WO2024259321A2

WO2024259321A2 - Systems and methods for diagnosis of tuberculosis and other infections

Info

Publication number: WO2024259321A2
Application number: PCT/US2024/034125
Authority: WO
Inventors: Aniruddh SARKAR; Sarah M. ALI; Asma HASHIM
Original assignee: Georgia Tech Research Institute; Georgia Tech Research Corp
Current assignee: Georgia Tech Research Institute; Georgia Tech Research Corp
Priority date: 2023-06-15
Filing date: 2024-06-14
Publication date: 2024-12-19
Anticipated expiration: 2025-12-15
Also published as: WO2024259321A3

Abstract

An exemplary embodiment of the present disclosure provides a composition for identifying a presence of tuberculosis in a subject. The composition comprises a molecule of interest configured to bind with a target molecule in a biological sample; a probe molecule configured to bind with the target molecule; and a metal particle configured to bind with the probe molecule. Another aspect of the present disclosure provides a method of diagnosing diagnosing tuberculosis in a subject. The method comprises combining the composition of the present disclosure with a sample solution comprising the biological sample to form a testing sample, imaging the testing sample, and determining, based on an image of the testing sample, whether the biological sample comprises an active tuberculosis infection.

Description

SYSTEMS AND METHODS FOR DIAGNOSIS OF TUBERCULOSIS AND OTHER

INFECTIONS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63/521,175, filed on June 15, 2023, which is incorporated herein by reference in its entirety as if fully set forth below.

GOVERNMENT LICENSE RIGHTS

[0002] This invention was made with government support under grant/award number 1R01AI152158-01 awarded by the National Institute of Health. The government has certain rights in the invention.

SEQUENCE LISTING STATEMENT

[0003] This application contains a computer readable Sequence Listing, which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on June 13, 2024, is named 011529 114557.xml and is 11 , 142 bytes in size.

FIELD OF THE DISCLOSURE

[0004] The various embodiments of the present disclosure relate generally to systems and methods for diagnosing tuberculosis and other infections.

BACKGROUND

[0005] Tuberculosis (TB) has and continues to be one of the world's deadliest diseases, with over 9 million new cases of infection and 1.7 million deaths each year by the disease-causing bacterium, Mycobacterium tuberculosis (Mtb). Despite centuries of research, the struggle to eradicate tuberculosis continues. Approximately 90% of adults infected with Mtb never develop symptoms of clinical disease, resulting in latent TB infections (LTBI).

[0006] Currently approved tuberculosis diagnostics approved are laboratory-based tests that require highly skilled individuals, expensive instruments and can take several weeks or months to produce diagnostic results. Most of these available diagnostics suffer from low sensitivity or specificity due to complications related to sample acquisition and processing as well as disease heterogeneity between active TB (ATB) and LTBI. TB tests can be broadly divided into tests that detect presence of Mycobacterium Tuberculosis (Mtb) pathogen or tests that detect the host immune response to the Mtb pathogen.

[0007] The gold standard diagnostic test is based on sputum culture which, due to slow growing nature of Mtb, can take weeks to months to return results. This requires significant infrastructure from BSL-3 culture laboratory facilities and highly trained personnel. Other laboratory tests include the sputum smear analysis using microscopy to detect Mtb bacilli directly in sputum. This requires highly difficult sample extraction (coughing up sputum for staining and culture) and processing techniques which especially difficult to apply for pediatric TB. Sensitivity can be moderate to low due to difficulty to detect bacilli in low-bacterial burden stages of the disease. This lab-based test can only detect ATB, not LTBI. Newer modalities for detecting bacilli in sputum have emerged such as the RT-PCR based GeneXpert (Cepheid Inc) test which also requires integrated sputum sample preparation. The RT-PCR method still requires sputum extraction from patients and us also limited to detecting ATB and not LTBI.

[0008] Additional diagnostic methods include TB skin test (TST or Mantoux skin test), or blood tests for Interferon Gamma Release Assay (IGRA: QuantiFERON-TB Gold Plus or T- Spot) which measures the T cell response to Mtb infection. Conversely to the sputum culture tests, the skin and blood tests only detect LTBI and not ATB. TST required 2 to 3 days while IGRAs require up to 24 hours to return results. Both require trained personnel and IGRAs require significant lab instrumentation such as cell culture, ELISA or ELISPOT readers. A biomarker-based diagnostic method includes measuring antigens such as Mtb LAM in urine; however, this method is limited to TB diagnosis only for HIV+ subjects.

[0009] Despite the prevalence of antiretroviral therapy, HIV remains the strongest risk factor for developing Tuberculosis (TB), which is the leading cause of death among people living with HIV (PLHIV). Lack of accurate yet rapid and inexpensive diagnostics is a critical bottleneck in control of TB. Key challenges in TB diagnostics include difficulty in discrimination of the heterogenous spectrum of TB disease, which is further complicated in HIV/TB co-infection, and difficulty in obtaining and processing sputum samples for point-of- care (POC) diagnosis. Earlier, antibody (Ab)-based tests have failed due to poor specificity in discriminating past infection from current infection as well as latent TB infection (LTBI) from active TB (ATB).

[0010] Recent work has pointed to the difference in glycosylation status of the Fc region of antigen-specific antibodies in LTBI - - ^{A rrn} "owe ver, measuring antigen-specific antibody glycosylation currently requires expensive instrumentation such as mass spectrometers, large amounts of sample and laborious sample preparation steps for the isolation of antigen-specific antibodies, and high levels of expertise for all the above steps.

[0011] Therefore, there is a need for fast, simple, and easy diagnostic methods that can accurately distinguish ATB from LTBI.

BRIEF SUMMARY

[0012] An exemplary embodiment of the present disclosure provides a composition for identifying a presence of tuberculosis in a subject. The composition comprises a molecule of interest configured to bind with a target molecule in a biological sample; a probe molecule configured to bind with the target molecule; and a metal particle configured to bind with the probe molecule.

[0013] In any of the embodiments disclosed herein, the molecule of interest comprises one or more of: an antigen, an epitope, an antibody, a protein, DNA, RNA, a virus, a bacterium, and a mammalian cell.

[0014] In some embodiments, the molecule of interest comprises one or more antigens.

