Multiplexed Protein Detection Research Articles

Abstract PO-045: Multiplex analysis of coronavirus and influenza antibodies using the ZIVA multiplex platform

Abstract Detection of antibodies to upper respiratory pathogens is critical to surveillance, assessment of the immune status of individuals, vaccine development, and basic biology. The urgent need for antibody detection tools has proven particularly acute in the COVID-19 era. We have used Arrayed Imaging Reflectometry (AIR™), a label-free protein microarray technology that relies on the target binding-induced perturbation of an antireflective coating on the surface of a silicon chip, to create a multiplex antibody detection array. ZIVA is a new multiplex protein detection platform that leverages AIR™ technology. We have used ZIVA technology to develop an acute respiratory virus panel capable of detecting the presence of antibodies specific for coronavirus and influenza including, but not limited to, detection of antibodies to SARS-CoV-2, SARS-CoV-1, MERS, three circulating coronavirus strains (HKU1, 229E, OC43), and multiple strains of influenza. We find that the array is readily able to distinguish uninfected from convalescent COVID-19 subjects and provides quantitative information about total IgG, as well as IgG- and IgM-specific responses. Samples may also be evaluated with ZIVA cytokine assays to monitor the cytokine storm in conjunction with viral antibody response. Citation Format: Jon Schmuke, Ben Miller, Linda Meeh, Byran Witherbee. Multiplex analysis of coronavirus and influenza antibodies using the ZIVA multiplex platform [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2020 Jul 20-22. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(18_Suppl):Abstract nr PO-045.

Regioselective SN2-Type Reaction for the Oriented and Irreversible Immobilization of Antibodies to a Glass Surface Assisted by Boronate Formation.

Antibodies have exquisite specificities for molecular recognition, which have led to their incorporation into array sensors that are crucial for research, diagnostic, and therapeutic applications. Many of these platforms rely heavily on surface-bound reactive groups to covalently tether antibodies to solid substrates; however, this strategy is hindered by a lack of orientation control over antibody immobilization. Here, we report a mild electrophilic phenylsulfonate (tosylate) ester-containing boronic acid affinity ligand for attaching antibodies to glass slides. A high level of antibody coupling located near the Fc region of the boronated antibody complex could be achieved by the proximal nucleophilic amino acid driven substitution reaction at the phenylsulfonate center. This enabled the full-length antibodies to be permanently tethered onto surfaces in an oriented manner. The advantages of this strategy were demonstrated through the individual and multiplex detection of protein and serum biomarkers. This strategy not only confers stability to the immobilized antibodies but also presents a different direction for the irreversible attachment of antibodies to solid supports in an orientation-controlled way.

Abstract 1127: Adarza's ZIVA multiplex protein detection platform utilizes a label-free technology to simultaneously monitor murine and human inflammation cytokines in a CAR-T model system

Abstract We have used Arrayed Imaging Reflectometry (AIR™), a label-free protein microarray technology that relies on the target binding-induced perturbation of an antireflective coating on the surface of a silicon biosensor, to create a multiplex antibody array for the detection of human and murine serum cytokines and inflammatory markers. The biosensor is capable of providing quantitative protein concentrations, with limits of detection in the low picograms per mL. Provided in a standard 96-well microplate format, the label-free nature of the array enables flexible creation of standard or custom panels for development of model systems. Here, we demonstrate a custom 22 plex panel developed in collaboration with WUGEN Inc. to provide multi-species cytokine analysis in humanized CAR-T cell models for the evaluation of biologic product efficacy and safety. Samples from in vitro and in vivo models developed for the evaluation of CAR-T activity were analyzed and demonstrated a novel platform for concurrent cross-species cytokine analysis on a single array. Results are evaluated for sensitivity, precision, and accuracy with ZIVA assays comparable to traditional ELISAs. With array capabilities from low to hundreds of plex combined with a hands-free, automation-friendly workflow enabled by the ZIVA instrument, the technology has been designed for the ultimate user-experience. Citation Format: Jon J. Schmuke, Alun Carter, You Zhou, Ernest Mueller, Matt Cooper. Adarza's ZIVA multiplex protein detection platform utilizes a label-free technology to simultaneously monitor murine and human inflammation cytokines in a CAR-T model system [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1127.

