Methods and related aspects for digital neutralizing antibody assays for disease detection and immunity assessment

Abstract

Provided herein are methods of analyzing antibody binding. In some embodiments, the methods comprise contacting a set of SARS-CoV-2 protein variant functionalized nanoparticles (MNPs) with a set of antibodies, and contacting a set of ACE2 protein functionalized nanoparticles (MNPs) with the set of antibodies and a set of competition probes that comprise the SARS-CoV-2 protein variant. In some embodiments, the methods also include detecting binding, if any, of the SARS-CoV-2 protein variant functionalized MNPs with the set of antibodies and binding, if any, of the ACE2 protein functionalized MNPs with the set of antibodies and the set of competition probes that comprise the SARS-CoV-2 protein variant. Related systems and kits are also provided.

Description

METHODS AND RELATED ASPECTS FOR DIGITAL NEUTRALIZING ANTIBODY ASSAYS FOR DISEASE DETECTION AND IMMUNITY ASSESSMENTCROSS-REFERENCE TO RELATED APPLICATONS

[0001] This application claims priority to, and the benefit of, U.S. Provisional Patent Application Ser. No. 63 / 653,356, filed May 30, 2024, the disclosure of which is incorporated herein by reference.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under R21 AH 69098 awarded by the National Institutes of Health. The government has certain rights in the invention.SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on May 29, 2025, is named 0391_0108-PCT_SL.xml and is 9,552 bytes in size.DETAILED DESCRIPTION

[0004] Infectious diseases, such as coronavirus disease 2019 (COVID-19), incurred devastatingly high levels of morbidity and mortality with substantive economic losses worldwide. To date, over 700 million people are estimated to have been infected, and about 7 million have died from COVID-19. The virions of SARS-CoV-2, the cause of COVID-19, are decorated with homotrimers of the spike (S) glycoprotein that enables cell entry of the virus by attaching to the human angiotensin-converting enzyme 2 (ACE2). Neutralizing antibodies (nAbs) are produced by the human immune response to infection or vaccination, or rationally engineered artificially, to block the ACE2-RBD binding and inhibit virus infection. Therefore, S proteins and their RBDs are the primary antigenic targets of nAbs, making them important for diagnostics and vaccine development. The S and RBD proteins are also under significant selective pressure to mutate in order to evade host immunity, as exemplified by the rapid and continued emergence of SARS-CoV-2 variants. For example, the Gamma (P1) variant underwent S protein conformation changes, and the Omicron B.1 .1 .529 variant contained 15 mutations in RBD with the immunity-escaping capability to over 200 nAbs designedagainst the wild-type variant. Subsequent Omicron variants (e.g., XBB.1 .5, EG.5.1 , and JN.1 ) continue to accrue mutations that enhance infectiousness and resistance to immunity. Given that vaccine development has been focused on raising nAbs against the viral RBD and S proteins, determining ways to rapidly identify the quality and quantity of the nAbs is important for establishing correlates of immunity and protection.

[0005] Commercially available serological assays such as anti-spike ELISA focus on quantifying total levels as a predictor of immunity. Despite the importance of antibody level quantification, these methods do not provide insight into nAb potency, which is crucial to the effectiveness evaluation of nAb treatment and vaccine protection of vulnerable populations. Hence, it is necessary to develop modular assay platforms that can detect the efficacy of nAbs through a platform modularly adapted to deal with newly emerging variants. Currently, the Plaque Reduction Neutralization Test (PRNT) is the gold standard for analyzing nAbs but is labor-intensive and semi-quantitative. Microneutralization assay (MNA) has improved speed but still requires several days for results, and importantly, similar to PRNT, also requires a Biosafety Level 3 (BSL3) setting. Pseudoviruses offer a safer alternative but face challenges, one of which is that the spike proteins displayed on pseudo-viruses may not be identical to those on the authentic virus, potentially affecting the accuracy of the results. Surrogate ELISA assays and competitive chemiluminescence immunoassay are promising alternatives in lower biosafety settings but require multi-day workflow and large equipment for readout. In comparison, the Lateral Flow Immunoassay (LFI), emerging microfluidic systems or electrochemical biodevices offer higher throughput but suffer from poor sensitivity, require complex enzymatic reactions, or face challenges in achieving rapid and accurate readout and data interpretation. Therefore, there is a need for low-cost, rapid, facile assays that can effectively address the neutralization measurement required for screening.SUMMARY

[0006] In some aspects, the present disclosure provides a low-cost, widely accessible, rapid, sensitive, digital neutralizing assay (Fig. 1 ) that can distinguish the nAb potency against different SARS-CoV-2 variants and quantify the neutralizing ability of human serum without the need for authentic viruses. This modular diagnosticplatform analyzes the immunity potency of samples against different variants of the SARS-CoV-2 virus within 20 minutes in a single test tube. It reports the results in a point-of-care (POC) setting using a semiconductor-based digital readout system. Specifically, this assay, a nanoparticle-supported rapid electronic detector for neutralizing antibody quantification (NasRED-nAb), is performed simply by mixing the target nAbs (or sera) with two testing reagents, i.e., ACE2 functionalized gold nanoparticles (ACE2-AuNPs) as the cell-mimicking signaling beacon and SARS-COV-2 S or RBD protein as the virus antigen, followed by an accelerated readout scheme. The quantity and quality of the target nAbs dictate the level of freely available S (or RBD) proteins that can bind to ACE2-AuNPs and, accordingly, the amount of clustering AuNPs. The readout signal is readily collected by probing the optical extinction of the free-floating ACE2-AuNPs using our PED system made of simple electronic circuitry (Fig. S1). Here, we demonstrate this integrated and modular sensing platform can quantify the neutralizing ability of human serum and distinguish the potency of nAbs against the different variants of SARS-CoV-2.

[0007] In another aspect, the present disclosure provides a method of analyzing antibody binding. The method includes contacting a set of SARS-CoV-2 protein variant functionalized nanoparticles (MNPs) with a set of antibodies, and contacting a set of ACE2 protein functionalized nanoparticles (MNPs) with the set of antibodies and a set of competition probes that comprise the SARS-CoV-2 protein variant. The method also includes detecting binding, if any, of the SARS-CoV-2 protein variant functionalized MNPs with the set of antibodies and binding, if any, of the ACE2 protein functionalized MNPs with the set of antibodies and the set of competition probes that comprise the SARS-CoV-2 protein variant, thereby analyzing antibody binding.

[0008] In another aspect, the present disclosure also provides a system for analyzing antibody binding. The system includes a sample container receiving structure configured to receive a sample container that contains a set of SARS-CoV-2 protein variant functionalized nanoparticles (MNPs) with a set of antibodies, and / or a set of ACE2 protein functionalized nanoparticles (MNPs) with the set of antibodies and a set of competition probes that comprise the SARS-CoV-2 protein variant; a light source operably connected to the sample container receiving structure, wherein the light sourceis configured to transmit light through the sample container; a photodetector operably connected to the sample container receiving structure, wherein the photodetector is configured to measure light intensity transmitted through the sample container from the light source; a controller operably connected to the light source and to the photodetector, wherein the controller is configured to effect transmission of the light through the sample container using the light source and measurement of the light intensity transmitted through the sample container that contains the set of SARS-CoV-2 protein variant functionalized MNPs with the set of antibodies, and / or the set of ACE2 protein functionalized MNPs with the set of antibodies and the set of competition probes that comprise the SARS-CoV-2 protein variant using the photodetector; a power source operably connected to the controller, wherein the power source is configured to selectively supply power to the controller; and, a housing structure operably connected to the sample container receiving structure, wherein the controller and power source are substantially housed in one or more cavities disposed within the housing structure.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain embodiments, and together with the written description, serve to explain certain principles of the methods, reaction mixtures, devices, kits, and related systems disclosed herein. The description provided herein is better understood when read in conjunction with the accompanying drawings which are included by way of example and not by way of limitation. It will be understood that like reference numerals identify like components throughout the drawings, unless the context indicates otherwise. It will also be understood that some or all of the figures may be schematic representations for purposes of illustration and do not necessarily depict the actual relative sizes or locations of the elements shown.

[0010] FIG. 1 A is a flow chart that schematically shows exemplary method steps of analyzing antibody binding according to some aspects disclosed herein.

