Methods, devices, and related aspects for detecting african swine fever virus

Abstract

Provided herein are methods of detecting an African swine fever virus (ASFV) biomarker in a sample. The methods include contacting the sample with a plurality of plasmonic metal nanoparticles (MNPs) that are conjugated with a set of ASFV proteins, or fragments thereof, that comprise an epitope that binds to an anti-ASFV antibody in the sample to produce bound anti-ASFV antibodies, or contacting the sample with a plurality of MNPs that are conjugated with a set of antibodies, or antigen binding portions thereof, that binds to an epitope of an ASFV protein in the sample to produce bound ASFV proteins. The methods also include detecting the ASFV biomarker in the sample when aggregations of the bound anti-ASFV antibodies form with one another or when aggregations of the bound ASFV proteins form with one another. Related compositions, reaction mixtures, devices, kits, and systems are also provided.

Description

METHODS, DEVICES, AND RELATED ASPECTS FOR DETECTING AFRICAN SWINE FEVER VIRUSCROSS-REFERENCE TO RELATED APPLICATONS

[0001] This application claims priority to, and the benefit of, U.S. Provisional Patent Application Ser. Nos. 63 / 530,679, filed August 3, 2023 and 63 / 532,501 , filed August 14, 2023, the disclosures of which are incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under AFRI 2022- 67021-37013 awarded by USDA / NIFA. The government has certain rights in the invention.BACKGROUND

[0003] African swine fever (ASF) is a highly contagious and often lethal infectious disease of swine, for which there is currently no effective treatment or vaccine. Currently, strict control strategies remain the only effective way of ASF containment, typically replying on early disease detection through rapid field suspicion and laboratory diagnosis followed by slaughtering all affected or exposed swine herds. Therefore, ASF has serious socioeconomic impact, especially in countries that export live pigs, pork and / or products, as well as in countries where these products are important sources of diet. To date, ASF has already spread from sub-Saharan Africa to multiple countries in Africa, Europe, and Asia. For example, since the introduction of ASFV in 2018 in China, an estimated ~50% of all pigs were wiped out. Fortunately, currently the U.S. is still ASF-free. However, as the world’s third largest pig producer, with over 1 1 .5 million tons of pork produced per year, and the world’s second largest pork exporter, with exports in 2017 valued at 4.6 billion USD, the U.S. may face far-reaching economic consequences if ASF is introduced. Preparedness and prevention measures to avoid the introduction of ASF, and other animal diseases, is highly important to food security. Given the absence of treatment and vaccines, rapid and accurate ASF diagnostics is crucial to border inspection, surveillance, and outbreak containment.

[0004] Typical ASF diagnosis requires identification of animals that are or have previously been infected with the ASF virus (ASFV). Thus, an appropriate diagnosis involves the detection and identification of ASFV-specific nucleic acids (double-stranded DNA), antigens, and / or antibodies. Real time polymer chain reaction (PCR), although highly sensitive and considered the ‘gold standard’ test for diagnostics, is limited to lab use due to required instrument, personnel training, and long turnaround readout time. Additionally, a negative PCR result is inconclusive whenever ASFV is suspected circulating in a swine population. Rather, serological assays to detect ASFV-specific antibodies is necessary to identify survivors that can act as potential virus carriers, typically using enzyme-linked immunosorbent assays (ELISA). However, ELISA is also costly, time-consuming and instrument-demanding, therefore difficult to operate for field tests. The use of rapid and portable diagnostic tests in the field and next to a pen, where the infected animal populations are, would therefore facilitate earlier and more effective disease control. In addition, reduced cost and simplified operation will facilitate high- frequency deployment of such tests for large-volume and in-time surveillance, which is important to disrupting transmission chain and ASF containment. Currently, there is still a lack of low-cost yet accurate antibody or antigen tests that can be deployed to pen sides for surveillance purposes.SUMMARY

[0005] The present disclosure relates, in certain aspects, to ultra-sensitive plasmonic metal nanoparticle (MNP)-based colorimetric assays for portable African swine fever virus (ASFV) biomarker detection. In some embodiments, the MNP surfaces are functionalized with high affinity antibodies, nanobodies, and / or other antigen binding portions to ASFV protein-related biomarkers or functionalized with ASFV proteins, or fragments thereof, that include an epitope that binds to an anti-ASFV antibody biomarker. In these embodiments, the biomarkers generally induce the crosslinking of MNPs leading to large MNP aggregate formation and precipitation, resulting in a decrease of MNP monomer concentration in solution and accordingly, a visible change in solution transparency.

[0006] In some embodiments, the technology disclosed herein includes portable electronic sensors and related systems that can be used to detect ASFV in areas that are prevalent for the disease. This technology allows for rapid and sensitive detection of ASFV-specific antibodies and antigens, which facilitates the containment of outbreaks of ASF. In some embodiments, the technology works by using MNP-based assays, which employ different optical characteristics of different ASFV biomarkers and generate optically distinguishable signals that detect such biomarkers. Some embodiments, for example, include the use of the antigen (p72) or the antibody (anti-p72) of ASFV to form complexes that can generate electric readouts that are determined by a photo detection chamber. These readouts are also proportional with the concentration of antibody concentration, which allows for readouts of how much antigen or anti-ASFV antibody is in a given sample.

[0007] The methods disclosed herein are better than current methods because they are quick, simple, and low cost. Once incubated, for example, a sample can be easily put into a photo detection tube in which a readout can determine if there is ASFV antigens or anti-ASFV antibodies in a sample. Other exemplary attributes of the methods and related aspects of the present disclosure include achieving a limit of detection (LOD) that is in the picomolar to femtomolar range, which is 1 -3 logs more sensitive than, for example, an ELISA assay as well as an assay dynamic range of at least about 6 logs. In addition, the assays of the present disclosure typically only need small sample volumes (e.g., <7 pL per test) to produce a given readout and only take about 10 minutes or less per test to perform with no intervening washing step needed. In some embodiments, the assays are performed accurately by portable electronic readout using simple and low- cost electronic equipment with a total cost per test being typically less than $1 .

[0008] In one aspect, the present disclosure provides a method of detecting an African swine fever virus (ASFV) biomarker in a sample. In some embodiments, the method includes contacting the sample with a plurality of plasmonic metal nanoparticles (MNPs) that are conjugated with a set of ASFV proteins, or fragments thereof, that comprise an epitope that binds to an anti-ASFV antibody in the sample to produce bound anti-ASFV antibodies; or contacting the sample with a plurality of MNPs that are conjugated with a set of antibodies, or antigen binding portions thereof, that binds to anepitope of an ASFV protein in the sample to produce bound ASFV proteins; and, detecting the ASFV biomarker in the sample when one or more aggregations of the bound anti- ASFV antibodies form with one another or when one or more aggregations of the bound ASFV proteins form with one another.

[0009] In some embodiments, the other MNPs comprise silver, copper, aluminum, platinum, palladium, or the like. In some embodiments, the sets of antibodies, or antigen binding portions thereof, comprise nanobodies. In some embodiments, a given antibody, or antigen binding portions thereof, comprises a mass of between about 10 kDa and about 20 kDa (e.g., about 1 1 kDa, about 12 kDa, about 13 kDa, about 14 kDa, about 15 kDa, about 16 kDa, about 17 kDa, about 18 kDa, or about 19 kDa). In some embodiments, a given antibody, or antigen binding portions thereof, comprises an equilibrium dissociation constant (KD) on the order of between about 1 nM and about 100 nM (e.g., about 5 nM, about 10 nM, about 15 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, or about 90 nM). In some embodiments, the set of ASFV proteins comprises a p72 protein, a p30 protein, a p54 protein, and / or a pp62 protein.