[0015] In some embodiments, the one or more antigens is selected from the group consisting of PPD, Ag85A, LAM, ESAT6, CFP10, PstSl, HspX, Rvl363c, Rv0826 or mixtures thereof. [0016] In some embodiments, the target molecule comprises one or more of: an antibody, a paratope, a protein, DNA, RNA, a virus, a bacterium, and a mammalian cell.

[0017] In some embodiments, the target molecule comprises one or more antibodies.

[0018] In some embodiments, the one or more antibodies are M. tuberculosis (Mtb)-specific antibodies.

[0019] In some embodiments, the probe molecule comprises an Fc receptor and/or immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM) and/or lectins including sambucus nigra lectin (SNA) and ricinis communis agglutinin 1 (RCA1).

[0020] In some embodiments, the probe molecule comprises monomeric FcR3B.

[0021] In other embodiments, the probe molecule comprises multimeric FcR3B.

[0022] In some embodiments, the monomeric FcR3B is conjugated to horseradish peroxidase (HRP).

[0023] In some embodiments, the multimeric FcR3B is conjugated to polymerized HRP.

[0024] In some embodiments, the immunoglobulin G is conjugated to HRP.

[0025] In some embodiments, the molecule of interest is attached to the surface of a bead. [0026] In some embodiments, the molecule of interest is attached to a polydimethylsiloxane (PDMS) fdm.

[0027] In some embodiments, the composition is with a point of care (POC) diagnostic kit, the POC diagnostic kit comprising an array of wells.

[0028] In some embodiments, the metal particle comprises silver.

[0029] In some embodiments, the composition is configured to distinguish an active tuberculosis infection from a latent tuberculosis infection.

[0030] Another embodiment of the present disclosure provides a method of diagnosing tuberculosis in a subject. The method comprises combining the composition described herein with a sample solution comprising the biological sample to form a testing sample; imaging the testing sample; and determining, based on an image of the testing sample, whether the biological sample comprises an active tuberculosis infection.

[0031] In some embodiments, the composition is with a point of care (POC) diagnostic kit, the POC diagnostic kit comprising an array of wells.

[0032] In some embodiments, the subject is HIV positive.

[0033] These and other aspects of the present disclosure are described in the Detailed Description below and the accompanying drawings. Other aspects and features of embodiments will become apparent to those of ordinary skill in the art upon reviewing the following description of specific, exemplary embodiments in concert with the drawings. While features of the present disclosure may be discussed relative to certain embodiments and figures, all embodiments of the present disclosure can include one or more of the features discussed herein. Further, while one or more embodiments may be discussed as having certain advantageous features, one or more of such features may also be used with the various embodiments discussed herein. In similar fashion, while exemplary embodiments may be discussed below as device, system, or method embodiments, it is to be understood that such exemplary embodiments can be implemented in various devices, systems, and methods of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The following detailed description of specific embodiments of the disclosure will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosure, specific embodiments are shown in the drawings. It should be understood, however, that the disclosure is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0035] FIG. 1 provides an illustrated view of a composition, in accordance with an exemplary embodiment of the present disclosure.

[0036] FIGs. 2A-2C show that multimerized Fc receptor probes provide high sensitivity and discrimination, in accordance with an exemplary embodiment of the present disclosure. FIG. 2A provides an example multimerized Fc receptor probe with high avidity binding to antibodies. FIG. 2B provides a polar plot of Mtb-specific Ab Fc profile showing higher FcR3B binding and lower sialic acid in TB vs LTBI. FIG. 2C provides a comparison of FcR3B binding signal in point-of-care (POC) format to the bead-based assay, which shows higher differences in TB vs LTBI in POC format.

[0037] FIG. 3A provides a comprehensive Antibody-omics workflow using Schisto antigen coated beads, patient samples, and labeled probes, in accordance with an exemplary embodiment of the present disclosure. FIG. 3B is a Volcano Plot indicatING top distinguishing features between Egg+ and Egg- samples based on a Log-fold Median Change >2 and p- value<0.05, in accordance with an exemplary embodiment of the present disclosure. FIG. 3C is a multivariate Least Absolute Shrinkage and Selection Operator (LASSO) Partial Least- Squares Discriminant Analysis (PLS-DA) model identifying a unique set of features to differentiate between Egg+ and Egg- samples, in accordance with an exemplary embodiment of the present disclosure.

[0038] FIG. 4A provides a multiplex metallization assay workflow, in accordance with an exemplary embodiment of the present disclosure. FIG. 4B is an example cellphone image of multiplexed metallization, in accordance with an exemplary embodiment of the present disclosure. FIG. 4C provides metallization outputs of each antigen for each patient sample, in accordance with an exemplary embodiment of the present disclosure. FIG. 4D provides ROC Curves that illustrate improvement after multiplexed detection implementing several Oncho antigens, in accordance with an exemplary embodiment of the present disclosure.

[0039] FIGs. 5A-5F show univariate and multivariate analysis of antibody Fc bio profiling data acquired on individual adult TB samples (ATB patients, n=16, LTBI patient, n=20), in accordance with exemplary embodiments of the present disclosure. FIG. 5A provides a conceptual overview of a Tuberculosis antibody profiling platform used to characterize and quantify serum antibodies (Abs) directed against Mtb antigens. FIG. 5B provides flower plots illustrating measured Ab features aga' — * - - FIG. 5C provides feature selection frequency of LASSO-SVM model. FIG 5D provides LASSO-selected features from model built using deep humoral profiles against Mtb specificities, ATB and LTBI. FIG. 5E provides performance of LASSO model to discriminate between ATB and LTBI built using deep humoral profiles against Mtb specificities. FIG. 5F provides PLS-DA using only the LASSO- selected features from the model in (FIG. 5D) to discriminate between ATB and LTBI.

