Highly scalable multiplex serology by mass spectrometry analysis of beads

Abstract

The present disclosure provides methods for determining the presence of at least two biomolecules in a sample. The present disclosure further provides methods for determining the presence of a two or more biomolecules in a sample using single particle mass spectrometry as a readout.

Description

MGB Ref: 33929 / 70816HIGHLY SCALABLE MULTIPLEX SEROLOGY BY MASS SPECTROMETRY ANALYSIS OF BEADSSTATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with Government support under contract AI100627 awarded by the National Institutes of Health. The Government has certain rights in the invention.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Application No. 63 / 707,356, filed on October 15, 2024, the entire contents of each of which are fully incorporated herein by reference.FIELD OF THE INVENTION

[0003] The present disclosure provides an enhanced bead-based assay platform that enables multiplexed detection of multiple samples, multiple targets, and multiple readout biomolecules in a single run.BACKGROUND

[0004] Traditional biomolecule detection methods, such as ELISA and fluorescently barcoded bead-based assays, are limited in multiplexicity and the need for multiple wash steps, restricting throughput and hindering automation. Additionally, many existing platforms are unable to simultaneously detect multiple biomolecules and differentiate between various immunoglobulin types, proteins, oligonucleotides, and functional readouts on the same assay at a high scale. A need exists for a highly scalable, high-throughput platform that can assay multiple samples, targets, and biomolecules for large-scale studies, diagnostics.

[0005] As one example, infectious disease outbreaks are a serious concern for public health. The pandemic of COVID- 19 caused by severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2) is an illustrative example of how a localized outbreak can quickly escalate to a worldwide pandemic if not rapidly controlled. Among important interventions, extensive population testing to identify individuals experiencing current or past infection is key to controlling an infectious disease outbreak.

[0006] To monitor and control the spread of an outbreak, there is a need for new strategies to escalate testing capabilities to detect biomolecules and provide deeper understanding of the tested sample. While high-throughput techniques such as Rapid Extracellular Antigen ProfilingMGB Ref: 33929 / 70816(REAP) have been described previously (Wang et al., medRxiv. 2020; 20030593) such methods were limited to protein detection and yeast protein barcode libraries.SUMMARY OF THE INVENTION

[0007] In various aspects, the present disclosure provides methods for determining the presence of at least one biomolecule in a sample. In one embodiment, a method for determining the presence of at least one biomolecule in a sample comprising: (a) obtaining a sample from a subject; (b) incubating the sample with a composition comprising a plurality of beads, wherein each bead is conjugated with (i) at least one barcode moiety, and (ii) at least one ligand capable of binding to a biomolecule, thereby producing a composition comprising a plurality of constructs, wherein each construct comprises a bead, a ligand and a bound biomolecule; (c) washing the composition of (b) under conditions that allow removal of unbound biomolecules; (d) optionally pooling the composition of (c) with one or more additional compositions comprising separate samples that have been incubated with separate compositions according to (b), thereby preparing a pooled composition; (e) incubating the composition or the pooled composition comprising bound biomolecules with a composition comprising a plurality of secondary constructs, wherein each secondary construct comprises a barcode moiety conjugated to a biomolecule capable of binding to bound biomolecules; (f) washing the composition or pooled composition of (e) under conditions that allow removal of unbound secondary constructs; and (g) determining the presence of the barcode moieties of the first and second constructs, thereby determining the presence of at least one biomolecule in a sample.

[0008] In one embodiment, a method for determining the presence of at least one biomolecule in a sample comprising: (a) obtaining a sample from a subject; (b) incubating the sample with a composition comprising a plurality of beads, wherein each bead is conjugated with (i) at least one barcode moiety, and (ii) at least one ligand capable of binding to a biomolecule, thereby producing a composition comprising a plurality of constructs, wherein each construct comprises a bead, a ligand and a bound biomolecule; (c) incubating the composition comprising the constructs of (b) with a fixing reagent, thereby fixing the bound biomolecules to their respective beads; (d) incubating the composition of (c) with a denaturing reagent, thereby denaturing the bound and unbound biomolecules; (e) optionally pooling the composition of (d) with one orMGB Ref: 33929 / 70816 more additional compositions comprising separate samples that have been incubated with separate compositions according to (b), thereby preparing a pooled composition; (f) removing unbound biomolecules; (g) incubating the composition or the pooled composition comprising bound, fixed and denatured biomolecules with a plurality of secondary constructs, wherein each secondary construct comprises a barcode moiety conjugated to a biomolecule capable of binding to a bound, fixed and denatured biomolecules: (h) removing unbound secondary constructs; and (i) determining the presence of the barcode moieties of the first and second constructs, thereby determining the presence of at least one biomolecule in a sample.

[0009] In one embodiment, a method for simultaneously detecting a plurality of biomolecules from a collection of samples comprising: (a) obtaining a collection of samples from a single subject or a plurality of subjects; (b) separately incubating the samples with compositions comprising a plurality of beads, wherein each bead is conjugated with (i) a plurality of barcode moieties, and (ii) at least one ligand capable of binding to a biomolecule, thereby producing a composition comprising a plurality of constructs, wherein each construct comprises a bead, at least one ligand and at least one bound biomolecule; (c) washing the compositions of (b) under conditions that allow removal of unbound biomolecules; (d) pooling the composition of (c) with one or more additional compositions comprising separate samples that have been separately incubated with separate compositions according to (b), thereby preparing a pooled composition; (e) incubating the pooled composition comprising bound biomolecules with a composition comprising a plurality of secondary constructs, wherein each secondary construct comprises a barcode moiety conjugated to a biomolecule capable of binding to bound biomolecules; (f) washing the pooled composition of (e) under conditions that allow removal of unbound secondary constructs; and (g) determining the presence of the barcode moieties of the first and second constructs, thereby determining the presence of at least one biomolecule in a sample.

[0010] In one embodiment, a method for simultaneously detecting a plurality of biomolecules from a collection of samples comprising: (a) obtaining a collection of samples from a single subject or a plurality of subjects; (b) separately incubating the samples with compositions comprising a plurality of beads, wherein each bead is conjugated with (i) a plurality of barcode moieties, and (ii) at least one ligand capable of binding to a biomolecule, thereby producing a composition comprising a plurality of constructs, wherein each construct comprises a bead, at least one ligand and at least one bound biomolecule; (c) incubating the composition comprisingMGB Ref: 33929 / 70816 the constructs of (b) with a fixing reagent, thereby fixing the bound biomolecules to their respective beads; (d) incubating the composition of (c) with a denaturing reagent, thereby denaturing the bound and unbound biomolecules; (e) pooling the composition of (d) with one or more additional compositions comprising separate samples that have been separately incubated with separate compositions according to (b), thereby preparing a pooled composition; (f) removing unbound biomolecules; (g) incubating the composition comprising bound, fixed and denatured biomolecules of (e) with a plurality of secondary constructs, wherein each secondary construct comprises a barcode moiety conjugated to a biomolecule capable of binding to a bound, fixed and denatured biomolecules; (h) removing unbound secondary constructs; and (i) determining the presence of the barcode moieties of the first and second constructs, thereby determining the presence of at least one biomolecule in a sample.

[0011] In yet another embodiment, an aforementioned method is provided wherein each bead of the first construct is conjugated to one ligand, wherein the ligand is an antigen capable of being bound by an antibody in the sample, and wherein the biomolecule of the secondary construct is a secondary antibody.

[0012] In yet another embodiment, an aforementioned method is provided wherein the isotype and / or subclass of antibody bound to the antigen is determined.

[0013] In yet another embodiment, an aforementioned method is provided wherein each bead of the first construct is conjugated to one ligand, wherein the ligand is an antigen capable of being bound by an antibody in the sample, and wherein the biomolecule of the secondary construct is a Fc gamma receptor (FcyR), a complement cascade-initiating protein, or a lectin.

[0014] In yet another embodiment, an aforementioned method is provided wherein each bead of the first construct is conjugated to one ligand, wherein the ligand is capable of being bound by a receptor in the sample, and wherein the biomolecule of the secondary construct is a receptor capable of binding to the ligand of the first construct.

[0015] In yet another embodiment, an aforementioned method is provided wherein the ligand and / or receptor is selected from the group consisting of an antigens, an analytes, and a proteins.

[0016] In yet another embodiment, an aforementioned method is provided wherein each bead of the first construct is conjugated to a plurality of ligands, wherein each ligand is an antibodyMGB Ref: 33929 / 70816 capable of binding one or more antigens in the sample, and wherein the biomolecule of the secondary construct is a secondary antibody.

[0017] In yet another embodiment, an aforementioned method is provided wherein the one or more antigens comprise biomolecules selected from the group consisting of antigens, peptides, analytes, and proteins.

[0018] In yet another embodiment, an aforementioned method is provided wherein each bead of the first construct is conjugated to one ligand, wherein the ligand is an oligonucleotide capable of being bound by a nucleic acid in the sample, and wherein the biomolecule of the secondary construct is a secondary oligonucleotide capable of being bound by the same nucleic acid in the sample in a distinct region.

[0019] In yet another embodiment, an aforementioned method is provided wherein each bead of the first construct is conjugated to a plurality of ligands, wherein each ligand is an oligonucleotide capable of binding one or more nucleic acids in the sample, and wherein the biomolecule of the secondary construct is a secondary oligonucleotide.

[0020] In yet another embodiment, an aforementioned method is provided wherein each bead of the first construct is conjugated to one ligand or a plurality of ligands, wherein each ligand is an antibody, a ligand capable of being bound by a receptor, or an oligonucleotide, and wherein the biomolecule of the secondary construct is a secondary antibody, a receptor capable of binding a ligand of the first construct, or a secondary oligonucleotide.

[0021] In yet another embodiment, an aforementioned method is provided wherein each bead is conjugated to 1, 2, 3, 4, 5, 6, 7. 8, 9, 10, 20. 30. 40. 50 60, 70, 80. 90. or 100 or more different ligands.

[0022] In yet another embodiment, an aforementioned method is provided wherein a plurality of samples are obtained from a plurality of subjects.

[0023] In yet another embodiment, an aforementioned method is provided wherein approximately 10, 100, 1000, 10,000, 20,000, 30,000, 40,000, 40,000, 50,000, 100,000 or more samples are obtained.

[0024] In yet another embodiment, an aforementioned method is provided wherein the subject is an animal, plant, fungi, protist. bacterium or archaea.MGB Ref: 33929 / 70816

[0025] Tn yet another embodiment, an aforementioned method is provided wherein the animal is selected from the group consisting of a human, a cow, a pig, a chicken, a sheep, a duck, and a goat.

[0026] In yet another embodiment, an aforementioned method is provided wherein the presence of approximately, 1, 10, 100, 1,000, 5,000, 10,000, 20,000 or more biomolecules are determined.

[0027] In yet another embodiment, an aforementioned method is provided wherein the sample or plurality of samples is a blood, serum, saliva, nasal / oropharyngeal swab, semen, urine, lymph, cerebrospinal fluid, interstitial fluid, spinal fluid, peritoneal fluid, pleural fluid, amniotic fluid, stool, bile, bone marrow, or skin sample.

[0028] In yet another embodiment, an aforementioned method is provided wherein a barcode moiety associated with the first construct identifies the incubated sample.

[0029] In yet another embodiment, an aforementioned method is provided wherein a barcode moiety associated with the first construct identifies the ligand or ligands.

[0030] In yet another embodiment, an aforementioned method is provided wherein the biomolecule that is determined is an antibody, a cytokine, a lectin, a receptor, an analyte, a protein or a nucleic acid.

[0031] In yet another embodiment, an aforementioned method is provided wherein the biomolecule that is determined is providing functional information of biomolecule binding.

[0032] In yet another embodiment, an aforementioned method is provided wherein the biomolecule is a nucleic acid, and wherein the nucleic acid is selected from the group consisting of a DNA, a viral DNA, a cell-free DNA (cfDNA), a tumor cell-derived DNA, a RNA, a viral RNA, and / or a tumor cell derived RNA.

[0033] In yet another embodiment, an aforementioned method is provided wherein the nucleic acid is an RNA from a pathogen.

[0034] In yet another embodiment, an aforementioned method is provided wherein the nucleic acid is a DNA from a pathogen.MGB Ref: 33929 / 70816

[0035] Tn yet another embodiment, an aforementioned method is provided wherein the protein is a protein from a pathogen.

[0036] In yet another embodiment, an aforementioned method is provided wherein the pathogen is a virus, a bacterium, a fungi, a protozoa, or a worm.

[0037] In yet another embodiment, an aforementioned method is provided wherein the ligand is an antigen or biomolecule-binding fragment of an antigen, a biotinylated protein, an antibody, a polymer or a nucleic acid.

[0038] In yet another embodiment, an aforementioned method is provided wherein the bead comprises an affinity binding system .

[0039] In yet another embodiment, an aforementioned method is provided wherein the affinity binding system comprises one or more of streptavidin and / or biotin.

[0040] In yet another embodiment, an aforementioned method is provided wherein the barcode moiety on the first construct and / or on the second construct comprises one or more isotopes, one or more peptides, one or more small molecules, one or more lipids, one or more glycans, one or more oligonucleotides or one or more fluorophores.

[0041] In yet another embodiment, an aforementioned method is provided wherein the barcode moieties present on the first and second constructs comprise dual isotope barcodes.

[0042] In yet another embodiment, an aforementioned method is provided wherein the bead comprises a polymer bead.

[0043] In yet another embodiment, an aforementioned method is provided wherein the polymer is selected from the group consisting of polystyrene, poly(methyl methacrylate) (PMMA), hydrogels, silica, poly(lactic-co-glycolic acid) (PLGA), and polyethylene glycol (PEG).

[0044] In yet another embodiment, an aforementioned method is provided wherein the fixing reagent is selected from the group consisting of paraformaldehyde (PF A), formaldehyde, acetone, glutaraldehyde, ethanol, glyoxal, and methanol.

[0045] In yet another embodiment, an aforementioned method is provided wherein the denaturing reagent is selected from the group consisting of sodium dodecyl sulfate (SDS),MGB Ref: 33929 / 70816 thiourea, lithium perchlorate, lithium acetate, magnesium chloride, urea, guanidine hydrochloride, methanol, and CHAPS.

[0046] In yet another embodiment, an aforementioned method is provided wherein the determining step (i) comprises using mass cytometry, flow cytometry, mass spectrometry, fluorescence microscopy, electron microscopy, isotope-based imaging, Raman spectroscopy, Nuclear Magnetic Resonance (NMR), fluorescence resonance energy transfer (FRET), X-ray fluorescence (XRF), or nucleic acid sequencing.

[0047] In yet another embodiment, an aforementioned method is provided wherein the presence of a signal from the barcode moiety of the first construct is indicative of a current or prior infection from a pathogen associated with the ligand.

[0048] In one embodiment, a method for determining the presence of antibodies targeting pathogens in a sample comprising: (a) obtaining a sample from a subject; (b) incubating the sample with a composition comprising a plurality of beads, wherein each bead is conjugated with (i) at least one barcode moiety, and (ii) at least one antigen capable of binding to a pathogen antibody, thereby producing a composition comprising a plurality of constructs, wherein each construct comprises a bead, an antigen and a bound pathogen antibody; (c) washing the composition of (b) under conditions that allow removal of unbound pathogen antibodies; (d) optionally pooling the composition of (c) with one or more additional compositions comprising separate samples that have been incubated with separate compositions according to (b), thereby preparing a pooled composition; (e) incubating the composition or the pooled composition comprising bound pathogen antibodies with a composition comprising a plurality of secondary constructs, wherein each secondary construct comprises a barcode moiety conjugated to a secondary antibody capable of binding to bound pathogen antibodies; (f) washing the composition or pooled composition of (e) under conditions that allow removal of unbound secondary constructs; and (g) determining the presence of the barcode moieties of the first and second constructs, thereby determining the presence of at least one pathogen antibody in a sample.

