ELISA question

BJ Allan rjallan at interchange.ubc.ca
Sun Oct 29 23:38:06 EST 2000

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

*                       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 *

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