The Lab Notebook

Colorimetric?  Fluorometric?  How to choose the right platform for your ELISA

Posted by Bindi M. Doshi, PhD on Sep 21, 2015 11:41:52 AM

What happens when you use a black well plate for a fluorometric ELISA assay reading?  What happens when you use a clear well plate for a colorimetric ELISA assay reading?  Well the short answer is that you made the right plate decision!

The concept of an ELISA assay is pretty straight forward, right? You have a sample (maybe some cells) and you want to know if something interesting is going on with your protein Y within that cell population.  You can either create your own ELISA (lots of work) or buy an ELISA kit (little work).  But what does it mean if a kit says it is colorimetric or fluorometric?

For a colorimetric assay, energy is absorbed.  You are able to visually see which wells had a reaction and which wells did not.  Sometimes, it’s a nice blue color or a nice yellow color- you get the gist.  The concept of the assay is this: You have a plate that has a particular antibody bound to the wells.  You add your sample.  Maybe your sample binds to this antibody.  Then you add a detection antibody and a substrate to react with that detection probe.  After adding a stop solution, you can take readings to see how much of a particular analyte is in your sample.  For this assay, it is OK to use clear wells because the reaction occurs with no bleed through.  The darker the color, the greater the analyte in that well.  Pretty gratifying. 

Only one wavelength is needed, however, some labs prefer to use two, one wavelength for the measurement reading and anther wavelength for reference.  This reference wavelength can compensate for well to well variations not having to do with the experiment (such as plastic difference, bubbles, etc.).  (However, if you have enough replicates, and you feel your physical well properties don’t vary too much, you may not need to run a reference wavelength.)  So in this case, you would subtract the reference values from the measurement value to give you the “real” value.

A fluorometric assay is very similar, you add a substrate to your cells and then detect a fluorescence response using a plate reader.  You can think of it as energy is emitted.  In order to have the most accurate reading, the fluorescence of each individual cell needs to be measured and there should not be any cross over between the cells.  Hence, the reason to use opaque plates with this type of assay.  You want to reduce the amount of light scatter and a black plate will meet this need.  Two wavelengths are needed, one for excitation, one for emission.

Fluorometric assays are a little more sensitive than colorimetric assays and they are able to detect more of an analyte than colorimetric assays.  They allow for a very high reading to be measured by widening the dynamic range.  Some labs will combine both techniques by using a clear bottom plate.  This allows for measuring a fluorescent probe for one analyte and a colorimetric probes for a second analyte.

Regardless of your preference for colorimetric, fluorometric, or both, MBL has a product that can meet your needs.  Below are just a few examples:

Colorimetric Fluorimetric
4800 APOPCYTO™ Caspase-3 Colorimetric Assay Kit   4815 APOPCYTO™ Caspase-3 Fluorometric Assay Kit      
4805 APOPCYTO™ Caspase-8 Colorimetric Assay Kit   4820 APOPCYTO™ Caspase-8 Fluorometric Assay Kit      
4810 APOPCYTO™ Caspase-9 Colorimetric Assay Kit   4825 APOPCYTO™ Caspase-9 Fluorometric Assay Kit      

 

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Topics: Kits and Assays