[0049] In yet another embodiment, an aforementioned method is provided wherein the pathogen is a coronavirus.MGB Ref: 33929 / 70816

[0050] Tn yet another embodiment, an aforementioned method is provided wherein the antigen is a Spike protein antibody from SARS-CoV-1 or SARS-CoV-2.BRIEF DESCRIPTION OF THE DRAWING

[0051] Fig. 1A-E shows a post-synthesis strategy for incorporation of stable isotopes on polystyrene beads, the figure comprising (a) schematic representation of streptavidin coated polystyrene beads loaded with isotope-conjugated biotinylated protein carrier; (b) representative histogram of162Dy intensity per bead in mono-labeled beads wherein162Dy per bead was measured by mass cytometry; (c) schematic representation of beads loaded with “odd” and “even” isotopes; (d) representative histograms of isotope intensity per bead for “odd” and “even” beads; and (e) heatmap of isotope intensities for “odd” and “even”, wherein color shows the median of the normalized isotope intensities for odd and even bead populations.

[0052] Fig. 2A-C shows a double barcoding strategy enabled generation of 18,840 isotopebarcoded beads, the figure comprising (a) schematic representation of the workflow for the generation of isotope-barcoded beads, wherein for target ID barcoding, beads are labeled with three of the six target ID isotopes (159Tb to164Dy) to generate 20 barcodes and for sample ID barcoding, beads are labeled with six of the 12 sample ID isotopes (165Ho to176Yb) to generate 924 barcodes; (b) left: UMAP of beads grouped by the intensity of the target ID isotopes and colored by target ID barcode and right: representative groups colored by the intensity of the target ID isotopes, wherein each dot represents a bead and data are from three independent experiments; (c) left: UMAP of beads from barcode 20 label in panel (b) grouped by the intensity of the sample ID isotopes and colored by sample ID barcode and right: representative barcodes (BC) 549 and 619 colored by the intensity of the sample ID isotopes, wherein each dot represents a bead and data are from three independent experiments.

[0053] Fig. 3A-F shows a two-bead strategy to detect specific antibodies in low volumes of plasma, the figure comprising (a) schematic representation of the two-bead strategy for detection of antibodies in low volumes of plasma; (b) schematic representation of the baseline and flow- through, wherein beads bound by anti-IgG magnetic beads are retained in the column whereas unbound beads are collected in the flow-through; (c) dot plot of the number of barcoded beads in the baseline versus number in the flow-through for 20 serum samples collected prior to theMGB Ref: 33929 / 70816COVID- 19 pandemic (green) and 20 samples from patients who had tested positive for COVID- 19 (red); (d) comparison of ELISA (y-axis) versus the two-bead assay (x-axis) in the 20 COVID- 19-positive samples shown in panel (c), wherein the two-bead assay result is shown as the ratio of the number of beads in the flowthrough to the number of beads in the baseline (Note that the two samples in panel (c) that behave as pre-pandemic samples also show low ELISA values); (e) two-bead assay on a set of 39 COVID- 19-positive samples and 55 pre-pandemic samples, wherein the dot color indicates the sample volume: 100 nL is red, 10 nL is green, and 1 nL is blue, each dot is the mean of 12 replicates (3 sample volume replicates and 4 target replicates per sample volume), and targets were COVID-19 Spike SI, Spike SI RBD, Spike S2, Nucleocapsid, and negative (no target); and (f) heatmap of the values of the 12 replicates for each red dot shown in panel (e).

[0054] Fig. 4A-F shows a high-throughput and wash-free strategy for multiplexed detection of antibodies, the figure comprising (a) schematic representation of the wash-free strategy to detect antibodies against 20 targets in 924 samples in a single mass cytometry analysis; (b and c) Heatmap of b) IgG levels and c) IgM levels against 19 targeted proteins (rows) in 542 samples (columns) from the FIND study, which collected samples from individuals with confirmed SARS-CoV-2 positive tests along with additional household members, wherein samples are labeled by day of sample collection after positive symptoms for the household’s index case (timepoint), PCR test, and vaccination status, IgG and IgM levels were normalized to values of beads not loaded with a target antigen (negative), and samples and targets are hierarchically clustered by IgG or IgM levels; (d) boxplot of the IgG levels against SARS-CoV-2 Spike Trimer per sample by timepoint, wherein each dot represents a sample; (e) dot plot of the levels of IgG against SARS-CoV-2 Spike SI Trimer obtained by mass cytometry versus those obtained using the Abbott AdviseDx SARS-CoV-2 IgG II assay, wherein each dot represents a sample, the orange line is a polynomial regression line of degree 2, and the R2is 0.58; and (f) dot plot of the levels of IgG against the Beta SARS-CoV-2 Spike Trimer versus the wild- type trimer quantified using mass cytometry, wherein each dot represents a sample.

[0055] Fig. 5A-J shows an illustration of bead generation, assay, and analysis.

[0056] Fig. 6A-L shows various embodiments of optional combinations of methods described herein, ligands capable of binding to a biomolecule, and secondary constructs, wherein theMGB Ref: 33929 / 70816 biomolecule is an antigen and the secondary construct is an antibody (Figs. 6A-B), wherein the biomolecule is an antigen and the secondary construct is an isotype-conjugated FcyR (Figs. 6C- D), wherein the biomolecule is a ligand and the secondary construct is an isotype-conjugated receptor (Figs 6E-F), wherein biomolecular is an antibody and the secondary construct is an isotope-conjugated anti-analyte (Figs. 6G-H), wherein the biomolecule is a nucleic acid and the secondary construct is an isotope-conjugated oligo (Figs. 61- J), and wherein the biomolecule is a ligand, antibody, and a nucleic acid and the secondary construct is an isotope-conjugated antibodies, olio, and protein (Figs. 6K-L). The illustrations show methods comprising a washing step (Figs. 6A, 6C, 6E, 6G, 61, and 6K). The illustrations show methods comprising a fixing and denaturalizing steps (Figs. 6B, 6D, 6F, 6H, 6 J, and 6L). (BTOF corresponds to beads by time of flight. For example, each bead is conjugated to a combination of isotope and are analyzed using time-of-flight mass spec).DETAILED DESCRIPTION

[0057] The present disclosure addresses the aforementioned unmet need by providing methods and materials for determining the presence multiple biomolecules in a sample or collection of samples. In various embodiments, the biomolecule is an antibody, protein or a nucleic acid.

[0058] Serology testing is a ubiquitous tool in diagnostics, drug discovery, and epidemiological surveillance. Each sample is typically tested individually, one antigen at a time, which is costly and time consuming. There is a pressing need for highly parallel, low-cost, fast, and recurrent serology measurement techniques that use small sample volumes. In some embodiments, the present disclosure comprises a highly scalable technology for multiplex serology testing using mass cytometry that vastly surpasses limitations of existing serology multiplexing techniques. In some embodiments, the methods described herein use polystyrene beads that are uniformly and robustly loaded post- synthesis with combinations of stable isotopes and analyzed via mass cytometry. In some embodiments and as shown herein, 18,840 unique isotopically-barcoded beads were generated to simultaneously detect antibody levels against multiple SARS-CoV-2 proteins in multiple blood-derived samples. In some embodiments, high- throughput specific detection of antibodies was demonstrated using sample volumes in the hundreds of nanoliters range including the simultaneous analysis of the levels of IgG and IgMMGB Ref: 33929 / 70816 against 19 proteins in 924 samples. Serology by mass cytometry is rapid, scalable, and cost- effective, and has tremendous potential to contribute to more effective management of public health emergencies originating from infectious diseases.

[0059] Rapid, scalable and convenient detection of pathogens is critical for clinical purposes and population surveillance. Testing can be aimed at detecting current or past infection as extensively reviewed elsewhere (Esbin et al. 2020, RNA 26 (7): 771-83; Ravi et al. 2020, Biosensors & Bioelectronics 165 (112454): 112454). PCR-based testing is a highly sensitive method that reveals current infection, but it is slow, expensive, and requires trained personnel (Esbin et al. 2020, RNA 26 (7): 771-83). Antigen testing is more rapid and less expensive than PCR testing; however, utility of this type of test is limited by sensitivity and specificity issues (Winter and Hegde 2020, The Lancet Infectious Diseases. 20 (7); p755-874, el48-el79). Other alternatives for the detection of current infection include CRISPR- and LAMP-based testing, which are fast, easy, and sensitive (Ackerman et al. 2020, Nature, 582, 277-282; Lamb et al. 2020, medRxiv 19.20025155). Reliabilities of these methods as diagnostic tests have not been extensively studied yet. A common characteristic of all these types of tests is the inability to inform about past infections. Serology testing by lateral-flow assay reveals past infection and is rapid and inexpensive. Like antigen testing, the utility of serology testing by lateral-flow assay is hindered by sensitivity and specificity issues (Krammer and Simon 2020, Science (New York, N.Y.) 368 (6495): 1060-61). ELISA-based serology testing has an improved sensitivity and specificity compared to lateral-flow assays, but it is slow and limited in throughput (Krammer and Simon 2020, Science (New York, N.Y.) 368 (6495): 1060-61).

[0060] Most testing strategies offer only a binary readout and have difficulties with scalability to even the population level of towns, cities and counties. This means that these strategies are limited in scope if the problem escalates from a local to a global scale, as in the case with the COVID- 19 pandemic. Robotic systems have been implemented to increase the throughput of PCR and ELISA, providing the ability to test samples from a few hundred patients simultaneously. However, the total number of tested patients is still far below of what is needed to control an active pandemic, limited by the low-throughput of these devices. Moving testing abilities from binary responses to more complex scenarios will allow assessment of crucial information, including testing for distinct molecular components in the same sample or detecting co-infection with other pathogens. State-of-the-art strategies that provide this type ofMGB Ref: 33929 / 70816 multiplexing capability include CRISPR-based combinatorial arrayed reactions for multiplexed evaluation of nucleic acids (Ackerman et al. 2020, Nature, 582, 277-282) and protein microarrays for serological testing (Zhu et al. 2006. PNAS 03 (11): 4011-16: de Assis et al. 2020, BioRxiv). Unfortunately, neither of these strategies can achieve the scale at which population level testing would be possible.

[0061] Serology immunoassays are powerful tools that measure specific antibodies in biological samples. These assays have a wide range of applicability, including monitoring vaccine efficacy, epidemiological surveillance, mapping viral transmission dynamics, infectious and autoimmune disease diagnosis, and drug discovery. In situations with large number of samples requiring assessment across multiple targets, the scalability of current serology assays is a major bottleneck. There is a pressing need, highlighted by the COVID- 19 pandemic, for precise, accurate, highly parallel, low-cost, fast, and recurrent serology measurements that use small sample volumes.

[0062] Classic immunoassays such as enzyme-linked immunosorbent assay (ELISA) are widely used because of simplicity, sensitivity, and specificity. However, in ELISA, each sample is tested individually for one molecular target at a time, which severely hampers high-throughput applications due to cost and time constraints. In recent years, automation strategies and multiplexed serology techniques have increased throughput. For example, fluorophore barcoded beads used in Luminex assays allow higher throughput by simultaneously detecting multiple analytes in a single sample. However, the spectral overlap of fluorophores constrains the available number of barcodes and thus limits the scalability of fluorescent bead-based serology assays. An elegant strategy has recently ameliorated a major issue arising from spectral overlap in beads, non-linear behavior of multicolor Forster resonance energy transfer and resulting cascades, but the barcoding space of this method is currently limited to the generation of 580 distinguishable fluorophore combinations 27. The maturity and widespread acceptance of beadbased serology assays have allowed these assays to become industry standards. However, scalable paradigms capable of greatly surpassing existing barcoding limitations, to the range of tens of thousands of uniquely barcoded beads, would be a significant breakthrough for research and clinical applications.MGB Ref: 33929 / 70816

[0063] Mass cytometry is a mass spectrometry, multi-parameter, and single-cell technology extensively used in the last decade for cell profiling 28. Over 50 parameters are routinely analyzed on each cell using antibodies conjugated to isotopically enriched rare earth metals 28. The instrument provides mass spectra on the fly, one event at a time, at a speed of about 1000 events per second. In mass cytometry, channel overlap is minimal, and thus the theoretical barcoding space when 38 stable isotope channels are employed is in the range of billions. These unique capabilities of a mass cytometer could be harnessed for high-throughput serology testing by assessing antibody binding to isotopically barcoded beads loaded with specific targets. Beadbased assays for cytokine profiling via mass cytometry have been recently reported but are limited to the simultaneous detection of nine analytes. This mismatch between the theoretical capability of mass cytometry for serology testing and its practical implementation is largely explained by the laborious experimental requirements for the production of isotope-loaded beads compatible with mass cytometry using current techniques. Therefore, the vast potential of mass cytometry for serology testing has remained largely untapped.

[0064] In some embodiments, the present disclosure comprises a practical, low cost, highly scalable bead-based multiplex serology testing using mass cytometry. In some embodiments, polystyrene beads can be uniformly and robustly loaded post-synthesis with stable isotopes and can be reliably quantified via mass cytometry. In some embodiments, the scalability comprises a method via the generation of 18,840 distinct isotopically barcoded beads. By harnessing this large barcoding space, the detection of antibody levels against multiple SARS-CoV-2 proteins in multiple clinical serum and plasma samples simultaneously in a one-tube assay. In some embodiments, an assay takes one operator approximately 8 hours to complete for 924 samples, and no automation equipment is necessary. In some embodiments, mass cytometry with barcoded beads facilitates serology testing of multiple antigens in samples from large sample cohorts. In some embodiments, this technology has the potential to contribute to management of public health and infectious disease pandemic response.

[0065] As described herein, various embodiments of the present disclosure provide simultaneous detection of multiple pathogens, pathogen characteristics, or host characteristics (for example, up to thousands per assay) on multiple patient samples, simultaneously (for example, up to hundreds of thousands per assay) to enable clinical diagnosis. While the present disclosure describes one embodiment relating to COVID- 19 testing, the methods disclosedMGB Ref: 33929 / 70816 herein are applicable to the detection of any biomolecule, a plurality of biomolecules, diagnosis of any infectious disease, cancer, autoimmune disease, or other systemic perturbations with molecular alterations.

[0066] In one embodiment, the presence of at least two biomolecules in a sample is determined by the following steps: (a) obtaining a sample from a subject; (b) incubating the sample with a composition comprising a plurality of beads, wherein each bead is conjugated with (i) at least one barcode moiety, and (ii) at least one ligand capable of binding to a biomolecule, thereby producing a composition comprising a plurality of constructs, wherein each construct comprises a bead, a ligand and a bound biomolecule; (c) washing the composition of (b) under conditions that allow removal of unbound biomolecules; (d) optionally pooling the composition of (c) with one or more additional compositions comprising separate samples that have been incubated with separate compositions according to (b), thereby preparing a pooled composition; (e) incubating the composition or the pooled composition comprising bound biomolecules with a composition comprising a plurality of secondary constructs, wherein each secondary construct comprises a barcode moiety conjugated to a biomolecule capable of binding to bound biomolecules; (f) washing the composition or pooled composition of (e) under conditions that allow removal of unbound secondary constructs; and (g) determining the presence of the barcode moieties of the first and second constructs, thereby determining the presence of at least one biomolecule in a sample.

[0067] In one embodiment, the presence of at least two biomolecules in a sample is determined by the following steps: (a) obtaining a sample from a subject; (b) incubating the sample with a composition comprising a plurality of beads, wherein each bead is conjugated with (i) at least one barcode moiety, and (ii) at least one ligand capable of binding to a biomolecule, thereby producing a composition comprising a plurality of constructs, wherein each construct comprises a bead, a ligand and a bound biomolecule; (c) incubating the composition comprising the constructs of (b) with a fixing reagent, thereby fixing the bound biomolecules to their respective beads; (d) incubating the composition of (c) with a denaturing reagent, thereby denaturing the bound and unbound biomolecules; (e) optionally pooling the composition of (d) with one or more additional compositions comprising separate samples that have been incubated with separate compositions according to (b), thereby preparing a pooled composition; (f) removing unbound biomolecules; (g) incubating the composition or the pooled compositionMGB Ref: 33929 / 70816 comprising bound, fixed and denatured biomolecules with a plurality of secondary constructs, wherein each secondary construct comprises a barcode moiety conjugated to a biomolecule capable of binding to a bound, fixed and denatured biomolecules; (h) removing unbound secondary constructs; and (i) determining the presence of the barcode moieties of the first and second constructs, thereby determining the presence of at least one biomolecule in a sample.