[0010] In some embodiments, the detection step comprises determining a change in absorbance at a resonance wavelength of the MNPs. In some embodiments, the method includes quantifying an amount of the ASFV, the ASFV proteins and / or the anti- ASFV antibody in the sample. In some embodiments, the method further includes centrifuging the aggregations of the bound anti-ASFV antibodies or the aggregations of the bound ASFV proteins prior to and / or during the detecting step. In some embodiments, the method further includes agitating the aggregations of the bound anti-ASFV antibodies or the aggregations of the bound ASFV proteins to resuspend unbound MNPs prior to and / or during the detecting step. In some embodiments, the method further includes freezing the aggregations of the bound anti-ASFV antibodies or the aggregations of the bound ASFV proteins prior to the detecting step. In some embodiments, the method includes drop casting the aggregations of the bound anti-ASFV antibodies or the aggregations of the bound ASFV proteins prior to the detecting step. In some embodiments, the method includes obtaining the sample from a subject (e.g., a pig). In some embodiments, the method includes administering one or more therapies to thesubject when the ASFV biomarker is detected in the sample. In some embodiments, the method includes detecting the ASFV biomarker within about 20 minutes or less of obtaining the sample from the subject. In some embodiments, the sample is not processed prior to the contacting step. In some embodiments, the sample is disposed in a liquid buffer. In some embodiments, the liquid buffer comprises one or more of: a phosphate buffered saline (PBS) buffer, swine serum, swine blood, or another biological buffer used for protein or antibody analysis. In some embodiments, the method includes repeating the method using one or more longitudinal samples obtained from the subject (e.g., to monitor a course of therapy and / or progression of the ASFV disease over time).

[0011] Essentially any sample type is used in performing the methods disclosed herein. In some embodiments, for example, the sample comprises blood, plasma, or serum. In some embodiments, the sample comprises saliva or sputum. In some embodiments, the sample comprises urine.

[0012] In some embodiments, the detecting step comprises measuring an extinction from an MNP extinction wavelength, which extinction is indicative of a presence and quantity of the ASFV biomarker in the sample. In some embodiments, the detecting step comprises measuring a colorimetric change when the aggregations of the bound anti-ASFV antibodies form with one another or when the aggregations of the bound ASFV proteins form with one another. In some embodiments, the method includes visually detecting the colorimetric change when the aggregations of the bound anti-ASFV antibodies form with one another or when the aggregations of the bound ASFV proteins form with one another. In some embodiments, the method includes detecting the colorimetric change when the aggregations of the bound anti-ASFV antibodies form with one another or when the aggregations of the bound ASFV proteins form with one another using a spectrometer. In some embodiments, the method includes detecting the colorimetric change when the aggregations of the bound anti-ASFV antibodies form with one another or when the aggregations of the bound ASFV proteins form with one another using a device that is configured to electronically quantify an MNP suspension color. In some embodiments, a concentration of ASFV proteins or a concentration of anti-ASFV antibodies in the sample is about 15 nM or less. In some embodiments, a concentration of ASFV proteins or a concentration of anti-ASFV antibodies in the sample is about 100pM or less. In some embodiments, the MNPs comprise a substantially spherical shape. In some embodiments, the MNPs comprise a cross-sectional dimension of between about 10 nm and about 100 nm.

[0013] In another aspect, the present disclosure provides a device, comprising at least one reaction chamber or substrate comprising a plurality of plasmonic metal nanoparticles (MNPs) that are (i) conjugated with a set of African swine fever virus (ASFV) proteins, or fragments thereof, that comprise an epitope that binds to an anti-ASFV antibody in the sample to produce bound anti-ASFV antibodies and one or more aggregations of the bound anti-ASFV antibodies to produce a colorimetric change in the reaction chamber or on the substrate; or (ii) conjugated with a set of antibodies, or antigen binding portions thereof, that binds to an epitope of an ASFV protein in the sample to produce bound ASFV proteins and one or more aggregations of the bound ASFV proteins to produce a colorimetric change in the reaction chamber or on the substrate.

[0014] In another aspect, the present disclosure provides a reaction mixture, comprising: a sample comprising an anti-African swine fever virus (ASFV) antibody and / or an ASFV protein; and, a plurality of plasmonic metal nanoparticles (MNPs) that are (i) conjugated with a set of ASFV proteins, or fragments thereof, that comprise an epitope that binds to the anti-ASFV antibody in the sample to produce bound anti-ASFV antibodies; or (ii) conjugated with a set of antibodies, or antigen binding portions thereof, that binds to an epitope of the ASFV protein in the sample to produce bound ASFV proteins.

[0015] In another aspect, the present disclosure provides a composition, comprising: a plurality of plasmonic metal nanoparticles (MNPs) that are (i) conjugated with a set of anti-African swine fever virus (ASFV) proteins, or fragments thereof, that comprise an epitope that binds to an anti-ASFV antibody in the sample to produce bound anti-ASFV antibodies; or (ii) conjugated with a set of antibodies, or antigen binding portions thereof, that binds to an epitope of an ASFV protein in the sample to produce bound ASFV proteins.

[0016] In another aspect, the present disclosure provides a system, comprising: a device, comprising at least one reaction chamber or substrate comprising a plurality ofplasmonic metal nanoparticles (MNPs) that are (i) conjugated with a set of African swine fever virus (ASFV) proteins, or fragments thereof, that comprise an epitope that binds to an anti-ASFV antibody in the sample to produce bound anti-ASFV antibodies; or (ii) conjugated with a set of antibodies, or antigen binding portions thereof, that binds to an epitope of an ASFV protein in the sample to produce bound ASFV proteins; and, an electromagnetic radiation detection apparatus positioned, or positionable, within sufficient proximity to the device to detect one or more colorimetric changes produced in or on the reaction chamber or substrate when one or more aggregations of the bound anti-ASFV antibodies form with one another in or on the reaction chamber or substrate or when one or more aggregations of the bound ASFV proteins form with one another in or on the reaction chamber or substrate. In some embodiments, the electromagnetic radiation detection apparatus comprises a spectrometer; wherein the electromagnetic radiation detection apparatus comprises a microscope; and / or wherein the electromagnetic radiation detection apparatus comprises a light-emitting diode (LED) that transmits light into and / or through the reaction chamber or substrate and a photodetector that detects light from the reaction chamber or substrate.

[0017] In another aspect, the present disclosure provides a kit, comprising: a set of plasmonic metal nanoparticles (MNPs); and, a set of African swine fever virus (ASFV) proteins, or fragments thereof, that comprise an epitope that binds to an anti-ASFV antibody when present in a sample; or a set of antibodies, or antigen binding portions thereof, that binds to an epitope of an ASFV protein when present in a sample to produce bound ASFV proteins. In some embodiments, one or more components of the kit are disposed in one or more containers. In some embodiments, the MNPs are (i) conjugated to the set of ASFV proteins, or fragments thereof, or (ii) conjugated to the set of antibodies, or antigen binding portions thereof. In some embodiments, the set of ASFV proteins comprises a p72 protein, a p30 protein, a p54 protein, and / or a pp62 protein.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 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.