[0011] FIG. 2A-2C: Design Scheme of NaSRED digital sensors, (a) The components of the portable electronic readout system comprise an LED, a photodetector, a tube chamber, signal digitalization customized circuitry, (b) NasRED-tAb comprising AuNps- RBD / S (tAbPs) formation by streptavidin-biotin reaction, and Ab-induced clusterization.The curve indicates SAD-S35 and CR3022 tested against WT-RBD. (c) NaSRED-nAb comprising AuNps-ACE-2 (nAbPs) formation by streptavidin-biotin reaction, Ab and nAbPs competition for attaching to the tetramer RBD / S protein (CPs) resulting in neutralization. The curve indicates SAD-S35 and CR3022 neutralization results against WT-RBD. Electronic readout is the normalized extinction relative to the Negative Control (NC).

[0012] FIGS. 3A-3H: NaSRED-tAb distinguishes the binding efficacy of monoclonal antibodies to different SARSCOV2 variant antigens, (a-d) Optical images of reaction tubes (mAb concentration and reaction groups indicated on each corresponding tube, NC=Negative Control), (e-h) Electronic readout of the reaction groups of mAbs versus the RBD or S proteins for SARS-COV-2 variants.

[0013] FIGS. 4A-4D: NaSRED-nAb engineering to enhance low potency mAb detection (a) Schematic representation of probe concentration effect on NaSRED-nAb. (b-c) (Optical picture of the NaSRED-nAb for SARS2-03 (low potency nAb against WT- S1 in LPM and HPM scenarios (mAb concentration and reaction groups indicated on each corresponding tube, NC=Negative Control), (d) Normalized electronic readout (Neutralization %) against the logarithm of SARS2-03, SARS2-10, SARS2-31 (low potency) in low (dashed lines) and high (solid lines) probe modes.

[0014] FIGS. 5A-5E: NaSRED-nAb quantitative discrimination of mAbs’ neutralization potency against different variants, (a) High-potency mAbs neutralization versus WT-RBD (solid lines), and P1 -RBD (dashed lines) at high probe mode, (b) NaSRED-nAb optical pictures for SARS2-71 (high potency) versus WT-RBD, P1 -RBD at high probe mode (mAb concentrations and reaction groups indicated on each corresponding tube, NC=Negative Control), (c) High- and low-potency mAbs neutralization versus WT-S1 (solid lines), and Omicron-RBD (dashed Lines) at low probe mode, (d) NaSRED-nAb optical pictures for SARS2-03 (low potency) versus WT- S1 , ORBD at moderate viral load design (mAb concentrations and reaction groups indicated on each corresponding tube, NC=Negative Control), e) Comparing the EC50 value of the SARS2-71 against wild type variant with hACE2 Binding Competitive ELISA (grayscale dashed line) and NASRED-nAb (black solid line, LPM) (EC50 values adjusted to the molar ratio of RBD concentration for comparison).

[0015] FIGS. 6A-6C: Correlation of NaSRED-nAb with hACE2 Binding Competitive ELISA in Immunity Screening, Contrasted with Total IgG Count, (a) Optical picture of the NaSRED-nAb tubes for 40 clinical sera samples (10 negatives + 30 positives) (tube labels indicate hACE2 Binding Competitive ELISA values), (b) NasRED-nAb calibration curve in accordance with hACE2 Binding Competitive ELISA values, (c) NaSRED-Cal versus WT-S1 against hACE2 Binding Competitive ELISA (grayscale fitting and stars) indicating NaSRED-Cal and hACE2 Binding Competitive ELISA agreement (dark and light grayscale shading around the fitting demonstrate the 95% confidence and prediction bands, respectively). Grayscale triangles represent total IgG ELISA assay measurements indicating the randomness of Total IgG count.

[0016] FIGS. 7A-7G: Application of NaSRED in Longitudinal Seroconversion Studies against Wildtype and Omicron Variant, (a) Optical images of the tubes organized with the color and pattern code of the box chart for NaSRED-tAb and NaSRED-nAb assays against WTRBD / ORBD of Sero 1 and Sero 2 samples. The grayscale color of some samples is the natural sample grayscale removed from the readout background, (b) Tukey two-way ANOVA analysis for serotype and variant variables indicating a nonsignificant difference (P>0.05) between total antibody levels and (c) Significant difference (P<0.001 ) between neutralization levels, d) NaSRED-tAb results of pseudolDs in serol (Fall 2021 ) (solid bars) and sero2 (Fall 2022) (checkered bars) and e) their changes between surveys against WTRBD (grayscale) and ORBD (grayscale) indicating lack of trend in total antibody quantification, f) NaSRED-nAb results of pseudolDs in serol (solid bars) and sero2 (checkered bars) and g) their changes between surveys against WTRBD (grayscale) and ORBD (grayscale) indicating a notable increase in neutralization.

[0017] FIG. 8: Shows an exemplary NaSRED-tAb Workflow.

[0018] FIGS. 9A-9C: NaSRED components binding affinity evaluation, a) Calculated limit of detection and binding affinity (Kd) of WT-RBD coated AuNps (tAbPS) to SAD- S35, (b) CR3022, and (c) Dimer-ACE2.

[0019] FIGS. 10A and 10B: Temperature independence, a) Optical Images and (b) Electronic readout of SARSCOV2-38 mAb concentrations in NaSRED-tAb against WT-RBD at Room Temperature and 37 C demonstrating the same color trend and measurement in both temperature indication temperature independence of the assay.

[0020] FIGS. 11 A and 1 1 B: NaSRED-tAb distinguishes between the binding efficacy of different monoclonal antibodies to different SARSCOV2 antigens (RBD / S1 ). Optical Picture of reaction tubes (mAb concentration and reaction groups indicated on each corresponding tube, NC=Negative Control).

[0021] FIGS. 12A and 12B: NaSRED-tAb distinguishes between the binding efficacy of different monoclonal antibodies to different SARSCOV2 variants (P1 / Omicron). Optical Picture of reaction tubes (IgG concentration and reaction groups indicated on each corresponding tube, NC=Negative Control).

[0022] FIG. 13: Shows an exemplary NaSRED-tAb Workflow.

[0023] FIGS. 14A and 14B: NaSRED-nAb measurements for high concentrations of mAbs against Omicron. a) Optical picture of IgG Max(1 pM) of single SARS2-02, SARS2-03, SARS2-10, SARS2-31 , SARS2-38, and SARS2-71 against ORBD at LPM (NC is IgG concentration^ nM, PC is CP=0 nM). b) Electronic readout (darker grayscale bars, darker grayscale left Y axis (A.U)) and Neutralizing Ability (lighter grayscale bars, lighter grayscale right Y axis (%)) of respective IgG Max(1 pM) of single SARS2-02, SARS2-03, SARS2-10, SARS2-31 , SARS2-38, and SARS2-71 against ORBD at LPM. Bar chart normalized between 0-100% based on the NC and PC respectively to analyze the potency of the antibodies against the omicron variant for sorting. SARSCOV2-03 showed about 50% neutralization against the omicron variant while against the wildtype variant, it is sorted as low potency. On the other hand, SARSCOV2-71 showed minimal potency against the omicron variant. At the same time, it is sorted as high potency against the wildtype variant indicating the mutations of the RBD in the S protein of the Omicron.

[0024] FIGS. 15A-15C: Sera NaSRED-nAb sensor calibration utilizing Mix mAbs (Artificial serum sample) in Human pooled serum (HPS). a) schematics of the analysis and calibration steps for the detection of sera samples. Subtraction of the before and after centrifugation steps for the samples to remove the induced background noise from the yellowish color of the serum and light absorbance interference. B) Optical picture of IgG Mixture (nM) of SARS2-02, SARS2-03, SARS2-10, SARS2-31 , SARS2-38, andSARS2-71 (equal ratio) spiked in human pooled serum against moderate viral load Wt- S1 (top picture) and ORBD (bottom picture) (NC is mAbs concentration^ nM). The top image indicates the grayscale of the tubes before the centrifugation step. The grayscale serum color is visible in the negative control samples, which leads to light absorbance and noise. C) Neutralizing ability of IgG Mix (%) versus ORBD (red) and WT-S1 (black) at LPM.