[0068] In one embodiment, the presence of at least two biomolecules in a sample is determined by the following steps: (a) obtaining a collection of samples from a single subject or a plurality of subjects; (b) separately incubating the samples with compositions comprising a plurality of beads, wherein each bead is conjugated with (i) a plurality of barcode moieties, and (ii) at least one ligand capable of binding to a biomolecule, thereby producing a composition comprising a plurality of constructs, wherein each construct comprises a bead, at least one ligand and at least one bound biomolecule; (c) washing the compositions of (b) under conditions that allow removal of unbound biomolecules; (d) pooling the composition of (c) with one or more additional compositions comprising separate samples that have been separately incubated with separate compositions according to (b), thereby preparing a pooled composition; (e) incubating the pooled composition comprising bound biomolecules with a composition comprising a plurality of secondary constructs, wherein each secondary construct comprises a barcode moiety conjugated to a biomolecule capable of binding to bound biomolecules; (f) washing the pooled composition of (e) under conditions that allow removal of unbound secondary constructs; and (g) determining the presence of the barcode moieties of the first and second constructs, thereby determining the presence of at least one biomolecule in a sample.

[0069] In one embodiment, the presence of at least two biomolecules in a sample is determined by the following steps: (a) obtaining a collection of samples from a single subject or a plurality of subjects; (b) separately incubating the samples with compositions comprising a plurality of beads, wherein each bead is conjugated with (i) a plurality of barcode moieties, and (ii) at least one ligand capable of binding to a biomolecule, thereby producing a composition comprising a plurality of constructs, wherein each construct comprises a bead, at least one ligand and at least one bound biomolecule; (c) incubating the composition comprising the constructs of (b) with a fixing reagent, thereby fixing the bound biomolecules to their respective beads; (d) incubating the composition of (c) with a denaturing reagent, thereby denaturing the bound and unbound biomolecules; (e) pooling the composition of (d) with one or more additionalMGB Ref: 33929 / 70816 compositions comprising separate samples that have been separately incubated with separate compositions according to (b), thereby preparing a pooled composition; (f) removing unbound biomolecules; (g) incubating the composition comprising bound, fixed and denatured biomolecules of (e) with a plurality of secondary constructs, wherein each secondary construct comprises a barcode moiety conjugated to a biomolecule capable of binding to a bound, fixed and denatured biomolecules; (h) removing unbound secondary constructs; and (i) determining the presence of the barcode moieties of the first and second constructs, thereby determining the presence of at least one biomolecule in a sample.

[0070] The methods of the present disclosure are based on a one-bead and optionally wash- free system and is therefore distinct from the methods described in WO 2022 / 150591. The following additional advantages relative to known techniques are also provided herein:

[0071] Multiplexing capability across multiple biomolecules, samples, and targets. Many current assays focus on detecting a single analyte, such as one antibody isotype. The present disclosure enables multiplexed detection across multiple biomolecules (e.g., antibodies, proteins, oligonucleotides) and can process multiple samples and multiple targets simultaneously.Whether configured for antibody detection (against multiple antigens), protein quantification, or oligonucleotide detection, this platform allows for comprehensive, multi-analyte analysis in a single assay. The ability to detect multiple biomolecules per bead offers unprecedented flexibility and efficiency in high-throughput applications.

[0072] High throughput. Existing technologies are limited in throughput due to manual handling and multiple wash steps. The optional wash-free system described herein facilitates massive scalability, allowing for the processing of multiple samples and multiple targets within the same assay. For instance, in one exemplary method described below it is demonstrated that the present methods are capable of running 36,960 tests in a single tube with minimal sample volumes (400 nL per test), making it suitable for large-scale studies.

[0073] Elimination of wash steps. Traditional serology assays, such as ELISA and fluorescently barcoded bead-based assays, require multiple wash steps to remove unbound antibodies. In some embodiments, the present disclosure eliminates the need for washing by using a fixation (PFA) and denaturation (SDS) process, making it more efficient and scalable.MGB Ref: 33929 / 70816

[0074] Sensitivity and specificity!. In one embodiment, mass cytometry-based detection provides high sensitivity and specificity. It can distinguish between different levels of biomolecule responses, even for closely related targets, such as SARS-CoV-2 variants or different protein isoforms, with minimal background noise.

[0075] Minimal sample volume. The methods described herein use significantly less sample volume than traditional methods, reducing the burden on patients and increasing the feasibility of longitudinal or large cohort studies. It is ideal for situations where sample availability is limited. Sample collection from finger-prick-derived dried blood spots and saliva are also viable and convenient options.

[0076] Automation. In some embodiments, the removal of wash steps and simplified workflow make the methods provided herein highly compatible with automation, overcoming the complexity and labor-intensiveness of current methods. Automation further increases throughput and reduces human error, making it highly attractive for commercial applications.

[0077] Quantitative and robust comparative analyses. The methods provided herein allows for robust, internally-controlled comparisons within samples, providing a high degree of accuracy and reliability for comparative studies. Whether used for immunoglobulin detection, protein quantification, or oligonucleotide detection, it offers flexibility without compromising sensitivity.

[0078] In various embodiments, the beads have distinct physical attributes (for example, size and magnetism or a combination of properties). In various embodiments, readout methods include, but are not limited to, flow cytometry, mass cytometry, fluorescence microscopy, electron microscopy, isotope-based imaging, and sequencing. Exemplary steps of the disclosed methods are illustrated in Fig. 4 and described in detail herein.

[0079] As described herein, one application of the present disclosure relates to the detection and / or quantitation of nucleic acid molecules from human patient samples where the patient is infected with a pathogen.Definitions

[0080] The plural herein shall equally denote the singular, and the singular shall equally denote the plural wherever reasonable. The words “for example” or “by way of example” orMGB Ref: 33929 / 70816 similar phrases shall be interpreted as equivalent to “for example but not by way of limitation”, such that any example shall not limit the generality to which the example pertains.

[0081] As used herein, the term “sample” or “biological sample” encompasses a variety of sample types obtained from a variety of sources, which sample types contain biological material. For example, the term includes biological samples obtained from a subject, e.g., a human subject, and biological samples obtained from a food, water, or other environmental source, etc. The definition encompasses blood and other liquid samples of biological origin, as well as solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as polynucleotides. The term “sample” or “biological sample” encompasses a clinical sample, and also includes cells in culture, cell supernatants, cell lysates, cells, serum, plasma, biological fluid, and tissue samples. “Sample” and “biological sample” includes cells, e.g., bacterial cells or eukaryotic cells; biological fluids such as blood, serum, saliva, nasal / oropharyngeal swab, semen, urine, lymph, cerebrospinal fluid, interstitial fluid, spinal fluid, peritoneal fluid, pleural fluid, amniotic fluid sample, and the like; stool; bile; bone marrow; skin sample (e.g., skin biopsy); and viruses or viral particles obtained from an individual.

[0082] Tn various embodiments, the methods disclosed herein provide for simultaneous testing of one or more patient samples to determine the presence of one or more biomolecule(s). In various embodiments 1. 2, 3, 4. 5, 6, 7, 8, 9, 10. 25. 50. 100, 500, 1000. 10000. 20000, 40000, 50000, 100000, 200,000 or more patient samples may be tested simultaneously using the methods disclosed herein.

[0083] As used herein, the term “biomolecule” refers to an antibody, polypeptide, a nucleic acid or fragments thereof which is detected in a sample.

[0084] As described more fully herein, in various embodiments the subject methods may be used to detect a variety of biomolecules from such samples. Biomolecules include, but are not necessarily limited to, nucleic acid (e.g., DNA and / or RNA), protein / peptide / polypeptide, cells (e.g., circulating cells and / or circulating tumor cells), viruses, and many other components that may be present in a biological sample.MGB Ref: 33929 / 70816

[0085] The term “nucleic acid" (e.g., as it relates to a biomolecule) refers to a polymer composed of a multiplicity of nucleotide units (ribonucleotide or deoxyribonucleotide or related structural variants) linked via phosphodiester bonds. A nucleic acid (or polynucleotide) can be of substantially any length, typically from about six (6) nucleotides to about 109nucleotides or larger. Nucleic acids include RNA, cDNA, genomic DNA. In various embodiments, the RNA, cDNA, or genomic DNA is RNA, cDNA, or genomic DNA from a pathogen. In one embodiment, the DNA is cell-free DNA (cfDNA) which may be, for example, of tumor cell origin and therefore useful in detecting cancer.

[0086] The term “protein” (e.g., as it relates to a biomolecule) refers to a polymer of amino acid residues, wherein a protein may be a single molecule or may be a multi-molecular complex. The term, as used herein, can refer to a subunit in a multi-molecular complex, polypeptides, peptides, oligopeptides, of any size, structure, or function. It is generally understood that a peptide can be 2 to 100 amino acids in length, whereas a polypeptide can be more than 100 amino acids in length. A protein may also be a fragment of a naturally occurring protein or peptide. The term protein may also apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid. A protein can be wild-type, recombinant, naturally occurring, or synthetic and may constitute all or part of a naturally-occurring, or non-naturally occurring polypeptide. The subunits and the protein of the protein complex can be the same or different. A protein can also be functional or non-functional.

[0087] As used herein, an "antibody" refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are typically classified as either kappa or lambda. Heavy chains are typically classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[0088] A typical full-length (intact) immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The N-MGB Ref: 33929 / 70816 terminus of each chain defines a variable region of about 100 to 1 10 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.

[0089] Antibodies exist as intact immunoglobulins or as a number of well-characterized fragments that can be produced, inter alia, by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2;a dimer of Fab which itself is a light chain joined to VH-CHI by a disulfide bond. The F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab')2 dimer into a Fab' monomer. The Fab' monomer is essentially a Fab with part of the hinge region (see, Fundamental Immunology, W.E. Paul, ed., Raven Press, N.Y. (1993), for a more detailed description of other antibody fragments). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fab' fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein also includes whole antibodies, antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies.

[0090] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or post-translation modifications (e.g., isomerizations, amidations, etc.) that may be present in minor amounts. Monoclonal antibodies are typically highly specific, being directed against a single epitope. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The term "monoclonal" indicates the character of the antibody as being obtained from, or one of, a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies may be made by a variety of techniques, including, but not limited to, the hybridoma method (see, e.g., Kohler and Milstein. (1975) Nature, 256:495-497; Hongo et al. (1995) Hybridoma, 14 (3): 253-260; Harlow et al. (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2d ed.); Hammerling et al. (1981) In: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y.)), recombinant DNA methods (see,MGB Ref: 33929 / 70816 e.g., U.S. Patent No. 4,816,567), phage-display technologies (see, e.g., Clackson et al. (1991) Nature, 352: 624-628; Marks et al. (1992) J. Mol. Biol. 222: 581-597; Sidhu et al. (2004) J. Mol. Biol. 338(2): 299-310: Lee et al. (2004) J. Mol. Biol. 340(5): 1073-1093; and the like), and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., PCT Patent Publication Nos: WO 1998 / 24893; WO 1996 / 34096; WO 1996 / 33735; and WO 1991 / 10741; U.S. Patent Nos: 5,545,807; 5,545,806; 5,569,825; 5,625, 126; 5,633,425; and 5,661,016; Jakobovits et al. (1993) Nature 362: 255-258; Bruggemann et al. (1993) Year in Immunol. 7: 33; Marks et al. (1992) Bio / Technology 10: 779-783; Lonberg et al. (1994) Nature 368: 856-859; Morrison (1994) Nature 368: 812-813; Fishwild et al. (1996) Nature Biotechnol. 14: 845-851); Neuberger (1996) Nature Biotechnol. 14: 826; Lonberg and Fluszar (1995) Intern. Rev. Immunol. 13 : 65-93; and the like).

[0091] In some embodiments, an aptamer or aptamers (e.g., single- stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules that bind to biomolecules such as protein targets by folding into a three-dimensional conformation, similar to antibodies) are used in the described methods.

[0092] In various exemplary embodiments, the antibody is a coronavirus antibody is an antiSpike protein antibody for SARS-CoV-1 or SARS-CoV-2.

[0093] As used herein, the term “subject” (e.g., as it relates to a biomolecule) refers to an animal, plant, fungi, protist, bacterium or archaea for which a sample is obtained from for use in the disclosed methods. Suitable subjects for the methods disclosed herein include animals, e.g., a human., an armadillo, an axolotl, a bird, a cat. a chicken, a chinchilla, a cow, a dog, a donkey, a duck, a fish, a frog, a gerbil, a goat, a guinea pig, a hamster, a horse, a lizard, a monkey, a mouse, a pig, a rabbit, a rat, a salamander, a sheep, a snake, a toad, and / or a zebrafish. In one embodiment, farm animals are contemplated. The subject may be one that exhibits clinical presentations of a disease condition or has been diagnosed with a disease. In certain embodiments, the subject may be one that has been diagnosed with an infection, e.g., COVID- 19, or cancer, exhibits clinical presentations of infection or cancer.

[0094] As used herein, the term “bead” refers to any type of solid phase particle of any convenient size, of irregular or regular shape, and which is fabricated from any number of knownMGB Ref: 33929 / 70816 materials such as polystyrene, cellulose, cellulose derivatives, acrylic resins, glass, silica gels, gelatin, polyvinyl pyrrolidone, co-polymers of vinyl and acrylamide, polystyrene cross-linked with divinylbenzene, or the like (as described, e.g., in Merrifield (1964) Biochemistry 3: 1385- 1390), polyacrylamides, latex gels, polystyrene, dextran, rubber, silicon, plastics, nitrocellulose, natural sponges, silica gels, controlled pore glass (CPG), metals, cross-linked dextrans (e.g., Sephadex™), agarose gel (Sepharose™), nanoparticle and other solid phase bead supports known to those of skill in the art. In various embodiments, the first or second or both beads is / are magnetic. In various embodiments the methods disclosed herein may comprise multiple beads. In related embodiments, the methods disclosed herein may comprise 2 beads (i.e. a first bead and a second bead). In various embodiments, the methods disclosed herein may comprise 1, 2, 3, 4, 5, 6, 7. 8, 9, 10, 50. 100, 500, 1000, 10000. 20000 or more beads. As described herein, beads according to various embodiments may be modified to allow conjugation of polymers and / or ligands and / or barcode moieties. By way of example, the beads may be biotinylated or modified to include maleimide-thiol chemistries to allow conjugation of conjugation of polymers and / or ligands and / or barcode moieties.

[0095] As used herein, the term “affinity binding system” is used to refer to a combination of two protein that bind each other (i.e.., streptavidin and / or biotin). In some embodiments, a bead is pre-coated with a binding protein, wherein the binding protein is streptavidin. In some embodiments, the bead is conjugated with biotinylated BSA. In some embodiments, the biotinylated BSA is used as carrier protein and it was conjugated to isotope-chelated polymers.

[0096] As used herein, the terms “barcode moiety” or “barcode moieties” are used to refer to a unique combination of molecular marker(s) or tag(s) which is attached to one or more beads. In various embodiments, the barcode moiety is an isotope, oligonucleotide or fluorophore barcode. In some embodiments, the barcode or barcodes, e.g., on a first construct and / or a second construct as described herein, comprise one or more isotopes (e.g., conjugated to a bead or conjugated to a secondary biomolecule, etc.). In some embodiments the isotype-chelated polymers comprise139La,140Ce,141Pr,142Nd,143Nd,144Nd,145Nd,146Nd,147Cm,148Nd,149Sm,150Nd,151EU,152Sm,153EU,154Sm,155Gd,156Gd,157Gd,158Gd,159Tb,160Gd,161Dy,162Dy,163Dy,164Dy,165Ho,166Er,167Er,168Er.169Tm,170Er,171Yb.172Yb,173Yb,174Yb,175Lu,176Yb or any combination thereof.MGB Ref: 33929 / 70816

[0097] Tn some embodiments, a set of 18 distinct isotopes are individually conjugated to biotinylated BSA using two types of barcoding: a) target ID barcoding, which contains isotopes from159Tb to164Dy to generate 20 unique combinations; and b) sample ID barcoding, which uses isotopes from165Ho to176Yb to generate 924 unique combinations.

[0098] In some embodiments, the barcoded beads are analyzed on a mass cytometer. In some embodiments, data acquired from the mass cytometer are max-min normalized and a threshold value is identified for each isotope. Based on the threshold, the beads are assigned a Target and a Sample ID. In some embodiments, table of the levels of IgG (isotope197Au) and IgM (isotope115In) per Target and Sample ID is generated and then analyzed.

