Configurations and methods for detecting insulin autoantibodies by enzyme-linked immunosorbent assay

Abstract

Disclosed herein are compositions, methods, devices, and systems for detection of T1D autoantibodies. Bridging complexes for use in enzyme-linked immunosorbent assays (ELISAs) are disclosed. The bridging complexes can be used to detect insulin autoantibodies (lAAs) in human and animal blood or serum. The disclosed compositions, methods, devices, and systems can form complexes between a biotin-labeled proinsulin molecule, an TAA, and a dinitrophenol (DNP)-labeled proinsulin molecule.

Description

CONFIGURATIONS AND METHODS FOR DETECTING INSULINAUTOANTIBODIES BY ENZYME-LINKED IMMUNOSORBENT ASSAYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 715,326, filed 1 November 2024, and U.S. Provisional Application No. 63 / 886,541, filed 19 August 2025, the entire disclosures of which are hereby incorporated by reference.BACKGROUND

[0002] Islet autoantibodies (lAbs) are reliable biomarkers for type 1 diabetes (T1D). lAbs can appear in the peripheral blood years before the clinical onset of T1D. Autoantibody to insulin (IAA) is a diagnostic marker that can be used in young children and is generally the first autoantibody to mark the initiation of islet autoimmunity.

[0003] In the United States, 1.6 million people have T1D and as many have lAbs or preclinical T1D. Of those with preclinical T1D, 84% will progress to clinical diabetes in 15 years with a remarkable consistency across populations. It is useful to identify T1D patients before the onset of symptoms to prevent life-threatening diabetic ketoacidosis and provide an opportunity to participate in trials to prevent T1D. While screening programs for T1D are underway worldwide and multiple candidate interventions are being proposed to abrogate or slow progression to diabetes among lAb positive individuals, mass screening of the general population faces obstacles. Current standard radio-binding assays (RBA), where each lAb is measured individually, are laborious and inefficient.

[0004] Enzyme-Linked Immunosorbent Assay (ELISA) is a common diagnostic method used in laboratories. Unfortunately, there are currently no workable ELISAs available for IAA detection due to several barriers. Currently, there is one workable ELISA for other islet autoantibodies. For the last three decades, laboratories have made efforts to find an ELISA method to detect insulin autoantibodies, but they have failed to produce a working assay.

[0005] ELISA is a common method in laboratories for rapid detection of a variety’ of biomolecules, which ranges from antigens and peptides to proteins and antibodies. An ELISA-based 3 Screen ICA™ kit recently became available. This assay measures three lAbs (GADA, IA-2A, and ZnT8A) in a single well. The kit has been used in general population screening for T1D in Germany. A notable disadvantage of this kit is the14916-3137-2662'1inability to detect a major lAb - IAA. IAA is a key lAb in young children since it is generally the first lAb to appear. Moreover, IAA has a high rate of positivity to mark the initiation of islet autoimmunity.

[0006] Accordingly, reagents, compositions, and methods for rapid detection of autoantibodies in diabetes are desirable.SUMMARY

[0007] An aspect of the present disclosure relates to a composition for use with an enzyme-linked immunosorbent assay (ELISA). The composition includes a first antigen with a first label molecule and a second antigen with a second label molecule. The first antigen and the second antigen have affinity for insulin autoantibody (IAA).

[0008] In one or all examples, at least one of the first label molecule or the second label molecule can be selected from biotin or dinitrophenol (DNP). At least one of the first antigen or the second antigen can be proinsulin. In one or all examples, the first antigen can include proinsulin. The first label molecule can include biotin. The second antigen can include proinsulin. The second label molecule can include DNP. In one or all examples, the second label molecule can be different from the first label molecule. The composition can be configured to form a bridge between the first antigen with the first label molecule, the IAA, and the second antigen with the second label molecule.

[0009] In one or all examples, the composition can be operable to detect type 1 diabetes (T1D) with a specificity of at least 98%. In one or all examples, the composition can further include a plurality of molecules to detect one or more of glutamic acid decarboxylase-65 (GAD A), insulinoma-associated antigen-2 (IA-2A). or zinc transporter- 8 (ZnT8A).

[0010] Another aspect of the present disclosure relates to a method of detecting an insulin autoantibody (IAA). The method includes forming a fluid by combining a biological fluid sample with a first antigen and a second antigen, adding the fluid to a well including a plate with a molecule with an affinity for at least one of the first antigen or the second antigen, adding a reagent to the well such that the reagent interacts with at least one of the first antigen or the second antigen to produce a signal and detecting the signal. The first antigen includes a first labeling molecule, the second antigen includes a second labeling molecule, and the first antigen and the second antigen have an affinity for the IAA.24916-3137-2662'1

[0011] In one or all examples, the first antigen and the second antigen can form a complex with the IAA in the fluid. In one or all examples, at least one of the first antigen or the second antigen can include proinsulin. In one or all examples, one of the first labeling molecule or the second labeling molecule can include biotin. The other of the first labeling molecule or the second labeling molecule can include dinitrophenol (DNP). In one or all examples, the molecule can be attached to the plate. The molecule can include an anti-DNP antibody. In one or all examples, the method can further include adding horseradish peroxidase (HRP)-conjugated streptavidin to the well before adding the reagent to the well. In one or all examples, the reagent can include a 3, 3', 5,5'- tetramethylbenzidine (TMB) substrate.

