The complete guide

to multiplex assays in
drug discovery
Everything you need to know when choosing a suitable
multiplex immunoassay for your drug discovery program

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Contents

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

– What does the guide cover? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

– The advantages of multiplex immunoassays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
– The importance of multiplexing for drug discovery. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Choosing a multiplex assay for use in drug discovery. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Review of multiplex assay techniques employed in screening. . . . . . . . . . . . . . 5

– Multiplex suspension assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

– Multiplex planar assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
– Comparison of standard multiplex assays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Partnering with Abcam on multiplex immunoassay solutions. . . . . . . . . . . . . . 8

– FirePlex multiplex assays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

– Fireplex-96 immunoassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
– High-throughput FirePlex immunoassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
– How can Abcam help you choose the right multiplex immunoassay
for your workflow?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
– Case study: Profiling of secreted proteins from iPSCs with FirePlex-384 . . . . . . . . . . . . . . . . . . 11

References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

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Introduction
What does the guide cover?

This guide aims to highlight the value of multiplexing in drug discovery and assist in
choosing the most suitable multiplex immunoassay for your drug discovery program.

The first chapters discuss the advantages of multiplexing and its value for drug discovery.
The following chapters highlight the key factors to consider when choosing a multiplex
assay and provide a comprehensive overview of various multiplex assay techniques
used in screening.

The advantages of multiplex immunoassays

Accurately quantifying protein levels is essential in research and diagnostics, whether

quantitatively analyzing the concentration of a particular target or qualitatively testing
for the relative presence of protein markers. Despite the robustness and popularity of
assays measuring a single analyte (ie, singleplex, such as ELISA or western blot), they may
not be the optimal choice for your research as it’s often more biologically informative to
analyze more than one target protein simultaneously.

Measuring multiple targets by ELISA involves performing several assay workflows in

parallel, which is time-consuming and increases the risk of error. Moreover, sample
volume requirements increase with the number of analytes measured, which is a crucial
challenge with scarce and precious biological samples.

Multiplex immunoassays enable us to measure multiple target analytes in a single

reaction volume reducing workflow and sample volume problems. For example, 25–50 µL
sample volume is required to test multiple markers compared with 100 µL per target
required for ELISA.

The importance of multiplexing for drug discovery

The demand for safe and efficacious drugs is steadily growing, increasing the need for
fast, efficient immunoassays for drug discovery and development. The drug discovery
process often starts with lead optimization using high-throughput screening (HTS) to
narrow down huge compound libraries to a few promising candidates. HTS requires
scalable, high-quality immunoassays to identify the candidates quickly, efficiently, and
with minimal costs.

Single analyte immunoassays, such as ELISA, were historically used and are still invaluable
in early discovery and measuring therapeutic responses in clinical trials. However, they
are inefficient in terms of cost, labor, time, and sample volume required when measuring
more than one target protein in HTS. Multiplex immunoassays address those challenges
by studying several targets in a single well using minimal amounts of sample and
exponentially increasing the number of data points collected in HTS.

Tackling more complex conditions such as cancer and autoimmune disease using
increasingly advanced techniques has led to more demand for multiple analyte
analyses. For this reason, multiplex assays have become a necessary part of drug
discovery as they help identify valid drug candidates earlier in the screening process.
Multiplexing achieves this by generating more biologically relevant data from limited
sample volume, as various plausible targets are monitored in a single well, eliminating
the need for separate sample/control for each target and reducing the coefficient of
variability in downstream analysis. Furthermore, multiplex assays can simultaneously look
at a target, downstream events, and parallel pathways, thus helping us better identify
the participating pathways and players, leading to better drug efficacy. This way,
screening can effectively eliminate toxic/ineffective compounds early in the discovery
pipeline, decreasing later-stage failures and avoiding costly mistakes1-3.

