I'm trying to conjugate an antibody to silver nanoparticles (20 nm diameter, citrate capped AgNPs). The conjugation protocol essentially involves centrifuging AgNP stock, resuspending at the same volume and adding 10 ug/mL antibody, incubating while mixing, then centrifuging, resuspending, and centrifuging a last time to wash away unbound antibody. Finally the pellet is resuspended. Using the resuspended pellet, I can get great data performing ELISAs with an anti-species, secondary antibody-HRP conjugate. However, whenever I try to detect the silver directly, I don't see anything.
My current hypotheses are: 1. Remaining unbound antibody is out-competing the conjugate and 2. The AgNP severely reduces antibody affinity for target.
Are there ways I can test my hypotheses, or in general find out why I can't detect silver in the assay? Thanks.
I'd like to update this by saying I've used magnetic beads that bind IgG at a high binding capacity to assay for silver after making a AgNP-antibody conjugate. I found lots of silver, so I'm assuming the antibody is binding.
One possibility is that your bioconjugation protocol is not directional. If the antigen binding sites of the antibody is bound to the nanoparticle, then you bioconjugated nanoparticles are not immunospecific. I would suggest using a directional conjugation protocol such as the one written by Kumar, Aaron, and Sokolov (Nature Protocols 2009) if your antibodies are monoclonal.
If your bioconjugated magnetic bead uses a secondary antibody then these typically bind to the light chain (base of the antibody), thus confirming loss of specificity.
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