[0025] FIGS. 16A and 16B: NaSRED-nAb distinguishes between the potency of different monoclonal antibodies against different SARSCOV2 antigens (RBD / S1 ). Optical Picture of reaction tubes (mAb concentration and reaction groups indicated on each corresponding tube, NC=Negative Control).

[0026] FIGS. 17A and 17B: NaSRED-nAb distinguishes between the potency of different monoclonal antibodies against different SARSCOV2 variants (PVOmicron). Optical Picture of reaction tubes (mAb concentration and reaction groups indicated on each corresponding tube, NC=Negative Control).

[0027] FIGS. 18A and 18B: Clinical Sera Samples ORBD neutralization potency compared to ELISA results against WTRBD. a) Optical picture of the NaSRED-nAb for ORBD in LPM design (Labels indicate the reported number by ELISA for neutralizing Antibody concentration (A.U.) in each sample)(the final concentration of the neutralizing antibody is the shown number divided by 4 for the assay dilution setup, b) Normalized electronic readout [0,100] (Neutralizing ability%, left Y-axis) against Elisa reported neutralizing antibody concentration of the samples against WTRBD (X-axis).DEFINITIONS

[0028] In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms may be set forth throughout the specification. If a definition of a term set forth below is inconsistent with a definition in an application or patent that is incorporated by reference, the definition set forth in this application should be used to understand the meaning of the term.

[0029] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and / orsteps of the type described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0030] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0031] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0032] 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. Further, 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 pertains. In describing and claiming the methods, reaction mixtures, devices, kits, and systems, the following terminology, and grammatical variants thereof, will be used in accordance with the definitions set forth below.

[0033] About. As used herein, “about” or “approximately” or “substantially” as applied to one or more values or elements of interest, refers to a value or element that is similar to a stated reference value or element. In certain embodiments, the term “about” or “approximately” or “substantially” refers to a range of values or elements that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 1 1 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value or element unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value or element).

[0034] Administer: As used herein, “administer” or “administering” a therapeutic agent (e.g., an immunological therapeutic agent) to a subject means to give, apply orbring the composition into contact with the subject. Administration can be accomplished by any of a number of routes, including, for example, topical, oral, subcutaneous, intramuscular, intraperitoneal, intravenous, intrathecal and intradermal.

[0035] Antibody: As used herein, the term “antibody” refers to an immunoglobulin or an antigen-binding domain thereof. The term includes but is not limited to polyclonal, monoclonal, monospecific, polyspecific, non-specific, humanized, human, canonized, canine, felinized, feline, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies. The antibody can include a constant region, or a portion thereof, such as the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes. For example, heavy chain constant regions of the various isotypes can be used, including: IgGi, lgG2, IgGs, lgG4, IgM, IgAi, lgA2, IgD, and IgE. By way of example, the light chain constant region can be kappa or lambda. The term “monoclonal antibody” refers to an antibody that displays a single binding specificity and affinity for a particular target, e.g., epitope.

[0036] Antigen Binding Portion: As used herein, the term “antigen binding portion” refers to a portion of an antibody that specifically binds to a target analyte (e.g., a SARS-CoV-2 protein, such as a receptor-binding domain (RBD) protein, Thrombospondin-2 (THBS2), etc.), e.g., a molecule in which one or more immunoglobulin chains is not full length, but which specifically binds to a target analyte. Examples of binding portions encompassed within the term “antigen-binding portion of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VLC, VHC, CL and CH1 domains: (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VHC and CH1 domains; (iv) a Fv fragment consisting of the VLC and VHC domains of a single arm of an antibody, (v) a dAb fragment, which consists of a VHC domain; and (vi) an isolated complementarity determining region (CDR) having sufficient framework to specifically bind, e.g., an antigen binding portion of a variable region. An antigen binding portion of a light chain variable region and an antigen binding portion of a heavy chain variable region, e.g., the two domains of the Fv fragment, VLC and VHC, can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VLC and VHC regions pair toform monovalent molecules (known as single chain Fv (scFV)). Such single chain antibodies are also encompassed within the term “antigen binding portion” of an antibody. The term “antigen binding portion” encompasses a single-domain antibody (sdAb), also known as a “nanobody” or “VHH antibody,” which is an antibody fragment consisting of a single monomeric variable antibody domain. These antibody portions are obtained using conventional techniques known to those with skill in the art, and the portions are screened for utility in the same manner as are intact antibodies.

[0037] Bind: As used herein, “bind,” in the context of target analyte detection, refers to a state in which a first chemical structure (e.g., a pathogenic particle or other biomarker) is sufficiently associated a second chemical structure such that the association between the first and second chemical structures can be detected.

[0038] Conjugate: As used herein, “conjugate” refers to a reversible or irreversible connection between two or more substances or components. In some embodiments, for example, gold nanoparticles (AuNPs) and / or other plasmonic metal nanoparticles (MNPs) are connected to antibodies and / or to antigen binding portions thereof. In some embodiments, AuNPs and / or other plasmonic metal nanoparticles (MNPs) are conjugated with antibodies and / or to antigen binding portions thereof via one or more linker compounds.

[0039] Detect: As used herein, “detect,” “detecting,” or “detection” refers to an act of determining the existence or presence of one or more target analytes (e.g., a SARS- CoV-2 protein, Thrombospondin-2 (THBS2), etc.) and / or a pathogen (e.g., a SARS- CoV-2) in a sample.

[0040] Epitope: As used herein, “epitope” refers to the part of an antigen (e.g., a SARS-CoV-2 protein) to which an antibody and / or an antigen binding portion binds.

[0041] Reaction Mixture: As used herein, "reaction mixture" refers a mixture that comprises molecules and / or reagents that can participate in and / or facilitate a given reaction or assay. A reaction mixture is referred to as complete if it contains all reagents necessary to carry out the reaction, and incomplete if it contains only a subset of the necessary reagents. It will be understood by one of skill in the art that reaction components are routinely stored as separate solutions, each containing a subset of the total components, for reasons of convenience, storage stability, or to allow forapplication-dependent adjustment of the component concentrations, and that reaction components are combined prior to the reaction to create a complete reaction mixture. Furthermore, it will be understood by one of skill in the art that reaction components are packaged separately for commercialization and that useful commercial kits may contain any subset of the reaction or assay components.

[0042] Sample. As used herein, “sample” means anything capable of being analyzed by the methods, devices, and / or systems disclosed herein.

[0043] Severe Acute Respiratory Syndrome Coronavirus-2-. As used herein, “severe acute respiratory syndrome coronavirus-2” or “SARS-CoV-2” refers to the coronavirus that emerged in 2019 to cause a human pandemic of an acute respiratory disease, now known as coronavirus disease 2019 (COVID-19).

[0044] Specifically Bind: As used herein, "specifically bind,” in the context of pathogen detection, refers to a state in which substantially only target chemical structures (e.g., target SARS-CoV-2 proteins, Thrombospondin-2 (THBS2), etc.) are sufficiently associated with a corresponding or cognate binding agent (e.g., an antibody, or antigen binding portion thereof), to the exclusion of non-target chemical structures, such that the association between the target chemical structures and the binding agent can be detected.

[0045] Subject: As used herein, “subject” refers to an animal, such as a mammalian species (e.g., human, dog, cat) or avian (e.g., bird) species. More specifically, a subject can be a vertebrate, e.g., a mammal such as a mouse, a primate, a simian or a human. Animals include farm animals (e.g., production cattle, dairy cattle, poultry, horses, pigs, and the like), sport animals, and companion animals (e.g., pets or support animals). In certain embodiments, the subject is a human. In certain embodiments, the subject is a companion animal, including, but not limited to, a dog or a cat. A subject can be a healthy individual, an individual that has or is suspected of having a disease or a predisposition to the disease, or an individual that is in need of therapy or suspected of needing therapy. The terms “individual” or “patient” are intended to be interchangeable with “subject.”

[0046] System-. As used herein, "system" in the context of analytical instrumentation refers a group of objects and / or devices that form a network for performing a desired objective.