[0040] FIGs. 6A-6M show high-throughput, multiplexed, sensitive and quantitative point-of- care diagnostic for optical detection of TB biomarkers using small serum samples point-of-care diagnostic platform, in accordance with exemplary embodiments of the present disclosure. FIG. 6A provides an overview of workflow for enzymatically amplified silver metallization. TB antigens are immobilized on laser cut PDMS microwells on a PDMS coated slide, a drop of patient sera is incubated on antigen immobilized slide, followed by HRP-labelled IgG and FcyR3B probe incubation. Silver substrate components added and incubated to generate silver metallization related to TB biomarker concentration. Enzymatic metallization based optical readout using cellphone camera is carried out to obtain quantitative and sensitive TB diagnostic outputs. FIG. 6B shows a custom microwell array created using laser-cut PDMS film on PDM- coated standard microscope glass slide showing silver metallization obtained in ATB (upper left), LTBI (right) and control n (lower left) groups. FIG. 6C shows individual patient silver darkness readout for ATB, LTBI and control. FIG. 6D shows serum dilution curves of ATB and LTBI for the FcyR3B probe. FIG. 6E shows evaluation of FcyR3B probe developments using different oligomers. FIG. 6F shows two layers of laser cut PDMS used to create an array of seven wells for immobilization of different Mtb antigens and a bigger well for sharing assay solutions between antigen wells. FIG. 6G shows distinct localization of silver deposition of positive control, negative control, various Mtb antigens for individual ATB (n=l ) and LTBI patients (n=l) probed with IgG and FcyR3B. FIG. 6H provides quantified multiplexed IgG antibody response. FIG. 61 provides quantified multiplexed FcyR3B antibody response. FIG. 6J shows LASSO selected features from a model built using POC diagnostic platform response to IgG and FcyR3B biomarkers. FIG. 6K shows performance of LASSO model to discriminate between ATB and LTBI using POC diagnostic platform response to IgG and FcyR3B biomarkers. FIG. 6L shows PLS-DA using only LASSO-selected features from the model in FIG. 6J. FIG. 6M shows performance of the model (built using primary TB cohort) on an orthogonal cohort. [0041] FIGs. 7A-7P provide dot plots showing deep humoral profiles against Mtb antigen specificities for ATB, LTBI and controls, in accordance with exemplary embodiments of the present disclosure. FIG. 7A provides a dot plot IgG. FIG. 7B provides a dot plot for IgGl. FIG. 7C provides a dot plot for IgG2. FIG. 7D provides a dot plot for IgG3. FIG. 7E provides a dot plot for IgG4, FIG. 7F provides a dot plot for IgA. FIG. 7G provides a dot plot for IgAl . FIG. 7H provides a dot plot for IgA2. FIG. 71 provides a dot plot for IgM. FIG. 7J provides a dot plot for FCR1. FIG. 7K provides a dot plot for FCR2A. FIG. 7L provides a dot plot for FCR2B. FIG. 7M provides a dot plot for FCR3A. FIG. 7N provides a dot plot for FCR3B. FIG. 70 provides a dot plot for SNA. FIG. 7P provides a dot plot for RCA.

[0042] FIGs. 8A-8B provide correlation heatmaps showing corresponding pairwise features correlations (Spearman) across ATB (FIG. 8A) and LTBI (FIG. 8B), in accordance with exemplary embodiments of the present disclosure. FIG. 8C provides P-value heatmap for all features, in accordance with an exemplary embodiment of the present disclosure. FIG. 8D provides median fold change heatmap for all features, in accordance with an exemplary embodiment of the present disclosure.

[0043] FIGs. 9A-9J provide correlation plots between Luminex and optical PoC Dx performance on individual adult TB patient samples (ATB patients, n=20, LTBI patient, n=20), in accordance with exemplary embodiments of the present disclosure. FIGs. 9A-9E provide antigen comparison of individual patient IgG response, with FIG. 9A showing Ag85A, FIG. 9B showing LAM, FIG. 9C showing PPD, FIG. 9D showing ESA T6, and FIG. 9E showing HspX. FIGs. 9F-9J provide antigen comparison of individual patient FcyR3B binding response, with FIG. 9F showing Ag85A, FIG. 9G showing LAM, FIG. 9H showing PPD, FIG. 91 showing ESA T6, and FIG. 9 J showing HspX.

[0044] FIGs. 10A-10D provide univariate analysis of a POC Dx Platform on a primary cohort, in accordance with exemplary embodiments of the present disclosure. FIG. 10A provides antigen comparison of individual patient IgG response on secondary patient cohort (ATB Vietnam, n=20 and LTBI South Africa, n=20). FIG. 10B provides antigen comparison of individual patient FcyR3B binding response on secondary patient cohort (ATB Vietnam, n=20 and LTBI South Africa, n=20). FIG. 10C provides AUROC curves showing performance of ATB vs LTBI Dx based on IgG response. FIG. 10D provides Area Under Receiver Operator Characteristic Curves (AUROC) showing performance of ATB vs LTBI Dx based FcyR3B binding response. [0045] FIGs. 11A-11D provide univariate analysis of a POC Dx Platform on secondary a cohort, in accordance with exemplary embodiments of the present disclosure. FIG. 11A provides an antigen comparison of individual patient IgG response on a secondary patient cohort (ATB Vietnam, n=20 and LTBI South Africa, n=20). FIG. 11B provides an antigen comparison of individual patient FcyR3B binding response on a secondary patient cohort (ATB Vietnam, n=20 and LTBI South Africa, n=20). FIG. 11C provides AUROC curves showing performance of ATB vs LTBI Dx based on IgG response. FIG. 11D provides AUROC curves showing performance of ATB vs LTBI Dx based FcyR3B binding response.

[0046] FIGs. 12A-12B provide Flower plots illustrating measured Ab features against Mtb antigens in ATB (FIG. 12A) and LTBI (FIG. 12B), in accordance with exemplary embodiments of the present disclosure. FIG. 12C shows performance of a LASSO model to discriminate between ATB and LTBI built using deep humoral profiles against Mtb specificities, in accordance with an exemplary embodiment of the present disclosure. FIG. 12D shows Variable Importance of top features selected by a LASSO-SVM model, in accordance with an exemplary embodiment of the present disclosure. FIG. 12E provides a PLSDA Loadings plot, in accordance with an exemplary embodiment of the present disclosure. FIG. 12F shows performance of a LASSO model to discriminate between ATB and LTBI built using deep humoral profiles against Mtb specificities, in accordance with an exemplary embodiment of the present disclosure. FIG. 12G provides PLS-DA using only the LASSO-selected features from the model in FIG. 12D to discriminate between ATB and LTBI, in accordance with an exemplary embodiment of the present disclosure.

DETAILED DESCRIPTION

[0047] To facilitate an understanding of the principles and features of the present disclosure, various illustrative embodiments are explained below. The components, steps, and materials described hereinafter as making up various elements of the embodiments disclosed herein are intended to be illustrative and not restrictive. Many suitable components, steps, and materials that would perform the same or similar functions as the components, steps, and materials described herein are intended to be embraced within the scope of the disclosure. Such other components, steps, and materials not described herein can include, but are not limited to, similar components or steps that are developed after development of the embodiments disclosed herein. [0048] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0049] Also, in describing the preferred exemplary embodiments, terminology will be resorted to for the sake of clarity. It is intended that each term contemplates its broadest meaning as understood by those skilled in the art and includes all technical equivalents that operate in a similar manner to accomplish a similar purpose.

