Selecting a Multiplex Platform

The number of multiplex platforms continues to grow. Since it would be impossible to cover every available option here, this editorial focuses on five different multiplex technologies and shares insights to help identify a platform that best meets your experimental needs.

The concept of a multiplex immunoassay was first proposed in 1961.¹ However, it was not until 1989 that assay miniaturization and the principles of microarray technology were described, paving the way for multiplex immunoassays as we know them today.² Now, researchers have access to a broad range of multiplex platforms that vary in terms of plex, sample compatibility, and whether specialized instrumentation and/or reagents are required for their use. Here, we look at five different multiplex technologies and the advantages they offer, and suggest when you might wish to consider choosing them.

• Antibody arrays from RayBiotech

Level of plex possible: 8000 proteins (semi-quantitative) or 1400 proteins (quantitative)

Compatible sample types: any biological fluid, cell culture medium, cell extract, or tissue lysate

Instrumentation required: depending on the chosen platform, RayBiotech’s arrays are compatible with any standard microarray scanner, CCD camera/x-ray film developer, or flow cytometer with red and blue lasers.

RayBiotech offers antibody arrays on several different platforms, including planar glass slide and membrane-based arrays, as well as cytometric bead-based arrays (CBA). All of these are based on immunocapture of proteins with a panel of pre-selected antibodies, followed by signal detection using a biotin tag (direct labeling of protein) or a biotinylated detection antibody (sandwich immunoassay).

“A main advantage of our technology is that it does not require extensive training—the workflow is similar to ELISA,” reports Valerie Jones, Ph.D., Marketing Director at RayBiotech. “Protein identity is known instantly by referring to the array map, while the included analysis tool simplifies signal normalization and calibration.”

Antibody arrays have utility for both large- and small-scale sample profiling. “When performing exploratory studies, such as biomarker discovery or secretome profiling, large arrays let you capture as many proteins as possible,” says Jones. “Alternatively, when targeting a subset of proteins involved in a certain pathway or area of interest, smaller arrays can represent a more budget friendly option.”

• Luminex xMAP^® Technology

Level of plex possible: up to 500 proteomic or genomic biomarkers

Compatible sample types: proteomic biomarkers can be measured in samples including clinical biospecimens (e.g., blood, serum, tissue, sputum), non-conventional matrices such as wound dressings, air/breath, and dried-blood spots, and in cell culture supernatants; sample types that can be analyzed for genomic biomarkers include formalin-fixed paraffin embedded (FFPE) tissue, liquid biopsy material, and conventional biospecimens.

Instrumentation/reagents required: Luminex fluorescence analyzer and xMAP microspheres

“xMAP assays are based on two major components: the xMAP microspheres (color-coded antibody-coated beads) and a portfolio of Luminex fluorescence analyzers to meet various multiplexing needs and throughput,” explains Dominic Andrada, Sr. Marketing Manager – Scientific Applications, at Luminex Corp (a DiaSorin company). “An extensive selection of off-the-shelf kits is available for xMAP immunoassay and genomic assays, as well as custom assay development services from a variety of vendors and service providers.”

When deciding whether to use Luminex xMAP technology, a key consideration is whether an assay with sufficient sensitivity and specificity is available for your chosen biomarkers. Vendors to investigate include Bio-Rad, Bio-Techne, MilliporeSigma (the U.S. and Canada Life Science business of Merck KGaA, Darmstadt, Germany), and Thermo Fisher Scientific, many of whom also offer custom assay development services. “In addition, subject matter experts in the LuminexPLORE Lab can help build an assay for any biomarker and sample type, meaning that Luminex technology is open to almost any application,” notes Andrada.

• SignalStar™ Multiplex IHC

Level of plex possible: 8

Compatible sample types: FFPE tissue

Instrumentation required: SignalStar protocols can be run manually at the lab bench or automated on the BOND RX Autostainer by Leica Biosystems; images can be captured with most fluorescent imaging microscopes.

