Hi All:
I am trying to set up an Elisa to quantitate the level of IgG in human
serum. I have tried to use goat anti-human IgG, Fab specific, to capture
the IgG. Then detect the bound IgG with goat anti-human IgG, heavy chain
specific conjugated with HRP. I have set up tests where I make up a
checkerboard with the capture antibody diluted to between 1:5000 to
1:100,000 and affinity purified human IgG diluted from 10 ug/ml to .5 ug/ml
. The result is that I see a strong response to dilution of the capture
antibody but no significant response with the dilution of the IgG target.
For example at 1:50,000 dilution of capture anti-fab, at ug/ml of IgG is
18% lower, at 2 ug/ml is 27% lower, at 1ug/ml is 20% lower and at 0.5 ug/ml
is 47% lower. The trend is toward lower signal with less IgG but I don't
know if I will be able to accurately quantitate my IgG levels with this
assay.
I would appreciate any suggestions would be helpfull. Can this capture
elisa be made specific? I purchased the capture antibody from Sigma, they
only test whether the antibody recognizes IgG not if it can be used to
quantitate the level of IgG.
I do not subscribe to this news group, so please email directly if you have
a suggestion.
Thanks in advance
Mike Gruidl
