Sensitivity and throughput are key considerations when selecting a multiplex immunoassay. This article explores how five leading multiplex technologies achieve both and looks at ways in which current capabilities are being expanded.

Luminex xMAP® Technology

How it works. xMAP Technology uses color-coded beads to capture up to 500 different protein analytes or genetic targets from solution. A fluorophore-labeled detection reagent is then added to enable quantification with a dedicated Luminex instrument.

How it achieves high sensitivity. “xMAP technology works with smaller sample volumes than most other methods while delivering more information and better sensitivity than conventional ELISAs,” reports Sherry Dunbar, Ph.D., MBA, Senior Director, Scientific Affairs Programs at Luminex. “This is accomplished by suspending beads in solution, which provides faster and more efficient binding kinetics for nucleic acids and proteins than being bound to an immobile substrate at the bottom of a well.”

How it provides high throughput. “Throughput has always been one of the major differentiators for xMAP Technology,” says Dunbar. “Because each bead serves as an individual test, a large number of different assays can be performed and analyzed simultaneously. Our instrument portfolio includes a range of platforms so scientists can choose the overall capacity that best meets their needs. Some of our users have even fully automated their xMAP workflows for industrial-scale throughput.”

Bio-Plex Multiplex Immunoassays (Bio-Rad)

How it works. Bio-Plex Multiplex Immunoassays are based on Luminex xMAP Technology. They are available as ready-to-use kits or custom configurations and allow for quantifying up to 48 biologically relevant biomarkers in a single well using as little as 12.5 µL sample.

How it achieves high sensitivity. “Bio-Plex assays have a sub-picogram per milliliter limit of detection (LOD) and a wide dynamic range (4 log), enabling both high and low concentrations of biomarkers to be quantified in the same assay without sacrificing accuracy,” explains Vanitha Margan, Global Product Manager, Immunoassays at Bio-Rad. “These performance characteristics are achieved by leveraging xMAP Technology and implementing the SMARTS approach during assay design, which includes rigorous antibody qualification.”

How it provides high throughput. The multiplex capability of Bio-Plex assays, combined with a significantly shorter protocol (≤3 hours) than a conventional ELISA, supports high-throughput analysis. Sample processing can be further streamlined with automation tools, such as the Bio-Plex Pro Wash Station, enhancing consistency and efficiency in the workflow. “The capacity for higher plex assays and faster protocols reduces manual labor while maintaining consistent performance, increasing both speed and accuracy in high-volume research environments,” says Margan.

Inkjet Microarray Technology (Arrayjet)

How it works. Inkjet Microarray Technology involves printing a library of samples onto a solid substrate. This is then treated with an analyte and selective binding events are detected using specialist imaging apparatus and software.

How it achieves high sensitivity. Arrayjet’s protein microarray-based immunoassays are primarily semi-quantitative designs with fluorescent detection, providing sensitivity down to the picomole range. “This high sensitivity is achieved through 1) our ability to immobilize a broad concentration range of proteins onto appropriate surface chemistries, 2) the use of bright and robust fluorophore labels, and 3) our capacity to tune assay readout sensitivity with advanced confocal based microarray scanners,” says Dr. Adam Buckle, Chief Scientific Officer at Arrayjet.

How it provides high throughput. “Because Arrayjet immunoassays accomplish multiplexing through miniaturization and spatial separation of the targets or analytes into distinct spots, the only factor limiting the number of sample-analyte interactions tested per condition is the physical constrains of the solid substrate,” explains Buckle. “The semi-quantitative format just described allows for testing as many as 50,000 sample-analyte interactions per condition, but additional levels of multiplexing can be achieved by using several fluorescent channels in parallel to label distinct test samples.”

Sequential immunofluorescence (seqIF™) on Lunaphore’s COMET™ platform

How it works. SeqIF uses non-conjugated primary antibodies in combination with fluorescently labeled secondary antibodies, or fluorescently labeled primary antibodies, for detecting analytes in cryopreserved or FFPE tissue sections.1 It involves repeated cycles of staining, imaging, and antibody elution on the COMET platform with a precision microfluidic chip technology for a fully automated workflow.

