Q&A: What is the reference wavelength used for in a plate reader?

Question by Dubious: What is the reference wavelength used for in a plate reader?

When you use a plate reader (for ELISA or some other type of colorimetric assay), you read the plate at 1 wavelength, but you also read it with a 2nd reference wavelength, what is this second reading used for?

I asked this question in Physics a while ago but I received no response.
When I Google it, I can't seem to find the answer.




Best answer:

Answer by gardengallivant
The reference value is subtracted from each reading to normalize the readings against a nonchanging value. One wavelength reports the change in absorption from enzyme action. The other should remain the same since it is set at a value unrelated to expected absorption values.

http://books.google.com/books?id=A3K3QQJFecIC&pg=PA89&lpg=PA89&dq=Elisa+why+dual+wavelength&source=bl&ots=92L5giqiKW&sig=7HARvi5zsM7Dz23cq3K1plHU_1Y&hl=en&ei=v4HtTNvMHpTGsAOaudCABw&sa=X&oi=book_result&ct=result&resnum=2&ved=0CCEQ6AEwAQ#v=onepage&q=Elisa%20why%20dual%20wavelength&f=false

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