[0099] In various embodiments, the oligonucleotide barcode moiety is from about 1 to about 100 or more nucleotides in length. In various embodiments, the oligonucleotide barcode moiety is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22. 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33. 34. 35. 36. 37. 38. 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50. 55. 60. 65, 70, 75, 80, 85, 90, 95, 100, or more nucleotides in length. In various embodiments, the oligonucleotide barcode moiety is 6-10 nucleotides in length. In various embodiments, the oligonucleotide barcode is detected by sequencing (See for example, Macosko et al. 2015, Cell 161 (5): 1202-14; Rodriques et al. 2019, Science 363 (6434): 1463-67; Liu et al. 2020, Cell 183 (6): 1665-1681. incorporated by reference in their entirety).

[0100] In various embodiments, the oligonucleotide barcode moiety is conjugated to a fluorophore (fluorophore barcode). Various fluorophore which may be used in the fluorophore barcode are known in the art. Fluorophores include, but are not limited to, rhodamines, fluorescein derivatives, Green Fluorescent Protein (GFP), BODIPY® dye, fluorescein, RHODAMINE GREEN™ dye, and OREGON GREEN® dye or derivatives thereof. In various exemplary embodiments, beads containing an oligonucleotide barcode sequences are conjugated by one or more rounds of hybridization-imaging-dehybridization which provide an image with the information of the barcoded beads which can be counted (See for example, co-detection by indexing (CODEX) described in Goltsev et al. 2018, Cell 174 (4): 968-981.el5, which is incorporated by reference in its entirety).

[0101] In various embodiments, one or more barcode moieties is associated with a bead. In various embodiments, a signal from one or more barcode moieties is used to determine theMGB Ref: 33929 / 70816 presence of one or more biomolecule(s). Examples of barcoded beads and barcoded moieties that may be used in the disclosed methods are known in the art and described in, for example, Dagher et al. 2018, Nature Nanotechnology 13 (10): 925-32; Juncker et al. 2014, Current Opinion in Chemical Biology 18 (February): 29-37; Gold et al. 2010, PloS One 5 (12): el5004 (all of which are incorporated by reference in their entirety). Multiple barcodes can be used on one or more beads as described herein. In some embodiments, one barcode is incorporated or conjugated to a bead in order to allow the identification of the subject (e.g., the subject from where the sample is obtained) where, for example, multiple subjects / samples are processed according to the methods described herein. Moreover, a second barcode moiety is likewise incorporated or conjugated to the bead which serves to identify the ligand that is associated with the bead. Thus, as used herein the phrase “wherein a barcode moiety identifies the subject” refers to a barcode as described herein that is unique to a specific sample or subject, such that following detection of a biomolecule can be using the beads described herein, the presence of the biomolecule can be attributed to the specific sample or subject. Likewise, the phrase “wherein a second barcode moiety identifies the ligand” refers to a barcode that uniquely identifies the ligand that is associated with the same band.

[0102] As used herein, the term “ligand” refers to an antigen or biomolecule-binding fragment of an antigen, biotinylated protein, polymer or a nucleic acid. In various embodiments, the ligand comprises a coronavirus spike or nucleocapsid protein or fragment thereof capable of binding to a coronavirus antibody. In various embodiments, the ligand is associated with a first bead. In various embodiments, the ligand is associated with one or more beads. In various embodiments, the ligand or bead is bioconjugated (for example, biotinylated or Maleimide-thiol reactions) to allow the conjugation of ligands and / or one or more barcode moieties as described herein.

[0103] As used herein, the term “agent” or “agents” refers to an antibody, protein or nucleic acid capable of binding to the biomolecule. In various embodiments, the agent is associated with one or more beads. In various embodiments, the agents capable of binding to the biomolecule of the first construct are antibodies. In related embodiments, the antibodies comprise the same immunoglobulins capable of binding to one or more biomolecules. In certain embodiments, the antibodies comprise different immunoglobulins capable of binding to one or more biomolecules.MGB Ref: 33929 / 70816

[0104] Tn various embodiments, the agents capable of binding to the biomolecule of the first construct are nucleic acids. In related embodiments, the nucleic acids are capable of binding to one or more nucleic acid sequences of the same biomolecule. In certain embodiments, the nucleic acids are capable of binding to one or more nucleic acid sequences of different biomolecules.

[0105] As used herein, the term “construct” refers to a mixture of two or more the following one or more bead(s), the ligand, the agent and the biomolecule. In various embodiments, a “first construct” refers to a mixture comprising the first bead, the ligand and the biomolecule. In various embodiments, a “second construct” refers to a mixture of comprising the first bead, the ligand, the biomolecule, the agent, and the second bead.

[0106] As used herein, the term “blocking agents” refers to agents used to bind any unbound antibody or protein or nucleic acid on the first bead or second bead or subsequent bead(s). In various embodiments, blocking agents are soluble synthetic proteins or subunits or antigens which block free sites on unbound antibody or protein or nucleic acid.

[0107] Generally, other nomenclature used herein and many of the laboratory procedures in cell culture, molecular genetics and nucleic acid chemistry and hybridization, which are described below, are those well-known and commonly employed in the art. (See generally Ausubel et al. (1996) supra; Sambrook et al, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, New York (1989), which are incorporated by reference herein). Standard techniques are used for recombinant nucleic acid methods, polynucleotide synthesis, preparation of biological samples, preparation of cDNA fragments, isolation of mRNA and the like. Generally enzymatic reactions and purification steps are performed according to the manufacturers' specifications.Beads

[0108] As described herein, the present disclosure provides methods that use beads. In some embodiments, a bead comprises polystyrene nanoparticles, also referred to as polystyrene latex nanoparticles. In some embodiments, these beads are nanometer-sized polystyrene beads made of polymer chains that form a spherical hydrophobic exterior. In some embodiments, the synthesis of polystyrene nanoparticles is via heterophase polymerization, such as emulsion, miniemulsion, suspension, and dispersion polymerization. In some embodiments, these processesMGB Ref: 33929 / 70816 are environmentally friendly, inexpensive, and easy to implement due to the low viscosity of the components.

[0109] Polystyrene particles present a flexible platform for applications in diagnostics and bio separations. In some embodiments, a biological ligands may be attached to a polystyrene particle via adsorption to plain polystyrene particles, covalent attachment to surface functionalized particles, and attachment of the ligand of interest to particles that are pre-coated with a binding protein. In some embodiments, a binding protein comprises Streptavidin, Protein A, Protein G, or any combination thereof.

[0110] In some embodiments, a bead is pre-coated with a binding protein, wherein the binding protein is streptavidin. In some embodiments, the bead is conjugated with biotinylated BSA. In some embodiments, the BSA is used as carrier protein and it was conjugated to isotope-chelated polymers. In some embodiments the isotype-chelated polymers comprise139La,140Ce,141Pr,142Nd,143Nd.144Nd,145Nd,146Nd.147Cm.148Nd,149Sm,150Nd,151Eu.152Sm.153Eu,154Sm,155Gd.156Gd,157Gd,158Gd,159Tb,160Gd,161Dy,162Dy,163Dy,164Dy,165Ho,166Er,167Er,168Er,169Tm,170Er,171Yb,172Yb,173Yb,174Yb,I 7LLI.176Yb or any combination thereof.

[0111] In some embodiments, a bead comprises a diameter of about 1 nm to about 1250 pm.In some embodiments, a bead comprises a diameter of about 3 pm. In some embodiments, a bead comprises available cross-linked polystyrene particles made of styrene / divinylbenzene, are stable in the presence of organic solvents. In some embodiments, the beads are impregnated visible or fluorescent dyes. In some embodiments, a bead comprises a plain polystyrene particle for protein adsorption. In some embodiments, a bead comprises surface modifications (COOH, NH2, Epoxy, Aldehyde-sulfate, NHS) for covalent ligand attachment. In some embodiments, a bead comprises NT A / Ni-NTA for the binding of histidine labeled proteins. In some embodiments, a bead comprises chitosan, collagen, UFH (Heparin), or any combination thereof showing special affinities to target surfaces. In some embodiments, a bead comprises Trialkylammonium groups (NR3+) exhibiting a positive zeta potential and sulfate (SO3H) exhibiting negative zeta potentials. In some embodiments, a bead comprises a hydrophobic Octadecyl (Cl 8) surface and Octyl (C8) surface having high selectivity for polar, neutral and moderately nonpolar pharmaceuticals, natural products, food additives, organic chemicals and biologicals.MGB Ref: 33929 / 70816

[0112] Tn some embodiments, a bead is used in scientific research, diagnostics, electronics, pharmaceuticals, and nanotechnology. In some embodiments, a bead is used as size standards, latex agglutination assays, lateral flow detection, solid phase immunoassays, markers, purification and separation, ion-exchange, or any combination thereof.

[0113] In some embodiments, a bead is used in lateral flow detection and latex agglutination tests. In some embodiments, a bead is used as markers to label biomolecules. In some embodiments, a bead comprises a carboxylated color-dyed polystyrene particles, wherein the carboxylated color-dyed particle allows for the covalent coupling of antibodies or antigens. In some embodiments, an anti-Escherichia coli antibody conjugated polystyrene particle (latex beads). In some embodiments, a bead can be utilized in agglutination assays often used in tandem with other bacteria identification assays to confirm the presence of specific bacteria in a sample.

[0114] In some embodiments, a bead comprises a fluorescent polystyrene particle, wherein the fluorescent polystyrene particle is used in imaging and diagnostic applications for the detection of binding events or signal enhancement. In some embodiments the bead can also be used for fluid tracing, fluid mechanics studies, cell tracking, phagocytosis studies, latex agglutination tests, fluorescence microscopy, and drug discovery research.Methods

[0115] The present disclosure provides methods and materials for determining the presence of biomolecules. As described herein, the sample obtained from the subject is incubated, conjugated with or separated from bead compositions. The methods also provide various means conjugating and separating the bead compositions described herein. In various embodiments, bead compositions can be separated using specific proteolytic cleavages, chemical-based disruption, mechanical disruption (e.g. mixing or vortexing), or heat-based dehybridization. Other means of incubating, conjugating and separating beads are known to those of ordinary skill in the art.

[0116] In various embodiments, the one or more steps of the disclosed methods may be optionally performed robotically / autonomously (e.g. automated and / or use of robot and / or machine). In exemplary embodiments, the following steps (a) through (g) may be performed robotically / autonomously: (a) obtaining a sample from a subject; (b) incubating the sample with a composition comprising a plurality of beads, wherein each bead is conjugated with (i) at leastMGB Ref: 33929 / 70816 one barcode moiety, and (ii) at least one ligand capable of binding to a biomolecule, thereby producing a composition comprising a plurality of constructs, wherein each construct comprises a bead, a ligand and a bound biomolecule: (c) washing the composition of (b) under conditions that allow removal of unbound biomolecules; (d) optionally pooling the composition of (c) with one or more additional compositions comprising separate samples that have been incubated with separate compositions according to (b), thereby preparing a pooled composition; (e) incubating the composition or the pooled composition comprising bound biomolecules with a composition comprising a plurality of secondary constructs, wherein each secondary construct comprises a barcode moiety conjugated to a biomolecule capable of binding to bound biomolecules; (f) washing the composition or pooled composition of (e) under conditions that allow removal of unbound secondary constructs; and (g) determining the presence of the barcode moieties of the first and second constructs, thereby determining the presence of at least one biomolecule in a sample.

[0117] In exemplary embodiments, the following steps (a) through (i) may be performed robotically / autonomously: (a) obtaining a sample from a subject; (b) incubating the sample with a composition comprising a plurality of beads, wherein each bead is conjugated with (i) at least one barcode moiety, and (ii) at least one ligand capable of binding to a biomolecule, thereby producing a composition comprising a plurality of constructs, wherein each construct comprises a bead, a ligand and a bound biomolecule; (c) incubating the composition comprising the constructs of (b) with a fixing reagent, thereby fixing the bound biomolecules to their respective beads; (d) incubating the composition of (c) with a denaturing reagent, thereby denaturing the bound and unbound biomolecules; (e) optionally pooling the composition of (d) with one or more additional compositions comprising separate samples that have been incubated with separate compositions according to (b), thereby preparing a pooled composition; (f) removing unbound biomolecules; (g) incubating the composition or the pooled composition comprising bound, fixed and denatured biomolecules with a plurality of secondary constructs, wherein each secondary construct comprises a barcode moiety conjugated to a biomolecule capable of binding to a bound, fixed and denatured biomolecules; (h) removing unbound secondary constructs; and (i) determining the presence of the barcode moieties of the first and second constructs, thereby determining the presence of at least one biomolecule in a sample.MGB Ref: 33929 / 70816

[0118] In exemplary embodiments, the following steps (a) through (g) may be performed robotically / autonomously: (a) obtaining a collection of samples from a single subject or a plurality of subjects; (b) separately incubating the samples with compositions comprising a plurality of beads, wherein each bead is conjugated with (i) a plurality of barcode moieties, and (ii) at least one ligand capable of binding to a biomolecule, thereby producing a composition comprising a plurality of constructs, wherein each construct comprises a bead, at least one ligand and at least one bound biomolecule; (c) washing the compositions of (b) under conditions that allow removal of unbound biomolecules; (d) pooling the composition of (c) with one or more additional compositions comprising separate samples that have been separately incubated with separate compositions according to (b), thereby preparing a pooled composition; (e) incubating the pooled composition comprising bound biomolecules with a composition comprising a plurality of secondary constructs, wherein each secondary construct comprises a barcode moiety conjugated to a biomolecule capable of binding to bound biomolecules; (f) washing the pooled composition of (e) under conditions that allow removal of unbound secondary constructs; and (g) determining the presence of the barcode moieties of the first and second constructs, thereby determining the presence of at least one biomolecule in a sample.

[0119] In exemplary embodiments, the following steps (a) through (i) may be performed robotically / autonomously: (a) obtaining a collection of samples from a single subject or a plurality of subjects; (b) separately incubating the samples with compositions comprising a plurality of beads, wherein each bead is conjugated with (i) a plurality of barcode moieties, and (ii) at least one ligand capable of binding to a biomolecule, thereby producing a composition comprising a plurality of constructs, wherein each construct comprises a bead, at least one ligand and at least one bound biomolecule; (c) incubating the composition comprising the constructs of (b) with a fixing reagent, thereby fixing the bound biomolecules to their respective beads; (d) incubating the composition of (c) with a denaturing reagent, thereby denaturing the bound and unbound biomolecules; (e) pooling the composition of (d) with one or more additional compositions comprising separate samples that have been separately incubated with separate compositions according to (b), thereby preparing a pooled composition; (f) removing unbound biomolecules; (g) incubating the composition comprising bound, fixed and denatured biomolecules of (e) with a plurality of secondary constructs, wherein each secondary construct comprises a barcode moiety conjugated to a biomolecule capable of binding to a bound, fixedMGB Ref: 33929 / 70816 and denatured biomolecules; (h) removing unbound secondary constructs; and (i) determining the presence of the barcode moieties of the first and second constructs, thereby determining the presence of at least one biomolecule in a sample.

[0120] “Detecting” or “determining” as used herein generally means identifying the presence of a biomolecule, such as a nucleic acid or protein or antibody, by the determining the presence one or more barcode moieties. In various embodiments, detection signals are produced by the methods described herein, and such detection signals may be optical signals which may include but are not limited to, colorimetric changes, fluorescence, turbidity, mass-to-charge ratio of ions, and luminescence. Detecting or determining, in still other embodiments, also means quantifying a detection signal, and the quantifiable signal may include, but is not limited to, transcript number, amplicon number, protein number, and number of metabolic molecules. In this way, sequencing or bioanalyzers are employed in certain embodiments. In various embodiments, the presence one or more barcode moieties is determined using flow cytometry, mass spectrometry, fluorescence microscopy, electron microscopy, isotope-based imaging, Nuclear Magnetic Resonance (NMR), fluorescence resonance energy transfer (FRET), X-ray fluorescence (XRF), or nucleic acid sequencing.

