hello Adam, well iam not that experinced yet, but the preferred method would be to use some linker molecule on to gold which has affinity towards gold and antibody or atleast the linking molecule should have thiol and aldehyde group at it ends for the antibody n gold to bind. e.g PEg,NHS ester linking molecules

i have read many people use the physiabsorb method of directly binding antibody on gold, what is your substrate? may be you should chk the UV-vis abs of the gold on substrate before and after antibody binding,if there is any change in spectra(spectral shift) that means there is antibody on gold, u may also increase the concentration of antibody and incubation time, i work with protein BSA on immobilized gold particles and it works for me quiet well., hope this info helps you !

Thanks guys. I use APTMS to link the gold to my glass substrate (a fiber optic cable). Badri, I can see the antibody binding in realtime to my sensor so I am confident that it's on there; however, I had read this thiol-aldehyde protocol before. I had wondered if the aldehyde protocol lends any advantage but I should probably test it myself.

Hello Adam,

Stable gold nanoparticle conjugates can be prepared by adsorption due to electrostatic and hydrophobic interactions between the antibody and the surface layer of the colloidal gold. One can achieve maximum binding/adsorption at a pH close to the pI of the antibody or protein to be conjugated. Another important factor is the amount of antibody bound to the colloidal gold. If too much or too little amount is adsorbed, it effects on the surface plasmon resonance and causes the aggregation upon addition of electrolytes present in standard buffers. A titration is recommended to determine the amount at which saturation and colloidal stability is reached.

I would also do the binding study of antibody to gold. For this purpose, NHS ester PEG is best solution at optimum pH.

All the best for your work

Ahsan Hashir

Thanks Ashan! This was very helpful. My np's shouldn't aggregated because they are affixed to a substrate, but I am certainly interested about the effect of electrolytes. You said NHS ester PEG is the best solution at optimum pH. The best solution fro what exactly?

Electrolytes play important role on the surface chemistry of gold colloidal.

NHS ester strongly binds with antibody. I mean that's the best solution of your conjugation problem.

hello adam, thats good that u can see the antibody bound to gold,checking for aggregation as mentioned before is good aldehydes for example gluteraldehyde are commonly used for binding proteins or antibody,ofcourse there r some disadvanges using gluteraldehyde but never the less it works, i have used it to bind my antibody using gluteraldehyde (not on gold) but the chemistry works,i guess u can use any good linking molecule to have specific n strong binding.. i would recommend you trying a good linking molecule using NHS ester

Hi Adam, just an observation to the previous comment of Badri. The glutaraldehyde coupling chemistry will work only if you have amino groups on the surface of your gold nanoparticles. If you have no functionality on the surface, I would go for thiol anchoring groups to gold and NHS activated ester for coupling the antibody molecule. It strongly depends on what you are trying to do. If you need a strong covalent binding go for NHS-PEG-SH type linkers which also provides a spacer between the gold surface and antibody molecule.

thanks Cristina,i forgot to mention that ,i was just giving him the an example of aldehyde ,ofcourse for his work he needs a linking molecule which has an aldehyde n a thiol group at its end which i told him before.

Hey guys, thanks for all of the nice advice. What I can't wrap my head around is this:

When I put the antibody in my system, I immediately see a response that is the antibody physiologically absorbing to the gold. Then I add antigen and nothing happens. Inversely, I've done the exact opposite. First I add the antigen and observe a signal. When I add the antibody, nothing. But I know this detection method works because I've observed biotin/avidin with huge success. Do you think it's possible that just absorbing proteins onto the gold surface is changing their structure, or messing up their primary binding sites? I say this because the biotin was tethered with a thiol linker, so maybe the linker also serves to preserve function?

Hi, have you checked the antigen-antibody binding in other system? I mean not on ur system but on some other where you are sure both works, also a stupid question may be - r u working with right antigen n its antibody?? If u r not getting little signal that surprising, i would at least expect weak signal.

Hi Badri,

My thoughts exactly. The group from which we've obtained the proteins is confident they work, but I'll have to see it with my own eyes on a western blot or Biacore until I'm comfortable saying for sure its working. As you said, it's strange to see no signal...

Thanks for the input

Dear Adam Hughes,

Hope you doing well,

I think I am too late to write my comment on this discussion but would like to give my inputs. I would like to know the status of your problem, if its been resolved then congratulation but if not then according to me possibly you should check the pH at which you are interacting your antibody to gold particle. probably if the antigen binding sites of your antibody would bind with the gold surface then it might produce signal of binding with gold surface but then it won't react with the antigen. So, I think you should make sure that your epitops of antibody are open for antigen.

With best wishes...

hallo friends

i just want to ask about references explain the nature of binding between gold and antibidy

and i cant apply that for nano silver