Multiplex single molecule assays for ultrasensitive detection of biomolecules

Abstract

Described herein are multiplex single molecule assays for ultrasensitive detection of biomolecules, based on an ultrasensitive multiplex digital ELISA platform that substantially reduces cross-reactivity, and methods of use thereof.

Description

[0001] Attorney Docket No. 29618-0504WO1 / BWH 2024-331

[0002] Multiplex Single Molecule Assays for Ultrasensitive Detection of Biomolecules

[0003] CLAIM OF PRIORITY

[0004] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 708,136, filed on October 16, 2024. The entire contents of the foregoing are hereby incorporated by reference.

[0005] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0006] This invention was made with Government support under Grant No. EB032826 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0007] TECHNICAL FIELD

[0008] Described herein are multiplex single molecule assays for ultrasensitive detection of biomolecules.

[0009] BACKGROUND

[0010] Protein-based biomarkers hold great promise for disease diagnostics, therapeutic monitoring, and treatment,1'3yet their low abundance in biofluids often poses detection challenges for traditional enzyme-linked immunosorbent assay (ELISA) techniques.

[0011] SUMMARY

[0012] Provided herein are near cross-reactivity-free digital ELISA methods that can achieve attomolar sensitivities — an order of magnitude improvement over the gold standard digital ELISA methods. We employed detector antibodies barcoded with unique DNA sequences and implemented a ‘parity check’ mechanism, where 'parity’ refers to the precise match between the bead type (determined by its color, size, or intensity) and the DNA barcode on the detector antibody, while ‘check’ refers to the verification of this match. This approach effectively identifies and eliminates cross- reactive binding events by detecting mismatched bead-probe pairs. We also integrated multispectral probe colors using ratiometric combinations, enhancing multiplexing capabilities to measure a panel of five biomarkers with minimal cross-reactivity. Attorney Docket No. 29618-0504WO1 / BWH 2024-331

[0013] Additionally, this approach has been expanded to quantify an eight-biomarker panel commonly used in cancer detection and monitoring, utilizing just under a quarter of a blood drop (approximately 9 pL). The methods described herein are thus well suited for early cancer detection, minimal residual disease monitoring, and longitudinal follow-up using low-volume biofluid samples.

[0014] Provided herein are methods of detecting the presence of an analyte of interest in a sample. The methods comprise: contacting the sample with capture beads coated with a capture antibody that binds to the analyte, wherein the capture beads have specific identifiable characteristics, optionally wherein the bead characteristics are color, intensity, and / or size, wherein the capture antibodies bind to any analytes present in the sample; contacting the sample with a detector antibody that binds to the same analyte, optionally in the presence of heparin, wherein the detector antibody is directly conjugated to a specific DNA template barcode sequence; performing rolling circle amplification using DNA probes comprising distinct fluorescent labels that bind to the DNA template barcode sequence, to produce double-labeled complexes; using flow cytometry to sort the double-labeled complexes based on the bead characteristics and the fluorescent label; and identifying double-labeled complexes that have the specific bead characteristics and the distinct fluorescent labels as comprising the analyte of interest. As used herein, “sort” need not include physical separation, but rather can be simple identification and optionally quantification.

[0015] Also provided herein are methods of detecting the presence of two or more analytes of interest in a sample. The methods comprise: contacting the sample with a plurality' of capture beads coated with capture antibodies that bind to each of the analytes, wherein the capture beads have specific identifiable characteristics for each of the analytes, optionally wherein the bead characteristics are color, intensity, and / or size, wherein the capture antibodies bind to any analytes present in the sample; contacting the sample with a plurality of detector antibodies that bind to each of the analytes, optionally in the presence of heparin, wherein the detector antibodies are directly conjugated to DNA template barcode sequences specific for each of the analytes; performing rolling circle amplification using DNA probes comprising distinct fluorescent labels that bind to each of the DNA template barcode sequences, to produce double-labeled complexes; using flow cytometry to sort the double-labeled complexes based on the bead characteristics and the fluorescent label; and identifying Attorney Docket No. 29618-0504WO1 / BWH 2024-331 double-labeled complexes that have both the specific bead characteristics and the distinct fluorescent labels as comprising an analyte of interest. As used herein, "‘sort” need not include physical separation, but rather can be simple identification and optionally quantification.

[0016] The methods can detect multiple analytes at the same time in a single sample, e.g., a unitary undivided portion of a sample.

[0017] In the present methods and compositions, binding agents other than antibodies, such as aptamers, nanobodies, engineered scaffolds, or synthetic polymers with affinity for analytes, and so on, can be used for either or both capture and detector.

[0018] In some embodiments, the DNA template barcode sequence comprises a circularized DNA template-primer DNA.

[0019] In some embodiments, for detecting a number A of analytes, N distinct capture bead types, each type having specific identifiable characteristics, are combined with N distinct DNA barcodes on detector antibodies.

[0020] In some embodiments, flow cytometry sorting is based on one or both of (1) bead-code fluorescence intensity, and (2) fluorescence intensity of single-colored fluorescent labels on a DNA probe, or on multispectral profiles from ratiometric combinations of fluorescent labeled probes that label individual analytes.

[0021] In some embodiments, the sample comprises a biological fluid, optionally blood, serum, or plasma, or stool, cervical fluids such as pap smears, uterine lavage, urine, cerebrospinal fluid (CSF), or sputum.

[0022] In some embodiments, the analytes comprise one, two, three, four, or more of Interleukin 6 (IL-6), IL-12p70, Human Epididymis Protein 4 (HE4, also known as WAP four-disulfide core domain 2 (WFDC2)), CA-125 (also known as Mucin-16 (MUC-16)), and ORF1 protein (ORF Ip), and optionally one or more of IL-7, IL- 18, and interferon gamma (IFN-y). In some embodiments, the analytes comprise cellular retinoic acid-binding protein 2 (CRABP2), optionally in combination with ORFlp. In some embodiments, the analytes comprise EV surface markers, optionally CD81 and / or CD9). and / or abundant serum proteins (e.g., albumin and / or apolipoprotein B (ApoB)).

[0023] Optionally the sample is from a subject suspected of or at risk of having cancer (e.g., at risk of cancer as a result of family or personal medical history, or cancer-predisposing genetics). Attorney Docket No. 29618-0504WO1 / BWH 2024-331

[0024] In some embodiments, the detection of a level of one or more of the analytes above a reference level in a sample from a subject indicates that the subject has cancer.

[0025] In some embodiments, the subject is a mammal, e.g., a human or non -human veterinary subject, e.g., a cat, dog, rabbit, horse, cow, or pig.

[0026] Also provided herein are kits comprising the reagents described herein.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0028] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0029] DESCRIPTION OF DRAWINGS

[0030] FIG. 1. Schematic illustrating the barcoded multiplex MOSAIC. In this format, each detector antibody in a multiplex MOSAIC assay is conjugated to a distinct DNA template, which is then paired with a corresponding fluorescent dye- conjugated probe. Each analyte corresponds to a specific pair of: (1) capture bead color, fluorescence intensity, and / or size; and (2) fluorescent probe color. Consequently, only “correct'’ matched pairs of capture bead and probe signals are classified as “on” beads for each analyte. Any mismatched “wrong” pairs of capture bead and probe colors, indicative of cross-reactive binding events, are eliminated from the analysis.

[0031] FIGs. 2A-B. Comparison of sensitivities between barcoded and nonbarcoded MOSAIC assays. (A) Schematic comparison of the barcoded to the nonbarcoded multiplex MOSAIC assays. In the latter, all detector antibodies are biotinylated and labeled with the same streptavidin-DNA conjugate and fluorescent probe, thereby masking cross-reactive binding events. Representative plots show beads differentiated by a series of gates in different fluorescence channels, with the Attorney Docket No. 29618-0504WO1 / BWH 2024-331

[0032] AMB for each bead type calculated based on probe-specific fluorescence intensities. Q750 and Q700 represent Quanterix Homebrew beads conjugated to 750 nm and 700 nm fluorescent dyes, respectively. SP denotes singleplex beads without an encoded dye. (B) Calibration curves for IL-8, IL-10, and IL-12p70 in the non-barcoded multiplex MOSAIC assay (left) and the barcoded multiplex MOSAIC assay (right). Curves are fitted using four-parameter logistic (4PL) regression, highlighting the overall attainment of attomolar sensitivities across assays. Error bars represent the standard deviation of triplicate measurements.

[0033] FIGs. 3A-B. Three-plex barcoded MOSAIC assay with markedly reduced cross-reactivity. (A) Protein dropout curves depicting increasing concentrations of each individual target protein for the non-barcoded multiplex MOSAIC (i) and the barcoded multiplex MOSAIC (ii). (B) Recoveries of spiked recombinant proteins for the three-plex barcoded MOSAIC assay and corresponding non-barcoded MOSAIC assay in buffer (i) and human plasma at 16-fold dilution (ii), showcasing only the recoveries of IL- 10 here; see detailed recoveries of other recombinant proteins summarized in Tables 3-4. Recoveries are calculated as the mean ± standard deviation from duplicate measurements and are defined as the ratio of the difference in interpolated concentrations post-spike to the baseline concentration — using the limit of detection (LOD) for buffer and the pre-spike plasma concentration at 16-fold dilution — divided by the indicated spiked protein concentration. The acceptable range of recoveries (70-130%) is highlighted in grey. Recovery rates higher than 100% can occur when the signal from the spiked sample exceeds the expected value, possibly due to matrix interference or non-specific binding, leading to overestimation of the analyte concentration.

[0034] FIGs. 4A-C. Five-plex barcoded MOSAIC with minimal cross-reactivity. (A) Schematic of multiplexing using barcoded MOSAIC. Beads coated with antibodies specific to different target analytes are differentiated by utilizing fluorescent dyes of varying wavelengths, intensities, and multiple bead sizes. Upon capture of the target analytes, single immunocomplex sandwiches are formed and labeled with detector antibodies that are barcoded with different DNA primer sequences (T14, 1 , 20, 21, 25, Table 10), hybridized with corresponding fluorophore-labeled DNA probes (yielding specific ratios of ATTO565 to ATTO647 fluorescence of 1:0, 0: 1, 4: 1. 1:4, and approximately 5: 1. The "approximately 5: 1" Attorney Docket No. 29618-0504WO1 / BWH 2024-331 ratio reflects the combined fluorescence contributions from ATTO590, ATTO565, and ATTO647, with a precise ratio of 3:2: 1, which can appear as roughly 5: 1 for ATTO565 to ATTO647. Table 10). RCA is carried out and the mixture of beads is analyzed by flow cytometry. Beads are differentiated by a series of gates in different fluorescence channels, and the AMB for each bead type is then determined from the intensities in the fluorescence channel corresponding to the probe color(s). (B) Calibration curves and LODs for five analytes. Reproducibility was evaluated by performing three independent calibration curves on separate days, which yielded consistent AMB values and LODs. The reported LOD values represent the [range] of averaged results from these independent measurements, reflecting day-to-day variability. The LODs and LLOQs in both units (fM and pg / mL) are summarized in Table 5. (C) Measured concentrations of five protein analytes in human plasma using a five-plex MOSAIC assay. Concentrations shown are the measured concentration values in the 8-fold diluted plasma samples. Assay LODs are denoted by the blue dashed lines.

