In the previous article I introduced the principles of and differences between 4 types of ELISA.
How do you decide which type of ELISA to use?
The following list consists of questions that can guide you as you devise an ELISA experiment:
Direct capture ELISA works like a Western blot by using a specially coated plate to capture the target antigen. While effective, there is a higher possibility of non-specific background signal, as many proteins in the heterogenous sample will bind to the coated plate.
Sandwich ELISA selectively captures and immobilizes the target antigen by binding an antibody to the plate that will specifically form a complex with your biomolecule under specific conditions. As a result, there will be less background signal. Many commercially available ELISA kits use indirect capture ELISA.
There are two major determinants for optimal sensitivity: the concentration of your target antigen in the total mixture and the antibody structure.
Conduct a literature search for the relative abundance of the target antigen in different cell lines. For example, Western blot analysis of the target antigen can give you a rough idea of how sensitive your ELISA must be in order to detect a signal.
Direct detection ELISA is less sensitive for the following reason: the immunoreactivity of the primary antibody is impaired because it is conjugated to the reporter enzyme. Indirect detection ELISA, on the other hand, is more sensitive because the primary antibody is not conjugated to the reporter enzyme. Rather, it is the secondary antibody that is conjugated with the reporter enzyme.
Sandwich ELISA is slower because the use of two different antibodies requires more incubations and washes.
For an assay as finicky as an ELISA, it’s imperative that your pipettes are accurate.
There should be at least a “blank” (no addition of antibody and reporter enzyme), “non-specific binding” (addition of antibody and reporter enzyme to buffer only), and “positive controls” (known concentrations of target antigen).
I have had the most experience with a specific type of indirect capture-indirect detection competitive ELISA, and I'd like to share my protocol using cyclic AMP (cAMP) as the target antigen:
This is a quantitative ELISA, so the optical density (OD) of the sample is compared to a standard curve, which is typically a serial dilution of a known concentration solution of the target molecule.
Here are my tips for reproducible quantification:
That's it!
I hope you found the ELISA tips here useful. Let me know in the comment section below if you have any question!
To help you find antibodies or kits for your next ELISA exerpiment, I'd suggest trying out BenchSci to review published data before making a decision, as a successful ELISA depends heavily on the specificity of the antibody used.