Hi Folks,
I am developing a LFA for detection of a specific disease. I have functionalised the gold nanopartciles with polyclonal antibodies against bacterial surface proteins (The antibodies are raised against the total surface proteins of the bacteria) and I made it sure that antibodies are attached to the gold surface (by checking aggregation and UV spectrum).
Later, I tried checking whether these antibodies functionalised nanopartciles (Ab-AuNPs) are active or not, so that I could use them in the downstream applications. Briefly, I am checking the activity of these Ab-AuNPs by dot blots. I spotted the intact bacterial cells/bacterial lysates on the nitrocellulose membranes along with the separate spotting for secondary antibodies against the antibodies used to functionalise the nanoparticles. In a separate set of experiments, by ELISA, I have confirmed that these antibodies are active against total bacterial lysates and intact bacterial cells.
When, I tried to detect the bacterial cells/lysates on these dot blots by Ab-AUNPs, it showed binding only with secondary antibodies. However, Ab-AuNPs did not show binding with bacterial lysates or intact bacterial cells.
I think after functionalization antibodies must be losing their activity. How to stabilize them? What might be the additional reasons for this inactivity? How to determine the activity of Ab-AuNPs? Unless, I solve this problem, I will not be able to use them in actual LFA. Help is appreciated! thanks in advance!!
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