[0035] FIGs. 5A-C. Enhanced multiplexing with barcoded MOSAIC technology.

[0036] (A) Schematic representation of the eight-plex barcoded MOSAIC assay for the accurate measurement of key oncological biomarkers such as ORF Ip, HE4, and CA- 125, where each analyte is paired with specific bead ty pes and probe colors to eliminate cross-reactivity. Additionally, auxiliary markers are measured using varied bead types that may share probe colors, thereby enhancing multiplexing capabilities while minimizing potential cross-reactive binding events among essential markers.

[0037] (B) Calibration curves for each of the eight analytes, illustrating assay sensitivity and dynamic range. LODs in IM are summarized in the accompanying table. Detailed calibration curves can be found in FIGs. 13A-H. (C) The measured concentrations of the eight protein analytes in human plasma, analyzed using this eight-plex MOSAIC assay. These concentrations are derived from measurements in 16-fold diluted plasma samples, with dashed blue lines indicating the LODs for each analyte. Error bars represent the standard deviation of replicate measurements (>2). LODs and LLOQs in both units (fM and pg / mL) for these analytes are provided in Table 7. The assay demonstrates reliable detection of ORFlp at sub-femtomolar concentrations. However, at the theoretical detection limit of 1 aM, the statistical variation due to Poisson noise becomes significant, as detecting such a small number of molecules Attorney Docket No. 29618-0504WO1 / BWH 2024-331 introduces substantial variability. This inherent variability may partly contribute to the day-to-day fluctuations observed in Figure 4B.

[0038] FIG. 6. Schematic of Cross-Reactivity Free Multiplex Ultrasensitive

[0039] Assay. The figure illustrates the workflow of a multiplex assay designed to minimize cross-reactivity7. Antibody-conjugated beads, vary ing in color, fluorescence intensity7, or size, are used to capture target analytes from a sample. Detector antibodies conjugated to unique DNA templates are added, followed by rolling circle amplification (RCA). Each amplified DNA product from the RCA reaction is labeled with a specific fluorescent dye-conjugated probe or a ratiometric combination of probes, corresponding to a unique analyte. Detection is performed using flow cytometry, where each analyte is identified by the unique combination of (1) bead capture characteristics (color, fluorescence intensity, or size) and (2) the specific fluorescent signal from the RCA reaction (either single-color or ratiometric combinations of dye-conjugated probes).

[0040] FIGs. 7A-B. Cross-reactivity analysis of protein dropout experiments. The relative signal-to-background ratio is defined as the ratio of the average molecules per bead (AMB) at a given sample concentration to the AMB of the blank sample (0 pg / mL), which serves as the background. The values of these relative signal-to- background ratios are summarized in Table 2.

[0041] FIGs. 8A-H. Screening ratiometric labeling probes for multiplexing expansion. Flow cytometry pseudocolor plots show fluorescence intensities and spatial distributions of fluorescent “on” and “off’ beads under blank control (A) and at 8 fM IL- 10 (B-H). The spatial distributions of the “on” beads correspond to detector antibodies barcoded with different DNA primer sequences (T14, T17, T18, T19, T20, T2L and T 15, from panels B-H, respectively), each paired with corresponding ratiometric combinations of fluorophore-labeled DNA probes. Specific primer-template and probe sequences used are provided in Table 10.

[0042] FIGs. 9A-B. Integration of ratiometric probe combinations to expand multiplexing capabilities. (A) Schematic illustration of the barcoded multiplex MOSAIC assay employing ratiometric combinations of dye-conjugated DNA probes, where the ratios of colors can be distinguished using multiple detection channels in flow cytometry. (B) Calibration curves for the three-plex barcoded MOSAIC assaytargeting IL-6, IL- 10, and IFN-y. with error bars representing the standard deviation Attorney Docket No. 29618-0504WO1 / BWH 2024-331 from duplicate measurements. The multiplexed assay was able to simultaneously measure three cytokines, showcasing sensitivity levels ranging from low to mid- attomolar concentrations. The LODs and LLOQs are detailed in the accompanying table.

[0043] FIGs. 10A-C. Dropout curves for each analyte in the three-plex barcoded MOSAIC assay. Each graph shows the signal response of distinct bead types with increasing concentrations of IL-6 (A), IFN-y (B), and IL- 10 (C), as shown in FIGs. 9A-B with minimal cross-reactivity across the measured concentration ranges. Error bars represent the standard deviation of duplicate measurements.

[0044] FIG. 11. Dilution linearity of the five-plex barcoded MOSAIC assay in an individual human plasma sample. The R2value from linear regression is shown for each analyte. Error bars denote the standard deviation of duplicate measurements. Reference lines representing the expected 1: 1 dilution relationship are included, originating from the lowest, highest, and midpoint concentrations, providing a visual comparison of the ideal linearity across dilutions.

[0045] FIG. 12. Dropout curves for each analyte in the five-plex barcoded MOSAIC assay. Each graph show s the signal response of each bead with increasing amounts of the individual analytes, with error bars representing the standard deviation of duplicate measurements.

[0046] FIGs. 13A-H. Eight-plex barcoded MOSAIC assay. Calibration curves for each of the eight analytes in the eight-plex barcoded MOSAIC assay are individually plotted (A-H). Dashed blue lines indicate the LOD for each analyte. The LODs and LLOQs are summarized in Table 7.

[0047] FIG. 14. Schematic illustrating the preparation of antibody-DNA conjugates. For each detector antibody, a 5' azide-modified primer is annealed to a specific DNA template, followed by ligation. The detector antibody is then modified with dibenzocyclooctyne-PEG4-N-hydroxysuccinimidyl ester (DBCO-PEG4-NHS) and conjugated to the primer- template complex.

[0048] FIGs. 15A-B. Improved Detection of Circulating CRABP2 in Plasma from Patients with High-Grade Serous Ovarian Carcinomas (HGSOC) Using Barcoded MOSAIC Assays. (A) Plasma CRABP2 concentrations w ere measured using a Simoa assay in 52 HGSOC patient samples and 30 control samples. CRABP2 was detectable in 15% of HGSOC cases and 12.5% of control cases. (B) The same Attorney Docket No. 29618-0504WO1 / BWH 2024-331 plasma samples were analyzed using the barcoded MOSAIC assay. The MOSAIC platform enabled CRABP2 detection in 100% of samples and demonstrated significantly higher CRABP2 levels in the HGSOC group (p < 0.01, Welch’s t-test). The bottom dashed line indicates the assay’s limit of detection (LOD). The top two dashed lines represent two independent thresholds used to define detection rates. Pie charts summarize the proportion of samples exceeding each threshold.

[0049] FIG. 16. Improved Detection of Pancreatic Cancer Using Barcoded MOSAIC Assays. (Right) Quantification of ORF Ip levels in plasma from 12 pancreatic cancer patients using the barcoded MOSAIC platform. Each data point represents a sample measured in duplicate. The enhanced sensitivity' of the MOSAIC assay — featuring an approximately 100-fold lower limit of detection (LoD) compared to Simoa — enabled detection in all patient samples (100% positive, 12 / 12), whereas Simoa detected ORF Ip in only 58% of cases.

[0050] FIG. 17. Multiplexed Detection of EV Markers and Abundant Plasma Proteins Using Barcoded MOSAIC Assays. Barcoded MOSAIC enables sensitive and quantitative detection of extracellular vesicle (EV) markers and plasma proteins across a broad dynamic range. Shown are calibration curves for CD81 and CD9 (tetraspanin EV markers, pg / mL range), ApoB, and albumin (abundant plasma proteins, ng / mL range). Average molecule per bead (AMB) is plotted against analyte concentration, demonstrating the platform's capability to multiplex diverse targets with wide-ranging abundance levels.

[0051] DETAIUED DESCRIPTION

[0052] Despite the discovery' of numerous potential biomarkers, there are multiple barriers to biomarker validation, particularly for multiplex biomarker assays. One key technological barrier has been the lack of multiplex immunoassays that efficiently utilize samples, provide robust quantitative data without increasing background or cross-reactions, are rapidly customizable without relying on difficult-to-access reagents and hardware.41

[0053] To address some of the analytical barriers limiting the use of protein-based biomarkers, various ultrasensitive methodologies such as digital ELISA have been developed, achieving up to 1000-fold increases in sensitivity.4'8Among these methods, a notable example is the Single-Molecule Arrays (Simoa) technology, which isolates individual protein molecules within femtoliter-scale reaction chambers, Attorney Docket No. 29618-0504WO1 / BWH 2024-331 facilitating signal generation at locally high concentrations for single molecule counting. While Simoa has markedly advanced biomarker detection, it often suffers from inefficient bead capture in a limited number of microwells,9which reduces analytical sensitivity and limits the sampling of rare molecules. Building upon Simoa, several digital ELISA platforms have been developed, including Molecular On-bead Signal Amplification for Individual Counting (MOSAIC).10dropcast single-molecule assays,11and droplet digital ELISA.7These platforms have improved sampling efficiencies and analytical sensitivity' tenfold by transforming single-molecule measurements into simplified assay formats through localizing non-diffusible signals on beads, thereby eliminating the need for microwells for signal compartmentalization.

[0054] Such advancements have enabled the detection of many rare molecules, thereby facilitating the analysis of complex multifactorial diseases, such as cancers, autoimmune diseases, and neurodegenerative diseases, which require the analysis of multiple biomarkers to effectively tailor therapeutic regimens.12Accordingly, the validation of novel biomarkers and their integration into multiplex immunoassay panels confer the attractive prospect of simultaneous measurements of multiple analytes from a single patient sample, significantly reducing assay costs and sample volume.13’15Notably, technologies like Simoa and MOSAIC have been adapted to simultaneously measure multiple low-abundance cytokines in biological fluids.9’10, 16’17enhancing the potential for comprehensive disease analysis for improved therapeutic outcomes. Nevertheless, the increased complexity of multiplex assays introduces greater potential for cross-reactivity, which can lead to false signals between binding antibodies and off-target proteins10, 18’19and compromise result accuracy.