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 Choosing a multiplex assay for
use in drug discovery
Assay specificity, sensitivity, and reproducibility

Robust analyte specificity is essential when screening thousands of compounds across

several targets to find the most promising hits because false positive or negative results
may result in wasting time on a false lead or missing a promising drug candidate.
Therefore, during screening studies with a high number of samples run in one-shot
approaches, specificity outweighs sensitivity, while both high throughput and
automation capabilities become extremely relevant.

Assay reproducibility is another critical factor to consider, and it depends on using

reliable antibody pairs with high batch-to-batch consistency. Recombinant monoclonal
antibodies overcome the issues observed with traditional monoclonal and polyclonal
antibodies in terms of consistency between batches and reproducibility. During
recombinant antibody manufacture, the antibody genes are cloned in high-yield
expression vectors, and the gene sequences used can be modified to improve antibody
sensitivity. More importantly, since the antibody gene sequences are controlled,
recombinant antibodies are produced with high batch-to-batch consistency, capable
of delivering highly reproducible results required by drug development research1,3.
ed
ed
High-quality, validated antibody pairs with:

Excellent batch-to-batch
Excellent batch-to-batch
consistency
consistency

Confirmed target specificity

Confirmed target specificity

mL Low pg/mL
mL High sensitivity Low pg/mL
ection High sensitivity Limit of Detection
ection Limit of Detection
Assay format:

Automation friendly
Automation friendly

Wash vs no-wash workflow

Wash vs no-wash workflow

Figure 1. The main factors to consider when choosing a multiplex assay for use in drug discovery.

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Automation

In recent years, automation has revolutionized how the drug discovery pipeline works
by streamlining certain decision-making processes4. As screening libraries contain
thousands of candidate molecules, multiple compounds need to be screened in a
single assay. To make this possible, automation has been implemented in compound
management, HTS and hit-to-lead generation in the last 20-30 years. Since some
screening processes are repetitive, monotonous, time-consuming, and prone to error,
automation has increased time savings while reducing errors and costs.

In the last ten years, the emergence of robotics, from cherry-picking the compounds to
carrying out routine procedures and assays, has transformed the pipeline process. For
instance, the use of liquid handlers for dispensing compounds and performing workflows
has reduced processing time, decreased the possibility of sample contamination, and
increased the efficiency of volume transfers. Advances in automation have encouraged
a transition to using smaller volumes of reagents and compounds, with experiments
being performed in microtiter plates of high density, usually in a 96-, 384, or 1536-plate
format. This process called assay miniaturization has dramatically improved the screening
processes by generating large data sets quickly and efficiently while reducing reagent
consumption and space5. Therefore, automation and assay miniaturization have
facilitated better-informed decisions and ultimately decreased costs in screening4.

When performing any HTS study, multiplexing while screening large numbers of
samples in a single assay is essential to help identify a few promising lead compounds
in a timely and cost-effective manner. Any screening study would benefit from an
automation-friendly assay because using liquid handlers/dispensers and robotics
will streamline the process, reducing errors in repetitive assays and making screening
thousands of compounds smoother and easier.

Wash vs no-wash assay

One of the automation challenges in a multiplex immunoassay is the need for multiple
wash steps during assay preparation. Although wash steps reduce the background,
increasing the signal-to-noise ratio and thereby sensitivity, they slow down the process
and introduce wash step-related errors. In contrast, a no-wash assay decreases the
number of steps between reagent handling and final data collection. Transitioning to a
non-wash assay workflow simplifies assay setup and automation implementation, which
would reduce potential errors and speed up the scale-up during HTS.

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Review of multiplex assay
techniques employed in screening
Protein identification and quantitation have become extremely valuable in advancing
our understanding of disease onset and progression, making the role of robust
immunoassays in drug discovery more prominent. A wide range of available assay
technologies makes it challenging to determine which platform would be best suited
for specific researcher’s needs and study design, allowing to collect the most data in
the shortest time. Each assay type has its advantages and disadvantages based on the
sample to be profiled and the required sensitivity.

Multiplex immunoassays are divided into two main categories: planar and suspension. Here
we describe the key principles, benefits and limitations of planar and suspension assays and
review the most common examples of multiplex immunoassays in both categories.

