Dissociation of Analyte

Dear SPR,

I just got a question in regards to the indirect method for KD analysis (ie. Immobilize Protein A unto a CM5 chip, then pass on the mAB, then pass on the Analyte (target), then regeneration using low pH glycine 1.5.)

Wouldn't the regeneration solution, glycine 1.5, knock off (dissociate) the mAB from the Protein A (since it is not covalently bound to the Protein A), during the regeneration step, instead of just knocking off just the Analyte to get the KD? Or would this be dependent and making sure that there is enough of the mAB that was passed through the Protein A surface to begin with. What I do know, is that after several rounds of regeneration will eventually dissociate the mAB from the Protein A surface, right? RL levels will slowly go down after some time...

Thanks.

Hi,

When using glycine pH 1.5 during regeneration all captured antibodies will be washed away from the protein A.
Sometimes it is possible to regenerate the protein-A-mab-analyte complex with less harsh solutions (e.g. high salt, pH 5.0) only dissociating the analyte from the mab.
The thing is that you should confirm that the analyte is totally removed from the mab before the next round (e.g. return to the baseline after mab capture). However, the mab will probably also dissociate from the protein A resulting in a lower baseline which is difficult to interpret since a lower base line could be a mix of free protein-A (mab is dissociated), protein-A-mab (analyte is dissociated) and prot-A-mab-analyte.
I would go for the total regeneration of the protein-A between cycles and capture each cycle fresh mab if there is no mild regeneration for the mab-analyte complex.

Kind regards
Arnoud