RE: Lack of immunostaining at high antibody concentration

From:Ralph Marcucio

Hi all--

Could someone explain this (Prozone effect on tissues, not in a diffusion
assay) in more detail.  I thought that this phenomenon had to do with
precipitation of Ab:Ag complexes that form at high of concentrations, and
thus limit the utility of a diffusion assay.  I am very interested in
understanidng how high antibody concentrations affect immunostaining.

Thanks in advance,

Ralph Marcucio, PhD
Department of Orthopaedic Surgery
University of California at San Francisco
San Francisco, Ca 94143

Phone: 415-502-4945
Fax: 415-476-1128

-----Original Message-----
Sent: Wednesday, March 20, 2002 1:43 AM
Subject: RE: Lack of immunostaining at high antibody concentration

Yep its well known that too high a concentration of a polyclonal ab can
cause the prozone effect.

Bob Quilty
Dept of Neuropathology
Frenchay Hospital
Bristol UK

-----Original Message-----
From: Jane Radford []
Sent: 20 March 2002 05:35
Subject: Lack of immunostaining at high antibody concentration

Dear Histonetters,

I have a researcher working in our lab that is obtaining unusual
results with his immunostaining.
The researcher is using a rabbit anti human TRAIL (H257) antibody on
human melanoma (intransit dermal metastasis) tissue. He has just
competed a dilution series of the primary antibody (20, 10 and 5
ug/mL) and found that the highest concentration (20ug/mL) actually
resulted in no staining at all.  The other two dilutions gave a
moderate level (10ug/mL) and very faint level (5ug/mL)  of staining
which is what I would have expected. I would have logically expected
the 20ug/mL concentration to have resulted in more intense staining.

He made serial dilutions of the primary antibody so we know it must
be in the solution and other than forgetting to apply the primary
antibody or other components to the slide in question (which he
assured me he didn't!) could there be a reason such as inhibition of
binding of the primary antibody due to it being present in too high a
concentration, or some other explanation?
Thank you in advance for your help,

Jane Radford

Jane Radford
Histopathology Laboratory Manager
Department of Pathology
University of Sydney
NSW Australia
Ph: 61 2 9351 6152
Fax: 61 2 9351 3429

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