[0047] Treat. As used herein, the term “treat,” “treating” or “treatment” are each used interchangeably herein to describe reversing, alleviating, or inhibiting the progress of a disease and / or injury, or one or more symptoms of such disease, to which such term applies. Depending on the condition of the subject, the term also refers to preventing a disease, and includes preventing the onset of a disease, or preventing the symptoms associated with a disease (e.g., viral infection). A treatment may be either performed in an acute or chronic way. The term also refers to reducing the severity of a disease or symptoms associated with such disease prior to affliction with the disease. Such prevention or reduction of the severity of a disease prior to affliction refers to administration of a treatment to a subject that is not at the time of administration afflicted with the disease. “Preventing” also refers to preventing the recurrence of a disease or of one or more symptoms associated with such disease.DETAILED DESCRIPTION

[0048] Reference will now be made in detail to example implementations. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention and it is to be understood that other embodiments may be utilized and that changes may be made without departing from the scope of the invention. The following description is, therefore, merely exemplary.

[0049] I. Introduction

[0050] Measurement of inhibitory immune responses against potential infections is crucial, due to the heterogeneity in human immunity and the constant emergence of immunity-evading variants. However, quantitative and specific assays for neutralizing antibodies (nAbs) often require high-level biosafety containment laboratories, sophisticated instrumentation, long detection time, and high cost. In this disclosure, we designed a nanoparticle-based, rapid, in-solution, nAb assay to quantify the neutralizing potency of monoclonal antibodies against the SARS-CoV-2 wildtype, Gamma, and Omicron variants. In some aspects, for example, human angiotensin-converting enzyme 2 (ACE2)-coated gold nanoparticles (AuNPs) were designed as cell-mimetic plasmonicbeacons, and mixed with target nAbs and SARS-CoV-2 variant spike or the receptor binding domain (RBD) proteins in a test tube. The presence of high-affinity, RBD- binding, and ACE2-competitive nAbs alters the spike (or RBD)-to-ACE2 binding, which triggers AuNP precipitation, thus leading to changes in the amount of AuNPs in solution resulting in light extinction. The optical extinction is subsequently digitized by a portable electronic detection (PED) system, comprised of inexpensive electronic components and circuitry, to assess the potency of nAbs. The assays disclosed herein can differentiate the neutralizing potency of different antibodies in buffer and serum samples, among other sample types, with a dynamic range of up to 4 logs (ng / ml- pg / ml) with femtomolar Limit of Detection (LoD). The modular nature of the assay design, rapid turn-around time (less than 20 minutes), portable and inexpensive (<$20) readout system, as well as low testing cost (~$2 cost of materials per test) make this nAb assay applicable to a wide variety of diagnostics such as vaccine potency, variant study, immunity assessment and re-infection prediction, and therapeutic applications.

[0051] II. Description of Example Embodiments

[0052] To illustrate aspects of the present disclosure, FIG. 1 provides a flow chart that schematically shows exemplary method steps of analyzing antibody binding. As shown, method 100 includes contacting a set of SARS-CoV-2 protein variant functionalized nanoparticles (MNPs) with a set of antibodies (step 102). Method 100 also includes contacting a set of ACE2 protein functionalized nanoparticles (MNPs) with the set of antibodies and a set of competition probes that comprise the SARS-CoV-2 protein variant (step 104). In addition, method 100 also includes detecting binding, if any, of the SARS-CoV-2 protein variant functionalized MNPs with the set of antibodies and binding, if any, of the ACE2 protein functionalized MNPs with the set of antibodies and the set of competition probes that comprise the SARS-CoV-2 protein variant (step 106).

[0053] In some embodiments, the SARS-CoV-2 protein variant comprises an RBD protein variant. In some embodiments, the SARS-CoV-2 protein variant comprises an S protein variant. In some embodiments, the set of antibodies comprises neutralizing antibodies. In some embodiments, the detecting step differentiates neutralizing antibody potency of different antibodies with a dynamic range of up to about 4 logs (ng / mL-pg / mL). In some embodiments, the detecting step differentiates neutralizing antibody potency of different antibodies with a femtomolar or lower Limit of Detection (LoD).

[0054] In some embodiments, a duration of method 100 is about 15 minutes or less. In some embodiments, In some embodiments, a sample comprising the set of antibodies comprises a volume of about 10 pL or less. In some embodiments, a sample comprising the set of antibodies comprises a sample type selected from the group consisting of: whole blood, serum, plasma, saliva, sputum, nasal fluid, cerebrospinal fluid, vaginal fluid, semen, and urine. In some embodiments, method 100 includes quantifying an amount of an antibody in the set of antibodies. In some embodiments, the detecting step comprises detecting one or more aggregations of the SARS-CoV-2 protein variant functionalized MNPs with the set of antibodies, and / or the ACE2 protein functionalized MNPs with the set of antibodies and the set of competition probes that comprise the SARS-CoV-2 protein variant. In some embodiments, method 100 includes obtaining a sample from a subject that comprises the set of antibodies. In some embodiments, method 100 includes administering one or more therapies to the subject when the binding of the SARS-CoV-2 protein variant functionalized MNPs with the set of antibodies, and / or the binding of the ACE2 protein functionalized MNPs with the set of antibodies and the set of competition probes that comprise the SARS-CoV-2 protein variant is detected in the sample.

[0055] In other aspects, the present disclosure provides a system for analyzing antibody binding. In some embodiments, the system includes a sample container receiving structure configured to receive a sample container that contains a set of SARS-CoV-2 protein variant functionalized nanoparticles (MNPs) with a set of antibodies, and / or a set of ACE2 protein functionalized nanoparticles (MNPs) with the set of antibodies and a set of competition probes that comprise the SARS-CoV-2 protein variant. In some embodiments, the system includes a light source operably connected to the sample container receiving structure, wherein the light source is configured to transmit light through the sample container, and a photodetector operably connected to the sample container receiving structure, wherein the photodetector is configured to measure light intensity transmitted through the sample container from the light source. In some embodiments, the system includes a controller operably connected to the lightsource and to the photodetector in which the controller is configured to effect transmission of the light through the sample container using the light source and measurement of the light intensity transmitted through the sample container that contains the set of SARS-CoV-2 protein variant functionalized MNPs with the set of antibodies, and / or the set of ACE2 protein functionalized MNPs with the set of antibodies and the set of competition probes that comprise the SARS-CoV-2 protein variant using the photodetector. In some embodiments, the system includes a power source operably connected to the controller, wherein the power source is configured to selectively supply power to the controller, and a housing structure operably connected to the sample container receiving structure, wherein the controller and power source are substantially housed in one or more cavities disposed within the housing structure.

[0056] In some embodiments, the controller is configured to quantify an amount of the antibody in the set of antibodies. In some embodiments, the SARS-CoV-2 protein variant comprises an RBD protein variant. In some embodiments, the SARS-CoV-2 protein variant comprises an S protein variant. In some embodiments, the set of antibodies comprises neutralizing antibodies. In some embodiments, the system differentiates neutralizing antibody potency of different antibodies with a dynamic range of up to about 4 logs (ng / mL-pg / mL). In some embodiments, the system differentiates neutralizing antibody potency of different antibodies with a femtomolar or lower Limit of Detection (LoD). In some embodiments, the sample container comprises sample having a volume of about 10 pL or less. In some embodiments, the sample container comprises a sample comprising the set of antibodies that comprises a sample type selected from the group consisting of: whole blood, serum, plasma, saliva, sputum, nasal fluid, cerebrospinal fluid, vaginal fluid, semen, and urine. In some embodiments, the controller is configured to detect one or more aggregations of the SARS-CoV-2 protein variant functionalized MNPs with the set of antibodies, and / or the ACE2 protein functionalized MNPs with the set of antibodies and the set of competition probes that comprise the SARS-CoV-2 protein variant. In some embodiments, kits comprise the systems of the present disclosure.