[0121] In various embodiments of the methods disclosed herein, the presence of 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10, 50. 100, 1000, 2000, 5000. 10000, 20000 or more biomolecules are determined.Pathogens

[0122] In various embodiments, the biomolecule is from a pathogen. In related embodiments the pathogen is a virus, bacterium, fungi, protozoa, or worm.

[0123] The present disclosure provides, in various embodiments, methods for detecting the presence of one or more of the following exemplary pathogens:

[0124] Exemplary pathogenic viruses or virus particles include, but is not limited to, for example, adenovirus , alphavirus , calicivirus (e.g., a calicivirus capsid antigen), coronavirus polypeptides, distemper virus , Ebola virus polypeptides, enterovirus , flavivirus , hepatitis virus (AE), herpesvirus, infectious peritonitis virus, leukemia virus, marburg virus, orthomyxovirus, papilloma virus, parainfluenza virus, paramyxovirus, parvovirus, pestivirus, picorna virus (e.g., a poliovirus), pox virus (e.g., a vaccinia virus), rabies virus, reovirus, retrovirus, rhinoviruses andMGB Ref: 33929 / 70816 rotavirus. Tn certain embodiments, the virus is SARS-CoV-1 , SARS-CoV-2, respiratory syncytial virus (RSV) human immunodeficiency virus (HIV), herpes simplex virus (HSV), human papillomavirus (HPV), influenza or parainfluenza virus.

[0125] Exemplary pathogenic bacterium include, but are not limited to, for example, members of the genus Actinomyces, Bacillus, Bacteroides, Bordetella, Bartonella, Borrelia (e.g., B. burgdorferi OspA), Brucella, Campylobacter, Capnocytophaga, Chlamydia, Corynebacterium, Coxiella, Dermatophilus, Enterococcus, Ehrlichia, Escherichia, Francisella, Fusobacterium, Haemobartonella, Haemophilus polypeptides, Helicobacter, Klebsiella, L-form bacteria, Leptospira, Listeria, Mycobacteria. Mycoplasma, Neisseria, Neorickettsi , Nocardia. Pasteurella, Peptococcus, Peptostreptococcus, Pneumococcus polypeptides (e.g., S. pneumoniae polypeptides), Proteus, Pseudomonas, Rickettsia, Rochalimaea, Salmonella, Shigella, Staphylococcus, group A streptococcus (e.g., S. pyogenes), group B streptococcus (S. agalactiae), Treponema, and Yersinia. In certain embodiments, the pathogenic bacterium is Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, Yersinia pestis (bubonic plaque), or Mycobacterium tuberculosis.

[0126] Exemplary pathogenic fungus includes, but are not limited to, for example, Aspergillus, Blastomyces, Coccidioides and Pneumocystis. In other related embodiments the fungus is a yeast, which in further embodiments is a Candida, wherein in still further embodiments the Candida is selected from C. albicans, C. glabrata, C. krusei, C. lusitaniae, C. tropicalis and C. parapsilosis. In certain embodiments, the pathogenic fungus / yeast is Candida, Tinea corporis (ringworm) Trichophyton, Microsporum, or Epidermophyton.

[0127] Exemplary protozoan pathogens include, but are not limited to, for example, Babesia, Balantidium, Besnoitia, Cryptosporidium, Eimeria, Encephalitozoon, Entamoeba, Giardia, Hammondia, Hepatozoon, Isospora, Leishmania, Microsporidia, Neospora, Nosema, Pentatrichomonas, Plasmodium. Examples of helminth parasites include, but are not limited to, Acanthocheilonema, Aelurostrongylus, Ancylostoma, Angiostrongylus, Ascaris, Brugia, Bunostomum, Capillaria, Chabertia, Cooperia, Crenosoma, Dictyocaulus, Dioctophyme, Dipetalonema, Diphyllobothrium, Diplydium, Dirofilaria, Dracunculus, Enterobius, Filaroides, Haemonchus, Lagochilascaris, Loa, Mansonella, Muellerius, Nanophyetus, Necator, Nematodirus, Oesophagostomum, Onchocerca, Opisthorchis, Ostertagia, Parafilaria,MGB Ref: 33929 / 70816Paragonimus, Parascaris, Physaloptera, Protostrongylus, Setaria, Spirocerca Spirometra, Stephanofilaria, Strongyloides, Strongylus, Thelazia, Toxascaris, Toxocara, Trichinella, Trichostrongylus, Trichuris, Uncinaria, and Wuchereria, Pneumocystis. Sarcocystis. Schistosoma, Theileria, Toxoplasma, and Trypanosoma. In certain embodiments, the protozoan pathogen is Entamoeba histolytica (Amoebiasis) or Plasmodium falciparum (malaria).

[0128] Exemplary worm pathogens include, but are not limited to, for example, platyhelminthes or flatworms (flukes and tapeworms) and nematoda or roundworms.Coronaviruses

[0129] Coronaviruses (CoV) have repeatedly emerged from wildlife hosts into humans and livestock animals to cause epidemics with significant morbidity and mortality. The emergence of SARS-CoV-2, the virus that causes COVID-19 in 2019 and the rapid, global spread of infection in humans highlights the need for developing therapeutics and vaccines to limit coronavirus epidemics (Wu F, et al., 2020, Nature 1-8; Zhou P, et al., 2020, Nature 1-4; and Zhu N, et al., 2020. N Engl J Med NEJMoa2001017).

[0130] Coronaviruses (Co Vs) are the largest group of viruses belonging to the Nidovirales order, which includes Coronaviridae, Arteriviridae, and Roniviridae families. The Coronavirinae comprise one of two subfamilies in the Coronaviridae family, with the other being the Torovirinae. The Coronavirinae are further subdivided into four groups, the alpha, beta, gamma and delta coronaviruses. The viruses were initially sorted into these groups based on serology but are now divided by phylogenetic clustering.

[0131] All viruses in the Nidovirales order are enveloped, non-segmented positive-sense RNA viruses. They all contain very large genomes for RNA viruses, with Coronavirinae having the largest identified RNA genomes, containing approximately 30 kilobase (kb) genomes. Other common features within the Nidovirales order include: i) a highly conserved genomic organization, with a large replicase gene preceding structural and accessory genes; ii) expression of many nonstructural genes by ribosomal frameshifting; iii) several unique or unusual enzymatic activities encoded within the large replicase-transcriptase polyprotein; and iv) expression of downstream genes by synthesis of 3' nested sub-genomic mRNAs. In fact, the Nidovirales order name is derived from these nested 3' mRNAs as nido is Latin for “nest”. The major differences within the Nidovirus families are in the number, type, and sizes of theMGB Ref: 33929 / 70816 structural proteins. These differences cause significant alterations in the structure and morphology of the nucleocapsids and virions.

[0132] Coronaviruses contain a non- segmented, positive-sense RNA genome of ~30 kb. The genome contains a 5' cap structure along with a 3' poly (A) tail, allowing it to act as a mRNA for translation of the replicase polyproteins. The replicase gene encoding the nonstructural proteins (Nsps) occupies two-thirds of the genome, about 20 kb, as opposed to the structural and accessory proteins, which make up only about 10 kb of the viral genome. The 5' end of the genome contains a leader sequence and untranslated region (UTR) that contains multiple stem loop structures required for RNA replication and transcription. Additionally, at the beginning of each structural or accessory gene are transcriptional regulatory sequences (TRSs) that are required for expression of each of these genes (see section on RNA replication). The 3 'UTR also contains RNA structures required for replication and synthesis of viral RNA. The organization of the coronavirus genome is 5 '-leader- UTR-replicase-S (Spike)-E (Envelope)-M (Membrane)-N (Nucleocapsid)-3'UTR-poly (A) tail with accessory genes interspersed within the structural genes at the 3' end of the genome. The accessory proteins are almost exclusively non-essential for replication in tissue culture; however some have been shown to have important roles in viral pathogenesis.

[0133] The present disclosure provides, in various embodiments, methods for determining the presence of one or more coronaviruses. Non-limiting examples of coronaviruses include SARS- Related coronaviruses, severe acute respiratory syndrome coronavirus-2, (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus 229E (HCoV-229E), human coronavirus 0C43 (HCoV-0C43), human coronavirus HKU1 (HCoV-HKUl), and human coronavirus NL63 (HCoV-NL63).Detection methods

[0134] In various embodiments, the determining steps (g) and / or (i) comprises using flow cytometry, mass spectrometry, fluorescence microscopy, electron microscopy, isotope-based imaging, Nuclear Magnetic Resonance (NMR), fluorescence resonance energy transfer (FRET), X-ray fluorescence (XRF), or nucleic acid sequencing.MGB Ref: 33929 / 70816

[0135] Before the present disclosure is further described, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0136] Unless defined otherwise, 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 disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0137] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0138] It must be noted that as used herein and in the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a conformation switching probe" includes a plurality of such conformation switching probes and reference to "the microfluidic device" includes reference to one or more microfluidic devices and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any element, e.g., any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.MGB Ref: 33929 / 70816

[0139] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. This is intended to provide support for all such combinations.EXAMPLES

[0140] Numerous embodiments of the present disclosure is demonstrated by the following non-limiting Example.Example 1: Highly scalable multiplex serology by mass spectrometry analysis of beads

[0141] As described herein, the present disclosure is an enhanced bead-based assay platform that enables multiplexed detection of multiple samples, multiple targets, and multiple readout biomolecules in a single run on an ICP-MS-TOF instrument. This system offers flexibility depending on the specific assay configuration: it can be set up for antibody detection (against a wide range of antigens), protein detection (for protein quantification), oligonucleotide detection (for oligonucleotide quantification), and functional readout. Each setup can simultaneously assay multiple targets per bead in multiple samples simultaneously, providing a comprehensive and scalable solution for high-throughput testing.

[0142] The methods provided herein use single-bead technology, where each bead is, in one embodiment, isotope-barcoded and configured to bind specific antigens, proteins, antibodies, and oligonucleotides depending on the intended assay. This allows the detection of multiple targets and specific biomolecules per bead, making it a powerful tool for broad- spectrum analysis. The assay is compatible with an optional wash-free process, for example, employing a fixation step with paraformaldehyde (PFA) and denaturation with sodium dodecyl sulfate (SDS) to stabilize bound molecules and remove unbound material, eliminating the need for traditional wash steps.

[0143] After the fixation and denaturation process, pooled samples are incubated with isotopically labeled secondary antibodies (for immunoglobulin and antigen detection), proteins (for functional readout), or complementary oligonucleotides (for oligonucleotide detection). TheMGB Ref: 33929 / 70816 system allows for quantitative analysis via mass cytometry, providing detailed data on multiple biomolecules depending on the assay configuration. This versatility allows for the simultaneous detection of multiple immunoglobulin isotypes, multiple proteins, or different oligonucleotides on the same bead in a single run.

[0144] The assay offers massive scalability, for instance, performing up to 36,960 tests in a single tube with a 30 pL reaction volume, using as little as 400 nL of sample per test. This capability enables large-scale studies, diagnostics, and high-throughput screening with exceptional precision, sensitivity, and specificity.

[0145] By providing the ability to detect multiple samples, multiple targets, and multiple specific targets per bead in a single assay on a single instrument, this disclosure represents a major advancement over traditional single-analyte assays. It offers unmatched flexibility, scalability, and efficiency for biomolecule detection across a variety of applications, including immunology, proteomics, and genomics.

[0146] The following Example describes materials and methods for determining the presence of multiple biomolecules using mass cytometry as a readout.Methods

[0147] Clinical samples. De-identified samples from the study (Wirz et al. Use of outpatient- derived COVID-19 convalescent plasma in COVID- 19 patients before seroconversion. Front. Immunol. 12, 739037 (2021)) were used for Fig. 3c-d, comprising individuals with confirmed SARS-CoV-2 infection through RT-PCR testing. The samples used here were collected from COVID- 19 convalescent plasma donors who donated plasma at the Stanford Blood Center between April 2020 and May 2020 (Wirz et al. Use of outpatient-derived COVID-19 convalescent plasma in COVID- 19 patients before seroconversion. Front. Immunol. 12, 739037 (2021)). This study was approved by the Stanford University Institutional Review Board (Protocols IRB-13952, IRB-48973, and IRB-55689).

[0148] De-identified COVID- 19-positive samples used for Fig. 3e-f were purchased fromDiscovery Life Sciences, comprising individuals with confirmed SARS-CoV-2 infection through RT-PCR testing.MGB Ref: 33929 / 70816

[0149] De-identified samples from FIND COVID were used for Fig. 4. FIND COVID is a CDC-funded longitudinal cohort of individuals recently diagnosed with SARS-CoV-2 infections in the San Francisco Bay Area, initiated in August 2020. Index cases were identified from individuals with a positive health provider- ordered SARS-CoV-2 nucleic acid amplification test at a UCSF-affiliated health facility. Index cases were enrolled with uninfected household contacts and followed at 5 field visits over the first 4 weeks of illness. Study visits occurred on the day of enrollment and days 9, 14, 21, and 28 post-index symptom onsets. Blood samples were collected form index cases and household contacts at each study visit. Additionally, participants self-collected anterior nasal swabs daily for the first 2 weeks following enrollment, then on days 17, 19, 21, and 28. All nasal specimens were used to quantify SARS CoV-2 RNA through RT-PCR targeting nucleocapsid (N) and envelope (E) genes to determine SARS-CoV-2 infection status of household participants. The study was approved by the UCSF Institutional Review Board (Protocol IRB# 20-30388) and given a designation of public health surveillance according to federal regulations as summarized in 45 CFR 46.102(d)(l)(2).

[0150] De-identified pre-pandemic samples used for Figs. 3 and 4 were purchased from Solomon Park, comprising samples collected before November 2019 (the official start of the COVID- 19 pandemic).

[0151] Conjugation of Bovine Serum Albumin (BSA) and antibodies to isotopes. Biotinylated BSA (Sigma, #A8549) was used as carrier protein and it was conjugated to isotope chelated Maxpar X8 polymers (Fluidigm, #201300). Briefly, 200 pg of biotinylated carrier proteins were reduced by TCEP treatment (Thermo Fisher Scientific, #77720) for 30 minutes at 37 °C. The reduced carrier proteins were reacted with isotope-chelated maleimide-containing polymers for 1.5 h at 37 °C and washed five times with IX PBS. Protein concentration was quantified using a Nanodrop 2000 (Thermo Fisher Scientific, #ND-2000) and all conjugates were diluted to 0.5 g / 1 in IX PBS. Isotope conjugation was confirmed after every conjugation experiment by loading isotope conjugated BSA into beads and analyzing them by mass cytometry. Anti-human IgM was conjugated to115In chelated Maxpar X8 polymers following the same procedure.

[0152] Bead loading with isotope-conjugated carrier proteins. Streptavidin-coated polystyrene beads with a diameter of 3 to 3.4 pm (Spherotech, #SVP-30-5) were washed thrice in IX PBS with 0.5% BSA and 0.001% Tween-20 (CSM-T). The beads where then incubated withMGB Ref: 33929 / 708162.5 pg / mL isotope-conjugated biotinylated carrier proteins for 30 minutes at room temperature, washed thrice in CSM-T, and stored at 4 °C.

[0153] Bead loading with isotopes by passive absorption. Streptavidin-coated polystyrene beads with a diameter of 3 to 3.4 pm (Spherotech, #SVP-30-5) were washed thrice in CSM-T. The beads where then incubated with 50 pM lanthanide chloride in IX PBS for 1 hour at room temperature, washed thrice in CSM-T, and stored at 4 °C until analysis.

[0154] Load Proteins on beads for 924 assays. For 1 mL of Streptavidin-coated polystyrene beads with a diameter of 3 to 3.4 pm (#SVP-30-5, Spherotech), 10 pL CSM with 0.1% Tween- 20 (Sigma, #P1379) were added. The beads were washed thrice with 500 pL CSM-T by centrifuging at 2800 relative centrifugal force (RCF) at room temperature. The supernatant was discarded. For each avi-tagged protein a 1:10 dilution was prepared (stock at 0.25 g / L) by adding water. The stock was quickly stored at -20°C after usage. The beads were resuspended in 165 pL CSM-T and then split in twenty tubes (7.5 pL each) labeled from 1 to 20. To each tube, 7.5 pL of an avi-tagged protein dilution were added to the beads. The beads with the avi-tagged protein were incubated for 30 minutes at room temperature. The beads were washed thrice with 100 pL CSM-T by centrifuging at 2800 RCF at room temperature. To each of the tubes 165 pL of CSM- T was added. Beads loaded with avi-tagged proteins were store at 4 °C until use.