[0012] In one or all examples, the signal can include a color signal that can be detected through an enzyme-linked immunosorbent assay (ELISA) plate reader. In one or all examples, the signal can be operable to detect type 1 diabetes (T1D) with a specificity of at least 98%. In one or all examples, the method can further include detecting at least one of glutamic acid decarboxylase-65 (GAD A), insulinoma-associated antigen-2 (IA-2A), or zinc transporter-8 (ZnT8A) using an additional molecule provided in the well.

[0013] Yet another aspect of the present disclosure relates to a method of forming a composition for detecting an insulin autoantibody (IAA). The method includes providing a first solution including dissolved proinsulin, labeling a first portion of the first solution with biotin, labeling a second portion of the first solution with dinitrophenol (DNP), and mixing the first portion of the solution with the second portion of the solution to form a second solution.

[0014] In one or all examples, the first portion of the first solution can be labeled using a molar ratio of proinsulin to biotin in a range from 1:3 to 1: 10. The second portion of the first solution can be labeled using a molar ratio of proinsulin to DNP in a range from 1:3 to 1: 10. In one or all examples, the method can further include incubating the first portion and the second portion of the first solution at room temperature with shielding from light for a period of greater than 1 hour and purifying the first portion and the second portion of the first solution to remove unbounded biotin and DNP. In one or all examples, the method can further include mixing the second solution with a sample of blood or serum including an IAA such that the IAA binds to biotin labeled proinsulin and DNP-labeled proinsulin.BRIEF DESCRIPTION OF THE DRAWINGS34916-3137-2662'1

[0015] The disclosure will be readily understood by the following detailed description in conjunction with the accompanying drawings, wherein like reference numerals designate like structural elements, and in which:

[0016] FIG. 1 illustrates a schematic view of a complex attached to a solid phase for use in an ELISA.

[0017] FIG. 2 illustrates a graph of positivity rates for IAA detected by the complex of FIG. 1 in an ELISA.

[0018] FIG. 3 illustrates a graph of positivity' rates for IAA detected by an ELISA-IAA and an RBA-IAA.

[0019] FIG. 4 illustrates a graph of positivity' rates for IAA detected by an ELISA-IAA and an ECL-IAA assay.

[0020] FIG. 5A illustrates a graph comparing positivity rates for IAA detected by an ELISA-IAA, an RBA-IAA, and an ECL-IAA.

[0021] FIG. 5B illustrates a graph comparing positivity rates for IAA detected by an ELISA-IAA, an RBA-IAA, and an ECL-IAA.

[0022] FIG. 6 illustrates a graph of an IAA affinity analysis of subjects with single IAA positivity'.DETAILED DESCRIPTION

[0023] Reference will now be made in detail to representative embodiments illustrated in the accompanying drawings. It should be understood that the following descriptions are not intended to limit the embodiments to one preferred embodiment. To the contrary, it is intended to cover alternatives, modifications, and equivalents as can be included within the spirit and scope of the described embodiments as defined by the appended claims.

[0024] Disclosed herein are compositions, methods, devices, and systems for detection of type 1 diabetes (T1D) autoantibodies. In some examples, bridging complexes for use in enzyme-linked immunosorbent assay (ELISA) autoantibody assays are disclosed. The bridging complexes can be useful in detecting insulin autoantibodies (lAAs) in human and / or animal serum. In particular, disclosed herein are compositions, methods, devices and systems for detection of a major islet autoantibody for type 1 diabetes.

[0025] The disclosed assay can be performed in a 96-well microplate such as an ELISA plate. The insulin autoantibodies (lAAs) may be present in the blood or serum of a human or animal patient or subject. The lAAs may be used to bridge two insulin and / or44916-3137-2662'1proinsulin antigen molecules. In one or all examples, the insulin / proinsulin can be labeled with a plurality of different labeling molecules. This can include two different labeling molecules. The labeled insulin / proinsulin can form a complex (e.g., including a first antigen-autoantibody-second antigen) in a fluid-phase. The first labeled antigen molecule in the complex can be captured on a solid phase which matches its corresponding conjugate. The second labeled antigen in the complex can produce a color signal through its labeled molecules upon binding with enzyme-substrates. The plate can be submitted to an ELISA plate reader for detection and quantitation of the color signal.

