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TRH- ELISA test

TRH- ELISA test - Protocols and Methods Forum

TRH- ELISA test - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 01-27-2006, 12:49 PM
Nada Vrkic
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Default TRH- ELISA test



Has anyone used an ELISA for TRH? We don't have a gamma counter, so I need an
alternative detection method.
Nada

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  #2  
Old 01-30-2006, 05:25 PM
Dr Engelbert Buxbaum
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Default TRH- ELISA test

Nada Vrkic wrote:


This largely depends on the equipment you do have and which antibodies.
For the latter, you'll need the detecting antibody and a labelled
secondary, available for example from Daco or Accurate (no affiliation).

Note that it is possible to count gamma-radiation with very high
efficiency in a beta-szintillation counter, if 10% ZnCl2 in water is
added to the szintillator. The gamma-rays produce Compton-electrons from
the Zn-ions, these are counted by the toluene/POPOP/PPO cascade. It is
sufficient to place the sample vials into the szintillator, no mixing is
required, which makes the method very oeconomical. For 125-I the
counting efficiency exceeds 80%.

Other than that you can use either fluorescence or, as you already
suggested, linked enzymes. In the latter case the use of
chemiluminescent substrates (available for both alkaline phosphatase and
horseraddish peroxidase) increases sensitivity by several orders of
magnitude compared to colourimetric detection.

Note that in ELISA the initial sample binding to the plate is very
inefficient (only 1-2%). Filtration over PVDF-membranes in a 96-well
manifold binds better than 90% of sample proteins, increasing the
sensitivity by almost 2 orders of magnitude. The membrane is developed
like a Western-blot, again using chemoluminescence detection.

If you do not have dedicated equipment for chemiluminescence counting,
X-ray film will do. The blackening can be later quantitated with a
conventional flatbed scanner, using the freely available NIH-Image
program. Unfortunately, that limits the linear range to about 1 order of
magnitude (similar to colourimetric detection), direct counting of
chemiluminescence is linear over at least 5-6 orders.
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