Adarza’s ZIVA multiplex protein detection platform demonstrates flexibility with custom targets for monitoring both inflammation and anti-drug antibody response

Abstract We have used Arrayed Imaging Reflectometry (AIR™), a label-free protein microarray technology that relies on the target binding-induced perturbation of an antireflective coating on the surface of a silicon chip, to create a multiplex antibody array. ZIVA is a new multiplex protein detection platform that leverages AIR™ technology. Understanding the immunogenicity of a therapeutic protein is critical to the development of bioactive stability and efficacy. ZIVA technology can be customized to monitor for multiple biomarker types including markers of inflammation as well as specific ADA targets. In this example, biosensors were custom printed to monitor for the presence of an anti-drug antibodies generated against a single chain antibody presented on the surface of engineered CAR-T cells. Samples that are positive for ADA response may also be evaluated for presence of neutralizing antibodies (NAB) by evaluating specific controls also included on the biosensors. With array capabilities from low to hundreds of plex combined with a hands-free, automation-friendly workflow enabled by the ZIVA instrument, the technology has been designed for the ultimate user-experience.

Adarza’s ZIVA multiplex protein detection platform offers format for ultra-high sensitivity

Abstract We have used Arrayed Imaging Reflectometry (AIR™), a label-free protein microarray technology that relies on the target binding-induced perturbation of an antireflective coating on the surface of a silicon chip, to create a multiplex antibody array for the detection of both low and highly abundant targets. ZIVA is a new multiplex protein detection platform that leverages AIR™ technology. Because of the label-free properties, sensitivity can be “fine-tuned” by leveraging a variety of mass enhancing options. We have previously demonstrated assays that respond in the nanogram to picogram/ml sensitivity using one format for enhancement. Herein, we demonstrate a newly developed mass enhancement option that increases sensitivity from very low pg/ml to mid-femtogram/ml levels. A custom designed biosensor with markers for inflammation and other custom needs was used to demonstrate this ultra-high sensitivity format in a multiplex array. With array capabilities from low to hundreds of plex combined with a hands-free, automation-friendly workflow enabled by the ZIVA instrument, the technology has been designed for the ultimate user-experience.

Adarza’s ZIVA multiplex protein detection platform utilizes a label-free technology to simultaneously monitor murine and human inflammation cytokines in a CAR-T model system

Abstract We have used Arrayed Imaging Reflectometry (AIR™), a label-free protein microarray technology that relies on the target binding-induced perturbation of an antireflective coating on the surface of a silicon biosensor, to create a multiplex antibody array for the detection of human and murine serum cytokines and inflammatory markers. The biosensor is capable of providing quantitative protein concentrations, with limits of detection in the low picograms per mL. The flexibility of this system can also reduce the need to generate a series of sample dilutions in order to get results in a limited detection range by enabling the development of each assay with sensitivity expectations for each target on a chip. Provided in a standard 96-well microplate format, the label-free nature of the array enables flexible creation of standard or custom panels for development of model systems. Here, we demonstrate a custom 22 plex panel developed in collaboration with WUGEN Inc. to provide multi-species cytokine analysis in humanized CAR-T cell models for the evaluation of biologic product efficacy and safety. Samples from in vitro and in vivo models developed for the evaluation of CAR-T activity were analyzed and demonstrated a novel platform for concurrent cross-species cytokine analysis on a single array. Results are evaluated for sensitivity, precision, and accuracy with ZIVA assays comparable to traditional ELISAs. With array capabilities from low to hundreds of plex combined with a hands-free, automation-friendly workflow enabled by the ZIVA instrument, the technology has been designed for the ultimate user-experience.

Phosphorylcholine-based encoded hydrogel microparticles with enhanced fouling resistance for multiplex immunoassays.

