>Marcia wrote:
>I was wondering if anyone has ever experimented with what protein
>concentrations are detectable in IHC. My problem: an ELISA performed on
>lysed supernatent from the same tissue I'm staining showed "off the scale"
>for TNF alpha levels, measured in pg/ml. My TNF IHC shows just about nill
>in terms of staining. I'm pretty much assuming it varies for each antibody,
>but does anyone know a general baseline?
-------------------------------
Dear Marcia,

Interesting question. I would like to share my thoughts on the above topic
and add some questions too. Hope others have the answers.

ELISAs differ in several aspects from IHC on a section a.o.:
- to visualize antigens on a section immunoenzymatically the concentration
of end-producthas to exceed the solubility constant of this product. Only
than you will obtain an on-site deposit that will be visible in the
microscope. For this you need a minimal concentration of enzyme. This
minimal concentration is related to the enzymatic activity and the
substrate used.

Q: How many enzyme molecules (e.g. peroxidase) are approx. needed to give
an on-site deposit. (with e.g.  DAB as substrate). Any literature available
on this topic? From this is might be possible to calculate the minimal
amount of antigen that can be detected.
Q: What about fluorescent markers. How many fluorescent molecules have to
be in close proximity of each other to generate a signal in the microscope?

When the concentration of end-product is below the solubility constant, in
IHC, the product diffuses and will bind aspecifically on your whole section
(i.e.  on proteins). This will happen in ELISAs too, but there this is not
a problem, because binding will happen on the tube wall, this signal will
be measured by the micro-plate reader.
- distribution of antigens is important in IHC and not in ELISAs.  When low
amounts of antigen are distributed over a large portion of your section it
may not become visible in the microscope (solubility constant again). When
the same amount is present in only a few cells you will not have a problem.
In an ELISA you will read the same value.
-  a spectrofotometer is far more sensitive than the eye.

Do you work on frozen non-fixed sections. You might have lost (washed out)
TNF. Or do you work on aldehyde fixed tissue. This might cause loss of
reactivity of your primary. Is your read-out still the same when you add an
aldehyde fixation step to your ELISA protocol? This might give you an
answer to your  'nill reaction' problem.

Regards, Peter




========================================
Peter van de Plas
AURION
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