ELISA question

William Joseph Kokolus cm006 at freenet.buffalo.edu
Mon Oct 30 17:28:42 EST 2000

50mM carbonate buffer, pH 9.5, is suitable for coating ELISA plates
with peptides - the original intention for that buffer, I believe. 

When coating with antibodies however, you could try a buffer with a
more neutral pH.

You could also use non fat dry milk as an alternative well blocker to BSA.
I had thought its composition less 'sticky' than BSA.  
A couple more washes in each washing step may also help.

BJ Kokolus,
Kenmore, N.Y.

On 30 Oct 2000, 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 *
William Joseph Kokolus, Ph.D., President,
Fountain's Rainbow Biomedicine, Inc.
69 Ferndale Ave.
Kenmore, New York, U.S.A. 14217-1003
phone: 716 873-6940
fax:   716 873-6940
email: cm006 at freenet.buffalo.edu
       kokolus at att.net


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