[0050] By ‘ ‘comprising” or “containing” or “including” is meant that at least the named compound, member, particle, or method step is present in the composition or article or method, but does not exclude the presence of other compounds, materials, particles, method steps, even if the other such compounds, material, particles, method steps have the same function as what is named.

[0051] Mention of one or more method steps does not preclude the presence of additional method steps or intervening method steps between those steps expressly identified. Similarly, it is also to be understood that the mention of one or more components in a device or system does not preclude the presence of additional components or intervening components between those components expressly identified.

[0052] The materials described as making up the various members of the invention are intended to be illustrative and not restrictive. Many suitable materials that would perform the same or a similar function as the materials described herein are intended to be embraced within the scope of the invention. Such other materials not described herein can include, but are not limited to, for example, materials that are developed after the time of the development of the invention.

[0053] In some embodiments, the test described herein provides a fast, simple and easy-to-use diagnostic test for identifying TB, and in particular, in distinguishing between LTBI and ATB disease. About 5-10 percent of people with latent infection will progress to active TB, which will require diagnosis and treatment to prevent additional spread of the pathogen. In some instances, when conversion from latent TB to active TB occurs, the host will not know the cause of their illness and may continue the spread. The methods and probes described herein can be used to monitor LTBI cases over time and check for ATB conversion. Further upon treatment these methods and probes can be used for treatment monitoring as well.

[0054] An embodiment of the present disclosure provides a polymeric probe to measure key antibody Fc features of antigen-specific antibodies from a sample of fluid. Because the antibody Fc has certain features that a"" * - - ctive infection that are different from the features during latent infection, the probe can distinguish ATB from LTBI. For instance, during active infection, the Fab end binds to the pathogen while the Fc end interacts with the immune system by driving cell-mediated antibody functions.

[0055] As shown in FIG. 1, an exemplary embodiment of the present disclosure provides a composition 100 comprising: a bead 110 having on its surface I l l a molecule of interest 120 configured to bind with a target molecule 131 in a biological sample 130, a probe molecule 140 configured to bind with the target molecule 131, and a metal particle 150 configured to bind with the probe molecule 140. Composition 100 can be configured to identify a presence of tuberculosis in a subject.

[0056] In any of the embodiments disclosed herein, the bead 110 can comprise an identifying characteristic 112. The identifying characteristic 112 can be one or more of: an electrical impedance signature, optical barcoding, shape, size, porosity, conductivity, permittivity, permeability, and stiffness.

[0057] In any of the embodiments disclosed herein, the metal particle 150 can be configured to modify an electrical property of the bead 110.

[0058] In any of the embodiments disclosed herein, the bead 110 can comprise one or more of: carboxylated polystyrene, aminated polystyrene, glass, silica, polymethyl methacrylate, and hydrogels.

[0059] In any of the embodiments disclosed herein, the bead 110 can be functionalized using polymerized horseradish peroxidase (HRP) streptavidin, or EDC-NHS(N-ethyl-N-(3- (dimethylamino)propyl)carbodiimide/N-hydroxysuccinimide) chemistry.

[0060] In any of the embodiments disclosed herein, the bead 110 can have a diameter of approximately 5 pm. In any of the embodiments disclosed herein, the bead 110 can have a diameter of approximately 10 pm. In any of the embodiments disclosed herein, the bead 110 can have a diameter of approximately 20 pm. In any of the embodiments disclosed herein, the bead 110 can have a diameter of from about 3 pm to about 20 pm.

[0061] In any of the embodiments disclosed herein, the molecule of interest 120 can comprise one or more of: an antigen, an epitope, an antibody, protein, DNA, RNA, virus, bacteria, and mammalian cell.

[0062] In any of the embodiments disclosed herein, the target molecule 131 can comprise one or more of: an antibody, a paratope, a protein, DNA, RNA, a virus, a bacterium, and a mammalian cell. In some embodiments, the target molecule 131 can comprise an antigenspecific antibody. In some embodiment - - ific antibody can bind an Fc-receptor. In some embodiments, the antigen-specific antibody is an M. tuberculosis (Mtb)-specific antibody.

[0063] In any of the embodiments disclosed herein, the probe molecule 140 can comprise horseradish peroxidase. In some embodiments, the probe molecule 140 can comprise an Fc receptor and/or immunoglobulin G (IgG). In some embodiments, the probe molecule 140 can comprise biotinylated Fc-receptors, such as, for example, FcR2A, FcR2B, FcR3A, and FcR3B. In some embodiments, the Fc receptor can be monomeric FcR3B. In other embodiments, the Fc receptor can be multimeric FcR3B. In some embodiments the monomeric or multimeric Fc3B can be conjugated to horseradish peroxidase (HRP) or polymerized HRP, respectively. [0064] In any of the embodiments disclosed herein, the biological sample 130 can comprise one or more of: serum, whole blood, saliva, urine, and cerebrospinal fluid.

[0065] In any of the embodiments disclosed herein, the composition 100 can further comprise a buffer. The buffer can be a phosphate buffer having a pH of from about 6.0 to about 7.0. The buffer can be a phosphate buffer comprising a pH of approximately 6.4.

[0066] As shown in FIGs. 2A-2C, multimerized Fc receptor probes provide high sensitivity and discrimination. FIG. 2A is a schematic of a composition 100 that shows a high avidity binding on Fc receptor multimers to Abs. FIG. 2B is a polar plot of Mtb-specific Ab Fc profile, showing higher FcR3b binding and lower sialic acid in TB versus LTBI. FIG. 2C is a comparison of FcR3b binding signal in point-of-care format compared to the bead-based assay, showing higher differences in TB versus LTBI in point-of-care format.

