ELISA

gitjye youwsh at earthlink.net
Thu Dec 12 18:52:26 EST 1996


sherry weppler wrote:
> 
> I am an undergraduate student taking an introductory immunology
> course. I have just finished a series of lab sessions in which
> we immunized mice with ovalbumin and an alum adjuvant, bled
> them, and preformed ELISA to calculate the concentration of
> ovalbumin antibodies in the plasma. It has now come time to
> write up the lab report, and we are to include in our
> discussion some information about capture ELISA and sandwich
> ELISA. The enzyme we used in the experiment was alkaline
> phosphatase with para-nitrophenylphosphate as the substrate.
> What other enzymes can be used, where do they come from, what
> is their substrate, and what is the detection colour?
> 
> I would really appreciate some information as I don't have much
> time to spend in the library.
> Thanks

A common enzyme-substrate system in use is Horse Radish Peroxidase 
(HRP).  The HRP molecules are conjugated onto the antibody and the 
substrate is usually 5-5-5-Tetramethylbenzidine (TMB).  The reaction 
turns the substrate to blue and then the reaction is stopped with 4M 
H2SO4.  After the stop solution is added, the ELISA wells turn yellow 
and is read in at A450.

You can get plenty of information by contacting Kirdegaard and Perry 
Labs and asking for their current catalog of products...it also includes 
introductions to their different enzyme sytems.  They're located in 
Bethesda, MD.

I hope that helps!

William Soo Hoo, PhD



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