Due to the growing interest in multiplex protein detection, encoded hydrogel microparticles have received attention as a possible path to high performance multiplex immunoassays through a combination of high multiplexing capability and enhanced binding kinetics. However, their practical operation in real complex samples is still limited because polyethylene glycol, which is the main component of hydrogel particles, suffers from oxidative damage and relatively high fouling properties in biochemical solutions. Here, we introduce poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC)-based encoded hydrogel microparticles to perform fouling-resistant multiplex immunoassays, where the anti-fouling characteristics are attributed to the zwitterionic PMPC. By applying a newly developed molding lithography technique, viscous PMPCs with low reactivity were successfully incorporated into the hydrogel network while maintaining uniformity and rigidity for use in multiplex immunoassays. Non-specific protein adsorption on the PMPC particles was reduced by about 37.5% compared to that of conventional PEG particles, which leads to better assay sensitivity. We also validate the multiplex capability of the PMPC particles by performing multiplex detection of two target proteins. Furthermore, we verify that the PMPC particles have a 70% enhancement in anti-fouling characteristics compared to PEG particles in human platelet-rich plasma, potentiating a practical immunoassay platform for clinical diagnosis.

Mixed Multiplex Staining: Automated RNAscope™ and OPAL™ for Multiple Targets.

The application of multiplex immunofluorescent detection of proteins, mRNAs, or both in combination has contributed to our understanding of biomarker expression and distribution within normal and pathological diseased tissues. In combination with whole-slide scanning these techniques permit the visualization of multiple biomarkers, their distribution within normal and pathological tissues, and their spatial and proximal distribution to each other in intact tissue sections. The expression of multiple biomarkers can now be used to accurately phenotype specific cell types, e.g., immune cells which rely on the expression of multiple biomarkers for precise identification. The availability of antibody reagents to detect specific protein targets, range of targets in specific technology platforms, and tissue fixation may mean that one technology may not be suitable for visualizing all biomarkers of interest. Here I demonstrate that a mixed multiplex approach for multiple protein and mRNA can be delivered on automated platforms suitable for high-throughput environments, and predict that mixed multiplex approaches will be important in future to compensate for tissue fixation differences, biomarker availability, and technology platform limitations.

Simultaneous Targeted Detection of Proteins and RNAs in Single Cells.

Simultaneous detection of both RNA and protein in individual single cells offers a powerful tool for genotype-to-phenotype investigations. Proximity extension assay (PEA) is a quantitative, sensitive, and multiplex protein detection system that has superb utility in single-cell omic analysis. We implemented PEA using the flexible microfluidic workflow of the Fluidigm® C1™ system followed by real-time quantitative polymerase chain reaction (RT-qPCR) on the Fluidigm Biomark™ HD system. With this workflow, targeted quantification of RNAs and proteins within individual cells is readily conducted.

Highly sensitive multiplex protein detection by droplet‐free digital ELISA

AbstractDigital enzyme‐linked immunosorbent assay (ELISA) is a single molecule counting technology and is one of the most sensitive immunoassay methods. The key aspect of this technology is to concentrate enzyme reaction products from a single target molecule in femtoliter droplets. On the other hand, we have developed a novel Digital ELISA that does not require droplets; instead, enzyme reaction products are concentrated using a tyramide signal amplification system. In this study, we present a method that enables the multiplex detection of proteins based on Droplet‐free Digital ELISA. First, two types of paramagnetic beads immobilized with antibodies specific to different target proteins were incubated with a sample solution, and target proteins were captured on their specific beads. Then, these beads were labeled with horseradish peroxidase (HRP), and tyramide substrate reacted with HRP on beads, resulting in products of this reaction deposited on those beads. This signal amplification on beads makes it possible to count the number of labeled beads digitally. We used this approach to simultaneously detect IL‐6 and HBs Ag with single molecule resolution. The obtained limit of detections were 0.1 pg/mL and 0.013 IU/mL, respectively. Our method has potential applications in simple in vitro diagnostic systems for simultaneous and high‐sensitive detection of protein biomarkers.

Visualization of HIV-1 RNA Transcription from Integrated HIV-1 DNA in Reactivated Latently Infected Cells.

We have recently developed the first microscopy-based strategy that enables simultaneous multiplex detection of viral RNA (vRNA), viral DNA (vDNA), and viral protein. Here, we used this approach to study the kinetics of latency reactivation in cells infected with the human immunodeficiency virus (HIV). We showed the transcription of nascent vRNA from individual latently integrated and reactivated vDNA sites appearing earlier than viral protein. We further demonstrated that this method can be used to quantitatively assess the efficacy of a variety of latency reactivating agents. Finally, this microscopy-based strategy was augmented with a flow-cytometry-based approach, enabling the detection of transcriptional reactivation of large numbers of latently infected cells. Hence, these approaches are shown to be suitable for qualitative and quantitative studies of HIV-1 latency and reactivation.