[0019] FIGS. 1A and 1 B show structural aspects of an African swine fever virus (ASFV). (a) shows a schematic view of an ASFV. (b) shows a transmission electron microscopy (TEM) image of an ASFV particle.

[0020] FIG. 2 is a flow chart that schematically shows exemplary method steps of detecting an ASFV biomarker in a sample according to some aspects disclosed herein.

[0021] FIGS. 3A-3D. Design concept portable electronic readout of ASF. (a,b) The sample preparation showing streptavidin-coated AuNPs react with biotinylated (a) antigens or (b) antibodies to form multivalent sensing probes. The detection step involves mixing the AuNP sensing probes with target antibodies or antigens in the target buffer or a titration of the swine sera samples. AuNP precipitation will occur due to the reaction, resulting in reduction of floating AuNPs. (c) Rapid Electronic Detection (RED) including condensation of probes at the bottom of the tube using centrifugation, followed by an incubation (10 min) to allow cross-linking, and vortexing to resuspend the non-reacting probes. The amount of floating AuNPs is characterized by using a portable electronic readout system, (d) The portable Electronic Readout (PED) comprises an integrated circuit board including LED, photodetector, battery connectors, and signal transmitters. The collected electronic signals are measured against the antibody and antigen concentrations for data analysis, to determine the detection limit and dynamic range.

[0022] FIGS. 4A-4G. Detection of anti-p72 and anti-p30 antibodies spiked in buffer and swine sera detection matrixes, (a) Schematic of p72 and p30 proteins attached onto AuNPs as the sensors, (b) Schematic showing commercially available anti- p72 and anti-p30 antibodies are spiked in negative ASF swine sera, together with ASF swine sera used as limited reference control and negative control, (c-f) Optical picturesof the testing tube samples in buffer (c,d) and swine sera (e,f). (g) Collected electronic signals when tested in serum (solid lines) and buffer (dotted lines) for anti-p72 (stars) and anti-p30 (triangles).

[0023] FIGS. 5A-5E. Antibody Detection of ASF-positive swine sera, (a) Schematic of p72 and p30 protein attached onto AuNPs as the sensors, (b) Schematic showing serially diluted ASF-positive swine sera, together with limited reference control and negative control. (c,d) Optical pictures of the testing tube samples, (e) Collected electronic signals from the ASF sera samples for anti-p72 (stars) and anti-p30 (triangles) showing the presence of 4 logs higher anti-p30 antibody in the tested samples than anti- p72.

[0024] FIGS. 6A-6F. Detection of p72 and p30 antigens spiked in buffer and swine sera detection matrix, (a) Schematic of anti-p72 and anti-p30 antibodies attached onto AuNPs as the sensors, (b) Schematic showing commercially available p72 and p30 proteins are spiked in buffer and negative ASF swine sera, together with ASF swine sera used as limited reference control and negative control, (c-d) Optical pictures of the test tube samples in buffer and p72 in swine sera (e). (f) Collected electronic signals when tested in serum (solid lines) and buffer (dotted lines) for p72 (stars) and p30 (triangles).

[0025] FIGS. 7A-7E. Antigen Detection of ASF-positive swine sera, (a) Schematic of anti-p72 and anti-p30 protein attached onto AuNPs as sensors, (b) Schematic showing serially diluted ASF-positive swine sera, together with semi-purified antigen and negative control. (c,d) Optical pictures of the testing tube samples, (e) Collected electronic signals from the ASF positive serum samples and semi-purified antigen measurements for p72 (stars) and p30 (triangles) showing the presence of over 4 logs higher amount of p30 in semi-purified samples.DEFINITIONS

[0026] 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.

[0027] 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 / or steps 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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).

[0032] Administer: As used herein, “administer” or “administering” a therapeutic agent (e.g., an immunological therapeutic agent) to a subject means to give, apply or bring 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.

[0033] African Swine Fever Virus: As used herein, the term “African swine fever virus” or “ASFV” is a large, double-stranded DNA virus in the Asfarviridae family. It is the causative agent of African swine fever (ASF). The virus causes a hemorrhagic fever with high mortality rates in domestic pigs. ASFV is not known to cause disease in humans.

[0034] 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.

[0035] Antigen Binding Portion-. As used herein, the term “antigen binding portion” refers to a portion of an antibody that specifically binds to a SARS-CoV-2 protein, such as a receptor-binding domain (RBD) protein, e.g., a molecule in which one or more immunoglobulin chains is not full length, but which specifically binds to a SARS-CoV-2 protein. 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 comprisingtwo 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 to form 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.

[0036] Bind. As used herein, “bind,” in the context of pathogen detection, refers to a state in which a first chemical structure (e.g., a pathogenic particle) is sufficiently associated a second chemical structure such that the association between the first and second chemical structures can be detected.

[0037] 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.

[0038] 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., an ASFV protein or an anti-ASFV antibody) and / or a pathogen (e.g., an ASFV particle) in a sample.

[0039] Epitope-. As used herein, “epitope” refers to the part of an antigen (e.g., an ASFV protein) to which an antibody and / or an antigen binding portion binds.

[0040] 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 for applicationdependent 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.

[0041] Sample: As used herein, “sample” means anything capable of being analyzed by the methods, devices, and / or systems disclosed herein.

[0042] 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 ASFV proteins) 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.

[0043] Subject: As used herein, “subject” refers to an animal, such as a mammalian species (e.g., pig, 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 havinga 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.”

[0044] System-. As used herein, "system" in the context of analytical instrumentation refers to a group of objects and / or devices that form a network for performing a desired objective.

[0045] 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

[0046] INTRODUCTION

[0047] In some aspects, the present disclosure shows the design and validation of a metal nanoparticle (MNP)-based assay platform, sometimes referred to herein as nanoparticle-supported rapid, electronic detection (NaSRED), and quantify its performance in ASFV diagnostics. In some embodiments, the NaSRED assay utilizes sets of MNPs of different optical characteristics to interrogate ASFV biomarkers and generates optically distinguishable signals that serve to detect such markers in a multiplexed fashion. As exemplified herein, the assays of the present disclosure can be used to detect ASFV antigens (e.g., p72, p30, etc.) and antibodies (e.g. anti-p72, anti- p30, etc.) in buffers and swine serum, and to determine the limit of detection (LOD) and dynamic range. The present disclosure also shows the use of portable and inexpensivesystems that can convert optical signals for electronic readout using simple electronic circuits. As additionally shown herein, rapid ASF detection can be performed in just a few minutes. These and other attributes will be apparent upon a complete review of this specification, including the accompanying figures.

[0048] AFRICAN SWINE FEVER VIRUS AND BIOMARKERS

[0049] ASF virus (ASFV) is a complex, large, icosahedral multi-enveloped DNA virus, which is -200 nm in diameter and constituted by complex multi-layered structures (FIGS. 1 A and 1 B). The viral particle contains more than 50 structural proteins, and nearly a hundred proteins have been identified on the target cells during ASFV infection. Both structural and infection-related proteins can regulate, inhibit and modulate virus replication, virus particle production and apoptosis. The major target cells of ASFV are swine monocyte-macrophages, but the virus is found in many other cells in the late phase of viral infection. The pathway of entry in target cells is a dynamin-dependent and clathrin- mediated macropinocytosis process, but exam mechanisms and the protein receptors remain unclear.

