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Multiplexing Assays: Building a Foundation for Optimal Performance

While multiplexing offers numerous efficiency advantages, it also introduces several technical challenges that make assay design more complicated. One of the biggest challenges is controlling for cross-reactivity and interference between all the antibodies and proteins in the assay.  Do you get the same results from IL-6 when run alone and when with 20 other analytes? 

Single-analyte assays are designed to be analyte-specific—meaning that every component, from buffer to antibody pairs and more, are custom-tailored for that specific analyte. Assays incorporating multiple analytes do not have this luxury.

multiplexing immunoassays

Multiplexing calls for carefully chosen compromises that allow analytes to play well together while still producing accurate, meaningful results. Interfering factors, no matter the mechanism, can result in either reduced or elevated signals relative to the actual concentration of target analyte.  

While validation protocols help ensure that antibodies are producing appropriate results, if the assay isn’t carefully developed and properly designed, achieving optimal performance will be difficult.

At Bio-Techne, we take great care to select the antibodies and proteins that can be plexed harmoniously to reduce interference and maximize performance across all analytes. Meanwhile, our rigorous quality control and validation efforts confirm the performance meets required standards and specifications lot after lot.

Immunoassays Including ELISA and multiplex assays

Ensuring assay precision and accuracy

A significant challenge in designing effective multiplex assays is identifying a buffer system that performs properly for all of the analytes of interest within a panel. Since it is not possible to customize the buffer to any individual analyte, optimizing buffers for peak performance, such as linearity or recovery, and managing matrix effects are key steps to ensuring sensitivity, precision, and accuracy of the resulting immunoassay.

Why is a sample dilution needed?

There are primarily two reasons for dilutions. In some assays most samples read above the standard curve, thus requiring a dilution for analyte levels to fall within the range of the assay. A second reason for dilution is to limit interference due to factors in complex matrices.

In the best scenario, an assay diluent works well with a broad range of analytes, allowing scientists to multiplex anywhere from a few to several dozen analytes within a single test. To avoid incorrect dilutions in multiplex immunoassays, you need to know the relative abundance of the analytes to be tested.

Higher-abundance analytes may require a different sample dilution than those of lower abundance. Grouping the analytes by common dilution is the most efficient means of creating multiplex panels.

No matter what dilution samples are run at, you should always derive the same concentration once back calculated, giving you consistency in results. As you dilute the sample, the interfering factors also dilute out, making the target analyte more accessible, and the concentration of the target analyte increases.

multiplexing immunoassay sample dilution
Sandwich ELISA antibody binding antigen in ELISA kit plate well

Multiplex with speed and efficiency

Confidence in immunoassay data depends on the accuracy and precision that is only possible from using high quality antibody reagents. This means selecting antibodies with not only high analyte affinity but also specificity, which is the ability to distinguish between the analyte and other structurally similar components.

While developing a multiplex assay presents several complexities compared to single analyte, plate-based ELISAs, the ability to generate high-quality data while dramatically reducing time and costs needed for testing provides researchers with a powerful advantage. From narrowing down drug candidates in preclinical studies to evaluating therapeutic responses during a clinical trial, multiplex assays provide several important benefits, including the ability to:

  • Maximize limited test samples: collect data from just 25μl or less of undiluted sample.
  • Minimize experimental variability: derive multiple data points from a single manipulation.
  • Optimize productivity: reduce sample preparation and processing time. 
  • Improve efficiency: examine multiple analytes in a single sample.

Meeting immunoassay performance goals is an ongoing challenge across research fields. The good news is that today’s scientists have access to an abundance of external experts who have deep technical experience and access to vast libraries of antibodies to help develop their assays. Engaging with an experienced supplier like Bio-Techne and paying attention to the technical factors outlined here will significantly improve your chances of developing high-quality, highly effective multiplex assays.

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