Multiplex suspension assays

In these assays, affinity capture agents are chemically affixed to populations of

characterized microscopic coded beads or particles (Figure 2). The subsequent
cognate immuno-affinity interactions are detected with fluorescent reporter probes and
interrogated by a laser in a flow cell, using flow cytometry principles.

H
1 2 3 4 5 6 7 8 9 10 11 12

Planar Suspension
B

Capture
A C
Y

A B C D
X D

Sample binding
A C
Y

A B C D
X D

Z
A

B
Detection Y
C

D
X

Figure 2. Planar and suspension assay formats. In planar assays, capture ligands are immobilized on a rigid
two-dimensional support and probed with a sample. X and Y coordinates are the fixed positions (solid phase)
for the binding to occur, with the Z axis resolving the fluorescent or chemiluminescent signal. In suspension
assays, capture ligands are immobilized on color- or size-coded microspheres. Suspension assays are
distinguished by coding attributes, and flow cytometry is used to detect assay-specific fluorescent signal.

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Since the suspension assays use a bead/particle immobilization approach, they need
to identify which analyte is being measured on which bead/particle. For this purpose,
suspension assays use combinations or levels of dyes to encode analyte-specific
capture beads/particles.

Benefits
– Flexible assay content

Limitations
– Chemical modification to biological capture agents
– Complex instrumentation
– Average sample volume required ~10-12.5 μL

The suspension assay workflow may include multiple wash steps for sensitivity and
dynamic range (eg, Luminex®) or have a no-wash format (eg, AlphaLISA®). Depending
on assay type, multiplex suspension assays can be read on a standard flow cytometer
(FirePlex®-96) or require specialized equipment (Luminex®, AlphaLISA®).

Popular suspension assays:

– Luminex® technology uses combinations of red and near-infrared dyes to encode

solid polystyrene microspheres, potentially generating codes for 500 different targets.
These codes are used to identify the bound capture antibodies, and a sandwich assay
with phycoerythrin-labeled detector antibodies is used to measure protein levels.

– AlphaLISA® assays use proximity-dependent chemical energy transfer to generate

a signal dependent on the occurrence of the desired biomolecular interaction.
The multiplexing capabilities with these assays are up to 3-plex and require specific
plate readers.

– Bead arrays represent a type of multiplex suspension assay that allows simultaneous
measurement of multiple analytes using beads containing different concentrations
of fluorescent dye. The beads are usually analyzed using standard flow cytometer
techniques. The workflow of such assays typically involves multiple wash steps to
improve sensitivity and dynamic range.

– Abcam FirePlex®-96 immunoassays use particle technology for multiplexing protein

targets. Affinity agents (antibodies or proteins) are immobilized on proprietary
barcoded hydrogel particles, providing the basis for target capture detected
through reporter fluorescence to quantify the target. FirePlex-96 measures up to 70
analytes per well, and the readout is achieved on standard flow cytometers.

– Abcam FirePlex®-384 and 1536 immunoassays use the same particle technology
as FirePlex-96® but employ static high content imaging to deconvolute stationary
particle codes and analyte-specific detector fluorescence at the bottom of a 384-
or 1536-plate well. These assays are compatible with high throughput workflows
and instrumentation and can be read with various high content imagers.

Multiplex planar assays

In these assays, immuno-affinity capture agents (antibodies or proteins) are affixed

co-locally to a planar substrate using various chemical or hydrostatic methods (Figure 2).
The subsequent cognate immuno-affinity interactions are detected with fluorescent,
chemiluminescent or chromogenic reporter methods and then interrogated with a
bi-focal fluorescent laser scanner or CCD camera.

Benefits
– Very high-density multiplexing
– Capture agents can be affixed without chemical modification
– Archivable source data

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Limitations
– Fixed assay content
– Complex data analysis from large data quantity

Popular planar assays:

– Plate-based electrochemiluminescence (ECL) assays use electro-chemilumines-

cent labels to generate a reporter signal decoupled from the stimulation method,
leading to a lower background and higher sensitivity. Measures up to 10-plex per
well in a 96-well format but require specific instrument installation.