[0055] To mitigate the higher risk of cross-reactivity inherent in multiplex assays due to the involvement of multiple antibodies, proximity -based detection approaches have been used to reduce cross-reactivity,19’20enabling the generation of detectable signals only when both the correct capture and detection antibodies, each conjugated with specific DNA sequences, specifically bind to the target protein in proximity. This intrinsic pairing mechanism ensures that signals are produced exclusively by correct antibody -DNA pairs, effectively minimizing cross-reactivity .21’22However, this approach entails multiple complex steps such as ligation, amplification, and Attorney Docket No. 29618-0504WO1 / BWH 2024-331 sequencing, which can be mid- throughput23and costly, especially for high-depth applications. Additionally, methods employing spatial or temporal separation have been developed, ensuring that each capture antibody is incubated only with its corresponding detection antibody, thereby reducing cross-reactivity24'26and enabling the accurate quantification of two or more biomarkers simultaneously without additional sample dilutions. Despite these advancements, adapting such approaches for ultrasensitive multiplex assays with minimal cross-reactivity’ remains challenging. Our lab has previously addressed this challenge by either running singleplex assays or by developing a multiplex Simoa assay utilizing sequential protein capture to mitigate cross-reactivity and enable higher-order multiplexing.27However, this method is limited by sample loss and bead carryover during multiple sequential incubations.

[0056] Development of Ultrasensitive Multiplex Digital ELISA Platform with Minimized Cross-reactivity.

[0057] We have developed an ultrasensitive multiplex digital ELISA platform that substantially reduces cross-reactivity’, leveraging the foundations of MOSAIC technology. The previous iteration involved the formation of single immunocomplex sandw iches on beads, comprising capture antibody-coated beads, protein analytes, and biotinylated detector antibodies. These complexes w ere then targeted with streptavidin-conjugated DNA primer-template pairs, followed by rolling circle amplification (RCA) to generate extended DNA concatemers. Subsequent incorporation of fluorescently labeled DNA probes during RCA facilitated in situ hybridization, producing robust fluorescent signals from beads bound w ith target molecules — these were designated as the "on" bead population. Flow cytometric counting of “on” and “off’ beads provided digital quantification of target molecules, quantified as “average molecules per bead” (AMB), measuring the average number of target molecules detected per bead. By localizing a non-diffusible signal to each captured target molecule on a bead, MOSAIC technology removes the need for traditional signal compartmentalization, which restricts the number of analyzable bead types due to physical constraints on compartment numbers. See, e.g., WO2023 / 059731.

[0058] The MOSAIC assay expands multiplexing capabilities by enabling the use of more bead types; however, it is still limited by antibody cross-reactivities as the level of multiplexing increases. Cross-reactivity primarily arises from non-specific Attorney Docket No. 29618-0504WO1 / BWH 2024-331 interactions, where detection antibodies bind to off-target analytes or beads, generating false-positive signals. This phenomenon is more pronounced in multiplex assays due to the presence of multiple antibody pairs and the increased potential for combinatorial interactions. However, it is less likely to occur in single-plex assays, where the absence of competing targets and detection antibodies reduces the likelihood of non-specific interactions.

[0059] In the methods described herein, instead of using the same streptavidin-DNA conjugate and the corresponding fluorescent probe for a single-color readout, we directly conjugate each detector antibody to a unique DNA template sequence (Figure 1). Each DNA template-barcoded detector antibody is paired with a capture bead, containing specific features such as color, intensity, or size, resulting in a unique identifier for each analyte. In flow cytometry', these pairs can be differentiated using multiple detection channels. Consequently, only “correct” matched pairs of capture beads and probe signals are classified as “on” beads for each analyte, with cross-reactive binding events, represented by mismatched pairs, being eliminated from analysis. Termed “barcoded” MOSAIC, this approach marks a significant advancement in reducing cross-reactivity. The sequences designed for this system can be integrated into existing sandwich ELISA configurations using simple copper-free click chemistry. Given the widespread availability of flow cytometers, the barcoded MOSAIC technology describe herein facilitates high-throughput, multiparametric single-molecule measurements and is adaptable to the dynamic landscape of molecular amplification methods. In the present methods and compositions, binding agents other than antibodies, such as aptamers, nanobodies, engineered scaffolds, or synthetic polymers with affinity for analytes, and so on, can be used for either or both of capture and detector. Antibodies can include full length or antigen-binding fragments.

[0060] FIG. 6 provides an exemplary' workflow of a multiplex assay designed to minimize cross-reactivity'. Antibody -conjugated beads, varying in color, fluorescence intensity, or size, are used to capture target analytes from a sample. Detector antibodies conjugated to unique DNA templates are added, followed by rolling circle amplification (RCA). Each amplified DNA product from the RCA reaction is labeled with a specific fluorescent dye-conjugated probe or a ratiometric combination of probes, corresponding to a unique analyte. Detection is performed using flow Attorney Docket No. 29618-0504WO1 / BWH 2024-331 cytometry', where each analyte is identified by the unique combination of (1) bead capture characteristics (color, fluorescence intensity, or size) and (2) the specific fluorescent signal from the RCA reaction (either single-color or ratiometric combinations of dye-conjugated probes).

[0061] The barcoded MOSAIC platform presented here markedly reduces crossreactivity and is compatible with standard immunoassay reagents, workflows, and instrumentation, thus enabling straightforward implementation in clinical and laboratory settings using a flow cytometer. Moreover, unlike commercially available multiplex systems that require extensive validation for new reagents due to potential cross-reactivity, barcoded MOSAIC supports a diverse array of detection affinity reagents, such as polyclonal antibodies that are more prone to cross-reactivity’. Additionally, barcoded MOSAIC provides a cost-effective and high-throughput solution, w ith eight-plex measurements costing less than three dollars in consumables and procedures taking less than three hours — only thirty' minutes of which involve hands-on time, along with 2.5 hours of incubation steps (Table 9). The current system is still limited by the fluorescence spectral overlap and the number of distinct detection probe colors that can be used simultaneously.

[0062] The assay relies on generating a concatemer or long continuous DNA molecule with multiple copies of the same sequence. Although the present methods exemplify using circular templates and rolling circle amplification, other approaches, such as linear rolling circle amplification (LRCA), multiple displacement amplification (MDA), or helicase-dependent amplification (HD A), can also produce concatemers. These methods don't require circular templates and can still yield long repetitive sequences. The assay could also be adapted for DNA-based readouts such as qPCR or sequencing to achieve even higher levels of multiplexing. Microfluidic cytometry or imaging cytometry can also be used. Although these higher-plex assays may show' increased cross-reactivity’ and reduced sensitivity7, implementing strategies like sequential target capture for spatial separation of detector antibodies can mitigate these effects, thereby enhancing the detection sensitivity’ and accuracy for critical analytes. Methods that include “sorting” need not include physical separation, but rather can be simple identification and optionally quantification.

[0063] Although the assay achieves attomolar detection limits, this does not imply that the antibodies themselves exhibit attomolar affinities. Rather, theoretical models Attorney Docket No. 29618-0504WO1 / BWH 2024-331 of digital ELISA demonstrate that antibodies with nanomolar to picomolar affinities, as measured by the dissociation constant (KD), which typically ranges from I O11to 109M, can still achieve attomolar sensitivities.42’43The KD values reported in previous studies for antibodies used in highly sensitive immunoassays44’43align well with the performance of the current assay (Table 10), supporting the notion that antibodies with affinities in this range can robustly capture and quantify rare molecules. Furthermore, the surface avidity effect created by bead-immobilized antibodies strengthens target binding through multivalent interactions, helping to stabilize the immune complexes and prevent the loss of captured targets during washing steps, thereby ensuring the assay's high sensitivity.

[0064] An additional application of the barcoded MOSAIC platform is its utility in streamlining the screening and optimization of antibody pairs for immunoassays. This platform enables simultaneous screening of multiple capture and detection antibody pairs in a single run, drastically reducing labor and resource expenditure. For instance, evaluating five antibodies as both capture and detection reagents would require 25 separate assays with a single-color readout. However, with the barcoded MOSAIC assay, this extensive process can now be accomplished in a single assay that utilizes 25 unique multispectral profiles, each pairing distinct beads w ith corresponding probe signatures. Barcoded MOSAIC not only accelerates the assay development process but also significantly cuts down on time and costs, making it a valuable tool for rapid assay optimization and validation.

[0065] In summary, the attomolar sensitivity, enhanced accuracy through reduced cross-reactivity, expanded multiplexing capabilities, streamlined workflow, and high- throughput flow cytometric readout of the barcoded MOSAIC technology’ hold vast potential for broad applications across various diseases. The platform also promises to enhance lab and clinic accessibility, potentially integrating into future point-of-care platforms.

[0066] Antibody-DNA template barcode sequence

[0067] The present methods make use of DNA template barcode sequences conjugated to a detection antibody. The templates include sequences complementary to DNA probes to produce concatemers, e.g., using RCA, linear rolling circle amplification (LRCA), multiple displacement amplification (MDA), or helicasedependent amplification (HD A). The DNA template barcode sequences can include a Attorney Docket No. 29618-0504WO1 / BWH 2024-331 circular template, e.g., for RCA, or can be linear, e.g., for LRCA, MDA, or HD A. The DNA template barcode sequences can be conjugated to the detection antibody using methods known in the art, including click chemistry.

