[Histonet] Re: Reason for lengthy primary antibody

From:"Johnson, Teri"

>From the Chemicon website

>>The time taken to reach equilibrium is dependent on the rate of
diffusion and the affinity of the antibody for the antigen, and can vary
widely. The affinity constant for antibody-antigen binding can span a
wide range, extending from below 105 mol-1 to above 1012 mol-1. Affinity
constants can be affected by temperature, pH and solvent. Affinity
constants can be determined for monoclonal antibodies, but not for
polyclonal antibodies, as multiple bondings take place between
polyclonal antibodies and their antigens. <<

Take the time to read the entire text from this website. It's very good
information regarding antibodies and antigens.

I agree with many that it's an issue of time convenience with research.
The more methods and materials you read in publications, the more you
realize that overnight at 4 degrees C is the standard. Why? Some because
of time management. I suspect mostly it's because it's always been done
that way.

It is very useful to only have to do an IHC once (if you are so lucky),
and if you have a better chance of getting binding overnight in the
refrigerator than 30-60 minutes at room temp, then do it. We have had
one antibody we could not get to work, until we incubated it 48 hours in
the refrigerator and used a polymer detection. Amazingly, 1 hour at RT
and 24 hours in the fridge did not produce any staining.

So, yes, it might well make a difference.

Having said all that, we routinely do many of our antibody procedures
using 1 hour at room temp. But we do not dismiss the need for longer,
extended incubation times in some cases. Additionally, all of our whole
mount staining is always done overnight.

Teri Johnson, HT(ASCP)QIHC
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64110

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