5 Tips for Multiplex Kit Users and Assay Developers

Woei Tan, PhD, is a Senior Staff Scientist at Bio-Rad Laboratories in the San Francisco Bay Area. Tan shares a few best practices that he’s learned while developing more than 400 Bio-Plex multiplex immunoassays over the past 15 years.

3 tips for multiplex assay users

Tip # 1 There is a difference between plasma and serumPlasma is processed from blood in the presence of an anticoagulant. Serum is processed from blood without an anticoagulant. The sample matrices are different for plasma and serum so conduct your study with one sample type to ensure data continuity.


Tip #2 Consistency in sample preparation and storage is critical for precisionBe consistent with the method of sample collection and storage as these factors can impact the consistency in sample measurement. For cytokine analysis, be sure to avoid multiple freeze-thaw cycles as this will decrease the stability of certain cytokines in the sample. Instead, prepare and freeze multiple aliquots of the same sample for single use.


Tip #3 Alway follow the assay protocolBio-Plex assays are specially optimized for the specific volume of the assay as written, not just the proportion of the reagents. If you must deviate from the default instructions, be sure you run a validation study. While diluting reagents or splitting kits might be tempting, it will compromise assay performance and data reliability, negating any small cost savings you may think you are gaining.


2 Tips for multiplex assay developers


Tip #1 Key development steps of multiplex sandwich immunoassays are:

  1. Selecting targets
  2. Sourcing of antibodies from trusted vendors and screening of antibody pairs
  3. Refining of buffer formulation
  4. Constructing and calibrating curves
  5. Optimizing assays to reduce cross-talk


Tip #2 Antibodies used in development of multiplexing assays require platform-specific validationDon’t rely solely on validation data from western blot or ELISA, as antibodies validated on these platforms don’t always perform as expected in the multiplex assay environment. For example, in ELISA, antibodies are attached via passive adsorption versus covalent coupling in Bio-Plex, which may result in altered antibody behavior. It’s essential to validate extensively and explicitly in a multiplex assay environment.


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