Highly Sensitive and Multiplexed Protein Measurements.

Proteins are involved in many biological processes. Misfolded, truncated, or mutated proteins as well as over- or underexpressed proteins have been implicated in many diseases. Therefore, detection and quantification of proteins is extremely important. Conventional techniques such as the enzyme-linked immunosorbent assay, Western Blot, and mass spectrometry have enabled discovery and study of proteins in biological samples. However, many important proteins are present at low concentrations, rendering them undetectable using conventional techniques. Furthermore, limited ability to simultaneously measure multiple proteins in a sample has constrained our ability to fully study the proteome. In this review, we comprehensively discuss approaches for protein detection. We first discuss the fundamentals of proteins and protein assays, including affinity reagents, surface functionalization, assay formats, signal detection, and multiplexing. We then discuss the challenges with these methods and review existing methods for highly sensitive and multiplexed protein detection. Finally, we review recent advances in protein detection from the literature and discuss challenges and future directions.

Highly Sensitive Multiplex Protein Detection by Droplet-free Digital ELISA

Highly Sensitive Multiplex Protein Detection by Droplet-free Digital ELISA

ELIPatch, a thumbnail-size patch with immunospot array for multiplexed protein detection from human skin surface.

Proteins secreted by skin have great potential as biomarkers for interpreting skin conditions. However, inconvenience in handling and bulky size of existing methods are existing limitations. Here, we describe a thumb-nail sized patch with the array of microdisks which captures multiple proteins from the skin surface. Microdisks with antibody on the surface enable multiplexed immunoassay. By self-assembly, microdisks are placed into 2-dimensional arrays on adhesive tape. The proposed Enzyme-Linked Immunospot array on a Patch shows sufficient sensitivity for IL-1α, IL1RA, IL-17A, IFN-g, and TNF-α, while IL-6 and IL-1β are non-detectable in some cases. As demonstrations, we quantified cytokines from different skin regions and volunteers in a high-spatial-resolution.

Multiplex protein detection on circulating tumor cells from liquid biopsies using imaging mass cytometry.

Molecular analysis of circulating and disseminated tumor cells (CTCs/DTCs) has great potential as a means for continuous evaluation of prognosis and treatment efficacy in near-real time through minimally invasive liquid biopsies. To realize this potential, however, methods for molecular analysis of these rare cells must be developed and validated. Here, we describe the integration of imaging mass cytometry (IMC) using metal-labeled antibodies as implemented on the Fluidigm Hyperion Imaging System into the workflow of the previously established High Definition Single Cell Analysis (HD-SCA) assay for liquid biopsies, along with methods for image analysis and signal normalization. Using liquid biopsies from a metastatic prostate cancer case, we demonstrate that IMC can extend the reach of CTC characterization to include dozens of protein biomarkers, with the potential to understand a range of biological properties that could affect therapeutic response, metastasis and immune surveillance when coupled with simultaneous phenotyping of thousands of leukocytes.

Low cost 3D microfluidic chips for multiplex protein detection based on photonic crystal beads.

Point-of-care testing (POCT) devices used in multiplex bioassays are in great demand for clinical, environmental and biomedical applications. Photonic crystal beads (PCBs), as structural color self-coding carriers, can be integrated with microfluidic chips to realize convenient and highly sensitive biomarker detection. Here we developed a three dimensional (3D) microfluidic chip based on PCBs, which is low cost and easy to manufacture for mass production and application. The chip was fabricated with polyethylene terephthalate, polymethyl methacrylate sheets, a Ni square mesh grid and transparent double-sided tape. In practice, the target molecules could be captured by PCBs immobilized with probes in a flow-through manner. It was found that the as-proposed chip needed less washing and its background was effectively reduced in comparison with a flow-over chip. Besides, the limit of detection (LOD) of anti-human alpha fetoprotein (AFP) was calculated to be 18.92 ng mL-1, which could meet the need of clinical detection of AFP. Furthermore, the chip demonstrated the feasibility of simultaneous detection of human immunoglobulin G, carcinoembryonic antigen and AFP, which suggests that it has a broad application prospect in multiplex bioassays.

Isotype-specific agglutination-PCR (ISAP): A sensitive and multiplex method for measuring allergen-specific IgE

Isotype-specific agglutination-PCR (ISAP): A sensitive and multiplex method for measuring allergen-specific IgE

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays.

