Think about it like that. You use the specific wavelength because you will absorb the maximum from your samples/standards fluorochrome emission. However, you still measure the non-specific emission from all the other materials in your wells (e.g. polysterene). Thus, you use an irrelevant wavelength where your samples/standards will not give out signal to subtract signal that comes from these materials. Hope this helps!

Thanks Farah, it does help !! Hope the sun is shinning in Algeria !!!

To eliminate the absorption of polystyrene, or any material that the ELISA plate is made with

Think about it like that. You use the specific wavelength because you will absorb the maximum from your samples/standards fluorochrome emission. However, you still measure the non-specific emission from all the other materials in your wells (e.g. polysterene). Thus, you use an irrelevant wavelength where your samples/standards will not give out signal to subtract signal that comes from these materials. Hope this helps!

... also turbidities in the solutions, scatterings due to scratches, plate coating (if you are using complete cells or particles as antigens in CELISA or Particle ELISA ...) ear eliminated by using a difference between the extinction at the color specific wavelength and the OD at an unspecific wavelength.

Just lock for the absorbance spectrum of your substrate. It has a peak at 492 nm in the case of oPD or TMB. optical failures have a spectrum which is more or less without special peaks in the visible part of the spectrum.

Theodore and Stephan are right, also is a way of monitoring the whole functioning of the spectophotometer...

I think that the response from Ahmed Djeghader is correct. In addition, If the main wavelengh is next to 350 nm and the wavelengh correction is in the order of 600 nm, you can also correct unspecfic turbidimetries.

Kind Regards.

Review_Dual-Wavelength Spectrophotometry

Authors

Dr. Shozo Shibata

First published: November 1976Full publication history

DOI: 10.1002/anie.197606731  View/save citation

Cited by (CrossRef): 15 articlesCheck for updatesCitation tools

Abstract

At present, dual-wavelength spectrophotometry is probably the least well known of the techniques of absorption spectrophotometry. However, recent improvements in application techniques have shown that for various organic and inorganic materials dual-wavelength spectrophotometry can provide higher sensitivity and selectivity than conventional spectrophotometry. This article reviews the principles and scope of dual-wavelength spectrophotometry, including derivative absorption spectrophotometry, in the hope that these techniques may be adopted more frequently in the future for resolving many analytical problems.

http://onlinelibrary.wiley.com/doi/10.1002/anie.197606731/pdf 

Dual wavelength is used in many microplate-based applications to reduce optical interference caused by scratches, fingerprints or other matter that absorb light equally at both wavelengths. For example, many investigators prefer to read microplate-based assays with lids or membrane seals in place to reduce biohazards, as well as evaporation. As a result of using lids, condensation may collect on the lid during the assay process. To illustrate the benefits of dual wavelength in these types of applications