– RayBio® antibody array platform has different antibody array options depending
on the studied protein activity and the assay readout.
– Quantibody® array quantitatively measures proteins using a fluorescent readout.
– C-Series arrays assess relative protein expression patterns across different sample
types and experimental conditions by chemiluminescence.
– L-Series array allows screening of many proteins per assay for target identification
using chemiluminescence or fluorescence.

– CDI HuProt™ protein microarray profiles proteins immobilized in duplicate on a

nitrocellulose-coated or epoxy glass slide. The protein interactions are detected by
a biotinylated streptavidin-conjugated fluorescent dye.

Comparison of standard multiplex assays

When selecting a multiplex assay, various factors should be considered, such as

requirements for specific instrumentation and sample volume, available plate
types, plex capability, wash or no-wash workflow, possibilities of automation and
high-throughput, and plate re-read option. Table 1 summarizes the main capabilities of
the most common multiplex planar and suspension assays.

Table 1. Technical capabilities of the most common multiplex assays.

Technical
Multiplex Planar Multiplex Suspension
requirements

Plate- FlexMap
CDI Bead Luminex® FirePlex®- FirePlex®- FirePlex®- MagPix® Alpha-
based RayBio® 3D®
HuProt™ Arrays 100/200 96 384 1536 (Luminex®) LISA®
ECL (Luminex )
®

Specific
Yes No No No Yes Yes No No No Yes Yes
iInstrument

Sample
10 µL <10 µL <10 µL <10 µL 25 µL 12.5 µL 12.5 µL 6.25 µL 2.5 µL 12.5 µL 10 µL
volume

Plate type
96-well 96-Well 2-Well 384-well 96-well 384-well 96-well 384-well 1536-well 96-well 1536-well
(max wells)

Plex size/well 10 100's 20,000 12-65 100 500 70 10 5 50 3

Workflow Wash Wash Wash Wash Wash Wash Wash No-Wash No-Wash Wash No-Wash

Low to
Throughput High High High Low Medium low High High Low High
medium

Plate re-read
No Yes Yes Yes No No Yes Yes Yes No No
possible

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Partnering with Abcam on
multiplex immunoassay solutions
Multiplex assays allow researchers to simultaneously measure multiple analytes in
each well, providing more insightful data to improve decision-making and accelerate
research and discovery studies. Abcam offers a comprehensive range of multiplex
assays, including high-throughput no-wash automatable formats.

FirePlex multiplex assays

Abcam FirePlex immunoassays have been designed to overcome the common challenges
in biomarker and drug screening studies. The FirePlex particle technology (Figure 3)
provides multiplex assays with high plex, sensitivity, and high-throughput capabilities.

A B C

Analyte
Code

Analyte
Quantity

D
Figure 3. FirePlex particle technology and its readout on a high content imager. A. Varying intensities of green
Field
and of View
yellow fluorescent dyes are Detection
used for particleColor
identityChannel Excitation
barcoding; yellow Emission
dye, which is co-locally separate
on the particle from the barcoding region, is used for analyte quantitation. B. Two images are captured per
well,
Entirewith approximately
single well area 20 particles
16-Bit analyzed per analyte using the FirePlex
Green Analysis
440-500 nmWorkbench500-535
[Link]
C.
Representative graph demonstrates analyte fluorescent intensity levels automatically output from scanned
of 384-well
wells plate
along with analyte CMOS
atstandard curves.
4X objective Camera Yellow 440-500 nm 570-630 nm

Compared to traditional multiplex assays, FirePlex multiplex assays offer a fast,

automatable workflow, small sample requirements, and easy readout on flow
cytometers and high-content imagers, saving hands-on time and sample. Furthermore,
using our high-quality antibodies with unrivaled reproducibility and excellent sensitivity,
FirePlex multiplex assays enable the generation of robust, consistent data.