[0050] A recent structural study with up to 4.1 angstroms resolution showed that the ASFV capsid structure is built from 17,280 proteins, including one major p72 and four minor capsid proteins organized into pentasymmetrons and trisymmetrons. The p72 protein is both an important antigenic and immunogenic target. It is located on the surface of the viral capsid, and its monoclonal antibodies were shown to partially neutralize virulent ASFV isolates. In addition, the ASFV isolates are usually genetically distinguished by the sequence of the C-terminus in the VP72 gene in molecular epidemiological research. Besides p72, p54 and p30 (also denominated p32) are believed to be the structural proteins involved in viral entry and serve as important ASFV antigenic structural proteins. The p54 protein is found located in the lipid outer membrane of virions, and also located by immunoelectron microscopy in the replication factories of infected cells. In addition, p30 and p54 have been clearly identified as highly antigenic during infection. Furthermore, antibodies against all these three proteins are involved in virus neutralization, inhibiting the attachment (p72 and p54) and internalization (p30) of virus. However, despite the potential of p72, p54 and p30 as serodiagnosis targets, these ASFVproteins are not sufficient for antibody-mediated protection against different virus strains. It was found that p30 is more efficient than p54 for antibody detection by ELISA. Similar findings were recorded in a comparative study using the p30, p54 and the pp62 proteins as antigens in antibody detection tests. The three recombinant proteins (p54r, p30r and pp62r) were also found to be reactive to sera from infected pigs by immunoblotting (IB) and effective when used as antigens in ELISA. Nevertheless, the combined use of p54 and p30 proteins for serological diagnosis of ASFV could improve the sensitivity.

[0051] PORTABLE ASF DETECTION

[0052] A wide range of laboratory tests are available to detect ASFV genome, antigens or antibodies against the virus. Real time polymer chain reaction (rt-PCR) is considered the ‘gold standard’ for earlier detection of the disease due to its superior sensitivity, specificity, robustness and high-throughput application to detect the ASFV genome in different kinds of clinical samples from domestic pigs, wild boar and ticks. As there is no vaccine against ASF, antibody presence is always indicative of infection. Animals usually develop antibodies within the second week after infection and can test positive for both ASFV and antibodies simultaneously for at least two months. Samples from animals surviving this period are usually positive for ASFV-specific antibodies, but negative for ASFV and its genome. Therefore, whenever ASFV is suspected of circulating in a swine population, a negative PGR result is inconclusive. Rather, serological assays are necessary for surveillance allowing the detection of survivors that can act as potential virus carriers. Serological tests were particularly important during ASF eradication on the Iberian Peninsula and in Brazil. ELISA is widely used as the workhorse for large-scale serological studies in serum samples. However, both PCR and ELISA are also comparatively more costly, time-consuming, and instrument- and resource-demanding, therefore difficult to operate for field tests. PCR tests require nucleic acid extractions and thermocycling processes for nucleic acids amplification. ELISA requires a series of washing steps, hours of incubation prior to readout, and a readout system dependent on conversion of a chemical substrate.

[0053] Additionally, the logistics of sending samples to official laboratories can further delay the time elapsed between the clinical suspicion and laboratory confirmation.The use of pen-side tests for a first front-line diagnosis under field conditions would have the advantage of providing additional support to the clinical assessment in the first instance and could reduce the time needed to confirm the test results in secondary cases of the disease. In particular, rapid, easy-to-operate and low-cost diagnostic tests that can be conducted at a high frequency would facilitate in-time surveillance, which is important to disrupting transmission chain and virus containment. Loop-mediated isothermal amplification (LAMP) and CRISPR technologies could be a cheaper diagnostic alternative to PCR in genetic identification, but still lack field validation to prove their effectiveness in ASFV detection. Currently, only lateral flow assays (LFAs) are commercially available for the detection of antibodies or the viral antigen in blood for ASF. However, LFAs have a lower sensitivity compared to the rt-PCR or ELISA test, respectively, and should not be used alone particularly for antigen detection. To date, there is still a lack of low-cost yet accurate antibody or antigen tests that can be deployed to pen sides for surveillance purposes.

[0054] The NaSRED assays of the present disclosure make use of nanoscale particles to interrogate ASF-specific antigens (e.g., p72 and p30 proteins) and antibodies (e.g., anti-p72 and anti-p30) to achieve improved diagnostic power for early and widely assessable ASF detection. The NaSRED assays and platforms present have features that distinguish them from previous technologies. To illustrate, the NaSRED assays are quantitative and accurate, with a LOD in the picomolar to femtomolar range, which is at least 1 to 3 logs more sensitive than ELISA based assays. The assays dynamic range is about 6 logs. Further, NaSRED assays need only small sample volumes (<7 pL per test) and do not need any washing steps, thus significantly simplifying assay operation. The assays are designed for rapid detection within a few minutes (e.g., ~10 min), thus much shorter than ELISA (typically 4 to 6 hours) and even better than LFAs. Additionally, the assay readout can be performed accurately by portable electronic readout using simple and low-cost electronic elements, estimated to be <$20. Moreover, the per test cost is estimated to be on the order of <$1 , making it easily accessible for high-frequency penside testing.

[0055] To illustrate, FIG. 2 is a flow chart that schematically shows exemplary method steps of detecting an ASFV biomarker in a sample according to some aspectsdisclosed herein. As shown, method 200 includes contacting the sample with a plurality of plasmonic metal nanoparticles (MNPs) that are conjugated with a set of ASFV proteins (e.g., a p72 protein, a p30 protein, a p54 protein, a pp62 protein, etc.), or fragments thereof, that comprise an epitope that binds to an anti-ASFV antibody in the sample to produce bound anti-ASFV antibodies, or contacting the sample with a plurality of MNPs that are conjugated with a set of antibodies, or antigen binding portions thereof (e.g., monoclonal antibodies, nanobodies, etc.), that binds to an epitope of an ASFV protein in the sample to produce bound ASFV proteins (step 202). In addition, method 200 also includes detecting the ASFV biomarker in the sample when one or more aggregations of the bound anti-ASFV antibodies form with one another or when one or more aggregations of the bound ASFV proteins form with one another (step 204). Additional exemplary aspects of the present disclosure are shown in FIGS. 3A-3D.

[0056] In some embodiments, method 200 includes obtaining the sample from a subject, such as a pig. Typically, the sample is not processed prior to the contacting step (e.g., (step 202)). In some embodiments, the sample comprises blood, plasma, serum, saliva, sputum, or urine. In some embodiments, the sample is disposed in a liquid buffer (e.g., a phosphate buffered saline (PBS) buffer, swine serum, swine blood, or another biological buffer used for protein or antibody analysis). Optionally, method 200 includes administering one or more therapies to the subject when the ASFV biomarker is detected in the sample. In some embodiments, method 200 includes detecting the ASFV biomarker within about 20 minutes or less (e.g., 15 minutes or less, 10 minutes or less, 5 minutes or less, or 1 minute or less) of obtaining the sample from the subject. In some embodiments, method 200 includes repeating the method using one or more longitudinal samples obtained from the subject.