[0067] Multimerized Fc receptor probes coupled to enzyme multimers enable highly sensitive and discriminative Mtb-specific Ab Fc profiling in POC-compatible formats. Fc receptor interaction with antibodies is modulated by both subclass differences in Abs as well as their differential glycosylation. Thus, Fc receptor binding can serve as an integrative signal in measuring the overall Fc modification state of Abs. Most human Fc receptors (except FcRl), however, have low to medium affinity to monomeric Abs. In-vivo, their function depends on highly avid binding to multiple Abs decorating immune complexes. As such, multimerized Fc receptor probes were developed which enable highly avid binding of even low affinity Fc receptors to antigen-specific Abs bound to antigens coated on assay surfaces (e.g. beads, microchips, FIG. 2A). Using these probes, in the bead-based Ab Fc profiling assay, a new FcR3B-binding signature difference between Mtb-specific Abs in TB and LTBI was found, with Abs in active TB showing higher FcR3B binding compared to LTBI as shown in the polar plot in FIG. 2B. Note than in this assc" ^{C N I A} ’--"ding still shows higher sialylation of Mtb-specific Abs in LTBI too (FIG. 2B). Additionally, to adapt these probes for the POC assay, the multimerized Fc receptors were bound to a polymerized enzyme multimer (polyHRP) as well. This new probe provides a highly discriminative signal accentuating the difference between TB and LTBI Mtb-specific Abs even more in the nanoscale enzymatic metallization based POC format compared to the bead-based format (FIG. 2C). Overall, this will enable both novel biomarker discovery as well as inexpensive yet sensitive and specific POC detection. [0068] In some embodiments, the advantages of the present diagnostic method include the ability to distinguish ATB from LTBI, the lack of sputum extraction and processing of same, rapid test compatibility, inexpensive and point-of-care compatibility, and broad applicability. Fragment crystallizable region (Fc)-based TB diagnosis can distinguish ATB from LTBI due to the unique differences in Fc features that vary according to disease state. The fragment crystallizable region (Fc region) is the tail region of an antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. This property allows antibodies to activate the immune system. In addition, because antibodies for Fc feature measurement are easily accessible in blood, serum, saliva, and other fluids, there is no need for sputum extraction. Antibody Fc features are measurable in a rapid binding assay which can provide results in 30 mins to a few hours instead of days to months. Antibody Fc features can be measured in inexpensive POC formats without expensive instrumentation or trained personnel. Additionally, this method is multiplexable with other antibody tests in multiplexable formats and can be applied to HIV+ or HIV- subjects.

[0069] It has been recently observed that the inflammatory state of Mtb-specific Abs, driven by changes in Fc-glycosylation, differs across LTBI and ATB. Disclosed herein is the application of a M. tuberculosis (Mtb)-specific Ab Fc profile -based biomarker for TB to distinguish ATB from LTBI and a method for its point-of-care (POC) detection using an inexpensive, multiplexed and high throughput optical biosensing method. First, highly multiplexed bead-based Ab biophysical profiling (both Fab and Fc) was applied to sera from a set of ATB and LTBI patients. Antigen-coated barcoded beads were incubated with serum and probed with fluorescently-labeled isotype and subclass probes, tetramerized Fc receptors and lectins. Machine-learning based methods (LASSO-SVM) applied to the resultant highdimensional dataset, revealed a minimal Ab Fc profile biomarker. Next, a POC detection method was developed where multiple TB antigens (Ag85A, PPD, Esat6, HspX) immobilized on laser cut PDMS microwells, are incubated with drops (<10pL) of TB patient serum. Horseradish peroxidase labelled Fc air¹ - ¹ - ? added, followed by silver substrates to produce a quantitative and sensitive enzymatic metallization based optical readout which can be read out using a cellphone camera. Application of this to two geographically distinct cohorts of TB patients (South Africa and Vietnam, n=40), achieved a high diagnostic accuracy (AUROC-O.9). Thus, a non-sputum based Ab Fc profile biomarker for TB and a method for its inexpensive yet accurate POC detection from a drop of serum was developed.

[0070] In any of the embodiments disclosed herein, composition 100 can be included in highly multiplexed ‘Abomics’ platforms for deep biophysical characterization (Fab & Fc) of broad sets of antigen-specific Abs, including their isotype, subclass, glycosylation, Fc receptor and complement binding. Machine learning applied to the high-dimensional data reveals unique Ab signatures predictive of disease state, as shown in FIG. 3A. Further, inexpensive optical and electronic detection techniques have been developed for multiplexed point-of-care (mPOC) detection of such immune signatures from a single drop of sample, using enzymatic silver metallization.

[0071] Here, the Ab-omics pipeline is applied and then mPOC detection to neglected tropical diseases (NTDs) - specifically schistosomiasis (schisto) and onchocerciasis (oncho). Serum samples from schisto patients (n=88, from Minais Gerais, Brazil), previously screened using Kato-Katz technique (Egg+/-) were characterized with multiple Schisto antigens (SEA, SM25, MEG, CD63, Calumenin) and probes (324 features/sample). This revealed that while SEA- specific IgG titers were equal, there was higher SEA- and CD63-specific IgG4 and FcyR2A binding of Calumenin-specific and FcyR3B binding of SM25-specific Abs, and Ab sialylation across antigens in Egg+ patients, as shown in FIG. 3B. A multivariate LASSO PLS-DA model identified the unique features to differentiate Egg+/- samples which can be used on the mPOC device in FIG. 3 C.

[0072] FIG. 4B demonstrates mPOC detection, oncho patient (n=56) and healthy (n=35) sera, which was measured for multiple antigen-specific (FAbP, OV-16, Ov33, OvMSA) IgG4 responses from a single drop of serum. As shown, Oncho+ samples exhibited dark metallization decreasing with serial dilution, while healthy controls showed low metallization. FIG. 4D provides multiple logistic regression with all antigens improved the AUC of ROC curves compared to a single antigen indicating the value of multiplexed detection.

[0073] A similar diagnostic strategy using Fc features of antigen-specific antibodies can be used for diagnosis of other endemic infectious diseases as well. As described above, this strategy is applicable to Schistosomiasis, which is a helminth infection endemic to parts of South America, Africa and Asia. [0074] It is to be understood that the embodiments and claims disclosed herein are not limited in their application to the details of construction and arrangement of the components set forth in the description and illustrated in the drawings. Rather, the description and the drawings provide examples of the embodiments envisioned. The embodiments and claims disclosed herein are further capable of other embodiments and of being practiced and carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein are for the purposes of description and should not be regarded as limiting the claims.

[0075] Accordingly, those skilled in the art will appreciate that the conception upon which the application and claims are based may be readily utilized as a basis for the design of other structures, methods, and systems for carrying out the several purposes of the embodiments and claims presented in this application. It is important, therefore, that the claims be regarded as including such equivalent constructions.

