Luminex® HP Assays 101 – Overcoming Multiplexing Challenges
Assay diluents matter because the simultaneous detection of multiple protein analytes presents many challenges. This includes assay non-specific binding, cross-reactivity, and antibody interference. R&D Systems recognizes these unique difficulties. The diluents provided with R&D Systems Luminex High Performance Assays have been tested and specifically formulated to overcome the challenges listed above that are unavoidable in multiplex assays.
Defining Interference, Non-specific Binding, and Cross-reactivity
Reagents provided in R&D Systems Luminex Assays are carefully designed and tested to minimize assay interference and ensure an optimally functional sandwich immunoassay (Figure 1). Immunoassay interference occurs when a substance within the assay prevents
Figure 1. Optimally Functional Sandwich Immunoassay
the accurate detection of the target analyte. It can occur due to non-specific antibody binding, antibody cross-reactivity, or antibody interference. In addition, complex sample matrices, such as serum and plasma, can often contain interfering factors. Interfering factors may also be introduced to biological samples via the reagents or equipment used. Interference, no matter the mechanism, can result in either reduced or elevated signal relative to the actual concentration of target analyte. This means untrustworthy data. Read below to learn how we carefully design our Luminex High Performance Assay diluents to overcome these challenges, ensuring that you can always trust the data you generate.
Non-specific binding occurs in an immunoassay when the antibody pair interacts with the sample container or other assay surfaces and contributes to background (Figure 2). Non-specific binding to microparticles, microplates, or other assay surfaces can be reduced by blocking. Diluents provided with R&D Systems Luminex High Performance Assays contain assay-optimized blocking reagents to ensure low non-specific binding.
Figure 2. Examples of Non-Specific Binding in an Immunoassay
Figure 3. Cross-Reactivity in an Immunoassay
Cross-reactivity, the interaction of the antibody pair with a molecule other than the targeted analyte, can be caused by either the capture antibody or the detection antibody and often occurs when proteins in the sample are structurally similar to the analyte of interest (Figure 3). Optimizing antibodies so as to avoid cross-reactivity with substances within the biological sample is often challenging. R&D Systems carefully selects and exhaustively tests our in-house developed antibody pairs used in our Luminex High Performance Assays to ensure analyte specificity.
Antibody interference occurs when endogenous antibodies within samples cross-link with assay antibodies (Figure 4; A-C) or substances within the sample (Figure 4; D, E) and prevent proper target-analyte binding to both the capture and detection antibodies. Common antibody interfering substances in biological samples include human anti-mouse antibodies (HAMA) and rheumatoid factor (RF) antibodies as well as any substance present in exceptionally high concentrations. Diluents provided with our Luminex High Performance Assays are designed to prevent antibody interference from HAMA and RF in biological samples.
Figure 4. Examples of Antibody Interference in an Immunoassay
See more in our educational series on Luminex Assays:
Lesson 1: The Devil is in the Diluents
Lesson 2: Sensitivity Matters
Lesson 3: Consistency is Key
Luminex® is a trademark of Luminex Corporation.