[0155] Generation of 20 barcodes for target ID. To create 20 barcodes, 3 isotope-conjugated biotinylated earner proteins from a collection of 6 were distributed in wells of 10 V-bottom 96- well plates (Costar, #3363) containing 94 pL of CSM-T. The concentration of each individual 3 isotope-conjugated biotinylated carrier proteins was 1 pg / mL. After dispensing, 50 pL of the beads loaded with avi-tagged proteins were placed on each well. The plates were incubated for 30 minutes at room temperature. Beads were then washed thrice in CSM-T by centrifugation at 2,000 RCF at room temperature and stored at 4 °C until use.

[0156] Generation of 924 barcodes for sample ID. High-throughput generation of barcodes was performed on a Tempest (Formulatrix). To create 924 barcodes, 6 isotope-conjugated biotinylated carrier proteins from a collection of 12 were distributed in wells of 10 V-bottom 96- well plates (Costar, #3363) containing 44 pL of CSM-T. After dispensing, the concentration of each individual 6 isotope-conjugated biotinylated carrier proteins was 1 pg / mL. Plates were manually transferred to a Bravo Automated liquid Handler Platform (Agilent) and 50 pL of theMGB Ref: 33929 / 70816 beads loaded with avi-tagged proteins were placed on each well. The plates were incubated for 30 minutes at room temperature. Beads were then washed thrice in CSM-T by centrifugation at 2,000 RCF at room temperature and stored at 4 °C until use.

[0157] Two-bead assay by flow cytometry. For each sample. 5 pL of fluorescently-labeled streptavidin polystyrene beads (Spherotech, #SVFP-0556-5 and #SVFP- 1068-5), were washed twice with CSM-T at a 1:1 ratio and pelleted by centrifuging at 21,000 RCF for 2 minutes after each wash and discarding the supernatant. Next, the beads were resuspended in 5 pl CSM-T per sample. Then, per sample, 1 pL of biotinylated Spike SI at 0.25 pg / pL (Sino biological, #40591- V27H-B) was added to 5 pL of beads and were incubated for 30 minutes at room temperature. The beads were washed three times by adding 100 pL of CSM-T, centrifuging at 21 ,000 RCF for 3 minutes and discarding the supernatant. Then the pellet was resuspended in 30 pL of CSM-T and transferred to a 96 well v-bottom plate (Costar, #3363). Next, 10 pL of 1:10 diluted plasma in CSM-T (or plasma titrated from 1, to 1:1000 pL) or an antibody titration was added to each condition. Beads were incubated for 30 minutes at room temperature and washed twice by adding 50 pL of CSM-T, centrifuging at 2800 RCF for 3 minutes and discarding the supernatant. The beads were then resuspended in 100 pL of CSM-T. In each well of the plate, 2 pL of Magnetic-IgG beads (RayBiotech, #801-101-1) were added. The beads were incubated for 30 minutes at room temperature with mixing by pipetting every 10 minutes. Subsequently, another 100 pL of CSM-T was added to each well and the plate was placed on a 96-well magnetic separator plate for 2 minutes to attach the magnetic beads. The unbound beads in solution were collected and added to another well. The collected beads were washed three times by adding 100 pL of CSM-T, centrifuging at 2800 RCF for 3 minutes and discarding the supernatant. Finally, the beads were resuspended in 100 pL of CSM-T and analyzed using a CytoFlex flow cytometer (Beckman Coulter). For each sample, acquisition was based on a specific volume, either 20 pL or 50 pL. Fluorescent Blue and Nile Red beads were initially identified and gated using forward and side scatter channels and subsequently gated based on their fluorescence signal. This volume-based acquisition approach enabled the counting of the total number of beads in each sample volume. The counts obtained were then analyzed and plotted using R.

[0158] Two-bead assay by mass cytometry. The beads from the barcoded 96 well v-bottom plates (Costar, #3363) loaded with biotinylated Spike SI (Sino biological, #40591-V27H-B) as described above were pelleted by centrifuging at 2800 RCF, the supernatant discarded, and theMGB Ref: 33929 / 70816 then resuspended in 30 pL of CSM-T. Patient plasma was diluted in CSM-T 1 : 10 and 10 pL of each sample was mixed with the barcoded beads. The plate was incubated for 30 minutes at room temperature. The beads were washed twice by adding 100 pL of CSM-T, centrifuging at 2800 RCF for 3 minutes and discarding the supernatant. Then, 100 pL of diluted Spike SI protein (2 ng / pL) in CSM-T (Sino biological, #40591-V02H) was added to each well and incubated for 10 minutes at room temperature. The plate was centrifuged at 2800 RCF, the supernatant discarded and another 50 pL of Spike protein (0.2 ng / pL) in CSM-T was added. All samples were combined in a reservoir and added to a 50 mL tube and spun down at 2800 RCF. Then 10% of the beads were collected and labeled as “baseline”. Samples were then diluted in 2.5x the number of samples in CSM-T (100 pL of CSM-T was added for 40 samples). MACS anti-IgG microbeads (Miltenyi Biotec, #130-047-501) were added to a 1:2 ratio and incubated at room temperature for 30 minutes. MACS LS columns (Miltenyi Biotec, #130-042-401) were prewashed with 1 mL CSM-T. Samples were diluted 10 times and added to a LS column - 1 mL of diluted sample was added to one LS column - and flow-through was collected in a 5 mL protein-low bind tube (Fisher Scientific, #0030122356). Another 4 mL were added and the flow- through was collected. The beads from the flow-through were pelleted at 2800 RCF and the supernatant discarded. The beads were resuspended in 1ml CSM-T and then then moved to a 1.5 ml protein-low bind tube (Fisher Scientific, #022431081) and pelleted at 21,000 RCF. The beads were washed by adding 1 mL of CSM-T, centrifuging at 21,000 RCF for 3 minutes and discarding the supernatant. The beads were then washed three times with 0.5 mL H2O before loading into CyTOF. The beads were analyzed using a CyTOF 2 mass cytometer (Fluidigm), at a rate of 500 events per second. The focus of this analysis was on isotopes within the mass range of 159 to 176.

[0159] Wash free assay by flow cytometry. The beads in 96 well v-bottom plates (Costar,#3363) loaded with biotinylated Spike SI (Sino biological, #40591-V27H-B) were pelleted by centrifuging at 2800 RCF, the supernatant discarded, and the then resuspended in 30 pL of CSM- T. Spike SI was detected by a human anti-Spike SI (Invivogen, #srbd-mabl) or patient plasma diluted 1:10 and 10 pL of each sample was mixed with the loaded beads. The plate was incubated for 30 minutes at room temperature. Next, 40 pL of PFA (3.2%) were added to each well of the respective samples and incubated for 15 minutes. Then, 80 pL of 4% SDS were added and incubated for 15 minutes. The plate was centrifuged at 2800 RCF, and the supernatant fromMGB Ref: 33929 / 70816 the beads was discarded. The wells were washed three times by adding 150 pL of CSM-T, centrifuging at 2800 RCF for 3 minutes and discarding the supernatant. Next 5 pL of (1:200 dilution) of anti-IgG-647 (Thermo Fischer Scientific. #A21445) was added to each well. Beads were incubated at room temperature for 15 minutes. The wells were then washed three times by adding 150 pL of CSM-T, centrifuging at 2800 RCF for 3 minutes and discarding the supernatant. Each well was resuspended in 150 pL of CSM-T and analyzed by a CytoFlex flow cytometer (Beckman Coulter). For each sample, a volume of 50 pL was analyzed. Beads were first distinguished and gated using forward and side scatter channels, followed by analysis based on the fluorescence intensity of anti-IgG conjugated with Alexa Fluor 647. Subsequent data analysis was performed utilizing R software

[0160] Wash free assay by mass cytometry. The beads from the barcoded 96 well v-bottom plates (Costar, #3363) loaded with biotinylated Spike SI (Sino biological, #40591-V27H-B) or 19 distinct SARS-CoV-2 protein variants were pelleted by centrifuging at 2800 RCF, the supernatant discarded, and the then resuspended in 30 pL of CSM-T. Patient plasma were diluted 1:10 and 10 pL of each sample was mixed with the loaded beads. The plate was incubated for 30 minutes at room temperature. Next, 40 pL of PFA (3.2%) was added to each well of the respective samples and incubated for 15 minutes. Then, 80 pL of 4% SDS were added and incubated for 15 minutes. Then all samples were combined in a reservoir by pipetting and then added to a 50 mL tube. The beads were washed three times with by adding 25 mL of CSM-T, centrifuging at 2800 RCF for 3 minutes and discarding the supernatant. Next, the beads were stained with 25 mL CSM-T containing 256 ng / L gold anti-IgG antibody (Nanoprobes, #2053) and 256 ng / L115In-conjugated anti-IgM antibody (BioLegend, #314502) for 30 minutes. The beads were pelleted 2800 RCF and resuspended in 200 pL CSM-T and transferred to a protein low-bind Eppendorf tube (Fisher Scientific, #022431081). Samples were washed thrice with 200 pL of CSM-T and pelleted by centrifuging at 21,000 RCF after each wash and discarding the supernatant. The beads were then washed three times with 0.5 mL H2O before loading into CyTOF. The beads were analyzed using a CyTOF 2 mass cytometer (Fluidigm), at a rate of 500 events per second. The focus of this analysis was on isotopes within the mass range of 159-176, 115 and 197.

[0161] The numbers to control and optimize the stoichiometry of biotin to streptavidin at the population level. The polystyrene used in this work (Spherotech, # SVP-30-5) have a size rangeMGB Ref: 33929 / 70816 of 3 to 3.4 pm diameter and they are at 0.5% w / v. Therefore, 1 mL (5 mg) of beads have a binding capacity of 29.41 to 66.67 pg of biotinylated BSA:

[0162] *One mg of a 4 pm Spherotech streptavidin particles typically binds 5 to 10 pg of biotinylated antibody. The binding capacity for biotinylated BSA is similar to the binding capacity of biotinylated antibody given the variability of the biotinylation process.

[0163] Particle number = (6W / 3.14PD3) x 1012

[0164] Total surface area (cm2) of bead population = (6W / PD) x 104

[0165] Where, W = Polymer Weight (in grams), P = Polymer Density (polystyrene density is 1.05), D = Particle Diameter (in pm)

[0166] The description of polystyrene particles can be found on the world wide web at spherotech .com / particle. html .

[0167] Guide to bead generation, assay, and analysis. Step 1 (Fig. 5A): Generation of isotopeconjugated BSA. A set of 18 distinct isotopes are individually conjugated to biotinylated BSA using two types of barcoding:1) Target ID barcoding, which contains isotopes from159Tb to164Dy to generate 20 unique combinations;2) Sample ID barcoding, which uses isotopes from165Ho to176Yb to generate 924 unique combinations.

[0168] Step 2 (Fig. 5b): Loading of antigens onto beads. Each selected Avi-tagged protein is loaded onto streptavidin beads.

[0169] Step 3 (Fig. 5c): Generation of 20 barcodes for Target ID. By selecting 3 out of the 6 isotope-conjugated BSA proteins from Step 1. all 20 possible barcode combinations wereMGB Ref: 33929 / 70816 generated. These combinations are then added to streptavidin-coated beads, containing Avi- tagged proteins of interest. Thus, each barcode is paired with a unique protein.

[0170] Step 4 (Fig. 5d): Pooling of beads and plate distribution. The 20 barcoded beads loaded with a protein target are pooled and aliquoted to 10 96-well plates.

[0171] Step 5 (Fig. 5e): Generation of 924 barcodes for Sample ID. By selecting 6 out of the 12 isotope-conjugated BSA proteins from Step 1, all 924 possible barcode combinations were generated. BSA combinations are added to the 10 96-well plates, one combination on each well. This process is automated using liquid handlers and plates are stored at 4o C until use.

[0172] Step 6 (Fig. 5f): Incubation of plasma samples with barcoded beads. Samples are incubated with barcoded beads in 96-well plates.

[0173] Step 7 (Fig. 5g): Sample fixation and denaturation. The samples are sequentially fixed with PFA and then denatured with SDS.

[0174] Step 8 (Fig. 5h): Secondary antibody labeling and mass cytometry acquisition. Samples are combined in one tube and thoroughly washed. Secondary antibodies, e.g. anti-IgG conjugated to197Au, are added to the mixture. After incubation, the pooled sample is washed and analyzed in a mass cytometer.

[0175] Step 9 (Fig. 5i): Bead debarcoding. The data acquired from the mass cytometer are max-min normalized and a threshold value is identified for each isotope. Based on the threshold, the beads are assigned a Target and a Sample ID.

[0176] Step 10 (Fig. 5j). Downstream analysis. A table of the levels of IgG (isotope197Au) and IgM (isotope115In) per Target and Sample ID is generated and then analyzed.

[0177] ELISA to detect anti-SARS-CoV-2 antibodies in plasma samples. The ELISA protocol performed for Fig. 3d in this study was previously described in Wirz et al. Use of outpatient- derived COVID- 19 convalescent plasma in COVID- 19 patients before seroconversion. Front. Immunol. 12, 739037 (2021) and Roltgen et al. Defining the features and duration of antibody responses to SARS-CoV-2 infection associated with disease severity and outcome. Sci.Immunol. 5, eabe0240 (2020). In short, 96-well Corning Costar high binding plates were coated with SARS-CoV-2 Spike-Si, RBD or N protein in phosphate-buffered saline (PBS) at a concentration of 0.1 pg per well overnight and at 4 °C. Plates were then blocked using PBS-MGB Ref: 33929 / 70816Tween 20 containing 3% non-fat milk powder for 1 hour. Plasma samples from COVID-19 convalescent plasma donors were incubated at a dilution of 1:100 in dilution buffer (PBS-T containing 1% non-fat milk powder) for 1 hour. Anti-SARS-CoV-2 IgG antibodies were detected using horseradish peroxidase-conjugated goat anti-human IgG (Thermo Fisher, #62-8420;1:5,000 dilution). Plates were developed using 3,3',5,5'-Tetramethylbenzidine. The reaction was stopped by adding sulfuric acid after 12 minutes. Optical density was measured at 450 nm and blank control values were subtracted. All samples were tested twice in independent experiments. In some experiments the ELISA kits: RAPID MAX™ SARS-CoV-2 Spike SI Human IgG ELISA Kit (BioLegend, cat. #447809), and LEGEND MAX™ SARS-CoV-2 Nucleocapsid Human IgG ELISA Kit (BioLegend, cat. #448107) were used. The protocol was implemented in strict accordance with the manufacturer’s instructions with 500 nl of plasma sample.

[0178] Abbot AdviseDx SARS-CoV-2 IgG II assay. SARS-CoV-2-specific antibodies were determined using the immunoassay AdviseDx SARS-CoV-2 IgG II for qualitative and semi- quantitative detection of IgG antibodies to the Spike S 1 RBD protein according to the manufacturer’s specifications. Serologic analyses for this study using the Abbott AdviseDx SARS-CoV-2 IgG II (Architect) were performed under protocols approved by the UCSF Institutional Review Board (IRB# 11-05519).

[0179] Automatic bead debarcoding. Beads were debarcoded using a custom R script. Briefly, all flow cytometry standard (FCS) data files of a CyTOF run were concatenated. Events were then arcsine transformed with a cofactor of 5 and scaled using min-max normalization. Data for each isotope were plotted in a histogram to confirm a bimodal distribution. Signal intensities for each isotope were automatically thresholded using auto_thresh of the autothresholdr R package (version 1.3.2) with IJDefault method. A vector of thresholded intensities was used to transform the detected events to a binary matrix in which “0” means signal lower than the threshold and “1” means signal higher than the threshold. Events with target and sample ID were selected for downstream analysis.

