Setting up a capture Elisa

Mike Gruidl gruidlme at moffitt.usf.edu
Fri Nov 5 08:10:50 EST 1999


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





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