[0076] Furthermore, the purpose of the foregoing Abstract is to enable the United States Patent and Trademark Office and the public generally, and especially including the practitioners in the art who are not familiar with patent and legal terms or phraseology, to determine quickly from a cursory inspection the nature and essence of the technical disclosure of the application. The Abstract is neither intended to define the claims of the application, nor is it intended to be limiting to the scope of the claims in any way.

EXAMPLES

[0077] The following Examples are presented to illustrate various aspects of the present application, but are not intended to limit the scope of the claimed application.

Example 1 - Materials and Methods for Examples 2 and 3

Materials

[0078] Tween 20 (97062-332) were obtained from VWR. Deionized water (DIW, LC267405), and phosphate buffer saline (PBS, 21-040-CVR) were obtained from Fisher Scientific. Bovine Serum Albumin (BSA) (A7030) was obtained from Sigma-Aldrich. Purified Protein Derivative (PPD), a mixture of proteins derived from Mycobacterium tuberculosis, was purchased from AJ Vaccines. Purified Ag85A, Mycobacterium tuberculosis Lipoarabinomannan (LAM), a- crystallin, CFP-10, MPT64, MPT32, GROES, PSTS1, ESAT6 and HspX Recombinant Reference Standard antigens were obtained from BEI Resources. Poly-HRP conjugated Streptavidin (N200) was obtained from ThermoFisher. Peroxidase AffiniPure Donkey AntiHuman IgG (H+L) (AB 2340495) were purchased from Jackson ImmunoResearch. Biotinylated Human FcyR3B/CD16b (NA2) Protein (CDB-H82Ea) (referred to throughout this disclosure as FcR3B or FcyR3B, and identified as SEQ ID NO: 8) was purchased from Aero Biosystems. EnzMet for General Research Applications (6010-45ML) was obtained from Cedarlane. Sambucus nigra lectin (SNA) and Ricinis communis agglutinin 1 (RCA1) were purchased from Vector Laboratories. Table 1 provides the sequences for SEQ ID NOs: 1-8.

TABLE 1. Seq ID NOs: 1-8

“Biotinylated Human FcyR3B/CD16b (NA2) Protein (CDB-H82Ea) contains a linker, and a polyhistidine tag at the C-terminus, followed by an Avi tag (Avitag™).

Clinical Samples

[0079] ATB (n=16) and LTBI (n=20) adult TB patient sera samples from South Africa used for Luminex and initial POC TB diagnostic development. ATB (n=20) and LTBI (n=20) adult TB patient sera samples from Vietnam and South Africa respectively were used as a secondary confirmatory cohort for the POC TB diagnostic.

Optical POC Diagnostic Platform Preparation

[0080] Thin poly dimethylsiloxane (PDMS) film (0.1mm thickness, Greene Rubber Company) was laser-cut to create an array of wells with diameters of 2mm. Another thin piece of PDMS was cut to fit the plane rectangular shape of a typical glass slide. The PDMS layers were soaked in 5% Alconox solution then rinsed thoroughly using DI water. Following air drying, scotch tape was used to remove any remaining dust particles. The two layers of clean PDMS were then visually aligned on a clean glass slide with the PDMS layer with the well array being placed onto of the plane PDMS. The two layers of PDMS were secured to the slide using parafilm on either side of the glass slide to prevent leakage.

Multiplexed TB Immunoassay

[0081] TB antigens (Ag85A, LAM, PPD, ESAT6 and HspX) were prepared at a concentration of 50pg/ml in PBS then added to each well of the prepared PDMS coated slide. Following overnight incubation at 4 °C in a humidified chamber, the slide was blocked using blocking buffer (1% BSA in 0.1% Tween 20 in PBS (0.1% PBST)). The antigen coated and blocked slide was then washed using 0.1% PBST, PBS and DI water. After spin-drying the slide, 3 pL of diluted serum sample (1 :20 dilution) was added to each well and incubated for 1 hr. Following washing and drying, an IgG probe solution (1 :800 dilution of HRP-conjugated IgG) or poly-HRP conjugated FcyR3B (Ipg/ml) was added to each well. After another 1 hr incubation, washing and drying, equal volumes of silver metallization substrate components A, B and C were sequentially added and incubated for 4, 4 and 5 minutes respectively. The reaction was stopped by quenching the slide in DI water and then drying it. Example 2 - Multiplexed Antigen-Specific Antibody Fc Profiling for Point-of-Care Diagnosis of Tuberculosis

[0082] A highly multiplexed “antibody-omics” biophysical profiling platform was employed to discover novel M. tuberculosis (Mtb)-specific Ab profile-based biomarkers for TB to distinguish ATB from LTBI (Fig. 5A). This process involved the probing of both the Fab and Fc domain profiles of antigen-specific antibodies, isotypes, subtypes, Fc receptor binding and glycosylation. This robust, sensitive, and cost-effective biophysical profiling was carried out using a custom Luminex assay. Sera samples were obtained from a cohort of ATB (n=l 6) and LTBI (n=20) adult patients from South Africa. This highly multiplexed bead-based assay resulted in the generation of multi-dimensional data which was subsequently analyzed using both univariate and multivariate analysis techniques for interpretation.

[0083] Univariate analysis of this multidimensional data revealed unique differences in the titer, Fc receptor binding and glycosylation trends between ATB and LTBI patients (Figures 5B, 7A-7P, and 8A-8D). Specifically, upregulation of FcyRl and FcyR3B binding was observed in ATB patients, while IgG2 titer and sialic acid upregulation was observed in LTBI patients. For both ATB and LTBI patients, IgA2 was upregulated.

[0084] Fc receptors (FcyRs) are a crucial component to the regulation and execution of antibody-mediated responses. FcyRl displays high monomeric antibody binding affinity while FcyR3B is a low affinity, GP-1 anchored FcyR, expressed on neutrophils (Nimmerjahn and Ravetch, “Fc-Receptors as Regulators of Immunity,” Adv Immunol, 96: 179-204 (2007). By altering the structure of the Fab domain, the Fc domain influences antigen binding affinity and avidity (Carpenter and Lu, “Leveraging Antibody, B Cell and Fc Receptor Interactions to Understand Heterogeneous Immune Responses in Tuberculosis,” Front Immunol, 13:830482 (2022). These traits combined with the ability of FcyRs to recruit and tune both humoral and cellular immune responses, demonstrates the potential for the unique FcyR features revealed in this analysis to transform TB diagnostics beyond initial titer-based analyses.

