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ELISA reader theory


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#1 SusanneInSwitz

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Posted 28 January 2011 - 07:17 AM

Can anyone explain to me why the readings on ELISA readers are considered linear from zero to up to OD 2 or 3 (give or take a couple % variance at the tail ends) when the Beer-Lambert law states that you are only in the linear range of an absorbance reading between 0.2 and 0.8 OD?

At least that's what the manufacturers say!!

#2 Gerard

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Posted 28 January 2011 - 02:02 PM

The statement that the Beer-Lambert law states a reading only in the linear range of an absorbance reading between 0.2 and 0.8 OD is correct isn't right, it states that in very diluted samples the concentration is linear to the absorbance.
This means that the range in with you can use the law depends on the molar absorbtion from the solution, with a high molar absorbtion the maximal absorbance of a solution can be much higher then in a solution with a low molar absorbtion.
Ockham's razor
Pluralitas non est ponenda sine necessitate
-- "You must assume no plural without necessity".

#3 SusanneInSwitz

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Posted 28 January 2011 - 02:44 PM

Thanks for that info, so it works because the chromogenic Substrates used have a high molar absorption?

Do you have a link or book reference for that? just to be able to make my point.

#4 Gerard

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Posted 29 January 2011 - 05:39 AM

Thanks for that info, so it works because the chromogenic Substrates used have a high molar absorption?

Do you have a link or book reference for that? just to be able to make my point.

Yes that's right and no I don't have a link or a book because it is simple reasoning.

What I didn't mention is the origin from the statement that the absorbance should be read between 0.200 en 1.000, this comes from the earlier photometers these were equiped with a analog logaritmic scale and tube amplifier. The ampliflier had a significant noise in the lower region and therefor the reading was less reliable in the lowerpart of the scale and a analog logaritmic scale is hard to read accurate above the 1.000
Ockham's razor
Pluralitas non est ponenda sine necessitate
-- "You must assume no plural without necessity".




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