We are optimizing the assay parameters for our TSH immunoassay. We had been using a 7.4 pH PBS buffer for antibody coating so far, but now want to evaluate if changing the pH of the coating buffer would enhance the binding of the TSH capture monoclonal antibody to the surface.
The surface is PMMA surface, modified with PEI to give amine-PMMA, further modified to give Glutaraldehyde as functional group on the surface.
Similarly, would changing the buffer for blocking buffer (currently using 1% BSA in PBS), improve binding?
Could you expand on the chemistry here? Do you have the PEI covalently bound (after PMMA oxidation plus EDC/NHS activation) or adsorbed? And is the glutaraldehyde a crosslinker (Schiff base rxn) between the PEI and NH2-antibody? Do you follow that with reductive amination?
If it's Schiff base rxn, you can look up pH optimization there.
Blocking buffer is going to help nonspecific binding. BSA is a good start, but look up blocking buffers for Western blotting. There are a lot of options.
Just an FYI, we work with antibody immobilization to thermoplastics a lot. We do surface oxidation followed by EDC/NHS direct immobilization to the surface, so one step reaction followed by blocking. Details are at http://pubs.rsc.org/en/content/articlehtml/2014/lc/c3lc50618e. There's another cleavable crosslinker chemistry for catch and release too, http://pubs.rsc.org/en/content/articlelanding/2015/cc/c4cc09765c/unauth#!divAbstract
Matt Jackson
Thanks a lot for your detailed answer
Our method is same as here-
https://pubs.acs.org/doi/abs/10.1021/la061123l?journalCode=langd5
As suggested by you we are exploring different blocking buffers, starting with the ones used with western blotting.
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