ELISA-style-binding assay

This entry is part 2 of 1 in the series ELISA

ELISA-style-binding-assays (here shortly called ELISA) are very robust and relatively simple established assays. They are extremely useful for making single points measurements, and thereby testing a lot of proteins for interactions. With a little bit more effort, it’s also possible to extract proper binding constants.

With a small series of posts, I’d like to help with the most important steps in planning and executing an ELISA assay.

Here the most important pro and cons of an ELISA assay in a table.

pros cons
No expensive equipment necessary No binding kinetics (no kON of kOFF)
Technically simple Very fast binding equilibriums might not be detectable
Scale-able to many tests (medium through-put) Sometimes a lot of problems with unspecific binding
Applicable to a wide array of binding constants

 

What do you need for an ELISA?

  • 96 well microtiterplates (ideally NUNC MaxiSorp)
  • A microplate reader with an absorption at 450nm
  • 3,3′,5,5′-Tetramethylbenzidine, DMSO, H2O2
  • Milk powder or BSA

Before you decide whether you establish an ELISA or not, you should answer the following questions:

  • Is a “simple” affinity constants (KD) enough to answer your question? (If you need kinetics you should use SPR or biolayer interferometer.
  • Do you have a rough idea about the expected dissociation constant (KD)? (potentially from homologue or similar interaction?)
  • Do you have an ELISA reader able to detect either absorbance (normally 450nm) oder fluorescence?
  • Optionally: Do you have the necessary equipment to help setting up an ELISA: It would be helpful to procure 8-channel pipette capable of handling 50-100µl. An ELISA plate reader would be great, although personally I never had the luxury to have one. (I do not recommend to us steppers as they are difficult to handle and you have no visual control about the amount you pipette!)

In the next post we will discuss shortly how an ELISA style binding assay works and what consideration we need to take in account regarding our two proteins of interest.

next episode: How does an ELISA work? >>

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