Hi all, I am asking for what reason most of the ELISA protocols states to make bichromatic absorbance reading at 450/620 after stopping the TMB reaction with acidic medium? Thanks :-)
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Let's say that you have two wells with identical samples (i.e. technical duplicate) and one of the two wells has some particulate artifact, such as dust floating, or a greasy fingerprint on the bottom. The artifact scatters light equally at both wavelengths (not really true, but a good approximation), less light enters the detector at 450 nm, and the spectrophotometer interprets this as extra absorbance at 450 nm (as well as 620 nm). If the reference wavelength is picked correctly in a wavelength region where your target molecule doesn't absorb, reference subtraction gives final absorbance which is closer to the other well...
Note that this correction doesn't help with soluble artifacts because molecules' absorption generally is very different at these wavelengths. Of course, scattering is also wavelength-dependent, but the approximation is good enough.
You can even test out Suola's explanation easily: the readings you get at 620 nm will closely approximate your buffer blank at 450!
Thanks Suola and Samm for help .
I know that this is an old post but I would appreciate an advice:
in the ELISA protocol they asked for the absorbance to be read in wavelength 450nm with 650nm as a reference. I read the microplate in both wavelengths. can I subtract the absorbance reading in wavelength 650nm from reading in 450nm? would this be right?
LA 2015,That's correct! You subtract the absorbance at 650 nm from the absorbance at 450 nm.