[0085] Least Absolute Shrinkage and Selection Operator (LASSO) regression analysis and Support Vector Machines (SVM) classification were employed to identify a minimal set of predictive biomarkers from the first South African dataset while reducing the influence of less relevant parameters. The efficiency of this model is demonstrated in Figure 5E where the AUC plots reveal a clear difference in SVM performance between the original (AUROC-O.9) and permuted (AUROC-O.6) data thus demonstrating limited overfitting of the model.

[0086] After 500 iterations in a cross-validation framework, top 5 most- frequently selected features were used to build the LASSO-SVM model (Figure 5C-D). Partial Least-Squares Discriminant Analysis (PLS-DA) was also carried out using the LASSO-selected features on the ATB and LTBI patients to further demonstrate the predictive capability of the constructed TB ML model (Figure 5F).

[0087] Following the discovery of these potentially useful TB biomarkers, their application in the field of point-of-care (PoC) diagnostics (Dx) was evaluated. A PoC diagnostic platform called the Multiplexed Optical Bioassay using Enzymatic Metallization (MO-BEAM) was engaged for this purpose (Rafat, et al., “Inexpensive High-Throughput Multiplexed Biomarker Detection Using Enzymatic Metallization with Cellphone-Based Computer Vision,” ACS Sens, 8:534-542 (2023). This low-cost, portable, and sensitive biosensing platform allows for the high-throughput, multiplexed and quantitative optical detection of biomarkers from small volumes of sera using a simple smartphone-based detection interface.

[0088] Briefly, multiple TB antigens (Ag85A, LAM, PPD, Esat6 & HspX) are immobilized on laser cut PDMS microwells and incubated with drops (<10pL) of TB patient serum. Horseradish peroxidase (HRP) labelled Fc and lectin probes are introduced, followed by silver substrates to produce a quantitative and sensitive enzymatic metallization based optical readout which can be read out using a cellphone camera (Figures 6A-D). The enzymatic metallization can be carried out by introducing a metallization substrate solution containing silver acetate, hydroquinone, and hydrogen peroxide. Samples which have bound the HRP labelled Fc and lectin probes can be deposited with silver metal creating dark spots that indicate the presence of ATB, where the darkness of the spots is proportional to the concentration of the analyte.

[0089] Additional evaluation of the performance of both monomeric and multimeric forms of the Fc/3B probe were carried out. Here the monomeric Fc,-3B was formed via reaction with monomeric HRP and the multimeric Fc,-3B using poly-HRP. It was observed that the multimeric Fc,-3 B showed significantly improved performance in distinguishing between ATB and LTBI patient groups (Figure 6E). The multiplexing ability of this robust platform is demonstrated in Figures 6F-I.

[0090] The performance of this POC TB diagnostic was evaluated using the same patient samples used in the previously described “antibody-omics” biophysical profiling experiments. It was observed that some correlatic" — — n the features selected by both the univariate and multivariate analysis techniques (Figure 9). Specifically, FcyR3B receptor binding stood out as a reoccurring factor in distinguishing ATB and LTBI. Thus, total IgG response and Fc,-R3B binding were evaluated on this PoC diagnostic platform (Figure 10).

[0091] PoC diagnostic studies revealed unique titer and Fc binding-based results. Individual patient data showed that both IgG response (Figure 10A) and Fc,-R3B (Figure 10B) binding successfully distinguished ATB and LTBI. Univariate analysis ROC curves of these studies showed AUROC>0.8 for Ag85A, LAM and PPD antigens for both IgG antibody titer (Figure 10C) and Fc,-R3B binding (Figure 10D). The improved titer-based discrimination between ATB and LTBI observed in these results juxtaposes the previously established convention of titer-based diagnostics being unsuitable for TB. Investigators believe that avidity unique to this platform may be influencing this behavior (Martinez-Veracoechea and Leunissen, “The Entropic Impact of Tethering, Multivalency and Dynamic Recruitment in Systems with Specific Binding Groups” Soft Matter, 9:3213-3219 (2013). Multivariate analysis was also carried out to both develop a new ML model specifically for the analysis of the current and future POC diagnostic results on this platform and compare these POC diagnostic results to the previously analyzed Luminex results to detect potential POC Dx platform specific features.

[0092] LASSO-SVM was again employed to develop the ML model for the POC Dx data and identify a minimal set of POC Dx platform specific predictive biomarkers. The efficiency of this model is demonstrated in Figure 6K where the AUC plots reveal a clear difference in SVM performance between the original (AUROC-O.85) and permuted (AUROC-O.65) data thus demonstrating limited model overfitting. The final LASSO-SVM model’s top 3 most- frequently selected features are shown in Figure 6J. The PLS-DA was also carried out (Figure 6L) using the LASSO selected features on the ATB and LTBI patients to further demonstrate the predictive capability of the constructed TB ML model.

[0093] To further validate the overall TB biomarker findings of this study, these results were tested using a secondary cohort of two sets of adults TB samples (ATB Vietnam, n=20 and LTBI South Africa, n=20). These experiments achieved high diagnostic accuracy (AUROC-0.9) for both titer and Fc,-R3B binding (Figure 11) outperforming the diagnostic accuracy reported for the initial training patient cohort (ATB, n=l 6, LTBI, n=20) .

[0094] To conclude, this study demonstrates the ability of the described antibody-omics pipeline to discover novel biomarkers for TB and translate them to a POC Dx platform across two cohorts of geographically isolated patients. It was revealed that the multimeric Fc,-3B measurement technique employed in - ^:marily responsible for the diagnostic performance of this POC Dx which surpasses the current standard for TB POC Dx set by the WHO (AuROC~0.8) and approach the standards set for laboratory Dx (AUROC-O.9).

Example 3 - Multiplexed Antigen-Specific Antibody Fc Profiling for HIV/Tuberculosis Coinfection

[0095] Herein is disclosed the discovery of a Mtb-specific Ab Fc profile-based biomarker to distinguish ATB from LTBI in PLHIV. Using the multiplexed ‘Ab-omics’ platform developed in lab, deep biophysical characterization (Fab and Fc) of a broad set of antigen-specific Abs including their isotype, subclass, glycosylation, and Fc receptor binding was carried out.

