I'm new to ELISA assays and have heard that multiplexing can be difficult. In your experience, what are some of the challenges that come with multiplexing? Is detecting up to 3 targets doable?
Dear Marcia,
In general, ELISA is not a multiplexing technique. Since you only obtain one read-out at one well of a plate (e.g., one optical density, fluorescence intensity, depending on the format). If you want to have a multiplexed immunoassay, you can either use immuno-microarrays or bead-based assays, like the SAFIA Assay (see www.safia.tech) or the Luminex system. With these systems, detecting three analytes is rather easy. It also depends on what you want to detect (proteins, small molecules, peptides...?) which system is best for you.
From my experience, the bead-based assays are quite easy to develop, but the multiplexing is limited. The microarrays can detect a large number of analytes/targets, but the development is much more complex.
Hi Marcia Chavez
With conventional ELISAs it will not be possible to multiplex as per the attached (all single-plex) for protein antigens:
Article Genetic control of antibody production during collagen-induc...
For cytokines, you may resort to bead based assays like Luminex with flourescence readout, which has limitations for sensitivity, scalability (in terms of number of targets) and dynamic range of the proteins detected.
To that end, I'd suggest Olink's qPCR based readouts either for the low or mid-plex portfolio. Please check here for targets covered:
https://olink.com/products-services/flex/
https://olink.com/products-services/target/
All the best & good luck with your experiments,
Michael
If you have the access to the readers (or a collaborator who does :) I recommend MSD or Luminex platforms for multiplexing.
https://www.mesoscale.com/en/products_and_services/assay_kits/u-plex_gateway/u-plex_technology
https://www.luminexcorp.com/xmap-technology/#instruments
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