[0068] Fluorescent Labels

[0069] Fluorescent labels that can be incorporated into the beads and / or DNA probes can include fluorophores and fluorescent proteins. Fluorophores can include Alexa Fluor® dyes (e.g., Alexa Fluor® 488. Alexa Fluor® 596. Alexa Fluor® 594, Alexa Fluor® 647), cyanine derivatives (e.g, Cy® dyes (e g, Cy3®, Cy5®), cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine), xanthene derivatives (e.g., fluorescein, rhodamine, Oregon green, eosin, Texas Red®), naphthalene derivatives (e.g., dansyl, prodan derivatives ), pyrene derivatives (e.g.. cascade blue), oxadiazole derivatives (e.g., pyridyloxazole, nitrobenzoxadi azole and benzoxadiazole), oxazine derivatives (e.g., Nile red, Nile blue, cresyl violet and oxazine 70), acridine derivatives (e.g., proflavin , acridine orange, acridine yellow), arylmethine derivatives (e.g., auramine, crystal violet, malachite green), tetrapyrrole derivatives (e.g, porphin, phthalocyanine, bilirubin), coumarin derivatives, 4', 6- diamidino-2-phenylindole (DAPI), fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), aniline blue, UBER Green, and UBER Red. A number of fluorescent proteins are known in the art, and include green fluorescent protein (GFP), variant of green fluorescent protein (GFP10), enhanced GFP (eGFP), TurboGFP, GFPS65T, TagGFP2, mUKGEmerald GFP, Superfolder GFP, GFPuv, destabilised EGFP (dEGFP), Azami Green, mWasabi, Clover, mClover3, mNeonGreen, NowGFP, Sapphire, T-Sapphire, mAmetrine, photoactivatable GFP (PA-GFP), Kaede, Kikume. mKikGR, tdEos. Dendra2. mEosFP2, Dronpa, blue fluorescent protein (BFP), eBFP2, azurite BFP, mTagBFP, mKalamal, mTagBFP2, shBFP, cyan fluorescent protein (CFP), eCFP, Cerulian CFP, SCFP3A, destabilised ECFP (dECFP), CyPet, mTurquoise, mTurquoise2, mTFPI, photoswitchable CFP2 (PS-CFP2), TagCFP. mTFPI, mMidoriishi-Cyan, aquamarine, mKeima. mBeRFP, LSS-mKate2, LSS-mKatel, LSS-mOrange, CyOFPl. Sandercyanin, red fluorescent protein (RFP), eRFP, mRaspberry, mRuby, mApple, mCardinal, mStable, mMaroonl, mGamet2, tdTomato, mTangerine, mStrawberry, TagRFP, TagRFP657, TagRFP675, mKate2, HcRed, t-HcRed, HcRed-Tandem, mPlum, mNeptune, NirFP. Kindling, far red fluorescent protein, yellow fluorescent Attorney Docket No. 29618-0504WO1 / BWH 2024-331 protein (YFP), eYFP, destabilised EYFP (dEYFP), TagYFP, Topaz, Venus, SYFP2, mCherry. PA-mCherry, Citrine, mCitrine, Ypet. IANRFP-AS83, mPapayal, mCyRFPl, mHoneydew, mBanana, mOrange, Kusabira Orange, Kusabira Orange 2, mKusabira Orange, mOrange 2, mKOK, mK02, mGrapel, mGrape2, zsYellow, eqFP611, Sirius, Sandercyanin, shBFP-N158S / L173I, near infrared proteins, iFP1.4, iRFP713. iRFP670, iRFP682, 1RFP702, iRFP720, iFP2.0. mIFP, TDsmURFP, miRFP670. Brilliant Violet (BV) 421, BV 605, BV 510, BV 711. BV786, PerCP, PerCP / Cy5.5, DsRed, DsRed2, mRFPl, pocilloporin, Renilla GFP, Monster GFP, paGFP, or a Phycobiliprotein, or a biologically active variant or fragment of any one thereof.

[0070] Beads

[0071] A number of different kinds of beads can be used in the present methods, including paramagnetic and polymeric beads. Suitable beads include, but are not limited to, paramagnetic beads, magnetic beads, non-magnetic beads, porous beads, plastic beads, ceramic beads, glass beads, polystyrene beads, methylstyrene beads, acrylic polymer beads, carbon graphited beads, titanium dioxide beads, latex or crosslinked dextrans such as SEPHAROSE beads, cellulose beads, nylon beads, crosslinked micelles, and TEFLON® beads. Non-spherical or irregularly-shaped beads may be used.

[0072] Methods of Use

[0073] The present methods provide tests for detection of one, two, three, or more analytes in a sample, e.g., low-abundance analytes. The methods can be used, e.g., for diagnosis cancer using circulating biomarkers. For detecting cancer, the one or more analytes can include one, two, three, four, or more of interleukin 6 (IL-6), IL-I2p70, Human Epididymis Protein 4 (HE4, also known as WAP four-disulfide core domain 2 (WFDC2)), CA-125 (also known as Mucin-16 (MUC-16)), and ORF1 protein (ORF Ip), and optionally one or more of IL-7, IL- 18, and interferon gamma (IFN-y).. The analytes can also or alternatively include one or more of cellular retinoic acidbinding protein 2 (CRABP2) (optionally in combination with ORFlp), e.g., to detect prostate cancer. The analytes can also include EV surface markers (e.g., CD81, CD9) and abundant serum proteins (e.g., albumin and / or apolipoprotein B (ApoB)). Preferably the sequences used are human sequences. Attorney Docket No. 29618-0504WO1 / BWH 2024-331

[0074] The following Table A lists analytes included in the present working examples, with corresponding human gene symbols and NCBI RefSeq accession numbers for mRNA (NM_) and protein (NP_).

[0075] TABLE A - Exemplary Analytes

[0076] For heterodimeric cytokines such as IL-12p70, RefSeq identifiers for both subunits (lL12A / p35 and IL12B / p40) are listed. ORFlp, encoded by human LINE-1 (L1HS) retrotransposons, does not have a single canonical RefSeq entry' due to sequence variability; instead, the human-specific L1HS consensus sequence (DFAM ID: DF0000225) is cited.

[0077] As used herein, the terms “cancer”, “hyperproliferative” and “neoplastic” refer to cells having the capacity for autonomous grow th, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. Hy perproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated w ith a disease state. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. “Pathologic hyperproliferative” cells occur in disease states characterized by malignant tumor growth. Examples of non-pathologic hyperproliferative cells include proliferation of cells associated with wound repair.

[0078] The terms "cancer” or “neoplasms” include malignancies of the various organ systems, such as affecting lung, breast, ovaries, cervix, thyroid, lymphoid, brain, soft Attorney Docket No. 29618-0504WO1 / BWH 2024-331 tissue, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas, which include malignancies such as most colon and colorectal cancers, kidney or renal-cell carcinoma, gallbladder, bile duct (cholangiocarcinoma), bladder, uterine, prostate cancer and / or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.

[0079] The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. In some embodiments, the disease is renal carcinoma or melanoma. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, kidney, gallbladder, bile duct (cholangiocarcinoma), bladder, uterine, colon / colorectal and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.

[0080] The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.

[0081] In some embodiments, the cancer is of origin in blood, bone marrow, brain, skin, or soft tissue in origin, especially lymphoma, leukemia, myeloma, glioblastoma, or melanoma.

[0082] As used herein the term “sample”, when referring to the material to be tested for the presence of an analyte using a method as described herein, includes inter alia a biofluid, e.g.. whole blood, plasma, or serum. In some embodiments, the sample is or comprises stool, cervical fluids such as pap smears, uterine lavage, urine, cerebrospinal fluid (CSF), or sputum. The sample can also be a tissue sample, e.g., from a biopsy (e.g., punch, needle, or shave biopsy, or surgical biopsy); for example tissue lysates can be used.

[0083] The methods can include incubating the sample, e.g., 0. 1 pL, 1 pL, 7.5 pl, 25 pl, 50 pl, 100 pl, 250 pl, 500 pl, 750 pl, 1 ml, 2 ml, 2.5 ml, or 10 ml of the sample, with beads coated with a capture antibody, as described herein. In some embodiments, the sample is diluted, e.g., 1 : 1, 1:2, 1:3, 1 :4, 1:5, 1:6, 1 :8, 1: 10, 1 :20, 1 :50, or 1: 100 and any ranges therebetween having the foregoing as endpoints, e.g., 1 : 1 to 1:20. or Attorney Docket No. 29618-0504WO1 / BWH 2024-331

[0084] 1 : 1 or 1 : 10. In some embodiments, the sample is diluted with a buffer; an exemplary sample diluent buffer is described herein, and can comprise a detergent, e.g., Triton- X 100, Tween 20, NP-40, Brij35, Brij58, or C12E8, for example, present at about 0.05%-2% of the sample. “About” as used herein means plus or minus 10%.

[0085] In some embodiments, the sample is contacted wi th the capture antibodies for a time sufficient for analytes present in the sample to bind to the capture reagent, e.g., for at least 5. 10, 15, 20, 30, or 45 minutes, or at least 1. 2, 3, 4. 5, or 6 hours, up to 1, 2, 3, 4, 5, 6, 8, 10, 12, 18, or 24 hours. In preferred embodiments, the bead is a paramagnetic bead, but other beads can be used, that have specific differentiatable characteristics.

[0086] The methods can also include comparing the presence and / or level of the analytes with one or more references, e.g., a control reference that represents a normal level of the analyte, e.g., a level in an unaffected subject, and / or a disease reference that represents a level of the proteins associated with cancer, e.g., a level in a subject having cancer.

[0087] In some embodiments, the presence and / or level of the analytes is comparable to the presence and / or level of the analytes in the disease reference, and the subject has one or more symptoms associated with cancer, then the subject has cancer. In some embodiments, the subject has no overt signs or symptoms of cancer, but the presence and / or level of one or more of the proteins evaluated is comparable to the presence and / or level of the protein(s) in the disease reference, then the subject has cancer or an increased risk of developing cancer. In some embodiments, once it has been determined that a person has cancer, or has an increased risk of developing cancer, then the subject can be selected or identified for further evaluation, e.g., using other blood-based diagnostics (e.g., biomarker panels), imaging, or biopsy to identify tumors or cancer, and / or a treatment, e.g., as known in the art or as described herein, can be selected and / or administered.

[0088] Suitable reference values can be determined using methods known in the art, e.g., using standard clinical trial methodology and statistical analysis. The reference values can have any relevant form. In some cases, the reference comprises a predetermined value for a meaningful level of the analytes, e.g., a control reference level that represents a normal level of the analytes, e.g., a level in an unaffected subject or a subject who is not at risk of developing a disease described herein, and / or Attorney Docket No. 29618-0504WO1 / BWH 2024-331 a disease reference that represents a level of the analytes associated with cancer, e.g., a level in a subject having cancer.

[0089] The predetermined level can be a single cut-off (threshold) value, such as a median or mean, or a level that defines the boundaries of an upper or lower quartile, tertile, or other segment of a clinical trial population that is determined to be statistically different from the other segments. It can be a range of cut-off (or threshold) values, such as a confidence interval. It can be established based upon comparative groups, such as where association with risk of developing disease or presence of disease in one defined group is a fold higher, or lower, (e.g., approximately 2-fold, 4-fold, 8-fold, 16-fold or more) than the risk or presence of disease in another defined group. It can be a range, for example, where a population of subjects (e.g., control subjects) is divided equally (or unequally) into groups, such as a low-risk group, a medium-risk group and a high-risk group, or into quartiles, the lowest quartile being subjects with the lowest risk and the highest quartile being subjects with the highest risk, or into n-quantiles (i.e., n regularly spaced intervals) the lowest of the n-quantiles being subjects with the lowest risk and the highest of the n- quantiles being subjects with the highest risk.