[0057] III. Examples

[0058] Example 1 : Nanoparticle-Supported, Rapid, Digital Quantification of anti-SARS-CoV-2 Neutralizing Antibodies Against COVID-19 Variants

[0059] Introduction

[0060] In this example, we develop a low-cost, widely accessible, rapid, sensitive, digital neutralizing assay (Fig. 2) that can distinguish the nAb potency against different SARS-CoV-2 variants and quantify the neutralizing ability of human serum without the need for authentic viruses. This modular diagnostic platform analyzes the immunity potency of samples against different variants of the SARS-CoV-2 virus within 20 minutes in a single test tube. It reports the results in a point-of-care (POC) setting using a semiconductor-based digital readout system. Specifically, this assay, a nanoparticle- supported rapid electronic detector for neutralizing antibody quantification (NasRED- nAb), is performed simply by mixing the target nAbs (or sera) with two testing reagents, i.e., ACE2 functionalized gold nanoparticles (ACE2-AuNPs) as the cell-mimicking signaling beacon and SARS-COV-2 S or RBD protein as the virus antigen, followed by an accelerated readout scheme. The quantity and quality of the target nAbs dictate the level of freely available S (or RBD) proteins that can bind to ACE2-AuNPs and, accordingly, the amount of clustering AuNPs. The readout signal is readily collected by probing the optical extinction of the free-floating ACE2-AuNPs using our PED29-31system made of simple electronic circuitry (Fig. 8). Here, we demonstrate this integrated and modular sensing platform can quantify the neutralizing ability of human serum and distinguish the potency of nAbs against the different variants of SARS-CoV-2.

[0061] Results

[0062] NasRED-nAb neutralizing and non-neutralizing antibodies differentiation

[0063] Our sensing platform utilizes electronic components and circuitry to collect, process, and transmit data of both total antibodies (tAbs) and nAbs measurements. The amount of free-floating AuNPs in a test tube serves as the readout beacons for both the tAbs or nAbs to be tested. The optical extinction due to the presence of AuNPs in solution is probed by a light emitting diode (LED) at their plasmonic resonance wavelength (~560 nm for 80 nm AuNPs) and collected by a photodetector. Both the LED and photodetector are mounted on a 3D printed chamber to minimize ambient noise and improve detection reproducibility and are designed with a supporting circuitryto produce stabilized electronic signals for minimized detection errors. The signals are digitized by an analog-to-digital converter (ADC) for signal processing and data analysis (Fig. 2a).

[0064] Based on a nanoparticle-supported, rapid electronic detection (NaSRED) and readout scheme, the total Ab assay (NaSRED-tAb) and the neutralization assay (NaSRED-nAb) differ in functionality. While NaSRED-tAb is based on the Ab binding to S protein (or RBD) (Fig. 2b), similar to our demonstrated antigen and antibody sensing methods, NaSRED-nAb analyzes the nAbs’ competition against ACE2 in binding to S protein (or RBD) (Fig. 2c). In NaSRED-tAb, AuNPs are coated with S or RBD proteins through a streptavidin-biotin reaction to create tAb Probes (tAbPs) (Fig. 8). Upon mixing with commercially available, high-affinity, RBD-binding antibodies, i.e. SAD-S35 and CR3022, the tAbPs bind to the antibodies and bridge into clusters, eventually precipitating to the bottom of the microcentrifuge tube. Subsequently, the amount of free-floating tAbPs and their plasmonic extinction signals decrease with the antibody concentration and affinity. The LoDs for the SAD-S35 and CR3022 were determined as ~3 and ~15 pM, respectively, due to their nearly identical binding affinity to the wild-type SARS-CoV-2 S protein RBD (Fig. 2b, 9) and found insensitive to incubation temperatures (tested under room temperature and 37°C, Fig. 10).

[0065] In NaSRED-nAb, in contrast, AuNPs are coated with ACE2 through a streptavidin-biotin reaction to create nAbs Probes (nAbPs). Uniquely, Competition Probes (CPs), created by binding biotinylated RBD or S proteins to streptavidin molecules, are also introduced into nAbPs solution. Upon mixing with nAbs, the CPs are neutralized and prevented from binding to the nAbPs, thus preserving the amount of free-floating nAbPs and their plasmonic extinction signals. Therefore, distinct from NaSRED-tAb, the AuNP extinction signals increase with the nAb concentration and potency. Differences in neutralization between the SAD-S35 and CR3022 indicate the former has an effective concentration (EC50) value of < 1nM against the Wuhan-1 (wild type) variant but the CR3022 is non-neutralizing (Fig. 2c, 13). The P384A mutation in the CR3022 epitope, found in the RBD of SARS-CoV-2 but not in SARS-CoV, impairs the neutralizing efficacy of CR3022 against SARS-CoV-2, despite its ability to bind to the RBD.

[0066] Detection of monoclonal antibodies (mAbs) by NaSRED-tAb.

[0067] We first tested performance of the NaSRED-tAb assay utilizing previously characterized high-potency (EC50 <10 ng / mL; SARS2-02, SARS2-71 , and SARS2-38) and low-potency (EC50 of 20-100 ng / mL SARS2-03, SARS2-10, and SARS2-31 ) neutralizing mAbs against SARS-CoV-2 variant antigens (RBD and S1 protein). The inhibitory activity of these mAbs, derived from immunized mice, had been determined using a focus reduction neutralization test (FRNT) on Vero cells with the WA1 -2020 isolate. Here, we used the mAbs to assess relative binding strength to different variant antigens (Wuhan-Hu-1 (Wild-type (or WT)), 20J / 501 Y.V3 (Gamma (or P1 )16and Omicron (BF.7 and BA.4.6) (Fig. 3). The colorimetric results of the naked eye detection for the tubes (Fig. 4a-d) established the ability of the NaSRED-tAb in distinguishing mAb binding to different variant antigens. To gain additional quantitative information, the tubes were analyzed by our electronic readout system (Fig. 2a). The electronic circuitry has stabilized LED illumination for reduced readout noise and highly sensitive detection. The results (Fig. 3e-h) showed a femtomolar sensitivity of NaSRED-tAb to quantify the mAbs over a broad (~ 7 Logs) detection range (26 nM for SARS2-31 against WT-RBD and 9 aM for SARS2-38 against WT-S1 (Table 3, and Fig. 11 ,12).

[0068] NaSRED-nAb sensor optimization

[0069] As a three-component competitive assay, including the nAbPs, nAbs, and CPs, the NaSRED-nAb reporter signals are dependent on the concentrations of both nAbPs and CPs. Therefore, the level of neutralization measured on NaSRED-nAb changes with the amount of antigens relative to the CPs for a given variant. This effect was studied from two experimentally designed scenarios for optimization of NASRED- nAb. In a Low Probe Mode (LPM), nAbPs were diluted to the minimum value to produce visible colors for experimental visualization (OD-0.4), and in a High Probe Mode (HPM), nAbs were at the maximum achievable concentration after AuNps functionalization with ACE2 (OD-0.8). CPs concentration at each mode was optimized relative to nAbPs concentration (HPM ~2 nM and LPM ~1 nM) to ensure aggregation of AuNps in the absence of nAbs. To evaluate the effect of probe density, we tested a few low potency neutralizing mAbs (SARS2-03 (Fig. 4b), SARS2-10 (Fig. 16), and SARS2-31 (Fig. 16) against WT-S1 in HPM and LPM scenarios. The factor of two in probes (nAbPs andCPs) difference produced a 10 to 20-fold difference in observed EC50-NasRED. Here the LPM produced EC50 values more consistent with those obtained from FRNT (Table 1). This showed that the NasRED-nAb assay could evaluate the antigen density effect, which can be clinically crucial to individualized patient treatment plans after infection. In our assay, this could be understood from the competitive binding mechanism of this multi-component assay, where the binding between multivalent nAbPs and CPs was influenced by the concentration, binding epitopes, and binding strengths of nAb to RBD or S protein.Table 1. Low Probe Mode is more consistent with FRNT. Comparison of the EC50 values for SARS2-03, SARS2-10, and SARS2-31 against WT-S1 in low and high probe modes and comparison with the Vero-TMPRSS2 FRNT data based on VanBlargan et al. (2021 )34(EC50 values adjusted to the molar ratio of RBD concentration for comparison)

