Anyone has a protocol regarding the conjugation of antibodies to the amine functionalized gold NPs (NH2-PEG-S-AuNPs)?
I need to activate the carboxyl group of antibody with EDC/NHS and conjugate it to the amine pegylated AuNPs.
I never recommend edc/nhs. You can use glutaraldehyde instead. Both protocols can be consulted in Hermanson’s book (bioconjugate techniques).
Activating the carboxyls on proteins which are carrying plenty of amino groups very likely will result in a huge blob of protein. As antibodies are glycosylated, you easily may oxidize the sugars with sodium metaperiodate without affecting their functionality and couple them to your amino-NPs by reductive amination (mix, let sit, then reduce with sodium cyanoborohydride, followed by borohydride to reduce unbound aldehydes which might interfere downstream). It's analog to the conjugation of antibodies with peroxidases (see Harlow and Lane, Using Antibodies for details). Might need some playing with parameters (ratios and concentrations of ABs and NPs), as you'll get inter-antibody conjugation, too.
Are unmodified gold NPs an option?
Wolfgang Schechinger Thank you so much for your answer. So Do you think it is better to activate gold NPs?
Regarding your question, I have conjugated NH2-PEG-SH to the gold NPs to make them biocompatible for the cell internalization. So maybe I can skip the conjugation of NH2-PEG-SH.
Hanieh Montaseri from a chemical point of view, activating the NPs would be optimal, as you are in full control of the functional groups that you are adding. If your experimental setup allows you to add carboxy groups instead of the amino groups, it is straightforward to activate them with EDC/NHS and (after a quick wash to remove excess reagents) to add the antibodies, so you may saturate the NPs with antibodies in a pretty well defined matter. Cleanup (removal of unbound ABs, if they should interfere with your downstream application) could be done by gel filtration (SEC) or possibly by centrifugation in a medium with suitable density, where the modified NPs precipitate and the ABs don't.
Wolfgang Schechinger I totally agree with you. The only thing is that I have seen in some papers that for the conjugation of NPs to the antibodies, we should leave the n' terminus of antibodies unreacted to so they can overexpress on the cell and attach to the receptors and we have to work on the conjugation of carboxyl groups of antibodies with the amine groups of NPs. Do you agree with this?
Omar Gonzalez-Ortega Thank you so much for your answer, Unfortunately I do not have access to this book. May you kindly please send me the related chapter if you have access?
Thank you
Hanieh Montaseri Not sure about the N-terminus accessibility. I'd assume that when you attach ABs to the NPs, that having free antigen binding sites is what is desired. But regardless of coupling method/strategy, there is always some chance to inactivate an AB by having a hit at the active site of individual proteins. I would not assume that all N- termini will have the same fate, so, in real life, there might be some trade-off, but it still should work. pH is influencing to some extent the ratio of N-terminal to in-chain coupling. It's terminal amino groups vs. lysine side chains, so you might go slightly alkaline, pH 8..8.5 instead of rather neutral to disfavor the terminal amines. When activating carboxyls on the AB, you also might block sites because coupling is happening nearby, but I'd anticipate that irreversible AB aggregation by inter-antibody coupling will cause much more problems than leaving the antibodies just to react with your activated NPs.
Wolfgang Schechinger Thank you so much for your advice. It is much appreciated.
Falk Fish Do you mean physical adsorption? I was trying to do chemical conjugation but I guess physical adsorption is also working.
Thank you so much
Falk Fish Does anti HER-2 antibodies have any amino acids with SH group?
Most antibodies do not have any free SH groups. Nevertheless, the adsorption of proteins, such as antibodies, is strong on plain gold particles. BTW: Disulfides bind to gold efficiently, too.
Dear Prof Michael G. Weller
Thank you so much for your answer. I also would like to ask which method can be used to confirm the physical adsorption of antibodies onto the surface of gold NPs?
The easiest way may be the NaCl test. If gold particles are completely surrounded and hence stabilized by proteins, they do not precipitate in a NaCl solution. This can be seen by the color of the solution:
https://www.cytodiagnostics.com/pages/gold-nanoparticle-properties
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