We offer FirePlex assays for multiplex proteins (FirePlex immunoassays) or miRNA (FirePlex
miRNA assays) analysis, including:

– FirePlex-96 immunoassay: up to 70-plex in 96-well plate, ideal for biomarker profiling

– FirePlex-384 and FirePlex-1536 immunoassays: up to 10-plex, or 5-plex respectively,
high-throughput (384- and 1536-well) formats with no-wash, automated workflow
– FirePlex miRNA assays: miRNAs profiling directly from small amounts of biofluid or
FFPE, without RNA purification or pre-amplification steps

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Fireplex-96 immunoassays

FirePlex-96 immunoassays are fully customizable and highly sensitive multiplex panels
enabling analysis of up to 70 analytes per well in a 96-well plate on standard flow
cytometers. These immunoassays enable comprehensive biomarker profiling directly
from biofluids.

– 0.5 pg/mL to 30,000 pg/mL detection

– Up 5-log dynamic range
– Only 12.5 µL of sample input, direct from biofluids
– Free data analysis software

Learn more about the advantages of FirePlex-96 immunoassays.

High-throughput FirePlex immunoassays

High-throughput FirePlex-384 and FirePlex-1536 immunoassays are ideal for discovery,

screening, and profiling projects providing a fast, easy and cost-effective tool to analyze
thousands of samples in a fraction of time.

Table 2. FirePlex-384 and FirePlex-1536 capabilities

FirePlex-384 FirePlex-1536
Multiplex capabilities 10 analytes/well 5 analytes/well
Assay format 384-well plate 1536-well plate
Data points per plate 3,840 7,680
Simple, no-wash workflow two-step one-step
Automated workflow with a < 20 min scan per plate <1 hour scan per plate
readout on high-content imagers

By providing a new, more effective biomarker analysis workflow (Figure 4, 5), FirePlex
immunoassays enable data-driven R&D to guide the most effective strategies and
minimize the risk of late-stage failures.

384-well plates High Content Imager Decoding software

1 Mix particles, detection antibodies, Image particles on Data analysis with FirePlex®
and 6.25 µL of sample high content imager Analysis Workbench software
2 Add samples and incubate overnight
3 Add imaging dye & image the plate

Figure 4. The FirePlex-384 simplified, no-wash assay workflow.

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 Incubate samples and particles Image Analyze

Shake for
5 min

Add
Shaker orbit imaging Collect
< 2mm dye data

FirePlex® Reagent mix

+ Incubate
Biological samples overnight at RT

Analytes captured Image particles Data analysis with

onto fluorescently on high content FirePlex® Analysis
labeled particles imager Workbench software

– Low volume sample input (2.5µl) – Read time 20 – 60 min

– One-step fully automatable workflow (depending on imager settings)
– High content imager used for signal acquisition – Large portfolio of analytes

Figure 5. An assay workflow of FirePlex-1536, the first 1536-well assay that enables up to 5-plex.

Find out more about high-throughput FirePlex multiplex immunoassays.

How can Abcam help you choose the right multiplex immunoassay
for your workflow?

FirePlex immunoassays are available in three different formats to provide the flexibility to
build an assay that best meets your needs.

– Choose from ready-to-use, pre-designed panels:

– Standard FirePlex-96 panels
– Standard FirePlex-384 panels

– Select analytes of interest from existing panels with Mix and Match assay designer

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Case study
Profiling of secreted proteins from
iPSCs with FirePlex®-384
From the paper:
Large-Scale Production of Human iPSC-Derived
Macrophages for Drug Screening
Simon Gutbier1, Florian Wanke1, Nadine Dahm1, Anna Rümmelin1, Silke Zimmermann1,
Klaus Christensen1, Fabian Köchl1 , Anna Rautanen1, Klas Hatje1, Barbara Geering1, Jitao
David Zhang1, Markus Britschgi1, Sally A Cowley2, Christoph Patsch1,3

Affiliations

1. Roche Pharma Research and Early Development, Roche Innovation Center

Basel, Switzerland.
2. James Martin Stem Cell Facility, Sir William Dunn School of Pathology,
University of Oxford, UK.
3. BlueRock Therapeutics, New York, USA.

