback with the monoclonal process is you are relying on the chances of a perfectly specific clone existing, and it may not.  Our recommendation for these cases is to pursue the best clone possible, then use purification techniques similar to those used for polyclonal serum to tweak reactivity to the desired levels.