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands of micro-scale immunoassays performed simultaneously on a single chip. Sandwich assay format improves assay specificity by detecting each target with two antibodies, but suffers from cross-reactivity between reagents thus limiting their multiplexing capabilities. Antibody colocalization microarray (ACM) has been developed for cross-reactivity-free multiplexed protein detection, but requires an expensive spotter on-site for microarray fabrication during assays. In this work, we demonstrate a snap chip technology that transfers reagent from microarray-to-microarray by simply snapping two chips together, thus no spotter is needed during the sample incubation and subsequent application of detection antibodies (dAbs) upon storage of pre-spotted slides, dissociating the slide preparation from assay execution. Both single and double transfer methods are presented to achieve accurate alignment between the two microarrays and the slide fabrication for both methods are described. Results show that <40 μm alignment has been achieved with double transfer, reaching an array density of 625 spots/cm2. A 50-plexed immunoassay has been conducted to demonstrate the usability of the snap chip in multiplexed protein analysis. Limits of detection of 35 proteins are in the range of pg/mL.

Microbead-Based Platform for Multiplex Detection of DNA and Protein.

We present a novel microbead-based detection platform as a simple and universal strategy for simultaneous determination of multiple biomolecules. This platform is composed of streptavidin coated uniform-sized polystyrene microbeads, dye and biotin-labeled ssDNA or aptamer probes, and quencher-labeled complementary sequences. By this method, upon target binding to the probes, quencher strand dissociation is triggered, which results in fluorescence reactivation of the microbead linked probes. The fluorescence variation is readily monitored by flow cytometry and with a high sensitivity. Explicitly, this microbead-based detection platform shows a high sensitivity for target DNA with a detection limit as low as 0.20 nM, alongside good selectivity from one-base mismatched DNA. This novel platform also shows good selectivity and high sensitivity for protein detection when aptamer is used as a probe. The detection limit for lysozyme is as low as 8.56 nM. Moreover, simultaneous detection of multiple targets has been achieved via incorporating different dye-labeled probes on the microbeads concurrently. We have also applied this developed strategy to the detection of target DNA in human serum. This strategy can be easily extended to other targets through simple probe and quencher variation.

A new approach for immuno-oncology biomarker discovery: High-plex, spatial protein profiling based on NanoString digital quantification.

27 Background: The immune response to cancer is shaped by the abundance and localization of immune cell populations, their activation status, and expression of immunomodulatory factors. To detect proteins at high multiplex with spatial resolution, NanoString optical barcoding technology was used to digitally profile protein expression in formalin fixed paraffin embedded (FFPE) samples and compared to traditional IHC. Methods: NanoString has enabled digital profiling of immuno-oncology protein targets, including immune cell markers and checkpoint proteins. Protein detection is enabled via primary antibodies (Abs) which are attached via a UV cleavable linker to DNA indexing oligos. FFPE samples are stained with a multiplex cocktail of labeled Abs, then DNA oligos are released by UV light exposure. Liberated oligos are then hybridized to optical barcodes for quantitation on a NanoString instrument. This technique enables quantitative, multiplex protein detection over 5 logs of dynamic range. IHC and NanoString spatial protein profiling were performed on alternating sections from blocks of tonsil, melanoma, and colorectal cancer. Sections were fluorescently labeled with panCK, Ki67, and Syto-83 to visualize morphology, and regions of interest (ROI) were selected for profiling using up to 30 oligo-tagged Abs. Results: NanoString counts strongly correlated with quantification of CD3, CD4, CD8, PanCK, Ki67, PD-1, and PD-L1 derived from IHC. To explore limits of detection, ROIs of 1, 2, 4, or 8 cells were profiled. Digital counts were detected above background at the single cell level and linearly correlated with cell count. Furthermore, quantification with two cell lineage markers and double positive cells were demonstrated using a 30-plex Ab cocktail. Conclusions: Multiplex, digital protein profiling with spatial resolution will enable deep characterization of immune responses in tumors. Additionally, single cell profiling enables inter-cellular characterization of variations in immune response. The strong correlation of NanoString data to IHC indicates the feasibility to spatially profile multiple key proteins with minimal consumption of patient tissue.