[0057] Method 200 includes various optional processing and / or detection embodiments. In some embodiments, for example, method 200 includes centrifuging the aggregations of the bound anti-ASFV antibodies or the aggregations of the bound ASFV proteins prior to and / or during the detecting step. In some embodiments, method 200 includes agitating the aggregations of the bound anti-ASFV antibodies or the aggregations of the bound ASFV proteins to resuspend unbound MNPs prior to and / or during the detecting step. In some embodiments, method 200 includes freezing theaggregations of the bound anti-ASFV antibodies or the aggregations of the bound ASFV proteins prior to the detecting step. In some embodiments, method 200 includes drop casting the aggregations of the bound anti-ASFV antibodies or the aggregations of the bound ASFV proteins prior to the detecting step.

[0058] Method 200 typically includes quantifying an amount of the ASFV, the ASFV proteins and / or the anti-ASFV antibody in the sample. In some embodiments, the detection step (step 204) comprises measuring an extinction from an MNP extinction wavelength, which extinction is indicative of a presence and quantity of the ASFV biomarker in the sample. In some embodiments, the detection step (step 204) comprises determining a change in absorbance at a resonance wavelength of the MNPs. In some embodiments, the detection step (step 204) comprises measuring a colorimetric change when the aggregations of the bound anti-ASFV antibodies form with one another or when the aggregations of the bound ASFV proteins form with one another. In some embodiments, method 200 includes visually detecting the colorimetric change when the aggregations of the bound anti-ASFV antibodies form with one another or when the aggregations of the bound ASFV proteins form with one another. In some embodiments, method 200 includes detecting the colorimetric change when the aggregations of the bound anti-ASFV antibodies form with one another or when the aggregations of the bound ASFV proteins form with one another using a device that is configured to electronically quantify an MNP suspension color.

[0059] As illustrated herein, the present disclosure shows the feasibility of testing the target p72 and p30 proteins and antibodies in centrifuge tubes in which gold nanoparticles (AuNPs, ~80 nm diameter) are surface-coated with streptavidin by first selfassembly thiolated carboxyl polyethylene glycol) linker via thiol-su If ide reaction and then functionalization of streptavidin via amine-carboxyl coupling by N-Hydroxysuccinimide / 1 - Ethyl-3-(3-dimethylaminopropyl) carbodiimide (NHS / EDC) chemistry. This chemistry ensures the reliable attachment of streptavidin on the AuNPs, which was found to be important for assay reproducibility in some embodiments. The mixing of biotinylated antigens or antibody creates antigen or antibody-bound AuNPs, which presents a reddish color in a microcentrifuge tube due to extinction from plasmonic resonance (e.g. at around 560 nm for 80 nm particles).

[0060] One potential rate-limiting factor is the MNP post-clustering precipitation time. AuNP clustering gradually leads to sedimentation (or precipitation) due to gravitational force. The sedimentation time is estimated by tsed= z / s ■ g, where z is the MNP precipitation distance ( / .e. height of the solution in microcentrifuge tube), s is sedimentation coefficient s =anc|gjsgravitational constant, pMis the MNPmass density and pwis buffer mass density. Assuming a solution height of 5 mm, we can estimate that tsedchanges from 38 hours for a single 80 nm AuNP to 1 .5 hours and 0.4 hour for a 400 nm and 800 nm AuNP clusters, respectively. This indicates that the large clusters are expected to precipitate given sufficient assay time, consistent with our experimental studies. It also reveals that MNP sedimentation can limit the assay speed. Accordingly, in some embodiments, a brief centrifugation step is introduced to minimize tsed. The basic principle is to use an extra brief centrifugation step (e.g. 3,500 rpm or 1 ,200 x g, 5 min) to concentrate the MNPs in its mixture with the analyte (antigens or antibodies) at the tube bottom, which serves to decrease the precipitation distance z from ~ 5 mm to an estimated a few pm. In some embodiments, the sample is incubated for a few minutes (around 10 min) and vortexed. This step is meant to re-suspend those AuNP monomers physically adsorbed at the tube bottom without strong chemical binding.

[0061] In some embodiments, to avoid the use of a laboratory-based spectrometer system for readout, a portable detector is used that electronically quantifies MNP suspension color for precise readout. In some embodiments, for example, a pair of low-cost LED and photodetector is attached to a 3D-printed microcentrifuge tube holder for miniaturized system integration (FIG. 3D). The basic working principle is very simple: a LED emits light at the MNP extinction wavelength, which is strongly absorbed and scattered by MNPs in the centrifuge tube, and the transmitted light will be then collected by a photodetector and read out as either the photodetector current or voltage on a serial resistor. In some embodiments, the device includes 3D-printed a black holder that snug- fits microcentrifuge tubes on the top and has windows to mount the LED and photodetector to its sides (FIG. 3D).

[0062] These embodiments have various advantages, including, for example, that the LEDs, photodetectors, as well other electronic components (such as batteries,resistors and ammeters, or voltmeters) are readily commercially available at very low cost. For example, at the current time, individual green LEDs can be purchased for as little as $0.50 each and photodetectors at $1.40 each. This can significantly lower the cost of the sensing system and make it easily accessible. The electronic readout system has very small footprint and weight. In addition, the electronic readout is much more accurate than bare-eye readout at low analyte concentration. Furthermore, the use of LED allows for the readout of analyte (e.g., p72 and anti-p72) signals at a particular narrow band without picking up interfering signals from other MNPs and is thus compatible with multiplexed detection. Moreover, the electronic readout data can be readily stored in computers or in an online database, thus automating data management.

[0063] Other characterization methods are also optionally used, including, for example, scanning electron microscopy (SEM) and dark field scattering imaging, among other techniques, can be applied for quantitative analysis in ASFV protein detection with less than one nM sensitivity and accuracy. In some embodiments, the present disclosure also demonstrates the feasibility of detecting ASFV protein in serum or other sample types with miniaturized portable UV-visible spectrometers for point-of-care detection. In some embodiments, for example, the MNP solution-based colorimetric assays and other aspects disclosed herein provide ultra-high sensitivity, low cost and electricity free colorimetric detection, which can be readily utilized for point-of-care detection of ASFV in remote pandemic regions.

[0064] EXAMPLES

[0065] EXAMPLE 1 : ASF ANTIBODY DETECTION

[0066] In the present example, we used a spherical AuNP based NaSRED assay for ASF antibody detection (FIGS. 4A-4G and FIGS. 5A-5E) and assessed the assay performance. The p72 protein was purchased from commercial vendors and biotinylated using a commercial biotinylating kit.

[0067] In the first test (FIG. 4A-4G), commercially purchased antibodies (Creative Biolabs, SUITE 203, 17 Ramsey Road, Shirley, NY 11967, USA) were spiked in swine sera (negative from ASF from Spain lab) at a concentration from 100 nM to 1 fM, and used to mimic swine sera infected by ASF. Such spiked sample allowed us to quantifythe sensor detection limit. In this test, limited reference control (LRC) sample and negative swine sera (NC) were also tested. The LRC served to compare our performance to other tests, such as ELISA, and the NC sample was used as a reference. Our analysis showed a detectable signal for the LRC compared to the NC, which means the NasRED is capable of detection more sensitively than ELISA (FIG. 4G). Furthermore, the ELISA determined negative samples were shown positive for Anti-p30 in the NaSRED assay which demonstrates the specificity of the NaSRED to target. All the tests were completed in 10 min. For testing in buffer, a centrifugation (3500 round per minute (rpm), or about 1200 g, for 5 min) was performed, followed by 10 min incubation, and a brief vortex mixing (1650 rpm for 5 seconds). For testing in sera, a centrifugation at slightly higher speed (4000 RPM ~ 1600 g, for 5 min) was performed, followed by 10 min incubation, and a brief vortex mixing (1800 rpm for 10 seconds).