[0090] In some embodiments, the predetermined level is a level or occurrence in the same subject, e.g., at a different time point, e.g., an earlier time point.

[0091] Subjects associated with predetermined values are typically referred to as reference subjects. For example, in some embodiments, a control reference subject does not have cancer, does not have a risk of developing cancer, or does not later develop cancer.

[0092] A disease reference subject is one who has (or has an increased risk of developing) cancer. An increased risk is defined as a nsk above the risk of subjects in the general population.

[0093] In some embodiments, the level of the analytes in a subject being greater than or equal to the reference level of the analytes is indicative of the presence or risk of developing cancer, and the level of the analytes in a subject being less than or equal to a reference level of the analytes is indicative of the absence of disease or normal risk of the disease.

[0094] Thus, in some embodiments, to assess whether a subject has cancer in the clinic, the method can include first log transforming the analyte values and then Attorney Docket No. 29618-0504WO1 / BWH 2024-331 assigning a predicted probability, e.g., using a logistic regression model, to produce a probability score. If a subject has a predicted probability’ score above a selected threshold, e.g., at least 50%, the subject would be predicted to have cancer (e.g., assigned to a cancer category). If the predicted probability score is below the selected threshold, e.g., 50%, the subject would be predicted to be healthy (e.g., assigned to a healthy category).

[0095] In some embodiments, the level of the analytes is used to calculate a score, e.g., along with one or more additional variable, e.g., age. The score can be calculated, e.g., using an algorithm such as summation, or weighted summation, of the (normalized) levels of the variables. Specific algorithms can be identified using known statistical methods including PCA. linear regression. SVM (support vector machine), decision tree, KNN (K-nearest neighbors), K-means, gradient boosting, or random forest methods.

[0096] In some embodiments, the amount by which the level (or score) in the subject is less than the reference level (or score) is sufficient to distinguish a subject from a control subject, and optionally is a statistically significantly less than the level (or score) in a control subject. In cases where the level (or score) of the biomarker(s) in a subject being equal to the reference level (or score) of the biomarker(s), the “being equal” refers to being approximately equal (e.g.. not statistically different).

[0097] The predetermined value can depend upon the particular population of subjects (e.g., human subjects) selected. For example, an apparently healthy population will have a different ‘normal’ range of levels of the biomarker(s) than will a population of subjects which have, are likely to have, or are at greater risk to have, a disorder described herein. Accordingly, the predetermined values selected may take into account the category (e.g., sex, age, health, risk, presence of other diseases) in which a subject (e.g., human subject) falls. Appropriate ranges and categories can be selected with no more than routine experimentation by those of ordinary’ skill in the art.

[0098] In characterizing likelihood, or risk, numerous predetermined values can be established.

[0099] In some embodiments, a plurality of assays are performed yvith different combinations of antibodies as described herein, e.g., to improve sensitivity and / or specificity. Attorney Docket No. 29618-0504WO1 / BWH 2024-331

[0100] Methods of Treatment, Screening, and Monitoring Efficacy of Treatment

[0101] As shown herein, plasma levels of the analytes determined at the time of diagnosis are prognostic of overall survival in cancer, including in colorectal and gastroesophageal cancers, and can be used to monitor treatment response over time. This application could allow patients to be stratified into high and low risk group to receive additional treatment, such as chemotherapy, more aggressive chemotherapy, or additional surgery, especially in colorectal, breast, or prostate cancers, where multiple treatments are available. Thus the methods described herein include methods for the treatment of cancer. Generally, the methods include selecting and optionally administering a therapeutically effective amount of a treatment for cancer to a subject who has been determined to be in need of such treatment by a method described herein. Treatments for cancer can depend on the type of cancer, and can include radiation, surgical resection, chemotherapy, hormone / endocrine therapy, and / or immunotherapy. In some embodiments, the cancer is a carcinoma, e.g., ovarian, breast, liver, colon / colorectal, lung, esophageal, prostate, gastric, head and neck, brain, soft tissue, kidney, gallbladder, bile duct (cholangiocarcinoma), bladder, uterine, or pancreatic cancer. In some embodiments, the cancer is of origin in blood, bone marrow, brain, skin, or soft tissue in origin, especially lymphoma, leukemia, myeloma, glioblastoma, or melanoma.

[0102] In some embodiments, where a subject is identified as likely to have ovarian cancer, the subject is treated with surgical resection and optionally with chemotherapy and / or immunotherapy. Chemotherapy can include, e.g., paclitaxel and carboplatin, docetaxel and carboplatin, or carboplatin and pegylated liposomal doxorubicin, gemcitabine, toptecan, etoposide, and / or bevacizumab; PARP inhibitors, e.g., olapanb; or hormonal therapy, e.g., tamoxifen or letrozole.

[0103] The methods can also include sending the subject for additional screening such as referral to additional workups e.g., trans-vaginal sonography, uterine lavage, or falloposcopy , based on an updated posterior probability of having ovarian cancer, optionally combining the analyte results with other clinical features and potentially other biomarkers (e g., CA125 for ovarian cancer).

[0104] The methods can also be used for monitoring response to a treatment, e.g., to radiation, surgical resection, chemotherapy, hormone / endocrine therapy, and / or immunotherapy. The methods can include determining a baseline level of the Attorney Docket No. 29618-0504WO1 / BWH 2024-331 analytes in the subject using a method described herein; administering a treatment, e.g., one or more doses of a treatment, and determining a subsequent level of the analytes in the subject, e.g., an on-treatment (when obtained while the treatment is ongoing) and / or post-treatment (when obtained after the treatment is completed. A decrease in the level of the analytes in the subject from the baseline to the subsequent level indicates that the subject is responding or has responded to the therapy. The methods can also be used to monitor a subject who is in remission, to determine whether the subject remains in remission (e.g., has levels of the analytes that are at or below a threshold, e.g., the level of detection in a sample, or a level in a subject who does not have cancer). If a treatment is effective, the methods can include continuing the treatment. If a treatment is not effective (e.g., the level of the analytes does not decrease, or increases), the methods can include selecting and optionally administering a different treatment.

[0105] Kits and Assay Reagents

[0106] Also provided herein are kits and assay reagents comprising the capture beads and / or DNA-conjugated detector antibodies (or other binding agents) as described herein.

[0107] EXAMPLES

[0108] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0109] METHODS

[0110] The following methods and materials were used in the examples below. Materials. All affinity reagents, recombinant proteins, and DNA oligonucleotides used in this work are listed in below (Tables 11-12). Buffers and paramagnetic beads were purchased from Quanterix Corporation and Bangs Laboratories. Custom DNA oligonucleotides were purchased from Integrated DNA Technologies.

[0111] Preparation of capture and labeling reagents. Capture antibodies were buffer exchanged with Bead Conjugation Buffer (Quanterix) using a 50K Amicon Ultra-0.5 mL centrifugal filter (MilliporeSigma). After adding Bead Conjugation Buffer to antibody solution in the filter up to 500 pL, buffer exchange was carried out by centrifuging three times at 14,000 xg for five minutes, with addition of 450 pL Attorney Docket No. 29618-0504WO1 / BWH 2024-331

[0112] Bead Conjugation Buffer between centrifugation cycles. The buffer-exchanged antibody was recovered by inverting the filter into anew tube, centrifuging at 1000 xg for two minutes, rinsing the filter with 50 pL Bead Conjugation Buffer, and centrifuging one more time at 1000 xg for two minutes. The concentration of the buffer-exchanged antibody was then measured using a NanoDrop spectrophotometer. For each bead type, indicated starting number of beads (Table 13) were washed three times with 300 pL Bead Wash Buffer (Quanterix) and two times with 300 pL Bead Conjugation Buffer (Quanterix) before resuspending in cold Bead Conjugation Buffer. Bead number and conjugation conditions for each analyte are shown in Table 13. A 1 mg vial of l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (Thermo Fisher Scientific) was dissolved in 100 pL cold Bead Conjugation Buffer, and the desired volume was added to the beads. The beads were shaken for 30 minutes at either room temperature or 4 °C. After EDC activation of the carboxyl groups on the beads, the beads were washed once with 300 pL cold Bead Conjugation Buffer before resuspension in the buffer-exchanged antibody solution. Antibody conjugation was carried out by shaking the beads for two hours at either room temperature or 4°C, followed by washing twice with 300 pL Bead Wash Buffer. The antibody-coupled beads were then blocked for thirty' minutes at room temperature with shaking in 300 pL Bead Blocking Buffer (Quanterix). After washing once each with 300 pL Bead Wash Buffer and Bead Diluent (Quanterix), the beads were resuspended in 200 uL Bead Diluent, counted with a Beckman Coulter Z1 Particle Counter, and stored at 4 °C. Detector antibodies in non-barcoded MOSAIC assay were obtained in biotinylated form as previously described.

[0113] Preparation of antibody-DNA conjugates. Preparation of circularized padlock DNA template-primer hybrid. For each detector antibody, a 5’ azide-modified primer was annealed to a unique DNA template by heating a solution of 30 pM primer and 30.3 pM template in NEBNext Quick Ligation Buffer (New England Biolabs) at 95 °C for two minutes and allowing to cool to room temperature over 90 minutes. Ligation was then performed with addition of T4 DNA ligase and incubation at room temperature for two hours. The ligation reaction was buffer exchanged into phosphate buffered saline (PBS) with 1 mM EDTA using a 7K MWCO Zeba spin desalting column (Thermo Fisher Scientific). Preparation ofDBCO-modified antibody. For conjugation, the detector antibody was either reconstituted into PBS Attorney Docket No. 29618-0504WO1 / BWH 2024-331 from lyophilized form or buffer exchanged into PBS using a 50K Amicon Ultra-0.5 mL centrifugal filter, incubated with a 20-fold molar excess of dibenzocyclooctyne- PEG4-N-hydroxysuccinimidyl ester (DBCO-PEG4-NHS, MilliporeSigma) for 30 minutes at room temperature, and purified with a 50K Amicon Ultra-0.5 mL centrifugal filter in PBS with 1 mM EDTA. The template-primer hybrid and DBCO- modified antibody can be prepared in either order. However, it is suggested that the DNA hybrid be prepared first, as the cooling (1.5 hours) and ligation (2 hours) steps provide a window for preparing the DBCO-modified antibodies. Conjugation of azide-modifled DNA to DBCO-modified antibody via copper free click chemistry. A two-fold molar excess of the ligated primer-template was then added to the DBCO- modified antibody and incubated overnight at 4°C. The conjugate was stored in aliquots at -80 °C in PBS with 5 mM EDTA, 0.1% BSA, and 0.02% sodium azide. (FIG. 14 provides a detailed schematic illustrating the workflow and key steps of the process.)