[0070] NaSRED-nAb to detect neutralization capabilities of mAbs

[0071] We applied the NaSRED-nAb assay on high- and low-potency mAbs against different antigens (WT-RBD / S1 , P1 -RBD, and Omicron (BF.7 and BA.4.6)-RBD to evaluate the NaSRED-nAb discrimination capability between different variant antigens (Fig. 5). NaSRED-nAb measurements for high potency mAbs (SARS2-02, SARS2-38, and SARS2-71 ) against the WT-RBD and WT-P1 at HPM design showed decreased neutralization of the SARS-02 against the P1 variant followed by SARS2-38 and SARS2-71 , respectively (Fig. 5a, b). This can be attributed to the P1 -RBD mutation on 446-448 sites and is consistent with the previous reports. Results of NaSRED-nAb for high and low potency mAbs (SARS2-02, SARS2-38, SARS2-71 , SARS2-31 , SARS2-03, and SARS2-10) against the WT-S1 and Omicron-RBD at LPM also showed much lowerneutralization activity of both the high potency mAb (SARS2-38) and low potency mAb (SARS2-03) against the Omicron variant (Fig. 5c, d). This can be attributed to several mutations on Omicron RBD binding sites that allow neutralization escape. The EC50 values calculation demonstrated the unique capability of NaSRED-nAb to quantitatively discriminate neutralization of different mAbs against different variants (Table 2, Fig. 16- 17). Furthermore, we confirmed the accuracy of NaSRED-nAb EC50 calculations in serum by hACE2 Binding Competitive ELISA utilizing SARS2-71 spiked Human Pooled Serum (HPS) samples. NaSRED-nAb EC50 demonstrated close agreement (17.3 ng / ml) compared to the results (15.8 ng / ml) from hACE2 Binding Competitive ELISA (Fig.5e), making the platform a reliable tool for SARS-CoV-2 antibody analysis.Table 2. Quantitative differentiation of mAbs’ potency across variants and antigens by NaSRED-nAb EC50 values. Comparison between mAbs’ EC50 values against wild type,Gamma, and Omicron variants in moderate viral load design and with Vero-TMPRSS2 FRNT data based on VanBlargan et al. (2021 )34(EC50 values adjusted to the molar ratio of RBD concentration for comparison).

[0072] NaSRED-nAb and quantification of neutralizing activity of clinical serum samples

[0073] We next utilized the neutralizing assay to measure the human sera to evaluate the feasibility of NasRED-nAb to detect nAbs in a complex and relevant medium. First, nAb-spiked HPS samples were used to optimize key parameters such as centrifugation, vortexing speed, sequential readout protocol, and light extinction data interpretation, which are affected by the viscosity and color of different biological fluids (Fig. 14,15). Subsequently, we tested 40 (10 negative and 30 positive) ELISA-validated clinical sera samples from either vaccinated, infected, or healthy participants of Serosurvey 1 in fall 2021 (STUDY00014505 under approval by Arizona State University’s institutional review board, Table 4). The NaSRED-nAb measurement of these samples was matched to their corresponding values measured from hACE2 Binding Competitive ELISA by an exponential fitting, following an empirically derived calibration equation (NasRED_Cal = 118.x In The statisticalanalysis of the calibration for NaSRED measurements indicated a highly reliable model, as evidenced by an extremely small residual sum of squares (RSS = 2.0964 x 10-27) and a perfect R-squared (R2= 1) (Fig. 6b). The fitting allowed us to directly compare the NaSRED-obtained signals to hACE2 Binding Competitive ELISA measurements and the two technologies in neutralizing ability / immunity measurement of the clinical sera (NaSRED-Cal = 0.95xELISA + 8.46, R2= 0.9) (Fig. 6c). In contrast, the same analytical strategy did not show any agreement between the total IgG and hACE2 Binding Competitive ELISA measurements producing a non-correlative fitting as evidenced by model statistics (RSS=2279, / ?2= 0.44). This comparison confirmed the use of total IgG count is not a reliable measure for neutralization. Furthermore, testing the neutralizing capability of these sera showed that they had a much weaker protection against the Omicron variant, which is consistent with the immunity escape capability of this variant (Fig. 18).

[0074] NaSRED-nAb for longitudinal seroconversion studies against WT and Omicron RBD

[0075] To analyze the total level and neutralizing potency of specific antibodies in serum (seroreactivity) using NasRED platform, we applied NaSRED-tAb and NaSRED- nAb against WT-RBD and Omicron-RBD to sera samples collected from 10 individuals participating in Serosurvey 1 and 2 (Fall 2021 and 2022). All subjects were vaccinated against the wild-type variant before sample collection in fall 2021 and reported infection by Omicron based on the symptoms before the sample collection in fall 2022 (Table 5). For assay calibration, commercially available HPS was used as the Negative Control (NC), and 1 pM high-potency SARS2-38 spiked in HPS was used as the Positive Control (PC) samples and set as 100 % neutralization. The electronic readout signal for each patient sample was then normalized between 0-100% to quantify the neutralization capability. We showed that the NasRED-tAb levels were not significantly different against both the WT and Omicron variants (P value>0.05) (Fig. 7b), whereas the neutralization levels from the same patient samples did show a significant difference (P value <0.001 ). The results also show that the Omicron infection increased neutralization of Omicron variants by over 50% but also ‘back-boosted’ immunity against the wild-type variant (Fig. 7c), consistent with previous studies demonstrating imprinting3637. Further analysis of the results indicated the randomness of the total amount of virus-binding IgG levels and their temporal changes (Fig. 7d-e). For example, subject 53 showed a strong reaction to both WT and Omicron in the first survey (Fall 2021 ) (-100%), but the total IgG level was reduced significantly in the second survey (Fall 2022), particularly against the WT that almost became non-detectable. In contrast, the NasRED-nAb showed significantly increased (~80%) neutralizing capability in the second survey against both WT and Omicron variants. In summary, the neutralization results showed an overall significant increase in Serosurvey 2 (Fall 2022) (Fig. 7f-g) that was consistent with the infection.

[0076] Discussion

[0077] We demonstrate a modular neutralizing assay featuring plasmonic nanoparticles as a promising tool for evaluating the potency of the nAbs against different variants of the SARS-CoV-2 virus. This assay was shown to distinguish theneutralizing potency of different mAbs in buffer and serum samples of broad concentration ranges (up to 4 logs). Furthermore, our NaSRED-nAb assay, after calibration using hACE2 Binding Competitive ELISA data, quantifies the neutralization capability in manner consistent with a hACE2-spike binding competitive ELISA (R2= 0.9). By comparing competitive and total IgG ELISA measurements of the same patient samples, we demonstrated detection of total IgG could not reliably predict the level of immunity (J?2= 0.44). Similarly in the longitudinal study, significant neutralization differences (P value <0.001 ) were observed between post-vaccination and post- Omicron infection samples, while total IgG levels were not indicative (P value >0.05). Compared to FRNT analysis, NasRED is advantageous in its rapid turn-around time (less than 20 minutes), portable and electronic readout system, low manufacturing cost, and potential applicability to a wide variety of pathogens due to platform modularity. Future studies will be conducted to validate the application of our methodology on whole blood and other body fluid samples. In addition, further automation and integration of the sample preparation and handling processes into a compact portable system will make the methodology more accessible to the POC settings.

[0078] Materials and methods

[0079] Antigen and commercial antibody selection

[0080] Biotinylated WT-RBD (Wuhan-Hu-1 ), WT-S1 (Wuhan-Hu-1 ), Gamma (P1 - 20J / 501 Y.V3)-RBD Omicron-RBD (BF.7 and BA.4.6), ACE-2 were purchased (ACROBiosystems, 1 Innovation Way, Newark, DE 19711 ) for probe creation (sequences in the supplementary material). The standard neutralizing (SAD-S35) and non-neutralizing (CR3022) antibodies were purchased from ACROBiosystems and Absolute Antibody, Oxford, United Kingdom, respectively. The antibodies were used to evaluate the sensing methodology as the standard controls for neutralization potency.

[0081] Nanoparticle functionalization (RBD / S1 or ACE2)

[0082] Commercially purchased (Cytodiagnostics, 919 Fraser Dr Unit 11 , Burlington, ON L7L 4X8, Canada) streptavidin-functioned gold nanoparticles (-0.13 nM 80 nm AuNPs, 50 pL) were first mixed with an excess amount of biotinylated RBD or ACE2 (about 1 .2 pM, 20 pL). The mixtures were incubated for 2 h, and then purified by centrifuge (accuSpin Micro 17, Thermo Fisher) at 10,000 rpm for 10 min and repeatedtwice to remove unbound biotinylated biomarkers. The purified functionalized AuNP colloid was measured by PED to determine the concentration. The concentration of AuNPs was subsequently adjusted to desired optical extinction level (e.g., in our case -0.048 nM for 80 nm AuNPs) and was aliquoted into 18 pL in Eppendorf tubes as explained in our previous studies.

