This message has been posted by: 612 right <i.hinners at REMOVE-THIS-TO-SENDicrf.icnet.uk>
Hi BJ,
what you call the capture antibody is that the very first antibody that you
use in the assay for coating the plates?
When we did this sort of assay we called it sandwhich ELISA and we found that
it is better to use 96well plates in tissue culture quality (!) because the
"real" ELISA plates have a surface designed so that antibodies don't bind
very good to reduce background binding in standard ELISAs. In your assay it
of course results in poor binding of the "capture" antibody. Try TC-plates.
Alternatively, have you tried to do a "normal" ELISA i.e. coating the antigen
rather than an antibody and then use only one specific antibody?
Maybe its not necessary to do an sandwhich assay?
Hope this helps, Ina
BJ Allan wrote:
> We have been having a problem with developing an ELISA. Here is the method
> we are using:
>> 1) We coat the plate with a goat polyclonal The antibody is in a 50mM
> carbonate/bicarbonate buffer, pH=9.5, and the coating is for 2 hrs @ 37C
> or overnight @ 4C. We are using EIA/RIA plates from CoStar.
>> 2) After 4 washes in PBS, we block for 1 1/2 hrs @ 37C with 2% BSA in PBS.
>> 3) The blocking buffer is dumped out and we add the antigen diluted in PBS
> + 1% BSA + 0.05% Tween-20. This is incubated for an hour @ 37C.
>> 4) Four more washes in PBS and then we add the primary antibody
> (monoclonals from mice) diluted in PBS + 1% BSA + 0.05% Tween-20. Another
> hour long incubation @ 37C.
>> 5) Four more PBS washes and then the secondary antibody (GAM-HRP from
> ProMega) in PBS + 1% BSA + 0.05% Tween-20 is added. One hour incubation @
> 37C follows.
>> 6) Four final washes in PBS, then we add ProMega's TMB One substrate and
> incubate for up to 30 min. at room temperature.
>> 7) The reaction is stopped with 1N HCl and the absorbance read at 450nm.
>> The problem is that I was trying to titrate the capture and primary
> antibodies against each other in a chess board pattern, with constant
> antigen and secondary. The primary behaved as expected, the signal dropped
> as the concentration decreased until, with no primary antibody, there was
> no product from the HRP.
> However, the capture antibody behaved exactly backwards, with the response
> increasing and the highest signal with no antibody at all. (The first
> time I wondered if I had the plate upside down.) I have now seen this with
> two different polyclonals, in combination with two different primaries.
> Does anyone have any idea what might be happening here? I'm wondering if
> our blocking isn't working, but that would suggest that the capture Ab is
> actually interfering with antigen binding. Any thoughts or tips would be
> appreciated.
>> * Robert (BJ) Allan *
> * University of British Columbia, Vancouver, BC *
> * "My suspicion is that the universe is not only queerer than we *
> * suppose, but queerer than we can suppose." J.B.S. Haldane *
--
Ina Hinners
ICRF
Secretory Pathways Laboratory
44 Lincolns Inn Fields
WC2A 3PX London
UK
email: I.Hinners at icrf.icnet.uk