[0114] Barcoded MOSAIC assays. MOSAIC assays were performed in a 96-well plate (Greiner Bio-One, 655096), with antibody-coated beads and detector antibodies diluted to the desired concentrations in Homebrew Sample Diluent (Quanterix). Assay conditions for each analyte are listed in Table 14. Sample volumes of 100 pL were used, with 10 pL of antibody-coated beads. The plate was sealed and shaken for one hour for target capture, followed by washing with System Wash Buffer 1 (Quanterix) using a BioTek 405 TS Microplate Washer. 100 pL detector antibody-DNA in Sample Diluent with 0.02 mg / mL heparin was added to the beads after target capture and washing steps. (Heparin, which mimics the poly anionic structure of nucleic acids, was included to mitigate non-specific interactions between the DNA-conjugated antibodies and non-target proteins.) The mixture was then incubated for 10 minutes. The samples were then washed with System Wash Buffer 1 for twelve cycles, transferred to a new 96-well plate, and washed an additional time with 180 pL System Wash Buffer 1 before being resuspended in 50 pL of the RCA reaction mixture. The RCA mixture consisted of 0.5 mM deoxynucleotide mix (New England Biolabs). 0.33 U / uL phi29 DNA polymerase, 0.2 mg / mL bovine serum albumin (BSA, Invitrogen), 1 nM fluorescently labeled DNA probe (Integrated DNA Technologies), and 0.1% Tween-20 in 50 mM Tris-HCl (pH 7.5), 10 mM (NH4)2SO4, and 10 mM MgCh. Dye labeled DNA probes (Table 14) were used for single target and multiplex MOSAIC Attorney Docket No. 29618-0504WO1 / BWH 2024-331 assays, respectively. Upon addition of the RCA mixture to each sample, the plate was shaken for 1.5 hours at 37 °C, followed by addition of 150 pL PBS with 0.1% Tween- 20 and 5 mM EDTA to stop the reaction. Samples were washed one time with 200 pL of the same PBS-Tween-EDTA buffer and resuspended in 100 pL of the buffer with added 0.1% BSA. Samples were measured using a NovoCyte Flow7Cytometer (Agilent Technologies) equipped with three lasers, in either tube or plate sampling mode. Bleach and buffer wells were included between different samples to minimize potential sample carryover. Multiplex MOSAIC assays were carried out following the same protocol as for the singleplex MOSAIC assays, with different fluorescent dye- encoded beads combined in the same sample. The incubation times are determined based on established protocols as well as prior theoretical modeling.10, 42

[0115] Plasma samples were diluted in 20% Sample Diluent (Quanterix) with 1% Triton X-100 in 80% StartingBlock™ Blocking Buffer (Thermo Fisher Scientific), with protease inhibitor (Halt™ Protease Inhibitor Cocktail, Thermo Fisher Scientific). Plasma samples were obtained from BioIVT and the Mass General Brigham Biobank. All human samples were de-identified, and experiments w ere performed under Institutional Review Board approval by Mass General Brigham. All plasma samples were centrifuged at 2000 xg for 10 minutes at 4 °C before diluting for measurements.

[0116] Calibration curves were employed to ensure accurate quantification in both singleplex and multiplex MOSAIC assays. Although concentrations can theoretically be determined through direct counting of molecules captured on beads, variations in the number of beads counted per sample introduce discrepancies in these counts. Additionally, practical factors such as bead occupancy variability, non-linear signal responses at extreme concentrations, and amplification inconsistencies necessitate calibration. These curves also account for non-specific binding, bead aggregation, surface coating heterogeneity, and matrix effects in biological samples, providing reliable and reproducible quantification that direct counting alone cannot achieve. Additionally, due to interactions between analytes and background noise as the number of analytes increases, separate calibration and curve fitting were performed for each specific multiplex panel. This ensures that assay -specific conditions, which may not be fully captured by a single calibration curve, are properly addressed to maintain accuracy across different multiplex configurations. Attorney Docket No. 29618-0504WO1 / BWH 2024-331

[0117] Non-barcoded MOSAIC assays. All non-barcoded assays were performed as previously described.10Assay conditions for each analyte are listed in Table 14. The same antibody -coated beads used in the barcoded MOSAIC assays were used for the non-barcoded MOSAIC assays, with the same detector antibody concentrations.

[0118] Barcoded MOSAIC assays for clinical analysis. Plasma samples from patients with high-grade serous ovarian carcinoma (HGSOC) and pancreatic cancer were analyzed using the barcoded MOSAIC assay. Capture antibodies were covalently conjugated to dye-encoded paramagnetic beads via EDC chemistry. For each analyte, a unique bead barcode — defined by fluorescence intensity7and / or color — was paired with a specific capture antibody. Plasma samples were diluted in buffer containing detergent and protease inhibitors, then incubated with the bead mixture in a 96-well plate for one hour at room temperature with gentle shaking to facilitate target binding.

[0119] Following incubation, beads were washed to remove unbound material and incubated with DNA-conjugated detector antibodies. Each detector antibody was site- specifically conjugated to a unique DNA template via copper-free click chemistry between DBCO-modified antibodies and azide-modified oligonucleotides. These antibody-DNA conjugates were matched to their corresponding bead populations, establishing a one-to-one mapping between bead type and detector barcode to enable downstream parity checking during analysis. After detector incubation and washing, beads were transferred to a new plate and resuspended in a rolling circle amplification (RCA) mixture containing phi29 DNA polymerase, dNTPs, and a fluorescently labeled probe complementary7to the DNA template. RCA was carried out at 37 °C for 1.5 hours. The reaction was quenched, beads were washed, and samples were resuspended in buffer for flow cytometric analysis on a NovoCyte flow cytometer equipped with multiple laser and detection channels. Beads w ere classified based on their barcode, and probe signals were quantified on a per-bead basis. Average molecules per bead (AMB) were calculated using Poisson statistics, and concentrations were determined from four-parameter logistic (4PL) calibration curves.

[0120] Multiplex barcoded MOSAIC panels for additional targets. Multiplex panels were designed to span a wide dynamic range by selecting analytes with differing endogenous abundances (e.g., CD9 and CD81 as low-abundance Attorney Docket No. 29618-0504WO1 / BWH 2024-331 extracellular vesicle (EV) markers, and albumin and apolipoprotein B (ApoB) as high-abundance plasma proteins). Probe concentrations and incubation conditions were optimized to balance signal intensity across targets. Standard curves for each analyte were generated in the multiplexed format to account for matrix effects and potential signal interdependencies.

[0121] Data analysis. Flow cytometry data were first analyzed with FlowJo™ Software (Becton, Dickinson and Company); beads were identified using gates on forward scatter, side scatter, and bead fluorescence. Single beads were additionally gated using forward scatter. The probe fluorescence intensities for each bead population were analyzed in as previously described10or using FlowJo™ Software. The fraction of "on” beads was then converted to an average number of analyte molecules bound per bead using Poisson statistics, and mapped to concentration using a four-parameter logistic calibration curve. Calibration curves were fitted using a four-parameter logistic (4PL) regression model, a standard choice for ELISA assays due to its capacity to handle sigmoidal curves. Since most curves in this study did not reach full saturation, the distinction between 4PL and 5PL models, which address symmetric versus asymmetric sigmoidal behavior, was not significant. The 4PL model was chosen for its compatibility7with the assay and aligns with prior work on similar immunoassay platforms.10-11, 27-28, 36The LOD and LLOQ were calculated as three and ten standard deviations above the background, respectively. The background refers to the baseline signal measured in the absence of the analyte, established by running multiple (>3) blank samples through the assay. The standard deviation of the background was calculated from these multiple blank measurements, with a correction factor C4(n) applied for unbiased estimation, as described previously in the literature.46

[0122] Example 1. Parity Matching of the Bead Type and Probe Color Greatly Reduced Cross-reactivity in Multiplex Assays.

[0123] To test the feasibility of pairing capture bead type and labeling probe color for the identification and elimination of mismatched cross-reactive binding events, we developed a proof-of-concept three-plex barcoded MOSAIC assay using three representative cytokines (Figure 2A): interleukins 8, 10, and 12p70 (IL-8, IL-10, and IL-12p70), which play important roles in regulating the immune response,28-29and are often present at low levels in many biological samples.10, 28In this assay, each Attorney Docket No. 29618-0504WO1 / BWH 2024-331 cytokine was assigned a unique fluorescent dye-coded bead, and a detector antibody conjugated to a distinct DNA template sequence matching a specific fluorescently labeled DNA probe. To test whether barcoded multiplex MOSAIC assays can successfully measure multiple protein analytes with high sensitivities, we compared the three-plex calibration curves in the barcoded MOSAIC assay against the corresponding non-barcoded MOSAIC assay (Figure 2B). We observed comparable sensitivities (attomolar levels) across both assay formats. Specifically, the barcoded approach exhibited a 4-fold and 16-fold reduction in limit of detections (LODs) for IL-10 and IL-12p70, respectively, whereas IL-8 displayed a 5-fold increase in LOD compared to non-barcoded MOSAIC (Table 1). The observed variations in LODs between the barcoded and non-barcoded assays may not signify a definitive advantage in sensitivity for either method. Rather, these differences may reflect intrinsic disparities in antibody performance, which are hypothesized to arise from modifications associated with specific labeling reagents. The covalent attachment of azide-DNA to DBCO-modified antibodies is posited to alter binding kinetics and antigen affinity, while the DNA template could introduce steric hindrance, affecting epitope accessibility compared to smaller biotin molecules. Additionally, barcoded MOSAIC reduces cross-reactive signals arising from mismatched detection antibodies binding to off-target analytes, which primarily improves the accuracy of the assay rather than the sensitivity. While this refinement improves quantification by reducing false positives, it does not imply that the LOD for non-barcoded MOSAIC is inherently worse — rather, the barcoded system reduces misleading signals caused by cross-reactivity, yielding more accurate and reliable measurements.