[0180] Data visualization. Plots were created using the ggplot2 R package as described in Wilkinson, L. Ggplot2: Elegant graphics for data analysis by WICKHAM, H. Biometrics 67, 678-679 (2011). For the boxplots: the center is the median, the minima and maxima bound of box are the 25thand 75thpercentiles, respectively, the whiskers extend from the minima andMGB Ref: 33929 / 70816 maxima bounds of box to the largest value no further than 1.5 times the inter-quartile range, and the outliers are shown as dots. Figs, la, 3a, and 4a, were created in part using BioRender.com. All figures were prepared using Illustrator (Adobe).Results

[0181] A post-synthesis strategy for robust incorporation of stable isotopes to polystyrene beads. Two requirements are necessary to enable high-throughput bead-based assays via mass cytometry: 1) uniform incorporation of a high load of isotopes per bead, and 2) a scalable strategy generating a large number of isotope barcoded beads. Dispersion polymerization of isotope-containing polystyrene beads is a standard approach for synthesis of beads containing a high load of isotopes 33, but this procedure is not ideal for high-throughput barcode generation because each synthesis is performed individually. Passive absorption of isotopes post-synthesis of the beads is an appealing strategy for barcoding due to being highly scalable, but in practice, isotope loading is non-uniform across beads thus impairing data decoding.

[0182] In some embodiments, binding of isotope-conjugated biotinylated proteins to streptavidin-coated polystyrene beads provides a high load of isotopes per bead and a uniform labeling, the latter understood as single beads in the population having similar isotope intensities (Fig. la), while being compatible with microfluidic technologies for high-throughput combinatorial dispensing. The stable isotope Dysprosium 162 (162Dy) was conjugated to biotinylated bovine serum albumin (BSA) and loaded to streptavidin-coated beads. Mass cytometry of these beads revealed a high load of isotopes per bead and a uniform labeling (Fig. lb). Next, the approach was extended to 37 additional stable isotopes, ranging in atomic mass from Lanthanum 139 (139La) to Ytterbium 176 (176Yb) by generating beads with two types of combinations of the isotopes. “Odd” beads were loaded with the stable lanthanide isotopes having an odd mass number, and “even” beads were loaded with the stable lanthanide isotopes having an even mass number (Fig. 1c). Using mass cytometry, the expected positive signal for each isotope for both “odd” and “even” beads was observed (Fig. Id), and the intensities for each isotope were similar despite the distinct transmission factors for each isotope typically observed in ICP-MS 34 (Fig. le). Analysis of a pool consisting of beads loaded with distinct amounts of biotinylated protein carriers conjugated to172Yb and176Yb revealed the expected 9 populations of beads, highlighting the tunability, flexibility, and robustness of the system. Together, theseMGB Ref: 33929 / 70816 analyses showed a uniform, tunable, and high load incorporation of 38 stable lanthanide isotopes to polystyrene beads post-synthesis and confirmed that this strategy is compatible with the loading of up to 19 distinct isotopes per bead.

[0183] A high-throughput strategy to create thousands of isotope-barcoded polystyrene beads. There are two main components involved in high-throughput serology assay applications: 1) the ability to analyze multiple molecular targets per sample, and 2) the ability to analyze large number of samples. To address both, a double barcoding strategy was designed wherein two sets of isotopes are present in each bead: one to identify the molecular target and another for identifying the sample. Each set of isotopes provides a barcode, and thus each bead has two barcodes. Each of the two barcodes consists of k isotopes from a total of n isotopes, where k = n / 2 for efficient generation of minimally redundant combinations and robust singlet detection 35. The use of two barcodes, instead of a single barcode that encodes both sample and molecular target identification, is also convenient because it enables harnessing post-synthesis bead incorporation of isotopes in sequential loading steps.

[0184] 18,480 barcodes were created using the double barcode strategy using 18 isotopes, ranging from Terbium 159 (159Tb) to176Yb. Each bead was first loaded with three out of the six isotopes from the first set (159Tb to164Dy) to generate 20 barcodes, pooled, and then loaded with six out of the twelve isotopes from the second set (Holmium 165 (165Ho) to176Yb) to generate 924 barcodes (Fig. 2a; 20 x 924 = 18,840 barcodes). As approximately half of the beads are expected to be positive any given isotope, when analyzed, each of the 18 isotopes showed the expected bimodal distribution in the global bead population. An automatic debarcoding pipeline capable of identifying bead events with a correct double-barcode signature was developed. From the analysis of three independent batches of barcoded beads, with a mean of 1.88±0.31 x 106 events per run, 72.9+10.97 % of bead events with a correct signature of six out of twelve isotopes from the second set were accurately debarcoded, and from those, 85.29+8.91 % of bead events had a correct signature of three out of six isotopes from the first set. Accurately debarcoded bead events grouped in one of the 20 groups from the first barcode set (Fig. 2b), and within each group, beads further separated in 924 groups from the second barcode set (Fig. 2c). Bead counts for each of the 924 barcodes from the second barcode set followed a normal distribution, with 100% recovery of the 18,480 barcodes. Together, these data demonstratedMGB Ref: 33929 / 70816 high-throughput generation of thousands of isotope-barcoded beads and their robust identification from a pooled mixture using mass cytometry.

[0185] Identification of thousands of barcoded beads from a mixture offers a versatile system for the design of different types of multiplexed serology assays with unique capabilities. As a demonstration of these capabilities, two systems were designed, the first for detection of serum antibodies using a very low sample volumes (Fig. 3), and the second system for rapid, wash-free, high-throughput detection of serum antibodies (Fig. 4).

[0186] An assay for detection of plasma antibodies using low sample volume. A two-bead selection system to detect the presence of specific antibodies in low volumes of human plasma was designed (Fig. 3a). In this system, one bead is barcoded with a predefined combination of stable isotopes and loaded with a biotinylated antigen of interest (e.g., a purified protein or a protein subunit). The other bead is magnetic and is conjugated to anti-human immunoglobulins. Cross-linking of barcoded beads to magnetic beads occurs only in the presence of antibodies recognizing target antigens. For example, in an assay leveraging the two-bead selection system, each clinical plasma sample is incubated in individual wells with barcoded beads loaded with distinct antigens to allow host antibodies to bind to antigens on beads. Barcoded beads are then washed to remove any unbound antibody and pooled across all samples to create a baseline bead mixture. The magnetic beads are then added providing an opportunity for cross-linking of magnetic beads to barcoded beads via anti-human immunoglobulins when host antibodies are present. Washes are performed using a magnet to retain only those barcoded beads from host antibody positive samples, and the flowthrough is then analyzed by mass cytometry. For each barcode, the ratio of the bead count in the flowthrough and the baseline bead mixture provides an estimate of host antibody levels, with low ratio values implying the presence of host antibodies specific to the antigen in a given sample.

[0187] As a demonstration of this method, first, barcoded beads loaded with the recombinant SARS-CoV-2 SI subunit of the spike protein (Spike SI) with 20 plasma samples from COVID- 19 convalescent donors and 20 negative control plasma samples collected prior to the emergence of COVID-19, all diluted 300-fold were incubated. The 40 distinct types of barcoded beads were similarly represented in the pooled baseline mixture, which is collected prior to magnetic bead selection (Fig. 3b; Baseline and Fig. 3c; X-axis). Consistent with expected patterns of serumMGB Ref: 33929 / 70816 immunity, after selection, all barcoded beads incubated with pre-COVID-19 pandemic samples were enriched (Fig. 3b; Flow-through and Fig. 3c; Y-axis, green circles) and most barcoded beads incubated with SARS-CoV-2 positive patient samples were depleted (Fig. 3c; Y-axis, red circles). Two of the SARS-CoV-2 positive samples resembled pre-COVID-19 samples (Fig. 3c). Consistently, independent ELISA analysis of the SARS-CoV-2-positive samples showed high anti-Spike SI levels for all samples except the two behaving similarly to pre-COVID-19 samples (Fig. 3d), suggesting these two patient samples may have low levels of SARS-CoV-2 antibodies despite being from individuals with prior SARS-CoV-2 positive PCR tests. Similar results were observed in three independent experiments. Furthermore, similar results were also observed when samples were analyzed individually in an independent flow cytometry assay rather than pooled together in the mass cytometry assay. Additionally proper alignment of a multiplexed assessment of the levels of antibodies were observed against SARS-CoV-2 Spike SI, the receptor binding domain of Spike SI (Spike SI RBD), and the Nucleocapsid protein to the results of an ELISA assay. To gauge sensitivity of the assay, beads loaded with Spike SI with increasing concentrations of a monoclonal antibody against Spike S 1 were incubated and observed detection down to 100 pg / mL for this particular antibody-antigen interaction. These results show that the mass cytometry results align with the results obtained with ELISA and flow cytometry, validating the two-bead system for antibody detection in patient plasma samples.

[0188] The extensive barcoding space of the isotope-tagged beads were leveraged to analyze an additional set of 39 plasma samples from individuals who had tested positive for SARS-CoV- 2 by PCR and 55 plasma samples collected prior to the COVID-19 pandemic. Each sample was diluted 1:300, 1:3000, and 1:30000, which is equivalent to using 100, 10, and 1 nL of sample, respectively, in a 30 pL reaction volume. The samples were incubated with barcoded beads loaded with recombinant SARS-CoV-2 Spike SI, Spike SI RBD, the S2 subunit of the spike protein (Spike S2). and the Nucleocapsid protein. Each sample and dilution were quantified 12 times using three sample replicates and four target replicates by calculating the ratio of the bead count in the flowthrough and the baseline bead mixture in each replicate. The two-bead selection system was able to distinguish all 39 COVID- 19 patient samples from negative controls with 100% success rate using 100 nL of sample volume. In some samples, antibodies against Spike SI were detected using only 1 nL of sample (Fig. 3e; Samples 21 and 36, Spike SI, blue dots). The assay also revealed patient- specific variability in SARS-CoV-2 antibody profiles. For instance,MGB Ref: 33929 / 70816 some samples had high levels of antibodies against Spike SI but not against Spike S I RBD, Spike S2, or Nucleocapsid (Fig. 3e; Samples 14 and 23, red dots). Others had high levels of antibodies against Spike SI, Spike SI RBD, Spike S2, and Nucleocapsid (Fig. 3e; Samples 16 and 28, red dots). Lastly some had high levels of antibodies against Spike SI and Nucleocapsid but not Spike SI RBD and Spike S2 (Fig. 3e; Samples 13 and 15, red dots). For each sample, each of the 12 replicates had similar values (Fig. 3f), and the results correlated well in two independently performed assays. Additionally, the levels of antibodies against SARS-CoV-2 Spike SI and Nucleocapsid had a strong correlation with independent ELISA assays conducted on the same set of samples. These data show that a two-bead strategy can detect host antibodies in low volume samples, down to 1 nL, and can provide unique profiles of the patient’s antibody responses against multiple antigens.

[0189] A high-throughput, w ash-free assay for multiplexed detection of antibodies. Secondary antibodies are advantageous for serology assays due to ease of use and have been previously used in low-throughput bead-based mass cytometry assays 32. The compatibility of secondary antibodies conjugated to isotopes was validated for the quantification of host immunoglobulins bound to our isotope-barcoded beads using positive and negative controls. Spike SI -loaded, isotope-barcoded beads were incubated with dilutions of a plasma sample from an individual positive for SARS-CoV-2 antibodies and with plasma samples from eight subjects collected prior to the COVID- 19 pandemic as negative controls. Beads were washed, pooled, and incubated with anti-human IgG conjugated to gold nanoparticles. A positive control signal was observed well above the background that spanned linearly from 8 pL to 62.5 nL in a 1:2 serial dilution series. Minimal non-specific binding of the secondary anti-IgG antibody to beads was observed and low non-specific binding of pre-pandemic samples. These results validated that antibodies bound to antigen-loaded, isotope-barcoded beads can be readily detected via secondary antibodies.

[0190] A major factor limiting the scalability of serology assays such as ELISA and fluorescently-barcoded bead-based assays is the need to perform multiple washes, one plate at a time, to remove unbound antibodies. Automation strategies can ameliorate the issue 36 but are not straightforward to implement. In some embodiments, a system consisted of consecutive fixation and denaturation steps to eliminate washing constraints for isotope-barcoded beads serology assays (Fig. 4a). In this wash-free system, antigen-loaded isotope-barcoded beads areMGB Ref: 33929 / 70816 first incubated with samples of interest to allow host antibodies to bind to bead loaded with antigens. Each well is then treated with paraformaldehyde (PFA) to fix host antibodies onto the beads. A subsequent sodium dodecyl sulfate (SDS) treatment denatures the bound and unbound immunoglobulins without removing isotopes from the beads. Samples are all then pooled and washed. Pooled samples are then incubated with isotopically labeled secondary antibodies and analysis is completed by mass cytometry to reveal the level of bound host antibodies on each barcoded bead.

[0191] To demonstrate bead-based multiplex serology testing by mass cytometry at scale in a wash-free system, the levels of IgG (Fig. 4b) and IgM (Fig. 4c) against 19 distinct SARS-CoV-2 protein variants were simultaneously analyzed in 542 longitudinal serum samples collected from a cohort of SARS-CoV-2 infected households in San Francisco (findcovidl9.org / ). As part of this study blood samples were collected longitudinally from index cases with positive SARS- CoV-2 tests and additional household members. Extensive epidemiological data was also captured that included prior SARS-CoV-2 vaccination status. Expectedly, anti-wild-type SARS- CoV-2 Trimer IgG levels increased over time across the cohort as a whole (Fig. 4d). and these increased levels were strongly related to both vaccination status and presence of a positive SARS-CoV-2 PCR test. In the same assay, triplicates or quadruplicates of 100 negative samples collected prior to the COVID-19 pandemic were included. The immunoglobulin levels against wild-type SARS-CoV-2 Trimer were below a given threshold in 97.5% of the tests for IgG and in 100% of the tests for IgM. In the samples collected at day 28 from infected and vaccinated individuals of the San Francisco study, all 23 samples were above the threshold for IgG, and 17 of the 23 samples were above the threshold for IgM. The levels of IgG against the wild-type SARS-CoV-2 Trimer obtained via mass cytometry correlated well with those obtained using Abbott AdviseDx SARS-CoV-2 IgG II (Architect) (Fig. 4e), an assay that received Emergency Use Authorization (EUA) by the Food and Drug Administration.

[0192] Because this assay provides internally controlled data on multiple targets for the same sample, it is feasible to make robust and direct comparisons within individuals across SARS- CoV-2 variants. A high correlation on the antibody response against SARS-CoV-2 variants for most samples was observed (Fig. 4f; Black dots) but identified a subset of samples with unique responses to certain variants (Fig. 4f; Colored dots) that could reflect variation in antibody repertoire driven by patient or virus-related factors. For example, samples #4567, #4582, andMGB Ref: 33929 / 70816#4599 showed lower IgG levels against the Beta, Gamma, Delta, and Omicron variants compared to the wild-type and alpha variant, and sample #4642 showed lower IgG levels against the Beta, Gamma, and Omicron variants compared to the wild-type, alpha, and delta variants (Fig. 4f; Colored dots).

[0193] These data show that bead-based multiplex serology testing by mass cytometry is quantitative, sensitive, and specific. This assay is capable of generating data comparable to existing authorized tests, but with massive improvements in scalability, performing 36,960 tests in one tube using 400 nL of sample volume in a 30 pL reaction volume.Discussion

[0194] Exemplified herein is an embodiment of a highly scalable and multiplexed serology technology with readout via mass cytometry. The generation of 18,840 unique barcodes by incorporating distinct combinations of stable isotopes on polystyrene beads post-synthesis was demonstrated. Further, an automatic pipeline to debarcode the raw data without the need to input user-defined parameters was provided. Using this technology and a bead- selection approach, IgGs against SARS-CoV-2 Spike SI using the equivalent of 100 nL of sample in a cohort of 55 pre-pandemic and 39 COVID- 19 samples were detected. In a subset of those samples, detection of IgGs using the equivalent of 10 and 1 nL of sample was shown. Using secondary detection antibodies, the simultaneous analysis of IgG and IgM levels against 19 proteins in 924 samples in an assay that took one operator approximately 8 hours to complete without the need of automation equipment was demonstrated. This is the equivalent of performing 36,960 ELISA- style serology assays in a single run. The ability to run tens of thousands of assays in parallel makes it a cost-effective technology. Moreover, compared to other strategies used in diagnostic labs such as ELISA and Luminex assays, the hands-on time of operators running the same number of assays is substantially reduced.

