Comparison of pre-processing methods for multiplex immunoassays

Traditional enzyme-linked immunosorbent assay (ELISA assay) recognizes and measures one analyte per plate. Multiplex immunoassays, such as Luminex xMAP technology, use the same principles and apply them to clearly identifiable beads. These beads allow the simultaneous detection of multiple analytes in a single well or reaction. Multiplex immunoassays provide a wealth of information on the role of multiple proteins and other biomolecules in various biological processes,  providing clinicians with insights into identifying and assessing disease progression. Multiplex cytokine assay is a chemiluminescent assay allowing concurrent measurement of four biomarkers or analytes. IL-6, TNFα, IFN-γ, IL-10.

Principle of Multiplex Immunoassays

This technique consists of a set of 100 different colored pearls made using two fluorescent dyes in different proportions. These beads can be further combined with reagents specific for a particular bioassay. Reagents can include antigens, antibodies, oligonucleotides, enzyme substrates, or receptors. This technique enables a multiplex immunoassay in which one antibody to a particular analyte binds to a series of beads of the same color and the second antibody to the analyte binds to a fluorescent reporter dye label. Beads of different colors can be used to multiplex detect many other analytes in the same sample at the same time. A dual detection flow cytometry is used to classify different assays by the pearl color of one channel and measure the fluorescence of the reporter dye  in another channel to determine the concentration of the analyte

Beads are internally colored with two different optical brighteners (red and infrared). Use different concentrations of red and infrared dyes to create up to 100 different areas of pearls. Each bead region binds to a specific target analyte, followed by a biotinylated detection antibody and the reporter dye streptavidin-bound phycoerythrin.

Comparison of Multiplex Immunoassays to Traditional ELISA

Traditional immunoassays, such as the enzyme-linked immunosorbent assay (ELISA), can only detect the presence or absence of one analyte in a single response. In a single response, multiplex immunoassays analyze dozens of distinct analytes. This is especially useful for valuable samples and when only a limited amount of material is gathered for analysis. Because numerous assays may be conducted at the same time, the multiplex immunoassay saves time.

Multiplex immunoassays are similar to ELISA in that they both rely on antigen-antibody binding specificity. A detecting system is also required. An enzymatic response is used in classic ELISAs, whereas fluorescence probes are used in most current multiplex experiments. Multiplex immunoassays are classified into two general groups based on the type of surface on which the antibodies or antigens are attached.

  1. A solid flat surface with strategically spaced targets for each analyte.
  2. Beads with an identifying color are coupled to an antigen or antibody.

The first method is to use spatially independent assays, which are identical to ELISAs except that they use the same reaction volume for several antibody pairs. There are a few variations on this topic, but the physical isolation of capturing antibodies on a shared solid surface is a common element.

Methods that integrate tests for several target analytes are required to solve workflow and sample capacity issues. Multiplex immunoassays integrate tests for many target analytes in a single reaction volume, minimizing sample volume and workflow issues.

When compared to singleplex assays, multiplex systems can provide significant benefits. The necessity for less sample input has already been mentioned; testing numerous markers typically requires 25–50  µL of sample volume, whereas ELISA requires 100  µL per target.

In addition, multiplex platforms frequently increase the test dynamic range. Multiplex assays have been found to maintain linearity across three or even five orders of magnitude, whereas ELISA rapidly loses linearity over a few orders of magnitude. Because panels frequently contain targets at wildly varying concentrations, linearity over a large range is critical for valid data.

Limitations of Single Analyte Assays

Although singleplex tests (assays that measure only one analyte) are reliable and popular, they may not be the optimal format for your research because measuring several target proteins is often more physiologically informative.

Multiple target ELISA measurement necessitates running numerous test procedures simultaneously, which takes time and raises the chance of mistakes. Furthermore, sample volume needs to rise in direct proportion to the number of analytes detected, posing a significant problem given the scarcity and value of biological samples.

To compensate for the increasing sample volume needs, assays can be downsized, although this generally necessitates the use of expensive specialist equipment. Microfluidics allows multiple assays to run in parallel with minimal manual processing; however, as more assays and samples are added, the complexity of the fluidics routes grows rapidly, limiting the number of samples and analytes that can be measured.

There are numerous advantages to using multiplex immunoassays over singleplex immunoassays, and different platforms are available, albeit specific equipment is now required in some circumstances.

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