[0068] Data analysis: For testing in buffer, 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(j=i to 12) as areference. The same tubes were then used to hold the AuNP solutions for antigen or antibody detection, during which 12 measurements were performed, and the absorption signals were averaged as to 12) for each antigen or antibodyconcentration C. Therefore, the antigen or antibody signal was normalized as 5(C) =The error of the measurement is the standard deviation (SD) of the 12 ref normalized signals, or 5D(C) = ( i =1 to 12). Furthermore, the limit ofdetection (LoD) in antigen / antibody concentration was calculated following S(LoD~) = S(NC) -1.645 x (5£) (NC) + SD ( Cmin)), where Cminis the lowest antibody or antigen concentration in the test.

[0069] Using the above analysis, we determined that the LoD of our NasRED sensor for ASF p72 and p30 antibody is about 2 fM and 15 fM, respectively, when spiked in buffer, and 8 fM when anti-p72 was spiked in swine sera. This could be attributed to the difference of swine sera colors across the samples, which could have caused higherreadout error for anti-p72 spiked swine sera. In addition, the dynamic range of the assay is found about 6 logs (FIG. 4G).

[0070] In the second test, we also performed testing of ASF-positive swine sera (no live virus, but with ASF proteins) (FIGS. 5A-5E). The purpose of this test is to determine the titer that can be evaluated by our NasRED assay. In this test, we serially diluted the swine sera by 7 logs, and measured the signals from all these samples, together with the LRF and NC samples. The results show that the detection method is capable of discrimination down to 4 logs of titration for Anti-P72 antibody and down to 8 logs of titration for Anti-p30 in real samples. Furthermore, positive samples regardless of titration factor indicated detectable signal as compared to the negative control, which is suitable for qualitative assay, however, quantification requires more investigation (FIG. 5E).

[0071] EXAMPLE 2: ASF ANTIGEN DETECTION

[0072] To prove the concept of antigen sensing, we have purchased anti-p72 antibodies and biotinylated them in our lab, and then mixed with AuNPs, followed by filtration to remove excessive antibodies. This creates the antibody-rendered AuNPs for antigen detection.

[0073] First, we analyzed commercial p72 and p30 antigens spiked at different concentrations in swine sera (FIGS. 6A-6F). The antigen tests were carried out by mixing the p72 or p30 antigens with the anti-p72 or anti-p30 conjugated AuNPs in centrifuge tubes. As expected, the antibody-antigen binding resulted in subsequent bridging of AuNP monomers to larger aggregates. As a result, higher antigen concentrations would result in smaller concentration of AuNP monomers in the supernatant and a clearer color. The experimental details, including speed and time for centrifugation, incubation and vortex mixing, and the data analysis methods, including calibration and LoD determination, were identical to what we described earlier in the antibody sensing section.

[0074] The antigen sensing results show about 7 logs in dynamic range when tested in buffer and about 5 logs in swine sera. The LoD was found to be 184 fM and 330 fM for p30 and p72, respectively, when tested in buffer and about 1 .7 pM for p72 in swine sera. The LRC served to compare our performance to other tests, such as ELISA, andthe NC sample was used as a reference. The analysis showed a detectable signal for the LRC compared to the NC which means the methodology is capable of detection more sensitively than ELISA method (FIG. 6F). The overall LOD for p72 and p30 antigen is significantly higher than p72 and p30 antibody detection. This can be attributed to the fact that the polyclonal antibody was used for p72 and p30 antigen sensing and they may have lower affinity towards the p72 and p30 antigen. In addition, their biotinylating is not site-specific, and therefore the binding epitopes of the polyclonal antibodies to the p72 and p30 antigens may not be accessible on the AuNPs. However, using polyclonal antibodies provided diverse binding sites against p30 which made its detection possible. As the assay is based on aggregation, the analyte has to be either comprising identical binding sites at different chains (as in p72 trimer) or targeted at different binding sites utilizing a variety of binders on the probes. The advantage of utilizing p30 polyclonal antibody in this assay is to provide multiple binding sites to p30 making the detection of monomer p30 antigen possible eliminating the need for multiple probes.

[0075] Further, we tested ASF-positive swine sera for p72 and p30 antigen (FIGS. 7A-7E). Clearly, ASF positive samples could be differentiated for their antigen content from the NC sample across a large dynamic range (10 logs). The NasRED readout for the Semi-Purified antigen was in the range of 7 logs and 3 logs in titration factor for p72 and p30, respectively. This may be due to the effect of impurities present in the sample due to purification method and the fact that P72 only represents a small portion of many ASF antigens present in the mixture of semi-purified antigen samples while p30 to be higher than p72 by 4 logs in the semi-purified antigen samples (FIG. 7E).

[0076] Overall, the p72 and p30 antigens obtained from swine sample showed a better detection by NasRED compared to p72 and p30 antigens from commercial manufacturers, which could be attributed to the presence of the porcine serum albumin from the swine samples instead of the bovine serum albumin (BSA) in commercial buffer. BSA is known to possibly interfere with the ASF antigens because of the presence of the anti-BSA antibody in swine sera due to the natural immune response of the swine immune system against the BSA containing products.

[0077] Some further aspects are defined in the following clauses:

[0078] Clause 1 : A method of detecting an African swine fever virus (ASFV) biomarker in a sample, the method comprising: contacting the sample with a plurality of plasmonic metal nanoparticles (MNPs) that are conjugated with a set of ASFV proteins, or fragments thereof, that comprise an epitope that binds to an anti-ASFV antibody in the sample to produce bound anti-ASFV antibodies; or, contacting the sample with a plurality of MNPs that are conjugated with a set of antibodies, or antigen binding portions thereof, that binds to an epitope of an ASFV protein in the sample to produce bound ASFV proteins; and, detecting the ASFV biomarker in the sample when one or more aggregations of the bound anti-ASFV antibodies form with one another or when one or more aggregations of the bound ASFV proteins form with one another.

[0079] Clause 2: The method of Clause 1 , wherein the set of antibodies, or the antigen binding portions thereof, comprise nanobodies.

[0080] Clause 3: The method of Clause 1 or Clause 2, wherein the set of ASFV proteins comprises a p72 protein, a p30 protein, a p54 protein, and / or a pp62 protein.

[0081] Clause 4: The method of any one of the preceding Clauses 1 -3, wherein the detection step comprises determining a change in absorbance at a resonance wavelength of the MNPs.

[0082] Clause 5: The method of any one of the preceding Clauses 1 -4, comprising quantifying an amount of the ASFV, the ASFV proteins and / or the anti-ASFV antibody in the sample.

[0083] Clause 6: The method of any one of the preceding Clauses 1 -5, further comprising centrifuging the aggregations of the bound anti-ASFV antibodies or the aggregations of the bound ASFV proteins prior to and / or during the detecting step.