[0096] The Ab-omics pipeline was applied to plasma from adults in South Africa with HIV and ATB (n=17) and LTBI (n=17) using multiple Mtb antigens (PPD, LAM, Ag85A, ESAT6, CFP10, HspX, PstSl) and non-Mtb antigens. Briefly, antigen- coated barcoded beads were incubated with plasma and probed with fluorescently labeled isotype probes, Fc receptors and lectins. With a total of 56 measured features (8 Ab Fc features X 7 antigens) from each participant, machine-learning based analytics (LASSO-SVM) applied to this high-dimensional dataset revealed a minimal Ab Fc profile biomarker (AUROOO.9) distinguishing ATB vs LTBI. This included increased specific Ab isotypes (IgM-PPD, IgA-HspX), and Fc receptor binding (FcyR3B-CFP10) and decreased Ab galacto sylation (PstSl), in ATB vs LTBI (Figures 12A-12G). These findings further support the results obtained in previous HIV-TB studies where FcyR3B played a major role in discriminating between ATB and LTBI in adult patient samples. Even in the presence of HIV coinfection, FcyR3B response remains a crucial factor for the differentiating between ATB and LTBI.

Claims

CLAIMS What is claimed is:

1. A composition for identifying a presence of tuberculosis in a subject, the composition comprising: a molecule of interest configured to bind with a target molecule in a biological sample; a probe molecule configured to bind with the target molecule; and a metal particle configured to bind with the probe molecule.

2. The composition of claim 1, wherein the molecule of interest comprises one or more of: an antigen, an epitope, an antibody, a protein, DNA, RNA, a virus, a bacterium, and a mammalian cell.

3. The composition of claim 1, wherein the molecule of interest comprises one or more antigens.

4. The composition of claim 3, wherein the one or more antigens is selected from the group consisting of PPD, Ag85A (SEQ ID NO: 1), LAM, ESAT6 (SEQ ID NO: 2), CFP10 (SEQ ID NO: 3), PstSl (SEQ ID NO: 4), HspX (SEQ ID NO: 5), Rvl363c (SEQ ID NO: 6), Rv0826 (SEQ ID NO: 7) or mixtures thereof.

5. The composition of claim 1, wherein the target molecule comprises one or more of: an antibody, a paratope, a protein, DNA, RNA, a virus, a bacterium, and a mammalian cell.

6. The composition of claim 1, wherein the target molecule comprises one or more antibodies.

7. The composition of claim 6, wherein the one or more antibodies are M. tuberculosis (Mtb)-specific antibodies.

8. The composition of claim 1, wherein the probe molecule comprises an Fc receptor and/or immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM) and/or lectins including sambucus nigra lectin (SNA) and ricinis communis agglutinin 1 (RCA1).

9. The composition of claim 8, wherein the probe molecule comprises monomeric FcR3B.

10. The composition of claim 8, wherein the probe molecule comprises multimeric FcR3B.

11. The composition of claim 9, wherein the monomeric FcR3B is conjugated to horseradish peroxidase (HRP).

12. The composition of claim 10, wherein the multimeric FcR3B is conjugated to polymerized HRP.

13. The composition of claim 8, wherein the immunoglobulin G is conjugated to HRP.

14. The composition of Claim 1, wherein the molecule of interest is attached to the surface of a bead.

15. The composition of Claim 1, wherein the molecule of interest is attached to a polydimethylsiloxane (PDMS) film.

16. The composition of claim 1, wherein the composition is with a point of care (POC) diagnostic kit, the POC diagnostic kit comprising an array of wells.

17. The composition of claim 1, wherein the metal particle comprises silver.

18. The composition of claim 1, wherein the composition is configured to distinguish an active tuberculosis infection from a latent tuberculosis infection.

19. A method of diagnosing tuberculosis in a subject, the method comprising: combining the composition of claim 1 with a sample solution comprising the biological sample to form a testing sample; imaging the testing sample; and determining, based on an image of the testing sample, whether the biological sample comprises an active tuberculosis infect' —

20. The method of claim 19, wherein the molecule of interest comprises one or more of: an antigen, an epitope, an antibody, a protein, DNA, RNA, a virus, a bacterium, and a mammalian cell.

21. The method of claim 19, wherein the molecule of interest comprises one or more antigens.

22. The method of claim 21, wherein the one or more antigens is selected from the group consisting of PPD, Ag85A (SEQ ID NO: 1), LAM, ESAT6 (SEQ ID NO: 2), CFP10 (SEQ ID NO: 3), PstSl (SEQ ID NO: 4), HspX (SEQ ID NO: 5), Rvl363c (SEQ ID NO: 6), Rv0826 (SEQ ID NO: 7) or mixtures thereof.

23. The method of claim 19, wherein the target molecule comprises one or more of: an antibody, a paratope, a protein, DNA, RNA, a virus, a bacterium, and a mammalian cell.

24. The method of claim 19, wherein the target molecule comprises one or more antibodies.

25. The method of claim 24, wherein the one or more antibodies are M. tuberculosis (Mtb)- specific antibodies.

26. The method of claim 19, wherein the probe molecule comprises an Fc receptor and/or immunoglobulin G (IgG) immunoglobulin A (IgA), immunoglobulin M (IgM) and/or lectins including sambucus nigra lectin (SNA) and ricinis communis agglutinin 1 (RCA1).

27. The method of claim 19, wherein the probe molecule comprises monomeric FcR3B.

28. The method of claim 19, wherein the probe molecule comprises multimeric FcR3B.

29. The method of claim 27, wherein the monomeric FcR3B is conjugated to horseradish peroxidase (HRP).

30. The method of claim 28, wherein the multimeric FcR3B is conjugated to polymerized HRP.

31. The method of claim 26, wherein the immunoglobulin G is conjugated to HRP.

32. The method of claim 19, wherein the molecule of interest is attached to the surface of a bead.

33. The method of claim 19, wherein the molecule of interest is attached to a polydimethylsiloxane (PDMS) film.

34. The method of claim 19, wherein the composition is with a point of care (POC) diagnostic kit, the POC diagnostic kit comprising an array of wells.

35. The method of claim 19, wherein the metal particle comprises silver.

36. The method of claim 19, wherein the composition is configured to distinguish an active tuberculosis infection from a latent tuberculosis infection.

37. The method of claim 19, wherein the subject is HIV positive.