My secondary antibody ( IgG goat anti rabbit) is immobilised on dextran-coated magnetic nanoparticles, primary antibodies (from rabbit serum) are bound.
The primary antibody must maintain its integrity because it will be used in other assays (mostly ELISA).
I thought of using either a dextranase to separate the secondary antibody from the nanoparticle?, or a pepsin to digest the antibodies but im not sure if its possible since they are immobilised?
if you have any ideas or suggestions, Ill be happy to read them
I cant find anything promising in the literature.
Thank you very much for your help.
You might be able to get the primary antibody away from the secondary antibody immobilized on the nanoparticles by competing it off with an excess of free secondary antibody, but I doubt that would work. Even if it did work, recovering active primary antibody from that mixture seems like an impossible task.
Dear Anais, as Don already pointed out, due to the very strong interaction between anti-rabbit IgG and rabbit IgG, it is quite unlikely to recover active primary antibody from these particles. Also digestion is likely to affect your antibody and should therefore not be performed. You may try acidic elution with 0.1M glycine HCl at pH 2.4 with subsequent neutralization of the supernatant but even these conditions might not be sufficient here.
For future work I would suggest you use Protein A or G coated particles or columns. These proteins do bind the rabbit antibodies very good, but the affinity is lower. Also, elution with acidic buffers works very well here. You can find commercial products including protocols from all major manufactures.
Thank you very much Don and Martin for your answers!
Yes I'm afraid I have reached a dead-end here, but your suggestions will surely be taken into consideration
(And I forgot to say that the secondary-antibodies were covalently immobilised by EDC/NHS if that changes anything..)
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