I'm facing some trouble with the dissociating of two antibodies. Short story behind it: I have patient sera with a complex of two antibodies. This complex I want to cut and use one of the antibodies later in a cellular based assay.
Therefore I use a double acid-dissociation. My setup works fine so far (tested by ELISA), so I have an outcome of the antibody I want. But this antibody seems not to work, it doesn't have a fine epitope anymore. That's what my cell-assay always shows. The antibody should neutralize a drug.
So I want to know, if anyone of you has experience with dissociating of antibodies and especially using them afterwards for a cellular based assay. The pH after the dissoziation is ok and the concentration (measured by ELISA) is also okay. An "empty" control (without antibodies) is not showing any effect of the dissociating-assay itself.
I am not sure I understand your issue. Do you have two different antibodies that are interacting?
yes.
Patient gets treated with drug, some patients make neutralizing antibodies. So I want to loosen the neutralizing antibody from the treatment-antibody. Then the neutralizing antibody should be tested in a cell based assay...
You mean, you treat patient with an antibody and some patients produce an anti-antibody. If this is case you can perform an affinity purification with the original antibody covalently crosslinked to protein A, protein G or protein L agarose or sepharose (depending on the source from where it has been produced). You can elute using your acidic buffer, which I guess is citrate. You should collect your sample in a buffer which allows for rapid acid neutralization to allow for your antibody to recover quickly its native conformation ….There are other elution protocols which are based on chaotropic salts elution but I only tried once. Acid elution always has worked for me and it is advised.
I'm also a bit confused. Do all patients make antibodies against this drug, but only some patients make neutralizing antibodies? And you want to isolate the neutralizing antibodies and the non-neutralizing antibodies?
Okay, I discribed it in a wired way... The drug itself is an anitbody. Some patients make neutralizing antibodies. These neutralizing antibodies I want to seperate and test in a cellular based assay.
My approach till now was an acid dissociation of the antibody complex. But the antibody at the end is not working - not neutralizing the drug, given to the cells, at all...
Hey, you might want to talk to an immunologist about this, but I do seem to remember that a neutralizing antibody doesn't have to physically block the activity a given target in vivo. Instead it can mediate the rapid clearance of the target from the circulation.
Maybe I'm wrong.
If I understand you correctly you are interested in the properties of this secondary antibody. I think you should consider studying the dissociation in a label-free biosensor, preferably in the Attana system where you can do biochemical, sera and cell-based assays. Then you can obtain information on kinetic rate constants like the dissociation rates and affinity and also obtain an understanding of how the secondary antibody affects the binding of the first antibody to its receptor in the cell
In addition YOU can dissociate even single antibodies, e.g in attachement.
Article Evaluation of intact-and fragmented-antibody based immunosen...
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