[0124] Table 1. Comparison of the limit of detection (LOD) and lower limit of quantification (LLOQ) values for the three-plex barcoded MOSAIC assay and the corresponding non-barcoded MOSAIC assay. LOD and LLOQ values were calculated as concentrations corresponding to three and ten standard deviations, respectively, above the background (established by running 3-6 blank samples through the assay). Reproducibility was evaluated by performing two independent Attorney Docket No. 29618-0504WO1 / BWH 2024-331 calibration curves on separate days, which yielded consistent AMB values and LODs. The reported LOD values represent the [range] of averaged results from these independent measurements, reflecting day-to-day variability.

[0125] We next assessed whether the barcoded multiplex MOSAIC assay reduces crossreactivity by performing protein dropout experiments, where increasing concentrations of a single interfering protein were measured to identify potential false positive “on” beads arising from cross-reactive binding events. In theory, any parity- mismatched color combinations were excluded from the analysis to enhance the accuracy of multiplex measurements in barcoded MOSAIC. We observed significant cross-reactivity for all three protein dropout assays in the non-barcoded multiplex MOSAIC assay, with observable false positive signals on off-target beads at protein concentrations above 8 fM (Figure 3A-i). We further assessed the specific binding and cross-reactivity for each analyte concentration by calculating the signal-to- background ratios (FIGs. 7A-B and Table 2), which we defined as the ratio of the AMB of a given sample concentration to the AMB of the blank sample, or background. Above 100 fM of IL12p70, for example, the false positive signal exceeds 28-fold and 42-fold above background for IL-8 and IL-10, respectively (Table 2). The non-barcoded multiplex MOSAIC, which utilizes a single-color readout through streptavidin-DNA conjugates targeting all biotinylated detectors, is prone to false positives primarily because proteins that bind cross-reactively to off-target beads may still be recognized by their specific detection antibodies, generating false signals. In contrast, the barcoded multiplex MOSAIC assay showed no detectable false signals arising on off-target beads across all three dropout assays, even at high protein concentrations, with the off-target bead signals remaining at the background levels (Figure 3A-ii). Thus, the inclusion of only correct capture bead-probe color pairs in barcoded multiplex MOSAIC enables the effective elimination of cross-reactive signals and thus ensures more accurate multiplex measurements.

[0126] To evaluate the measurement accuracy of the three-plex barcoded MOSAIC assay compared to that of the corresponding non-barcoded MOSAIC assay, we measured various spiked recombinant protein mixtures at different concentration levels of three analytes in both buffer and human plasma, where we compared recoveries according to the actual and measured protein concentrations. Initial evaluations in buffer established a baseline for cross-reactive performance without matrix interference. The barcoded MOSAIC assay consistently demonstrated higher Attorney Docket No. 29618-0504WO1 / BWH 2024-331 accuracy and reduced cross-reactivity (Table 3). For instance, recovery rates for IL-

[0127] 10 in the barcoded assay ranged from 72% to 113%, while the non-barcoded assay showed extreme variations from 4% to 853% (Figure 3B-i). Notably, in conditions with low IL-10 and high concentrations of the other two proteins (IL-10, IL-12p70, IL-8 = 0.1, 40, 200 fM), the barcoded assay accurately recovered IL-10 at 105%, while the non-barcoded assay showed significant cross-reactivity, reaching a recovery of 853%. Subsequent tests in biological fluids reinforced these findings. Spike and recovery assays in plasma typically achieved recoveries within the acceptable range of 70-130% for both assays, except under conditions of low cytokine concentrations or significant concentration disparities exceeding three logarithmic scales (Figure 3B-

[0128] 11 and Table 4). With a low spiked concentration of IL-10 alongside comparable levels of IL-12p70 and IL-8 (0.2, 0.2, 4 fM respectively), the non-barcoded assay exhibited extreme variability7, with recovery rates fluctuating from -28% to 28,400%. Negative recovery7rates in the non-barcoded MOSAIC assay likely arise from background interference or cross-reactivity7, where blank signals exceed those of spiked samples, leading to underestimation of analyte concentrations. In stark contrast, the barcoded assay displayed markedly7stable recovery rates, spanning from 95% to 280%. With a spiked concentration of IL-10, IL-12p70, IL-8 at 0.2, 40, 180 fM, the barcoded assay demonstrated relatively consistent recoveries, while the non- barcoded assay showed poor performance, illustrating the barcoded MOSAIC assay’s substantial reduction in cross-reactivity and its ability to provide accurate measurements across varied concentration levels, even when lower concentrations of measured analy te coexist with much higher concentrations of other analytes.

[0129] Although the barcoded system is highly effective in minimizing false positive signals from cross-reactivity, non-barcoded multiplex MOSAIC remains a useful approach in less complex multiplex assays with fewer analytes, particularly when carefully selected antibody pairs with low cross-reactivity7are employed. However, as the degree of multiplexing increases, the likelihood of cross-reactivity7between detection and capture antibodies rises, compromising assay accuracy.

[0130] Example 2. Development of Higher-Order Multiplexed Barcoded MOSAIC assay with Minimal Cross-reactivity.

[0131] To develop a higher-order multiplexed panel in barcoded MOSAIC for N analytes, we require N spectrally distinct signatures, which translates to N distinct Attorney Docket No. 29618-0504WO1 / BWH 2024-331 bead types — characterized by properties such as color, fluorescence intensity , and size — combined with N distinct DNA barcodes on detector antibodies. To expand beyond the number of available laser colors in conventional flow cytometry', instead of measuring the fluorescence intensity of a single-colored probe, we employ' multispectral profiles resulting from ratiometric combinations of probes that label individual molecules derived from RCA. By differentiating DNA template sequences conjugated to antibodies using multispectral probes, we generate distinguishable optical barcodes, facilitating high-dimensional multiplexing via multispectral signatures. We screened various DNA sequences capable of hybridizing with different ratiometric combinations of probes (FIGs. 8A-H). Utilizing two probe colors allowed us to generate four distinct non-overlapping signals. This strategy of employing ratiometric combinations of probes enabled a greater degree of multiplexing within a constrained spectral range, while retaining low to mid-attomolar sensitivities and minimal cross-reactivity for low-abundance cytokines (FIGs. 9A-B and 10A-C).

[0132] Utilizing an expanded palette of ratiometric probe combinations, we conducted proof-of-concept experiments to quantify a panel of five cancer-relevant markers — IL-6,30-32IL-12p7O,30HE4.33”4CA-125,35and ORF1 protein (ORFlp)36’37— in plasma samples (Figure 4A). This approach, leveraging distinct bead and probe colors for each target, effectively minimizes false signals from cross-reactive binding, enabling accurate quantification of each analyte. Calibration curves for this five-plex assay indicated sufficient sensitivities to measure all analytes at concentrations well below their physiological levels, although the LOD for IL-12p70 (around 20 aM) approached the measured concentrations (Figure 4B). Notably, the UOD for LINE1 ORFlp, a recently identified biomarker for multiple cancers,36’37improved by two orders of magnitude compared to the previously reported LOD from a singleplex Simoa assay.7-36By' decreasing the detector concentration to moderate the assay’s sensitivity' for HE4, which is highly abundant in plasma in healthy conditions (hundreds of pM) and further elevated in diseases such as ovarian cancer, we extended the assay’s dynamic range to accommodate both low and high abundance markers. This demonstrates the barcoded MOSAIC platform’s ability to quantify biomarkers with varying concentrations, addressing the typical variability in dynamic range required for clinical samples. We applied the assay across various dilutions — 8-, 12-, and 16-fold — in human plasma samples, observing acceptable Attorney Docket No. 29618-0504WO1 / BWH 2024-331 recoveries in spike and recovery experiments and consistent dilution linearity (Table 6 and FIG. 11), with minimal cross-reactivity across the target analyte concentration ranges (FIG. 12). The exceptional sensitivity of barcoded MOSAIC enables the use of higher dilution factors to minimize matrix interference and potential false signals due to cross-reactivity7. In conjunction wi th parity checking, these features collectively further enhance the assay ’s resistance to cross-reactivity-related false signals. We further measured the five analvtes in two healthy plasma samples, diluted eight-fold and requiring less than 25 pL across duplicates. Despite the low sample volume, the assay's performance remained robust, with quantification of analytes (Figure 4C) comparable to those previously obtained using Simoa28’36and non-barcoded MOSAIC methods10. This multiplexing capability, coupled with minimal crossreactivity and reduced sample volume requirements, underscores the assay’s potential as a valuable tool for accelerating biomarker signature discovery, particularly in applications with limited sample volumes like fingerpick blood.

[0133] To enhance the utility of our platform in cancer diagnostics, where high multiplexing is essential for delineating both sensitive and specific biomarker signatures, we expanded the assay to include three additional cytokine markers — IL- 7,38IL-18,39and IFN-y,40— resulting in an eight-plex panel. This assay incorporates three primary markers (CA-125, HE4, and ORFlp) with minimal cross-reactivity using distinct bead types and probe signatures. The auxiliary cytokines were analyzed using distinct bead types, with shared probe colors selectively applied to enhance multiplexing efficiency, particularly for less cross-reactive pairs or secondary markers where relative quantification is sufficient (Figure 5A). The LODs for the three primary markers remained comparable to those measured from the five-plex assay, even with the inclusion of additional cytokines (Figure 5B). After confirming satisfactory recovery for each analyte (Table 8), we measured these eight proteins in a cohort of 10 plasma samples using less than 17 pL of plasma, inclusive of two replicates. The eight-plex barcoded MOSAIC assay successfully quantified endogenous proteins across a broad concentration spectrum, from picomolar to attomolar (Figure 5C), demonstrating consistency with the general concentration ranges of these markers in plasma, as observed in previous measurements.10’11’28‘36The strategic use of shared probe colors for auxiliary markers underscores the flexibility of the barcoded system to adapt to varying degrees of cross-reactivity, Attorney Docket No. 29618-0504WO1 / BWH 2024-331 enabling efficient multiplexing while maintaining high specificity where necessary. Thus, the expanded multiplexing capability of the barcoded MOSAIC platform not only allows for accurate quantification of primary markers but also broadens the diagnostic potential by enabling the profiling of a more comprehensive spectrum of clinically relevant biomarkers.