[0083] NaSRED-tAb and NaSRED-nAb

[0084] Commercial antibodies or mAb stock solutions (6 pM, in 1 x PBS) underwent a 10-Log serial dilution to target concentrations (400 fM to 4 pM) in selected detection media. All the buffers contained 1 x PBS, 20% v / v glycerol and 1 % wt BSA, pH~7.4, while the volume ratio of HPS (Human Pooled Serum), and clinical serum in their corresponding detection media was all 100%. This resulted in a final 25% HPS or clinical sera samples in the detection assay. For example, for commercial antibody NaSRED-tAb against WT-RBD, solutions of WT-RBD-functionalized AuNPs (tabPs) and antibodies were mixed at a ratio of 3:1 and thoroughly vortexed. As for the neutralizing assay for the same antigen-antibody pair, solutions of ACE2-functionalized AuNPs (nAbPs), WT-RBD CPs and nAbs were mixed at a ratio of 2:1 :1 and thoroughly vortexed. CPs of any given variant in the neutralizing assay were created by mixing the biotinylated viral antigen with streptavidin (Sigma-Aldrich) at a molar ratio of 3:1 and incubated at room temperature for 3 hours. For rapid detection, the assay solution was centrifuged (accuSpin Micro 17, Thermo Fisher) at 3,500 rpm (1 ,200xg) for 5 min, 20 min incubated, and vortexed (analog vortexer, Thermo Fisher) at 2250 RPM for 5 seconds prior to readout.

[0085] Electronic readout with rapid test

[0086] An electronic readout system similar to our previous studies29-31, consisted of three key components: a light-emitting diode (LED) light source, a photodiode, and a microcentrifuge tube holder. The centrifuge tube holder was 3D printed using ABSplus P430 thermoplastic to snuggly fit a standard 0.5 mL Eppendorf tube. Holes were opened on the microcentrifuge tube holder to align a LED (597-3311 -407NF, Dialight), the upper-level assay liquid, and a photodiode (SFH 2270R, Osram Opto Semiconductors). The LED was powered by two AA batteries (3 V) through a serially connected resistor. The photodiode was reversely biased by three AA batteries (4.5 V)and serially connected to a 7 MO load resistor, which converted the photocurrent to voltage output.

[0087] Data analysis

[0088] LOD was calculated such that the measured signal distinguishes from the reference signal by 1 .645 times the standard deviation of the reference and the lowest concentration sample. For the data analysis of the samples, a background signal calibration was performed. Each tube was filled with the buffer, and 12 measurements were collected, and averaged to establish the background absorption Aref=(i=1 to 12) as a reference. The same tubes were then used to hold the AuNP solutions antibody detection, during which 12 measurements were performed, and the absorption signals were averaged as Atest(C) =(j=i to 12) for each antibodyconcentration C. Therefore, the antibody signal was normalized as S(C) =Aref Atest(c)_ refThe error of the measurement is the standard deviation (SD) of the 12 normalized signals, or SD(C) = (j=ito 12). Furthermore, the limit of detection (LoD)in antibody concentration was calculated following S(LoD)=S(NC) -1 .645x(SD(NC) + SD(C_min)), where C_min is the lowest antibody or antigen concentration in the test. For Sera analysis the data analysis method reference was changed from buffer to AuNps mixture (See Figure 15 methodology).Table. 3. NaRED-tAb sensitivity for detection of different mAbs against various SARSCOV2 variants / antigensTable 4. Randomly selected sera samples from Serosurvey 1 and corresponding Total antibody and competitive ELISA assay results. The highlighted rows show the randomly selected samples for the NaSRED-nAb against the ORBD.Table 5. Chronological clinical sera sample information table. Samples were collected from the same individuals with identical conditions in September 2021 in Serosurvey 1 when all subjects had been vaccinated against the wildtype variant (Wuhan-Hu-1 ) and in October 2022 in Serosurvey 2 after the same individuals’ infection by the Omicron Variant (BF.7 & and BA.4.6). Pseudo ID has been assigned to individuals and Serum aliquot number for collected samples.

[0089] Protein sequences0090] Some further aspects are defined in the following clauses:

[0091] Clause 1 : A method of analyzing antibody binding, comprising: contacting a set of SARS-CoV-2 protein variant functionalized nanoparticles (MNPs) with a set of antibodies; contacting a set of ACE2 protein functionalized nanoparticles (MNPs) with the set of antibodies and a set of competition probes that comprise the SARS-CoV-2 protein variant; and, detecting binding, if any, of the SARS-CoV-2 protein variant functionalized MNPs with the set of antibodies and binding, if any, of the ACE2 protein functionalized MNPs with the set of antibodies and the set of competition probes that comprise the SARS-CoV-2 protein variant, thereby analyzing antibody binding.

[0092] Clause 2: The method of Clause 1 , wherein the SARS-CoV-2 protein variant comprises an RBD protein variant.

[0093] Clause 3: The method of Clause 1 or Clause 2, wherein the SARS-CoV-2 protein variant comprises an S protein variant.

[0094] Clause 4: The method of any one of the preceding Clauses 1 -3, wherein the set of antibodies comprises neutralizing antibodies.

[0095] Clause 5: The method of any one of the preceding Clauses 1 -4, wherein the detecting step differentiates neutralizing antibody potency of different antibodies with a dynamic range of up to about 4 logs (ng / mL-pg / mL).

[0096] Clause 6: The method of any one of the preceding Clauses 1 -5, wherein the detecting step differentiates neutralizing antibody potency of different antibodies with a femtomolar or lower Limit of Detection (LoD).

[0097] Clause 7: The method of any one of the preceding Clauses 1 -6, wherein a duration of the method is about 15 minutes or less.

[0098] Clause 8: The method of any one of the preceding Clauses 1 -7, wherein a sample comprising the set of antibodies comprises a volume of about 10 pL or less.

[0099] Clause 9: The method of any one of the preceding Clauses 1 -8, wherein a sample comprising the set of antibodies comprises a sample type selected from the group consisting of: whole blood, serum, plasma, saliva, sputum, nasal fluid, cerebrospinal fluid, vaginal fluid, semen, and urine.

[0100] Clause 10: The method of any one of the preceding Clauses 1 -9, comprising quantifying an amount of an antibody in the set of antibodies.

[0101] Clause 11 : The method of any one of the preceding Clauses 1 -10, wherein the detecting step comprises detecting one or more aggregations of the SARS-CoV-2 protein variant functionalized MNPs with the set of antibodies, and / or the ACE2 protein functionalized MNPs with the set of antibodies and the set of competition probes that comprise the SARS-CoV-2 protein variant.

[0102] Clause 12: The method of any one of the preceding Clauses 1 -1 1 , comprising obtaining a sample from a subject that comprises the set of antibodies.

[0103] Clause 13: The method of any one of the preceding Clauses 1 -12, comprising administering one or more therapies to the subject when the binding of the SARS-CoV- 2 protein variant functionalized MNPs with the set of antibodies, and / or the binding of the ACE2 protein functionalized MNPs with the set of antibodies and the set of competition probes that comprise the SARS-CoV-2 protein variant is detected in the sample.

[0104] Clause 14: A system for analyzing antibody binding, comprising: a sample container receiving structure configured to receive a sample container that contains a set of SARS-CoV-2 protein variant functionalized nanoparticles (MNPs) with a set of antibodies, and / or a set of ACE2 protein functionalized nanoparticles (MNPs) with the set of antibodies and a set of competition probes that comprise the SARS-CoV-2 protein variant; a light source operably connected to the sample container receiving structure, wherein the light source is configured to transmit light through the sample container; a photodetector operably connected to the sample container receiving structure, wherein the photodetector is configured to measure light intensity transmitted through the sample container from the light source; a controller operably connected to the light source and to the photodetector, wherein the controller is configured to effect transmission of the light through the sample container using the light source andmeasurement of the light intensity transmitted through the sample container that contains the set of SARS-CoV-2 protein variant functionalized MNPs with the set of antibodies, and / or the set of ACE2 protein functionalized MNPs with the set of antibodies and the set of competition probes that comprise the SARS-CoV-2 protein variant using the photodetector; a power source operably connected to the controller, wherein the power source is configured to selectively supply power to the controller; and, a housing structure operably connected to the sample container receiving structure, wherein the controller and power source are substantially housed in one or more cavities disposed within the housing structure.