[0084] Clause 7: The method of any one of the preceding Clauses 1 -6, further comprising agitating the aggregations of the bound anti-ASFV antibodies or the aggregations of the bound ASFV proteins to resuspend unbound MNPs prior to and / or during the detecting step.

[0085] Clause 8: The method of any one of the preceding Clauses 1 -7, further comprising freezing the aggregations of the bound anti-ASFV antibodies or theaggregations of the bound ASFV proteins prior to the detecting step.

[0086] Clause 9: The method of any one of the preceding Clauses 1 -8, comprising drop casting the aggregations of the bound anti-ASFV antibodies or the aggregations of the bound ASFV proteins prior to the detecting step.

[0087] Clause 10: The method of any one of the preceding Clauses 1 -9, comprising obtaining the sample from a subject.

[0088] Clause 1 1 : The method of any one of the preceding Clauses 1 -10, wherein the subject is a pig.

[0089] Clause 12: The method of any one of the preceding Clauses 1 -1 1 , wherein the sample is not processed prior to the contacting step.

[0090] Clause 13: The method of any one of the preceding Clauses 1 -12, wherein the sample is disposed in a liquid buffer.

[0091] Clause 14: The method of any one of the preceding Clauses 1 -13, wherein the liquid buffer comprises one or more of: a phosphate buffered saline (PBS) buffer, swine serum, swine blood, or another biological buffer used for protein or antibody analysis.

[0092] Clause 15: The method of any one of the preceding Clauses 1 -14, comprising administering one or more therapies to the subject when the ASFV biomarker is detected in the sample.

[0093] Clause 16: The method of any one of the preceding Clauses 1 -15, comprising detecting the ASFV biomarker within about 20 minutes or less of obtaining the sample from the subject.

[0094] Clause 17: The method of any one of the preceding Clauses 1 -16, comprising repeating the method using one or more longitudinal samples obtained from the subject.

[0095] Clause 18: The method of any one of the preceding Clauses 1 -17, wherein the sample comprises blood, plasma, serum, saliva, sputum, or urine.

[0096] Clause 19: The method of any one of the preceding Clauses 1 -18, whereinthe detecting step comprises measuring an extinction from an MNP extinction wavelength, which extinction is indicative of a presence and quantity of the ASFV biomarker in the sample.

[0097] Clause 20: The method of any one of the preceding Clauses 1 -19, wherein the detecting step comprises measuring a colorimetric change when the aggregations of the bound anti-ASFV antibodies form with one another or when the aggregations of the bound ASFV proteins form with one another.

[0098] Clause 21 : The method of any one of the preceding Clauses 1 -20, comprising visually detecting the colorimetric change when the aggregations of the bound anti-ASFV antibodies form with one another or when the aggregations of the bound ASFV proteins form with one another.

[0099] Clause 22: The method of any one of the preceding Clauses 1 -21 , comprising detecting the colorimetric change when the aggregations of the bound anti- ASFV antibodies form with one another or when the aggregations of the bound ASFV proteins form with one another using a device that is configured to electronically quantify an MNP suspension color.

[0100] Clause 23: A device, comprising at least one reaction chamber or substrate comprising a plurality of plasmonic metal nanoparticles (MNPs) that are (i) conjugated with a set of African swine fever virus (ASFV) proteins, or fragments thereof, that comprise an epitope that binds to an anti-ASFV antibody in the sample to produce bound anti-ASFV antibodies and one or more aggregations of the bound anti-ASFV antibodies to produce a colorimetric change in the reaction chamber or on the substrate; or (ii) conjugated with a set of antibodies, or antigen binding portions thereof, that binds to an epitope of an ASFV protein in the sample to produce bound ASFV proteins and one or more aggregations of the bound ASFV proteins to produce a colorimetric change in the reaction chamber or on the substrate.

[0101] Clause 24: A reaction mixture, comprising: a sample comprising an antiAfrican swine fever virus (ASFV) antibody and / or an ASFV protein; and, a plurality of plasmonic metal nanoparticles (MNPs) that are (i) conjugated with a set of ASFV proteins, or fragments thereof, that comprise an epitope that binds to the anti-ASFV antibody in thesample to produce bound anti-ASFV antibodies; or (ii) conjugated with a set of antibodies, or antigen binding portions thereof, that binds to an epitope of the ASFV protein in the sample to produce bound ASFV proteins.

[0102] Clause 25: A composition, comprising: a plurality of plasmonic metal nanoparticles (MNPs) that are (i) conjugated with a set of anti-African swine fever virus (ASFV) proteins, or fragments thereof, that comprise an epitope that binds to an anti- ASFV antibody in the sample to produce bound anti-ASFV antibodies; or (ii) conjugated with a set of antibodies, or antigen binding portions thereof, that binds to an epitope of an ASFV protein in the sample to produce bound ASFV proteins.

[0103] Clause 26: A system, comprising: a device, comprising at least one reaction chamber or substrate comprising a plurality of plasmonic metal nanoparticles (MNPs) that are (i) conjugated with a set of African swine fever virus (ASFV) proteins, or fragments thereof, that comprise an epitope that binds to an anti-ASFV antibody in the sample to produce bound anti-ASFV antibodies; or (ii) conjugated with a set of antibodies, or antigen binding portions thereof, that binds to an epitope of an ASFV protein in the sample to produce bound ASFV proteins; and, an electromagnetic radiation detection apparatus positioned, or positionable, within sufficient proximity to the device to detect one or more colorimetric changes produced in or on the reaction chamber or substrate when one or more aggregations of the bound anti-ASFV antibodies form with one another in or on the reaction chamber or substrate or when one or more aggregations of the bound ASFV proteins form with one another in or on the reaction chamber or substrate.

[0104] Clause 27: The system of Clause 26, wherein the electromagnetic radiation detection apparatus comprises a spectrometer; wherein the electromagnetic radiation detection apparatus comprises a microscope; and / or wherein the electromagnetic radiation detection apparatus comprises a light-emitting diode (LED) that transmits light into and / or through the reaction chamber or substrate and a photodetector that detects light from the reaction chamber or substrate.

[0105] Clause 28: A kit, comprising: a set of plasmonic metal nanoparticles (MNPs); and, a set of African swine fever virus (ASFV) proteins, or fragments thereof, that comprise an epitope that binds to an anti-ASFV antibody when present in a sample;or a set of antibodies, or antigen binding portions thereof, that binds to an epitope of an ASFV protein when present in a sample to produce bound ASFV proteins.

[0106] Clause 29: The kit of Clause 28, wherein one or more components of the kit are disposed in one or more containers.

[0107] Clause 30: The kit of Clause 28 or Clause 29, wherein the MNPs are (i) conjugated to the set of ASFV proteins, or fragments thereof, or (ii) conjugated to the set of antibodies, or antigen binding portions thereof.

[0108] Clause 31 : The kit of any one of the preceding Clauses 28-30, wherein the set of ASFV proteins comprises a p72 protein, a p30 protein, a p54 protein, and / or a pp62 protein.

[0109] While the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be clear to one of ordinary skill in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the disclosure and may be practiced within the scope of the appended claims. For example, all the methods, kits, reaction mixtures, devices, and / or systems or other aspects thereof can be used in various combinations. All patents, patent applications, websites, other publications or documents, and the like cited herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference.

