Hey all,
I'm working on synthesizing Gold nanoparticles (GNP) - antibodies conjugates for biomedical purposes. We are struggling to find an efficient method to sperate the unconjugated antibodies from the Gold nanoparticles - Antibodies conjugates from final solution.
We tried centrifugation (Following many assays that suggest so, probably relaying on the fact that antibodies will sink later then GNPs) and measuring the supernatant for excess antibodies (assuming there will be less antibodies in the GNP - Antibodies reaction) but the results of our controls didn't add up (antibodies - only control has shown very few amounts of antibodies in the supernatant, which means that the unconjugated antibodies and the GNP-Antibodies conjugates have the same sedimentation properties, we assume) (The GNP-Antibodies reaction contained EDC and the antibodies-only control did not).
Does anyone familiar with a different separation method, or can enlighten me, what could be the cause of this problem?
Thanks in advance!
You can try using Protein A Sepharose beads to chelate the unbound antibodies after EDC-NHS coupling. They are inexpensive and the Protein A component of these beads have IgG binding domains. Since the beads recognize the Fc region, the Ab-GNPs won't bind. And rather than centrifuging the mixture, if you let the solution sit the Protein A Sepharose beads would sediment faster than the nanoparticles. You may lose some nanoparticles but the overall amount of unbound antibody in solution should reduce after a couple of passes with the beads.
Praveesh Valissery Thanks a lot for answering. The method that you mention did cross my mind but it is proven that the conjugation process of antibodies to GNPs doesn't necessarily involve the FC region, moreover there are less amine residues (that can be attacked y the carboxylic groups on the nanoparticle's linkers) on the antibodies' FC region comparing to the hinge or Fab regions, that's why the A protein purification may not be so valid for this approach? what your thought on it?
You are right. After conjugation you can expect a mixture of non-oriented (the ones where the Fab/ hinge is bound) and directed (Fc bound and Fab exposed) populations. And within each individual particle you can expect a non-uniform mixture of these two types of conjugation. So by incubating the formulation with ProteinA, the undesirable non-oriented ones will be removed (because they will sediment via the exposed Fc) while enriching the ideal directed Ab-GNP populations.
Secondly, the free Abs will bind with better efficiency than the Ab-GNPs because of steric effects. So a few passes with small amounts of ProteinA Sepharose should improve the purity of the formulation.
Overall, I think its worth a shot. But, to be on the safe side, you can check the orientation of the antibody by using fluorophore-labeled anti-IgG Fab secondary antibody before purification and after. Ideally the fluorescence should remain the same before and after purification. This ensures that you did not lose a lot of the properly oriented Ab-GNPs during purification.
I know that most papers mention that by centrifuging/washing with blocking buffer, the unbound Abs can be removed. But that wasn't the case with our lab as well.
To selectively separate your particles from unbound antibodies, centrifugal concentrators/buffer exchange devices with a cut-off in the high MW range (I'd try in the 250kDa to 1 MDa range) should work.
Depending on what's available to you, you could also try size exclusion chromatography (e.g. Sephacryl S300) or preparative ultracentrifugation, using a density gradient or a stepwise "gradient" that reflects the properties of your products and educts.
Regarding Protein A/G, I'd cautiously try with a small aliquot, but I'd expect that everything will bind, and you won't be able to elute your target (pH change or chaotropic salts required) unless the antibodies have been covalently bound.