Multiplex IHC Assay Development Guide
Multiplex immunohistochemistry (mIHC) enables the identification of different immune cell subsets, their activation state, and their spatial distribution in the tumor microenvironment (TME) allowing maximum data collection from precious clinical samples.
In practice, the simultaneous detection of multiple protein biomarkers on a single tissue section can be challenging therefore in a clinical setting it is essential to implement a rigorous optimization and validation process to ensure the assay can be trusted and is fit for purpose.
Step 1
Optimize the single IHC of each target. Identify antibody clone, antigen retrieval conditions and antibody incubation times for each target using positive control tissues. Multiplex immunohistochemistry (mIHC) enables the identification of different immune cell subsets, their activation state, and their spatial distribution in the tumor microenvironment (TME) allowing maximum data collection from precious clinical
samples.
In practice, the simultaneous detection of multiple protein biomarkers on a single tissue section can be challenging therefore in a clinical setting it is essential to implement a rigorous optimization and validation process to ensure the assay can be trusted and is fit for purpose.
Step 1
Optimize the single IHC of each target. Identify antibody clone, antigen retrieval conditions and antibody incubation times for each target using positive control tissues.
Step 2
Assemble the multiplex panel. Determine the order of antibody target and chromogen deposition.
Step 3
Evaluate the optimized staining conditions in the multiplex protocol. Perform calibration experiments to ensure the signal is specific and optimally balanced for each target across the multiplex panel.
Step 4
Compare the mIHC assay to its respective validated single plex IHC counterparts. Serial slides stained for each validated single plex IHC are compared to the multiplex staining.
Step 5
Precision testing. Serial slides are sequentially stained with the antibodies or an IgG isotype matched control (negative control).