[0195] The assessment of humoral immunity in the FIND cohort allowed benchmarking of the serology assay via mass cytometry versus an immunoassay approved for clinical use. The data indicate that the mass cytometry assay can detect unique SARS-CoV-2 variant specific signatures in a robust fashion. This detection is made possible due to internal control resulting from pooling and simultaneous processing of all samples. This serology technology is immediately extensible into additional antigen variants from SARS-CoV-2 37 and other targetsMGB Ref: 33929 / 70816 by producing the required recombinant proteins. Comprehensive characterization of immunoglobulin subclasses coupled to longitudinal testing campaigns can reveal immune mechanisms that might inform vaccination campaigns, as illustrated by a preferential class switch to IgG4 after booster shots of an mRNA vaccine for SARS-CoV-2 38. A future development could harness part of the remaining isotope space to include simultaneous assessment of IgGl. IgG2, IgG3. IgG4, IgM, IgAl, and IgA2 on each bead for deeper sample profiling. Other antibody locations with key biological relevance, such as the nasal mucosa 39 or gingival crevice 40, could be also interrogated by expanding the assay to additional sample sources.

[0196] From a technical standpoint, the current iteration of the isotope-based serology assay is mainly constrained by the number of available barcodes and acquisition speed. Over 50 isotopes are available off-the-shelf for mass cytometry 28, and the present example reports the simultaneous use of 20 of them in the serology assay. The remaining isotopes could be harnessed to increase the number of barcodes, but careful validation should be performed given the distinct transmission factors for each isotope in ICP-MS. For instance, using 16 instead of 12 isotopes for sample ID would generate 12,870 combinations, and 8 instead of 6 isotopes for target ID would generate 70 combinations, for an aggregate of 900,900 barcodes. An increase in the number of barcodes should also be accompanied by further assay miniaturization and automation. The inductively coupled plasma time-of-flight mass spectrometry instrument used in this report has a relatively slow sample acquisition speed of 1,000 events per second, which limits the number of samples that can be analyzed, but a higher speed could be achieved using alternative instruments 41, smaller particles, and multiplex signal amplification systems 42,43. The beads used in the present example have a diameter variation of 3 to 3.4 pm, with larger beads having a higher number of streptavidin molecules on their surface. In some embodiments, the use of beads with a narrower range of diameter variation improve the throughput of the assay by reducing the number of required beads for analysis. Additionally, the assembling of antigen panels is constrained by the scalability of protein purification. While the present technology scales faster than the process of integrating new purified proteins into the panel, thus posing a challenge in fully leveraging the multiplexing potential of the technology, the commercial availability of antigens ameliorates the issue, although validation will be required when assembling new antigen panels.MGB Ref: 33929 / 70816

[0197] Tn summary, the present example demonstrated an embodiment which designed and implemented a fast, high-throughput, and cost-effective serology technology that opens new opportunities for profiling humoral immune responses at population scales. In some embodiments, the present technology will have a broad range of eventual applications, such as in government- sponsored serology surveillance, drug-discovery efforts, monitoring of vaccination campaigns, diagnosis of autoimmune diseases and cancer, and monitoring of immune correlates of protection during infectious disease outbreaks. In some embodiments these applications of mass cytometry-based serology will help reveal humoral immune mechanisms at a systems level, which may guide development of therapeutic strategies and enable prediction of clinical outcomes.

Claims

MGB Ref: 33929 / 70816CLAIMSWhat is claimed is:

1. A method for determining the presence of at least one biomolecule in a sample comprising:(a) obtaining a sample from a subject;(b) incubating the sample with a composition comprising a plurality of beads, wherein each bead is conjugated with (i) at least one barcode moiety, and (ii) at least one ligand capable of binding to a biomolecule, thereby producing a composition comprising a plurality of constructs, wherein each construct comprises a bead, a ligand and a bound biomolecule:(c) washing the composition of (b) under conditions that allow removal of unbound biomolecules;(d) optionally pooling the composition of (c) with one or more additional compositions comprising separate samples that have been incubated with separate compositions according to (b), thereby preparing a pooled composition;(e) incubating the composition or the pooled composition comprising bound biomolecules with a composition comprising a plurality of secondary constructs, wherein each secondary construct comprises a barcode moiety conjugated to a biomolecule capable of binding to bound biomolecules;(f) washing the composition or pooled composition of (e) under conditions that allow removal of unbound secondary constructs; and(g) determining the presence of the barcode moieties of the first and second constructs, thereby determining the presence of at least one biomolecule in a sample.

2. A method for determining the presence of at least one biomolecule in a sample comprising:(a) obtaining a sample from a subject;(b) incubating the sample with a composition comprising a plurality of beads, wherein each bead is conjugated with (i) at least one barcode moiety, and (ii) at least one ligand capable of binding to a biomolecule, thereby producing a composition comprising aMGB Ref: 33929 / 70816 plurality of constructs, wherein each construct comprises a bead, a ligand and a bound biomolecule;(c) incubating the composition comprising the constructs of (b) with a fixing reagent, thereby fixing the bound biomolecules to their respective beads;(d) incubating the composition of (c) with a denaturing reagent, thereby denaturing the bound and unbound biomolecules;(e) optionally pooling the composition of (d) with one or more additional compositions comprising separate samples that have been incubated with separate compositions according to (b), thereby preparing a pooled composition;(f) removing unbound biomolecules;(g) incubating the composition or the pooled composition comprising bound, fixed and denatured biomolecules with a plurality of secondary constructs, wherein each secondary construct comprises a barcode moiety conjugated to a biomolecule capable of binding to a bound, fixed and denatured biomolecules;(h) removing unbound secondary constructs; and(i) determining the presence of the barcode moieties of the first and second constructs, thereby determining the presence of at least one biomolecule in a sample.

3. A method for simultaneously detecting a plurality of biomolecules from a collection of samples comprising:(a) obtaining a collection of samples from a single subject or a plurality of subjects;(b) separately incubating the samples with compositions comprising a plurality of beads, wherein each bead is conjugated with (i) a plurality of barcode moieties, and (ii) at least one ligand capable of binding to a biomolecule, thereby producing a composition comprising a plurality of constructs, wherein each construct comprises a bead, at least one ligand and at least one bound biomolecule;(c) washing the compositions of (b) under conditions that allow removal of unbound biomolecules;(d) pooling the composition of (c) with one or more additional compositions comprising separate samples that have been separately incubated with separate compositions according to (b), thereby preparing a pooled composition;MGB Ref: 33929 / 70816(e) incubating the pooled composition comprising bound biomolecules with a composition comprising a plurality of secondary constructs, wherein each secondary construct comprises a barcode moiety conjugated to a biomolecule capable of binding to bound biomolecules;(f) washing the pooled composition of (e) under conditions that allow removal of unbound secondary constructs; and(g) determining the presence of the barcode moieties of the first and second constructs, thereby determining the presence of at least one biomolecule in a sample.

4. A method for simultaneously detecting a plurality of biomolecules from a collection of samples comprising:(a) obtaining a collection of samples from a single subject or a plurality of subjects;(b) separately incubating the samples with compositions comprising a plurality of beads, wherein each bead is conjugated with (i) a plurality of barcode moieties, and (ii) at least one ligand capable of binding to a biomolecule, thereby producing a composition comprising a plurality of constructs, wherein each construct comprises a bead, at least one ligand and at least one bound biomolecule;(c) incubating the composition comprising the constructs of (b) with a fixing reagent, thereby fixing the bound biomolecules to their respective beads;(d) incubating the composition of (c) with a denaturing reagent, thereby denaturing the bound and unbound biomolecules;(e) pooling the composition of (d) with one or more additional compositions comprising separate samples that have been separately incubated with separate compositions according to (b), thereby preparing a pooled composition;(f) removing unbound biomolecules;(g) incubating the composition comprising bound, fixed and denatured biomolecules of (e) with a plurality of secondary constructs, wherein each secondary construct comprises a barcode moiety conjugated to a biomolecule capable of binding to a bound, fixed and denatured biomolecules;(h) removing unbound secondary constructs; andMGB Ref: 33929 / 70816(i) determining the presence of the barcode moieties of the first and second constructs, thereby determining the presence of at least one biomolecule in a sample.

5. The method according to any one of claims 1-4, wherein each bead of the first construct is conjugated to one ligand, wherein the ligand is an antigen capable of being bound by an antibody in the sample, and wherein the biomolecule of the secondary construct is a secondary antibody.

6. The method of claim 5, wherein the isotype and / or subclass of antibody bound to the antigen is determined.

7. The method according to any one of claims 1-4, wherein each bead of the first construct is conjugated to one ligand, wherein the ligand is an antigen capable of being bound by an antibody in the sample, and wherein the biomolecule of the secondary construct is a Fc gamma receptor (FcyR), a complement cascade-initiating protein, or a lectin.

8. The method according to any one of claims 1-4, wherein each bead of the first construct is conjugated to one ligand, wherein the ligand is capable of being bound by a biomolecule in the sample, and wherein the biomolecule of the secondary construct is a receptor capable of binding to the ligand of the first construct.

9. The method of claim 8, wherein the ligand and / or receptor is selected from the group consisting of an antigens, an analytes, and a proteins.

10. The method according to any one of claims 1-4, wherein each bead of the first construct is conjugated to a plurality of ligands, wherein each ligand is an antibody or aptamer capable of binding one or more antigens in the sample, and wherein the biomolecule of the secondary construct is a secondary antibody or aptamer.

11. The method of claim 10, wherein the one or more antigens comprise a biomolecules selected from the group consisting of antigens, peptides, analytes, and proteins.MGB Ref: 33929 / 7081612. The method according to any one of claims 1-4, wherein each bead of the first construct is conjugated to one ligand, wherein the ligand is an oligonucleotide capable of being bound by a nucleic acid in the sample, and wherein the biomolecule of the secondary construct is a secondary oligonucleotide capable of being bound by the same nucleic acid in the sample in a distinct region.

13. The method according to any one of claims 1-4, wherein each bead of the first construct is conjugated to a plurality of ligands, wherein each ligand is an oligonucleotide capable of binding one or more nucleic acids in the sample, and wherein the biomolecule of the secondary construct is a secondary oligonucleotide.

14. The method according to any one claims 1-4, wherein each bead of the first construct is conjugated to one ligand or a plurality of ligands, wherein each ligand is an antibody, a ligand capable of being bound by a receptor, or an oligonucleotide, and wherein the biomolecule of the secondary construct is a secondary antibody, a receptor capable of binding a ligand of the first construct, or a secondary oligonucleotide.

15. The method of any one of the previous claims, wherein each bead is conjugated to 1, 2, 3, 4, 5, 6, 7. 8, 9, 10, 20, 30, 40. 50 60, 70, 80, 90, or 100 or more different ligands.

16. The method of any one of the previous claims, wherein a plurality of samples are obtained from a plurality of subjects.

17. The method of claim 16, wherein approximately 10, 100, 1000, 10,000, 20,000, 30,000. 40,000,50,000, 100,000 or more samples are obtained.

18. The method of any one of the previous claims, wherein the subject is an animal, plant, fungi, protist, bacterium or archaea.MGB Ref: 33929 / 7081619. The method of claim 18, wherein the animal is selected from the group consisting of a human, a cow, a pig, a chicken, a sheep, a duck, and a goat.

20. The method of any one of the preceding claims, wherein the presence of approximately, 1, 10, 100, 1,000, 5,000, 10,000, 20,000 or more biomolecules are determined.

21. The method of any one of the preceding claims, wherein the sample or plurality of samples is a blood, serum, saliva, nasal / oropharyngeal swab, semen, urine, lymph, cerebrospinal fluid, interstitial fluid, spinal fluid, peritoneal fluid, pleural fluid, amniotic fluid, stool, bile, bone marrow, or skin sample.

22. The method of any one of the preceding claims, wherein a barcode moiety associated with the first construct identifies the incubated sample.

23. The method of any one of the preceding claims, wherein a barcode moiety associated with the first construct identifies the ligand or ligands.

24. The method of any one of the previous claims, wherein the biomolecule that is determined is an antibody, a cytokine, a lectin, a receptor, an analyte, a protein or a nucleic acid.

25. The method of any one of the previous claims, wherein the biomolecule that is determined is providing functional information of biomolecule binding.

26. The method of claim 24, wherein the biomolecule is a nucleic acid, and wherein the nucleic acid is selected from the group consisting of a DNA, a viral DNA, a cell-free DNA (cfDNA), a tumor cell-derived DNA a RNA, a viral RNA, and / or a tumor cell derived RNA.

27. The method of claim 26, wherein the nucleic acid is an RNA from a pathogen.

28. The method of claim 26, wherein the nucleic acid is a DNA from a pathogen.MGB Ref: 33929 / 7081629. The method of claim 24, wherein the protein is a protein from a pathogen.

30. The method of any one of claims 27-29. wherein the pathogen is a virus, a bacterium, a fungi, a protozoa, or a worm.

31. The method of any one of the previous claims, wherein the ligand is an antigen or biomolecule-binding fragment of an antigen, a biotinylated protein, an antibody, a polymer or a nucleic acid.

32. The method of any one of the preceding claims, wherein the bead comprises an affinity binding system.

33. The method of claim 32, wherein the affinity binding system comprises one or more of streptavidin and / or biotin.

34. The method of any one of the preceding claims, wherein the barcode moiety on the first construct and / or on the second construct comprises one or more isotopes, one or more peptides, one or more small molecules, one or more lipids, one or more glycans, one or more oligonucleotides or one or more fluorophores.

35. The method of any one of the previous claims, wherein the barcode moieties present on the first and second constructs comprise dual isotope barcodes.

36. The method of any one of the preceding claims, wherein the bead comprises a polymer bead.

37. The method of claim 36, wherein the polymer is selected from the group consisting of polystyrene polytmethyl methacrylate) (PMMA), hydrogels, silica, poly(lactic-co-glycolic acid) (PLGA), and polyethylene glycol (PEG).MGB Ref: 33929 / 7081638. The method of any one of claims 2 and 4, wherein the fixing reagent is selected from the group consisting of paraformaldehyde (PF A), formaldehyde, acetone, glutaraldehyde, and ethanol, glyoxal, methanol.

39. The method of any one of claims 2 and 4, wherein the denaturing reagent is selected from the group consisting of sodium dodecyl sulfate (SDS), thiourea, lithium perchlorate, lithium acetate, magnesium chloride, urea, guanidine hydrochloride, methanol, and CHAPS.

40. The method of any one of the preceding claims, wherein the determining step (i) comprises using mass cytometry, flow cytometry, mass spectrometry, fluorescence microscopy, electron microscopy, isotope-based imaging, Raman spectroscopy, Nuclear Magnetic Resonance (NMR), fluorescence resonance energy transfer (FRET), X-ray fluorescence (XRF), or nucleic acid sequencing.

41. The method of any one of the previous claims, wherein the presence of a signal from the barcode moiety of the first construct is indicative of a current or prior infection from a pathogen associated with the ligand.

42. A method for determining the presence of antibodies targeting pathogens in a sample comprising:(a) obtaining a sample from a subject;(b) incubating the sample with a composition comprising a plurality of beads, wherein each bead is conjugated with (i) at least one barcode moiety, and (ii) at least one antigen capable of binding to a pathogen antibody, thereby producing a composition comprising a plurality of constructs, wherein each construct comprises a bead, an antigen and a bound pathogen antibody;(c) washing the composition of (b) under conditions that allow removal of unbound pathogen antibodies;(d) optionally pooling the composition of (c) with one or more additional compositions comprising separate samples that have been incubated with separate compositions according to (b), thereby preparing a pooled composition;MGB Ref: 33929 / 70816(e) incubating the composition or the pooled composition comprising bound pathogen antibodies with a composition comprising a plurality of secondary constructs, wherein each secondary construct comprises a barcode moiety conjugated to a secondary antibody capable of binding to bound pathogen antibodies;(f) washing the composition or pooled composition of (e) under conditions that allow removal of unbound secondary constructs; and(g) determining the presence of the barcode moieties of the first and second constructs, thereby determining the presence of at least one pathogen antibody in a sample.

43. The method of claim 42, wherein the pathogen is a coronavirus.

44. The method of claim 43 wherein the antigen is a Spike protein antibody from SARS- CoV-1 or SARS-CoV-2.

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