[0105] Clause 15: The system of Clause 14, wherein the controller is configured to quantify an amount of the antibody in the set of antibodies.

[0106] Clause 16: The system of Clause 14 or Clause 15, wherein the SARS-CoV-2 protein variant comprises an RBD protein variant.

[0107] Clause 17: The system of any one of the preceding Clauses 14-16, wherein the SARS-CoV-2 protein variant comprises an S protein variant.

[0108] Clause 18: The system of any one of the preceding Clauses 14-17, wherein the set of antibodies comprises neutralizing antibodies.

[0109] Clause 19: The system of any one of the preceding Clauses 14-18, wherein the system differentiates neutralizing antibody potency of different antibodies with a dynamic range of up to about 4 logs (ng / mL-pg / mL).

[0110] Clause 20: The system of any one of the preceding Clauses 14-19, wherein the system differentiates neutralizing antibody potency of different antibodies with a femtomolar or lower Limit of Detection (LoD).[0011 1 ] Clause 21 : The system of any one of the preceding Clauses 14-20, wherein the sample container comprises sample having a volume of about 10 pL or less.

[0112] Clause 22: The system of any one of the preceding Clauses 14-21 , wherein the sample container comprises a sample comprising the set of antibodies that comprises a sample type selected from the group consisting of: whole blood, serum, plasma, saliva, sputum, nasal fluid, cerebrospinal fluid, vaginal fluid, semen, and urine.

[0113] Clause 23: The system of any one of the preceding Clauses 14-22, wherein the controller is configured to detect one or more aggregations of the SARS-CoV-2protein variant functionalized MNPs with the set of antibodies, and / or the ACE2 protein functionalized MNPs with the set of antibodies and the set of competition probes that comprise the SARS-CoV-2 protein variant.

[0114] Clause 24: A kit comprising the system of any one of the preceding Clauses 14-23.

[0115] Although this disclosure contains many specific embodiment details, these should not be construed as limitations on the scope of the subject matter or on the scope of what may be claimed, but rather as descriptions of features that may be specific to particular embodiments. Certain features that are described in this disclosure in the context of separate embodiments can also be implemented, in combination, in a single embodiment. Conversely, various features that are described in the context of a single embodiment can also be implemented in multiple embodiments, separately, or in any suitable sub-combination. Moreover, although previously described features may be described as acting in certain combinations and even initially claimed as such, one or more features from a claimed combination can, in some cases, be excised from the combination, and the claimed combination may be directed to a sub-combination or variation of a sub-combination.

[0116] Particular embodiments of the subject matter have been described. Other embodiments, alterations, and permutations of the described embodiments are within the scope of the following claims as will be apparent to those skilled in the art. While operations are depicted in the drawings or claims in a particular order, this should not be understood as requiring that such operations be performed in the particular order shown or in sequential order, or that all illustrated operations be performed (some operations may be considered optional), to achieve desirable results.

[0117] Accordingly, the previously described example embodiments do not define or constrain this disclosure. Other changes, substitutions, and alterations are also possible without departing from the spirit and scope of this disclosure.

Claims

WHAT IS CLAIMED IS:1 . A method of analyzing antibody binding, comprising: contacting a set of SARS-CoV-2 protein variant functionalized nanoparticles (MNPs) with a set of antibodies; contacting a set of ACE2 protein functionalized nanoparticles (MNPs) with the set of antibodies and a set of competition probes that comprise the SARS-CoV-2 protein variant; and, detecting binding, if any, of the SARS-CoV-2 protein variant functionalized MNPs with the set of antibodies and binding, if any, of the ACE2 protein functionalized MNPs with the set of antibodies and the set of competition probes that comprise the SARS- CoV-2 protein variant, thereby analyzing antibody binding.

2. The method of claim 1 , wherein the SARS-CoV-2 protein variant comprises an RBD protein variant.

3. The method of claim 1 , wherein the SARS-CoV-2 protein variant comprises an S protein variant.

4. The method of claim 1 , wherein the set of antibodies comprises neutralizing antibodies.

5. The method of claim 1 , wherein the detecting step differentiates neutralizing antibody potency of different antibodies with a dynamic range of up to about 4 logs (ng / mL-pg / mL).

6. The method of claim 1 , wherein the detecting step differentiates neutralizing antibody potency of different antibodies with a femtomolar or lower Limit of Detection (LoD).

7. The method of claim 1 , wherein a duration of the method is about 15 minutes or less.

8. The method of claim 1 , wherein a sample comprising the set of antibodies comprises a volume of about 10 pL or less.

9. The method of claim 1 , wherein a sample comprising the set of antibodies comprises a sample type selected from the group consisting of: whole blood, serum, plasma, saliva, sputum, nasal fluid, cerebrospinal fluid, vaginal fluid, semen, and urine.

10. The method of claim 1 , comprising quantifying an amount of an antibody in the set of antibodies.11 . The method of claim 1 , wherein the detecting step comprises detecting one or more aggregations of the SARS-CoV-2 protein variant functionalized MNPs with the set of antibodies, and / or the ACE2 protein functionalized MNPs with the set of antibodies and the set of competition probes that comprise the SARS-CoV-2 protein variant.

12. The method of claim 1 , comprising obtaining a sample from a subject that comprises the set of antibodies.

13. The method of claim 12, comprising administering one or more therapies to the subject when the binding of the SARS-CoV-2 protein variant functionalized MNPs with the set of antibodies, and / or the binding of the ACE2 protein functionalized MNPs with the set of antibodies and the set of competition probes that comprise the SARS- CoV-2 protein variant is detected in the sample.

14. A system for analyzing antibody binding, comprising: a sample container receiving structure configured to receive a sample container that contains a set of SARS-CoV-2 protein variant functionalized nanoparticles (MNPs)with a set of antibodies, and / or a set of ACE2 protein functionalized nanoparticles (MNPs) with the set of antibodies and a set of competition probes that comprise the SARS-CoV-2 protein variant; a light source operably connected to the sample container receiving structure, wherein the light source is configured to transmit light through the sample container; a photodetector operably connected to the sample container receiving structure, wherein the photodetector is configured to measure light intensity transmitted through the sample container from the light source; a controller operably connected to the light source and to the photodetector, wherein the controller is configured to effect transmission of the light through the sample container using the light source and measurement of the light intensity transmitted through the sample container that contains the set of SARS-CoV-2 protein variant functionalized MNPs with the set of antibodies, and / or the set of ACE2 protein functionalized MNPs with the set of antibodies and the set of competition probes that comprise the SARS-CoV-2 protein variant using the photodetector; a power source operably connected to the controller, wherein the power source is configured to selectively supply power to the controller; and, a housing structure operably connected to the sample container receiving structure, wherein the controller and power source are substantially housed in one or more cavities disposed within the housing structure.

15. The system of claim 14, wherein the controller is configured to quantify an amount of the antibody in the set of antibodies.

16. The system of claim 14, wherein the SARS-CoV-2 protein variant comprises an RBD protein variant.

17. The system of claim 14, wherein the SARS-CoV-2 protein variant comprises an S protein variant.

18. The system of claim 14, wherein the set of antibodies comprises neutralizing antibodies.

19. The system of claim 14, wherein the system differentiates neutralizing antibody potency of different antibodies with a dynamic range of up to about 4 logs (ng / mL-pg / mL).

20. The system of claim 14, wherein the system differentiates neutralizing antibody potency of different antibodies with a femtomolar or lower Limit of Detection (LoD).21 . The system of claim 14, wherein the sample container comprises sample having a volume of about 10 pL or less.

22. The system of claim 14, wherein the sample container comprises a sample comprising the set of antibodies that comprises a sample type selected from the group consisting of: whole blood, serum, plasma, saliva, sputum, nasal fluid, cerebrospinal fluid, vaginal fluid, semen, and urine.

23. The system of claim 14, wherein the controller is configured to detect one or more aggregations of the SARS-CoV-2 protein variant functionalized MNPs with the set of antibodies, and / or the ACE2 protein functionalized MNPs with the set of antibodies and the set of competition probes that comprise the SARS-CoV-2 protein variant.

24. A kit comprising the system of claim 14.

Images