[0134] Table 2. Cross-reactivity analysis of protein dropout experiments. The signal-to-background ratio is defined as the ratio of the AMB at a specified sample concentration to that of the blank (0 , or background. Atorney Docket No. 29618-0504WO1 / BWH 2024-331

[0135] Table 3. Recoveries of spiked recombinant proteins in the three-plex barcoded MOSAIC assay and the corresponding MOSAIC assay in buffer. Recoveries are calculated as the mean ± standard deviation from duplicate measurements and are defined as the difference in interpolated concentrations postspike relative to the LOD, divided by the indicated spiked protein concentration. The LOP is defined as the average of blank values plus three standard deviations. Atorney Docket No. 29618-0504WO1 / BWH 2024-331

[0136] Table 4. Recoveries of spiked recombinant proteins for the three-plex barcoded MOSAIC assay and corresponding MOSAIC assay in human plasma at 16-fold dilution. Recoveries are reported as the mean ± standard deviation of duplicate measurements. Atorney Docket No. 29618-0504WO1 / BWH 2024-331

[0137] Table 5. Analytical sensitivities for five-plex barcoded MOSAIC assay. Limit of detection (LOD) and lower limit of quantification (LLOQ) values of the five-plex arcoded MOSAIC assay are calculated in both pg / mL and fM,

[0138] Table 6. Recoveries of spiked recombinant proteins for the five-plex barcoded MOSAIC assay in human plasma at 8-, 12-, and 16-fold dilutions.

[0139] Recoveries are reported as the mean ± standard deviation of duplicate measurements. Attorney Docket No. 29618-0504WO1 / BWH 2024-331

[0140] Table 7. Analytical sensitivities for eight-plex barcoded MOSAIC assay.

[0141] Limit of detection (LOD) and lower limit of quantification (LLOQ) values of the eight-plex barcoded MOSAIC assay are calculated in both pg / rnL and fM,

[0142] Table 8. Recoveries of spiked recombinant proteins for the eight-plex barcoded MOSAIC assay in human plasma at 16-fold dilution. Recoveries are reported as the mean ± standard deviation of duplicate measurements. The inclusion of auxiliary analytes sharing probe colors in the 8-plex panel introduces higher variability in recovery rates for these markers due to unavoidable overlap. In contrast, the cross- reactivity-free panel for primary markers, which utilizes distinct bead types and probe signatures, demonstrated less variability. Atorney Docket No. 29618-0504WO1 / BWH 2024-331

[0143] Table 9. Summary of Time Required for Barcoded and Non-barcoded MOSAIC Assay Protocols. This table outlines the duration of each step in the barcoded and non-barcoded MOSAIC workflows. Additionally, the incubation times can be adjusted depending on the specific antibodies used. *For a typical 96-well Atorney Docket No. 29618-0504WO1 / BWH 2024-331

[0144] Table 10. Estimated dissociation constants (KD) for analytes based on IC50 measurements using 4-parameter logistic (4PL) curve fitting. This table presents the IC50 values obtained through 4PL curve fitting, used to estimate KD values for different analytes in the multiplex barcoded MOSAIC assay. IC50 values provide an approximation of KD, representing the concentration of the analyte that produces 50% of the maximal signal. KD estimation was not possible for HE-4 and ORFlp due to the absence of a saturation point. The IC50 values for CA-125, IL-6, and !L-12p70 suggest relative binding affinities, falling within the low picomolar range ( 1011to 1010M), consistent with the KD values required for such high sensitivity. While IC50 values are approximations of KD and may be influenced by assay-specific factors such as antibody concentration and detection method, the inferred KD values from the response curves align well with the expected binding affinities, confirming the assay's robustness.

[0145] Table 11. Antibodies and recombinant protein standards used in the non- barcoded and barcoded MOSAIC assays.

[0146] Table 12. Sequences of the DNA primer-template, and probe utilized in the barcoded MOSAIC assays in this study. Bolded sequences denote complementary regions in the primer and template. Asterisks (*) indicate the presence of Atorney Docket No. 29618-0504WO1 / BWH 2024-331 phosphorothioated nucleic acid bases, incorporated to inhibit exonuclease degradation. The term “invdT” signifies an inverted dT at the 3 ’-end of the probes, creating a 3 ’-3’ linkage that prevents degradation by 3’ exonucleases and hinders extension by DNA polymerases. Table 13. Coupling conditions for the antibody-coated capture beads used in this work.

[0147] The parameters were selected based on prior studies.lu-11 28-36 Attorney Docket No. 29618-0504WO1 / BWH 2024-331 Attorney Docket No. 29618-0504WO1 / BWH 2024-331

[0148] Table 14. Assay conditions used for the barcoded and non-barcoded MOSAIC assays in this work. The same concentrations of detector antibodies and target capture times were applied in corresponding barcoded and non-barcoded MOSAIC assays. A 3-step assay was performed in the non-barcoded MOSAIC assay. The listed incubation times encompass various stages: (1) incubation of the target analyte with beads, (2) binding of the detector to the bead-protein complex, (3) the additional time (specified in parentheses) utilized in the non-barcoded MOSAIC assay for applying streptavidin DNA labeling reagents, along with (4) the incubation period for the RCA reaction. The parameters were selected based on prior studies.10, 42

[0149] Example 3. Barcoded MOSAIC assays for clinical analysis of samples.

[0150] To evaluate the clinical and analytical performance of the barcoded MOSAIC platform, we quantified two cancer-associated biomarkers — CRABP2 and ORF Ip — in plasma samples from patients with high-grade serous ovarian carcinoma (HGSOC) and pancreatic cancer, respectively. In the HGSOC cohort (n = 52), CRABP2 was detected in 100% of samples using barcoded MOSAIC, compared to only 15% with the Simoa platform (FIGs. 15A-B). The signal distribution showed clear separation Attorney Docket No. 29618-0504WO1 / BWH 2024-331 between HGSOC patients and healthy controls (n = 30), with mean concentrations significantly different between groups (p < 0.01, Welch's t-test). This improvement reflects both the platform’s enhanced sensitivity and its ability to reduce background signal through bead-detector barcode matching.

[0151] In a separate study, ORFlp was quantified in plasma from 12 patients with pancreatic cancer. Barcoded MOSAIC detected ORFlp in all 12 cases (100% sensitivity), whereas Simoa detected the marker in only 58% of the same samples (FIG. 16). This enhanced sensitivity — corresponding to an approximately 100-fold lower limit of detection — is partly attributable to the barcoded MOSAIC platform’s improved specificity. By requiring concordance between the bead barcode and the detector DNA barcode, barcoded MOSAIC minimizes false positives from nonspecific binding and strengthens signal assignment through dual-recognition logic. This capability is especially valuable in complex biological matrices like plasma, where off-target interactions can otherwise obscure true signal.

[0152] To demonstrate the assay’s broader utility, we applied barcoded MOSAIC to a multiplexed panel including both low- and high-abundance plasma proteins. EV surface markers (CD81, CD9) and abundant serum proteins (albumin, ApoB) were simultaneously quantified in multiplexed assays, spanning concentrations from pg / mL to ng / mL (FIG. 17). Calibration curves generated in both buffer and plasma showed log-linear responses across 4-6 orders of magnitude. All targets exhibited signal-to- background ratios greater than 10-fold at physiological levels, with limits of detection for CD81 and CD9 in the low-pg / mL range — comparable to prior singleplex Simoa results.

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[0203] OTHER EMBODIMENTS

[0204] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Attorney Docket No. 29618-0504WO1 / BWH 2024-331WHAT IS CLAIMED IS:1 . A method of detecting the presence of an analyte of interest in a sample, the method comprising: contacting the sample with capture beads coated with a capture agent, optionally an antibody or aptamer, that binds to the analyte, wherein the capture beads have specific identifiable charactenstics, optionally wherein the bead characteristics are color, intensity, and / or size, wherein the capture agents bind to any analytes present in the sample; contacting the sample with a detector agent, optionally an antibody or aptamer, that binds to the same analyte, optionally in the presence of heparin, wherein the detector agent is directly conjugated to a specific DNA template barcode sequence; performing amplification, preferably using rolling circle amplification, using DNA probes comprising distinct fluorescent labels that bind to the DNA template barcode sequence, to produce double-labeled complexes; using flow cytometry to sort the double-labeled complexes based on the bead characteristics and the fluorescent label; and identifying double-labeled complexes that have the specific bead characteristics and the distinct fluorescent labels as comprising the analyte of interest.

2. A method of detecting the presence of two or more analytes of interest in a sample, the method comprising: contacting the sample with a plurality of capture beads coated with capture agents, optionally antibodies or aptamers, that bind to each of the analytes, wherein the capture beads have specific identifiable characteristics for each of the analytes, optionally wherein the bead characteristics are color, intensity, and / or size, wherein the capture agents bind to any analytes present in the sample; contacting the sample with a plurality of detector antibodies that bind to each of the analytes, optionally in the presence of heparin, wherein the detector antibodies are directly conjugated to DNA template barcode sequences specific for each of the analytes; performing rolling circle amplification using DNA probes comprising distinct fluorescent labels that bind to each of the DNA template barcode sequences, toAtorney Docket No. 29618-0504WO1 / BWH 2024-331 produce double-labeled complexes; using flow cytometry to sort the double-labeled complexes based on the bead characteristics and the fluorescent label; and identifying double-labeled complexes that have both the specific bead characteristics and the distinct fluorescent labels as comprising an analyte of interest.

3. The method of claim 1 or 2, wherein the DNA template barcode sequence comprises a circularized DNA template-primer DNA.

4. The method of claim 2, wherein for detecting a number N of analytes. N distinct capture bead types, each type having specific identifiable characteristics, are combined with N distinct DNA barcodes on detector antibodies.

5. The method of claim 2, wherein flow cytometry sorting is based on (1 ) bead-code fluorescence intensity, and (2) fluorescence intensify' of single-colored fluorescent labels on a DNA probe, or on multispectral profiles from ratiometric combinations of fluorescent labeled probes that label individual analytes.

6. The method of claims 1-5, wherein the sample comprises a biological fluid, optionally blood, serum, or plasma.

7. The method of claim 1 or 2, wherein the analytes comprise one. two, three, four, or more of IL-6, IL-12p70, HE4, CA-125, and ORF1 protein (ORFlp), and optionally one or more of IL-7, IL-18, and IFN-y.

8. The method of claim 1 or 2, wherein the analytes comprise CRABP2. optionally in combination with ORFlp.

9. The method of claim 1 or 2, wherein the analytes comprise EV surface markers, optionally CD81 and / or CD9, and / or abundant serum proteins, optionally albumin and / or ApoB.

10. The method of claim 7, wherein the detection of a level of one or more of the analytes above a reference level in a sample from a subject indicates that the subject has cancer.Atorney Docket No. 29618-0504WO1 / BWH 2024-33111. The method of any of claims 1-8, wherein the subject is a mammal, optionally a human or non-human veterinary subject.

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