We've developed a biosensor based on plasmonic resonance of nanoparticles and would like to perform antibody-antigen conjugation experiments. With almost no formal biology experience, it's really unclear to me how to select a antibody to try. Because we are characterizing our sensor, I'd like to do the following:
1. Measure binding between a very well understood, strongly interacting and affordable antibody-antigen pair.
2. Measure nonspecific binding.
Although it's not a certainty, I had planned to deposit the antigen onto the sensor surface and detect the antibody (rather than the other way around). Therefore, in choosing a non-specific binding protein, what considerations should be in play? I had thought that either BSA or cassein would be sufficient (any ideas?).
Cost is certainly an issue, so how important is purity in your opinion of the samples? Again, we're only testing our system, so the more simple the antibody-antigen, the better.
Most simple affordable high affinity couple is BSA - antiBSA or HSA - antiHSA. The antibodies should be polyclonal then you will get a high signal for testing your plasmonic nanoparticle detection system. A complicating factor for a nanoparticle detection system is precipitation of the nanoparticles caused by the immunochemical reaction. This definitely will happen when the ratio of antigen-antibody concentration are in the critical range. For signal generation it is always better to immobilize the antigen and detect the antibody and not vice versa. The higher the molecular weight of the analyte the higher the signal. In order to check non-specificity a "dirty" sample is normal standard serum (human, sheep is cheaper) can be applied. Success!
Most simple affordable high affinity couple is BSA - antiBSA or HSA - antiHSA. The antibodies should be polyclonal then you will get a high signal for testing your plasmonic nanoparticle detection system. A complicating factor for a nanoparticle detection system is precipitation of the nanoparticles caused by the immunochemical reaction. This definitely will happen when the ratio of antigen-antibody concentration are in the critical range. For signal generation it is always better to immobilize the antigen and detect the antibody and not vice versa. The higher the molecular weight of the analyte the higher the signal. In order to check non-specificity a "dirty" sample is normal standard serum (human, sheep is cheaper) can be applied. Success!
Thanks Richard.
One thing about our setup is that our AuNPs are fixed to a sensor, so they aren't free to precipitate.
I hadn't considered antiBSA, thanks! If you have any more suggestions, let me know.
Another common combination is biotin - streptavidin - biotinylated protein.
""our AuNPs are fixed to a sensor, so they aren't free to precipitate."
Apparently, you also have to conjugate your antibody to your probe, in a kind of sandwich, your antigen being immobilized on the solid phase. How easy is that coupling? Do you need an aggressive coupling agent, potentially harmful for the antibody function of your immunoglobulin. or not ?
By the way, you don't need to purify the antigen you'll coat on the solid phase.
My first idea to robustly address your problem would be to use normal human serum as an antigen, and a goat anti IgG polyclonal antiserum. Cost will remain quite low using that very basic reagent.
Another way would be to find some biologist or immunochemist near by your lab.
But you can succeed alone with the on-line help of the scientific community!
Good luck!
J.
Hi Jacques, thanks for your reply.
The antigen conjugation is fairly straightforward, and should not require an aggressive coupling agent. Furthermore, we pump out the coupling agents and run a clean buffer through at the time of anti-body conjugation.
Thanks for your suggestions. I was actually going to try the BSA as you recommended because our colleauges have high-purity, protease-free/IgG-free BSA that they'll lend us. Is the serum/IgG polyclonal antiserum preferable to this?
In Europe, Goat anti human IgG form Pierce are in the range of 100€ for 1 or 2 mg of purified Ab. And normal human serum is quite inexpensive!
But both BSA/ anti BSA or NHS/ anti IgG will work.
Keep us posted...!
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