Multiplex Immunoassays Powered by Luminex xMAP Technology

For almost 50 years, immunoassays have allowed sensitive and highly specific detection of analytes of interest in biological samples for use in both life science research and clinical diagnostics. Traditional enzyme-linked immunosorbent assays (ELISA assays) detect and measure a single analyte per plate. Newer bead-based immunoassays, like those based on Luminex xMAP technology, utilize the same principle and apply it to uniquely identifiable beads, enabling the simultaneous detection of multiple analytes in a single well or reaction. Multiplex immunoassays yield a wealth of information on the roles of multiple proteins and other biomolecules in diverse biological processes, thereby providing clinicians with insight into the identification and assessment of disease progression.

Early Immunoassays

The first immunoassay, developed by Yalow and Berson in 1960, used radiolabels for detection and became the standard due to its high sensitivity, despite health risks. In the early 1970s, ELISA emerged as a safer alternative, using enzymatic reactions for detection. While initially less sensitive than RIA, advances in monoclonal antibodies and chemiluminescence improved ELISA’s sensitivity. Early immunoassays, typically single-plex, were foundational for life sciences and clinical diagnostics but required substantial time and sample volume for comprehensive analysis.

Luminex xMAP Technology

Luminex xMAP® Technology is at the forefront of multiplex immunoassay techniques. This innovative technology combines advanced fluidics, optics, and digital signal processing to deliver high-throughput, high-precision results. It leverages the principles of bead-based assays but amplifies the capabilities by allowing the simultaneous analysis of up to 500 analytes in a single reaction, depending on system design.

The Bio-Plex® Multiplex Immunoassay System utilizes xMAP technology licensed from Luminex to permit the multiplexing of up to 100 different targets within a single sample. This technique involves 100–500 distinctly colored bead sets created by the use of two fluorescent dyes at distinct ratios. These beads are conjugated with a reagent specific to a particular bioassay. The reagents may include antigens, antibodies, oligonucleotides, enzyme substrates, or receptors. xMAP technology enables multiplex immunoassays in which one antibody to a specific analyte is attached to a set of beads with the same color, and a second antibody to the analyte is attached to a fluorescent reporter dye label. The use of different colored beads enables the simultaneous multiplex detection of many other analytes in the same sample. A dual-detection flow cytometer is used to sort out the different assays by bead colors in one channel and determine the analyte concentration by measuring the reporter dye fluorescence in another channel (Figure 1).

• 

  A. xMAP bead regions 1–100.

• 

  B. Each bead region maps to a target analyte.

Figure 1. Luminex xMAP Multiplex immunoassay technology. Beads are colored internally with two different fluorescent dyes (red and infrared). A. Different concentrations of red and infrared dyes are used to generate up to 100 distinct bead regions. B. Each bead region is conjugated to a specific target analyte followed by binding with a biotinylated detection antibody and a reporter dye, streptavidin-conjugated phycoerythrin. Each bead region enables multi-analyte detection in a single well.

Luminex xMAP Technology vs. Other Immunoassay Techniques

Luminex xMAP technology is frequently compared to the traditional ELISA technique, which is limited by its ability to measure only a single analyte. The inability of traditional ELISA to multiplex presents additional limitations in sampling volume, cost, and labor (Figure 3).

While the basic concepts of detection are similar between ELISA and Luminex xMAP technology, there are some important differences that center mainly on the capture antibody support. Unlike with traditional ELISA, Luminex xMAP capture antibodies are covalently attached to a bead surface, effectively allowing for a greater surface area as well as a matrix or free solution/liquid environment to react with the analytes. The suspended beads allow for assay flexibility in a singleplex or multiplex format.

Comparison of Bio-Plex Multiplexing vs. Traditional ELISA

Figure 3. Protocol comparison between traditional ELISA and the Bio-Plex System. The Bio-Plex Multiplex Immunoassay System, using Luminex xMAP technology, offers high-performance readers, industry-leading software, and sensitive assays in ready-to-use or custom configurations, enabling you to quantify over 48 different protein and peptide targets simultaneously in a single 12.5 µl sample.

Advantages of the Bio-Plex Multiplex System with Luminex xMAP Technology

Luminex xMAP technology offers numerous advantages that make it a preferred choice for multiplex immunoassays: