ELISA problems

Gys de Jongh GysdeJongh at compuserve.com
Tue Sep 14 17:23:32 EST 1999


Goran Pavlakovic <goran.pavlakovic at uni-essen.de> wrote in message
news:37DDDBF2.FE2F03F8 at uni-essen.de...
> Hi there,
>
> I am trying to measure a fatty acid binding protein in serum.
>
> When I do the purified standard protein dilution in water,
> ELISA is very good.  The moment I dilute my protein in serum
> (immediately before the first incubation), I am not getting any
> signal. I do a classical ELISA (coating with protein of interest,
> blocking, primary Ab, secondary Ab).

Hi,
i am not sure if i understand you correctly , but if your first step
is to coat fabp onto plastic then then you will only get an elisa
signal if you use the standard fabp dilution in water. The absorbtion
of protein on plastic is non-specific. If you dilute the fabp in serum
, which contains lots of globulins , then most of the protein on the
plate will be globulins. One way to detect fabp in serum is to use to
different antibodies (a and b) . Incubations would be 1) antibody-a in
buffer 2) block with an overdose of a-specific protein like bsa 3)
antibody-b 4) secondary antibody (against Fc part of antibody-b)
detection etc.
hth

--
Gys





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