SignalStar Multiplex IHC is a technology for spatial biology research that uses high-throughput, mid-plex IHC assays to label up to eight targets in FFPE tissues simultaneously. Importantly, it eliminates the need for antibody cycling, which risks epitope loss and masking, and provides results up to 70% faster than other traditional mIHC methods.

“Another advantage of SignalStar is that, by using a combination of oligonucleotides and fluorophores to amplify the antibody signal, it allows for detecting even low-expressing targets,” explains Sarah Klein, Ph.D., Associate Director, Multiplex Assays at Cell Signaling Technology. “SignalStar also provides the flexibility to switch out targets as required, with no need for additional panel or protocol optimization. Researchers simply use the SignalStar Multiplex IHC Panel Builder to select from a menu of IHC-validated antibodies that work out of the box in FFPE tissues.”

Being a multiplex IHC technology, SignalStar is ideally suited for investigating how complex and constantly evolving relationships between cellular proximity, organization, and the timing of signaling events can affect disease progression and therapeutic response. However, what sets SignalStar apart from traditional mIHC methods is its capacity to maximize the data acquired from limited samples without compromising sample quality.

• Ella™ instrument and Simple Plex Assays™—automated ELISA

Level of plex possible: 8

Compatible sample types: complex samples including serum, plasma, and cell culture supernatants

Instrumentation required: Ella instrument and Simple Plex cartridges

Traditional ELISAs detect just a single analyte and have a largely manual workflow that can introduce operator errors. The automated Ella instrument, coupled with Simple Plex Assays, was developed to address these limitations by simultaneously quantifying up to 8 analytes in a single sample, as well as reducing or removing many manual steps to deliver more reproducible results in just 90 minutes.

“A key feature of the Ella platform is the elimination of cross-reactivity when multiplexing,” reports Marissa Beck, Senior Product Manager at Bio-Techne. “This is achieved through the use of distinct microfluidic channels that allow for separate measurement of each analyte. Other advantages of the Ella platform include sub-picogram sensitivity and a broad dynamic range spanning four logs, which allow for accurately detecting analytes with different expression levels in the same sample.”

Beck suggests that the Ella platform is particularly suited for scenarios demanding high-throughput, accurate biomarker quantification with limited sample volume, where precision, low variability, and an efficient workflow are required.

• Simple Western—automated western blotting

Level of plex possible: Varies by assay; Bio-Techne has demonstrated multichannel detection of 6 targets plus total protein detection in 8 samples with 1 run

Compatible sample types: complex samples including cell, viral, and tissue lysates

Instrumentation required: Simple Western instrument and capillaries

The Simple Western platform was designed to overcome known challenges for traditional western blotting, including poor reproducibility, a lack of accurate quantitation, and extensive time to results. It represents the only fully automated solution for western blot analysis, providing gel-free, blot-free, hands-free results in 3 hours with just 3 µL samples.

“Simple Western is a capillary immunoassay platform that combines protein separation with sensitive chemiluminescence and fluorescence immunodetection using conventional primary and conjugated secondary antibodies,” explains Brett Houser, Senior Product Manager at Bio-Techne. “Specifically, automated in-capillary size separation and covalent immobilization of target analytes is followed by washing, antibody incubation, and detection steps.”

There are several approaches to multiplexing with Simple Western. One is to add several primary antibodies and multiplex by size in a single run. Additional infrared (IR) and near-infrared (NIR) channels can be used for multiplexing targets of similar molecular weights. You can also use the instrument’s RePlex module for stripping and re-probing, which allows for detecting additional targets or total protein for data normalization. Both provide reproducible and quantitative results, more quickly, than established methods for western blotting.

References

1. Feinberg JG. A 'microspot' test for antigens and antibodies. Nature. 1961 Dec 9;192:985-6. 

2. Ekins RP. Multi-analyte immunoassay. J Pharm Biomed Anal. 1989;7(2):155-68.