muhero at globalnet.co.uk
Mon Oct 30 16:14:04 EST 2000
"BJ Allan" <rjallan at interchange.ubc.ca> wrote in message
news:8titve$c4n$1 at nntp.itservices.ubc.ca...
> 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
It sounds like your antigen is binding directly to the plate OR to the BSA
on the plate. Your capture antibody is reducing the amount of antigen
available to take part in the reaction - either by competing for binding
sites in the antigen or by orienting the antigen in such a way that the
primary antibody doesn't get access to them. Try direct antigen binding to
the plate and if you get a usable result (try a dilution series of the
prinary antibody and ses if you get a decent curve) thenyou can try some
inhibition assays - keep one antibody constant and vary concentration of the
other. You can try detecting either antibody this way if you have some
antio-goat enzyme conjugate.
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