[0026] The sensitivity and specificity of the presently disclosed ELISA-IAA assay has been compared with the current standard IAA assay, which is based on a radio-binding assay (RBA). The selected serum samples included 43 samples from newly diagnosed patients with type 1 diabetes who were positive at differing levels of IAA. This group included a group of very low IAA levels - that is levels just above the assay cut-off of RBA. With the 100% specificity in 11 healthy control samples, the presently disclosed ELISA-IAA identified nearly all the IAA positive samples - 42 of 43. In one or all examples, the disclosed ELISA-IAA's sensitivity is greater and identifies patients that are negative by RBA. In one or all examples, the disclosed ELISA-IAA has a sensitivity that is greater than or equal to the currently standard RBA method for IAA detection.

[0027] Generally, it has been found that four major islet autoantibodies (lAbs) are useful for T1D prediction. This includes autoantibodies to insulin (IAA), glutamic acid decarboxylase-65 (GADA), insulinoma-associated antigen-2 (1A-2A), and zinc transporter-8 (ZnT8A). The multiplex enzyme-linked immunosorbent assay (ELISA) disclosed herein is designed to detect a complete panel of the 4 major islet autoantibodies, including IAA, GADA, IA-2A, and ZnT8A in human and / or animal serum or plasma for type 1 diabetes (T1D). In one or all examples, this can be referred to as a complete panel of lAbs. In one or all examples, the disclosed multiplex 4-IAb ELISA can be a useful tool for general population screening for T1D. The disclosed multiplex 4-IAb ELISA can have higher throughput, lower cost, greater portability to transfer to other laboratories, than existing assays.

[0028] The disclosed multiplex 4-IAb ELISA can be performed on a 96-well plate such as an ELISA plate. The multiplex 4-IAb ELISA can detect each lAb present in a blood and / or serum sample of a human and / or animal patient or subject. Each lAb may bridge the same two of its specifically recognized antigen molecules labeled with two different labeling molecules and form a complex (first antigen-autoantibody-second antigen) in a54916-3137-2662'1fluid-phase. The first labeled antigen molecule in the complex can be captured on a solid phase that matches its corresponding conjugate. The second labeled antigen in the complex can produce a color signal through its labeled molecules upon binding with enzyme-substrates. The plate can be counted on a standard ELISA plate reader. The positive signal can be detected if one or multiple lAbs present in the blood. The positive sample can be repeated with 4 single ELISAs to determine what specific IAb(s) the positive signal in the multiplex ELISA belongs to. In the general population, over 95% of people are negative for all lAbs. This multiplex 4-IAb ELISA can be used as a screening tool in a non-diabetic population with high throughput, low cost, and easily applicable in all laboratories.

[0029] The sensitivity and specificity of the multiplex 4-IAb ELISA have been compared with the current standard radio-binding assay (RBA). The selected serum samples included 24 samples from newly diagnosed patients with diabetes. 7 samples were negative for all lAbs and 17 samples were positive by RBA (e.g, 5 samples were positive for a single lAb and 12 samples were positive for multiple lAbs). The results from methods using the present multiplex 4-IAb ELISA correlated well with the current RBA method. The disclosed multiplex 4-IAb ELISA demonstrated 100% specificity in 8 healthy control samples and identified all but one of the 17 positive samples detected by RBA. The single exception was a sample with a single IAA alone at a low level. The disclosed multiplex 4-IAb ELISA was negative for all 7 patients who were negative for all lAbs detected by RBA.

[0030] A large proportion of insulin autoantibodies (IAA) detected by current standard radio-binding assay (RBA) are not predictive for ty pe 1 diabetes (T1D) due to low affinity. The most common laboratory method, enzyme-linked immunosorbent assay (ELISA) lacks sensitivity and specificity, specifically toward IAA. The ELISA-IAA configurations and methods disclosed herein are bridging assays for IAA detection with differential risk. The ELISA-IAA configurations and methods can detect high-affinity IAA with high sensitivity and specificity, providing an improved detection for IAA. The ELISA-IAA can be combined with ELISA configurations and methods used to detect GADA, 1A-2A, and ZnT8A to provide a complete ELISA panel.