How it achieves high sensitivity. According to Dr. Andreas Wiesner, Head of Product Management at Lunaphore, a Bio-Techne brand, the choice of technology during staining and imaging can impact challenges in sensitivity caused by tissue sourcing and processing. “An outstanding advantage of seqIF is the gentle, buffer-based elution process, which avoids harsh temperature cycles or bleaching,” he says. “This preserves epitope stability and tissue integrity, ensuring high sensitivity up to high plex and over multiple runs with the same tissue. This means far more data per sample than before.”

How it provides high throughput. COMET’s unique Fast Fluidic Exchange (FFeX™) technology decreases the antibody incubation time from several hours to a few minutes. “In addition, COMET automatically detects the sample and only images areas covered by the tissue, to significantly reduce scanning time compared to conventional methods,” comments Wiesner. “Further time is saved with ready-to-use antibody panels.”

Orion™ Multiplex Immunofluorescence Imaging (RareCyte)

How it works. The Orion platform combines advanced instrumentation and signal extraction technology with exceptionally bright photostable ArgoFluor™ dyes for rapid single round, whole slide staining and scanning.2 Panels are designed using RareCyte’s catalog of directly conjugated primary antibodies, and dye kits that enable custom biomarker addition though simple and direct amine conjugation chemistry.

How it achieves high sensitivity. “The high sensitivity of the Orion platform comes from the novel design of the instrument, and hardware and software matched to the exceptional brightness and photostability of the ArgoFluor dyes,” reports Joshua Nordberg, Ph.D., VP of Customer Success at RareCyte Inc. “Laser-based illumination provides powerful excitation for maximum fluorophore response, while the 16-bit camera has up to 95% quantum efficiency, maximizing the dynamic range of the system. As a result, Orion can simultaneously image 20 fluorescent channels in a single, very fast whole slide scan.”

How it provides high throughput. “Orion’s key advantage for spatial biology is providing high-plex data in a single staining and imaging round,” says Tad George, Ph.D., SVP of R&D. “The single-round approach has been specifically engineered to detect biomarkers revealed by direct conjugates, along with hardware/software enabling the extraction of spectrally overlapping signals. The system enables sub-cellular resolution imaging at very high rates (~75 minute per cm2) and can access the entire slide (up to 10 cm2) with unattended, multi-slide, multi-ROI scanning to accommodate large tissues and TMAs.”

Looking to the future

With researchers continuing to seek further improvements in both sensitivity and throughput, multiplex technologies are evolving. Luminex’ latest platform, the xMAP INTELLIFLEX® DR-SE System (RUO), includes a second reporter laser to double the amount of data collected in an experiment. And Arrayjet has developed its ArrayPlex multilayered microarray platform, which can identify binding interactions between two libraries without prior knowledge of either ligand or target and allows for up to 2 million interactions to be assayed in one 10-day screening cycle.

“At RareCyte, we’ve heard from customers that quantitative analysis is starting to be a bottleneck with the high throughput already provided by the Orion platform,” comments George. “As such, we have been developing advanced imaging tools with key opinion leaders in the spatial biology field that we will be integrating into near-future versions of software.”

Lastly, Wiesner emphasizes the demand for obtaining as many actionable insights from one tissue sample as possible. “This includes pulling in further omics data and capturing biological interactions that happen only in concrete locations in very short timeframes,” he says. “By integrating seqIF with RNAscope™ HiPlex Pro on COMET, we’re enabling researchers to generate true multiomic data that shows the tissue landscape and its interactions at a molecular level with precision.”

References

1. Rivest F. et al. Fully automated sequential immunofluorescence (seqIF) for hyperplex spatial proteomics. Sci Rep. 2023 Oct 9;13(1):16994. doi: 10.1038/s41598-023-43435-w.

2. Lin J.R. et al. High-plex immunofluorescence imaging and traditional histology of the same tissue section for discovering image-based biomarkers. Nat Cancer. 2023 Jul;4(7):1036-1052. doi: 10.1038/s43018-023-00576-1.