Claims

WHAT IS CLAIMED IS:1 . A method of detecting an African swine fever virus (ASFV) biomarker in a sample, the method comprising: contacting the sample with a plurality of plasmonic metal nanoparticles (MNPs) that are conjugated with a set of ASFV proteins, or fragments thereof, that comprise an epitope that binds to an anti-ASFV antibody in the sample to produce bound anti-ASFV antibodies; or, contacting the sample with a plurality of MNPs that are conjugated with a set of antibodies, or antigen binding portions thereof, that binds to an epitope of an ASFV protein in the sample to produce bound ASFV proteins; and, detecting the ASFV biomarker in the sample when one or more aggregations of the bound anti-ASFV antibodies form with one another or when one or more aggregations of the bound ASFV proteins form with one another.

2. The method of claim 1 , wherein the set of antibodies, or the antigen binding portions thereof, comprise nanobodies.

3. The method of claim 1 , wherein the set of ASFV proteins comprises a p72 protein, a p30 protein, a p54 protein, and / or a pp62 protein.

4. The method of claim 1 , wherein the detection step comprises determining a change in absorbance at a resonance wavelength of the MNPs.

5. The method of claim 1 , comprising quantifying an amount of the ASFV, the ASFV proteins and / or the anti-ASFV antibody in the sample.

6. The method of claim 1 , further comprising centrifuging the aggregations of the bound anti-ASFV antibodies or the aggregations of the bound ASFV proteins prior to and / or during the detecting step.

7. The method of claim 1 , further comprising agitating the aggregations of the bound anti-ASFV antibodies or the aggregations of the bound ASFV proteins to resuspend unbound MNPs prior to and / or during the detecting step.

8. The method of claim 1 , further comprising freezing the aggregations of the bound anti-ASFV antibodies or the aggregations of the bound ASFV proteins prior to the detecting step.

9. The method of claim 1 , comprising drop casting the aggregations of the bound anti-ASFV antibodies or the aggregations of the bound ASFV proteins prior to the detecting step.

10. The method of claim 1 , comprising obtaining the sample from a subject.11 . The method of claim 10, wherein the subject is a pig.

12. The method of claim 10, wherein the sample is not processed prior to the contacting step.

13. The method of claim 10, wherein the sample is disposed in a liquid buffer.

14. The method of claim 13, wherein the liquid buffer comprises one or more of: a phosphate buffered saline (PBS) buffer, swine serum, swine blood, or another biological buffer used for protein or antibody analysis.

15. The method of claim 10, comprising administering one or more therapies to the subject when the ASFV biomarker is detected in the sample.

16. The method of claim 10, comprising detecting the ASFV biomarker within about 20 minutes or less of obtaining the sample from the subject.

17. The method of claim 10, comprising repeating the method using one or more longitudinal samples obtained from the subject.

18. The method of claim 10, wherein the sample comprises blood, plasma, serum, saliva, sputum, or urine.

19. The method of claim 1 , wherein the detecting step comprises measuring an extinction from an MNP extinction wavelength, which extinction is indicative of a presence and quantity of the ASFV biomarker in the sample.

20. The method of claim 1 , wherein the detecting step comprises measuring a colorimetric change when the aggregations of the bound anti-ASFV antibodies form with one another or when the aggregations of the bound ASFV proteins form with one another.21 . The method of claim 20, comprising visually detecting the colorimetric change when the aggregations of the bound anti-ASFV antibodies form with one another or when the aggregations of the bound ASFV proteins form with one another.

22. The method of claim 20, comprising detecting the colorimetric change when the aggregations of the bound anti-ASFV antibodies form with one another or when the aggregations of the bound ASFV proteins form with one another using a device that is configured to electronically quantify an MNP suspension color.

23. A device, comprising at least one reaction chamber or substrate comprising a plurality of plasmonic metal nanoparticles (MNPs) that are (i) conjugated with a set of African swine fever virus (ASFV) proteins, or fragments thereof, that comprise an epitope that binds to an anti-ASFV antibody in the sample to produce bound anti-ASFV antibodies and one or more aggregations of the bound anti-ASFV antibodies to produce a colorimetric change in the reaction chamber or on the substrate; or (ii) conjugated with a set of antibodies, or antigen binding portions thereof, that binds to an epitope of anASFV protein in the sample to produce bound ASFV proteins and one or more aggregations of the bound ASFV proteins to produce a colorimetric change in the reaction chamber or on the substrate.

24. A reaction mixture, comprising: a sample comprising an anti-African swine fever virus (ASFV) antibody and / or an ASFV protein; and, a plurality of plasmonic metal nanoparticles (MNPs) that are (i) conjugated with a set of ASFV proteins, or fragments thereof, that comprise an epitope that binds to the anti-ASFV antibody in the sample to produce bound anti-ASFV antibodies; or (ii) conjugated with a set of antibodies, or antigen binding portions thereof, that binds to an epitope of the ASFV protein in the sample to produce bound ASFV proteins.

25. A composition, comprising: a plurality of plasmonic metal nanoparticles (MNPs) that are (i) conjugated with a set of anti-African swine fever virus (ASFV) proteins, or fragments thereof, that comprise an epitope that binds to an anti-ASFV antibody in the sample to produce bound anti-ASFV antibodies; or (ii) conjugated with a set of antibodies, or antigen binding portions thereof, that binds to an epitope of an ASFV protein in the sample to produce bound ASFV proteins.

26. A system, comprising: a device, comprising at least one reaction chamber or substrate comprising a plurality of plasmonic metal nanoparticles (MNPs) that are (i) conjugated with a set of African swine fever virus (ASFV) proteins, or fragments thereof, that comprise an epitope that binds to an anti-ASFV antibody in the sample to produce bound anti-ASFV antibodies; or (ii) conjugated with a set of antibodies, or antigen binding portions thereof, that binds to an epitope of an ASFV protein in the sample to produce bound ASFV proteins; and, an electromagnetic radiation detection apparatus positioned, or positionable, within sufficient proximity to the device to detect one or more colorimetric changesproduced in or on the reaction chamber or substrate when one or more aggregations of the bound anti-ASFV antibodies form with one another in or on the reaction chamber or substrate or when one or more aggregations of the bound ASFV proteins form with one another in or on the reaction chamber or substrate.

27. The system of claim 26, wherein the electromagnetic radiation detection apparatus comprises a spectrometer; wherein the electromagnetic radiation detection apparatus comprises a microscope; and / or wherein the electromagnetic radiation detection apparatus comprises a light-emitting diode (LED) that transmits light into and / or through the reaction chamber or substrate and a photodetector that detects light from the reaction chamber or substrate.

28. A kit, comprising: a set of plasmonic metal nanoparticles (MNPs); and, a set of African swine fever virus (ASFV) proteins, or fragments thereof, that comprise an epitope that binds to an anti-ASFV antibody when present in a sample; or a set of antibodies, or antigen binding portions thereof, that binds to an epitope of an ASFV protein when present in a sample to produce bound ASFV proteins.

29. The kit of claim 28, wherein one or more components of the kit are disposed in one or more containers.

30. The kit of claim 28, wherein the MNPs are (i) conjugated to the set of ASFV proteins, or fragments thereof, or (ii) conjugated to the set of antibodies, or antigen binding portions thereof.31 . The kit of claim 28, wherein the set of ASFV proteins comprises a p72 protein, a p30 protein, a p54 protein, and / or a pp62 protein.

Images