International Journal of Molecular Sciences, 2020, 21, 4808; doi:10.3390/ijms21134808

Summary

FirePlex multiplex immunoassays provide a no-wash, automation-friendly workflow in

384- and 1536-well high-throughput formats. Utilizing your existing high-content imager
and powered by batch-to-batch consistent recombinant antibodies, FirePlex enables
you to achieve high-throughput without sacrificing assay performance or data quality.
In this study, a large pharmaceutical company used FirePlex-384 to characterize
iPSC-derived macrophages for use in drug screening.

Power your progress

with more insightful data

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your next discovery:
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 Characterization of iPSC-derived macrophage cell line
using FirePlex

– Macrophages are used in drug discovery, but their quantities

are limited.

– Induced pluripotent stem cells (iPSCs) were utilized to produce a new cell line
with a macrophage-like phenotype.

– FirePlex-384 was used to profile the cytokines secreted from the newly
developed cell line to confirm its resemblance to macrophages.

Intermediate storage Storage and collection

Myeloid factories Macrophages
in spinner culture up to X weeks

Harvest of Media change

Macrophage and addition of Differentiation
progenitors new harvests

60000
30000
20000
15000
10000
pg/ml

5000

600
400
200
0
IL-8 MCP1 IL-6 TNFA IL10 IL1B INFG
Key: Spinner Spinner+LPS Harvest Harvets+LPS

Cytokine release of cells derived from suspension storage and directly

differentiated after harvesting in unstimulated state and stimulated with
100 ng/mL lipopolysaccharide (LPS) for 18 h was assessed.
Images adapted from Gutbier et al. Int. J. Mol. Sci. 21, 4808 (2020).

– The researchers were able to show that the generated iPSC-derived

macrophages closely resemble primary macrophages.

– The iPSC-derived macrophages recapitulate key functional characteristics,

including cytokine release, phagocytosis, and chemotaxis.

– The approach used in this study can be helpful to any researchers who need to
make detailed characterization and multi-phenotypic profile of any cell type
that they isolate, generate, or engineer.

For more information: [Link] 12

References
1 Ahsan, H. and Ahmad, R. Multiplex Technology for Biomarker Immunoassays. In book:
Innate Immunity in Health and Disease. Publisher: IntechOpen, London, UK (2020)
[Link]

2. Ray, C.A. et al. Development, validation and implementation of a multiplex

immunoassay for the simultaneous determination of five cytokines in human serum. J
Pharm Biomed Anal. 36(5):1037-1044 (2005).

3. Hudacek, A. et al. Multiplexing Immunoassay: Technical Considerations for Optimal

performance-drug discovery and development (2017)
[Link]
siderations-for-optimal-performance/.

4. Schneider, G. Automating Drug Discovery. Nat Rev Drug Discov. 17, 97-113. (2018).

5. Chai, S.C., Goktung A.N., Chen T. Assay validation in high-throughput screening

– from concept to application. In book: Drug Discovery and Development - From
Molecules to Medicine. Publisher: IntechOpen, London, UK (2015).
[Link]

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[Link]
Copyright © 2021 Abcam, All rights reserved.

Luminex®, Luminex® 100™, Luminex® 200™, MAGPIX®, FLEXMAP 3D® are trademarks
of Luminex Corporation, registered in the U.S. and other countries.

RayBio® is a trademark of RAYBIOTECH LIFE INC.

HuProt™ is a trademark of CDI Laboratories , Inc., (“CDI”) in the United States

and/or other countries.

AlphaLISA® is a registered trademark of PerkinElmer, Inc.

FirePlex® is a registered trademark of Abcam plc in the United States and is an

unregistered trademark elsewhere.

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