[0031] While traditional ELISA assays for IAA lack sensitivity and specificity, the current standard fluid phase RBA for IAA has proven difficult for many laboratories to implement. Though IAA are usually of high affinity, capacities are low and assay signals in most patients are very low, close to assay threshold except for younger children64916-3137-2662'1developing diabetes. Islet Autoantibody Standardization Program (IASP) workshops have demonstrated that though most laboratories have good specificity and sensitivity when measuring GADA, IA-2A, and ZnT8A, similar performance has not been achieved for IAA. Insulin is a relatively small protein of only 51 amino acids with disulfide linked A and B chains and thus it was not surprising that insulin directly bound to plates did not allow detection of IAA by traditional ELISA format. Given the desire for improved IAA assays and the hypothesis that the binding of insulin to solid phases can obscure a key determinant for recognition by human autoantibodies, Applicants have developed a non- radioactive electrochemiluminescence (ECL)-IAA assay that immobilizes insulin to a solid phase that preserved critical determinants. The ECL-IAA assay has shown a top performance in multiple IASP workshops for its excellent sensitivity and specificity. However, the ECL assay platform uses special equipment and plates that makes it difficult to use across laboratories. The disclosed bridging ELISA-IAA assay adopted the ECL assay format that allows the interaction of IAA with insulin antigen completely in a fluid-phase and the present study data has illustrated its high sensitivity and specificity. The disclosed ELISA-IAA assay, built on an ELISA format, is low-cost and easy to implement across laboratories. It holds strong potential as a practical tool for both population-based screening and clinical diagnosis of T1D.

[0032] Mass screening for lAbs in the general population is being conducted globally to prevent T1D. In screening studies of either relatives of T1D patients or the general population, the most detected lAbs are isolated single lAbs, typically GADA or IAA. These single lAbs are low-affinity in 60-80% of cases, and low-affinity autoantibodies typically do not mature into high-affinity forms over time. A large proportion of IAA detected by RBA in both relatives of T1D patients and the general population are low- affinity and not predictive of disease. The currently standard method of RBA demonstrated good sensitivity and specificity in T1D patients, compared with healthy controls but lacks specificity in non-diabetic population screening due to its inability to distinguish high-affinity from low-affinity antibodies, resulting in an overall poor predictive value. Applicant’s ECL assay format, with its unique feature of high-affinity antibody binding, has demonstrated a surprising ability to discriminate high-affinity from low- affinity lAbs with differentiating the risk, and enhancing the predictive value of single lAbs. Adopted from the ECL-IAA assay mechanism, the presently disclosed bridging ELISA-IAA assay retains the unique ability to detect high-affinity antibody binding, enabling discrimination of high- from low-affinity autoantibodies. Applicant’s74916-3137-2662'1study demonstrated the ELISA-IAA assay selectively identified individuals associated with the disease, including newly diagnosed patients with T1D, high-risk subjects positive for multiple lAbs, and the subjects with high-affinity single IAA detected by ECL assay.

[0033] These and other examples are discussed below with reference to FIGS. 1 through 6. However, those skilled in the art will readily appreciate that the detailed description given herein with respect to these figures is for explanatory purposes only and should not be construed as limiting. Furthermore, as used herein, a system, a method, an article, a component, a feature, or a sub-feature including at least one of a first option, a second option, or a third option should be understood as referring to a system, a method, an article, a component, a feature, or a sub-feature that can include one of each listed option (e.g.. only one of the first option, only one of the second option, or only one of the third option), multiple of a single listed option (e.g., two or more of the first option), two options simultaneously (e.g., one of the first option and one of the second option), or combination thereof (e.g.. two of the first option and one of the second option).

[0034] FIG. 1 illustrates a schematic view of a complex 100 attached to a solid phase 102. The complex 100 can be attached to the solid phase 102 in an enzyme-linked immunosorbent assay (ELISA). The ELISA can be used to detect an antibody 104, such as insulin autoantibody (IAA), through detecting the complex 100. The complex 100 can be formed between the antibody 104, a labeled capture antigen 106, and a labeled detection antigen 108. The capture antigen 106 can be attached to the solid phase 102 through a capture antibody 110 and a labeling molecule 112. The detection antigen 108 can be attached to a substrate 114 and an enzyme 116 through a binding agent 120 and a labeling molecule 118. A signal can be generated through the substrate 114 and the enzyme 116 when the antibody 104 is present in a sample such that the complex 100 can be used to detect the antibody 104. The complex 100 can be formed between the capture antigen 106, the antibody 104, and the detection antigen 108 in the fluid phase, such as in a well of an ELISA. The complex 100 or portions thereof (e.g., the capture antigen 106, the detection antigen 108. or other portions of the complex 100) can be used as or in compositions in an ELISA.

[0035] The antibody 104 can be present in blood or serum of a human or animal patient. The antibody 104 can be or include IAA. IAA is an important islet autoantibody (IAb) for detecting type 1 diabetes (T1D) and can be used to detect T1D in young children.84916-3137-2662'1IAA is usually the earliest islet autoantibody detected in children at risk for T1D. The antibody 104 can be present in a fluid sample and can be provided to an ELISA in a fluid form.

[0036] The solid phase 102 can be a surface of a well (e.g., a bottom surface) of a microplate in an ELISA. The solid phase 102 can be a plate. In one or all examples, the solid phase 102 can be a single plate of a 96-well plate. However, any type of ELISA plate or apparatus can be used and can include the solid phase 102. The solid phase 102 can include materials to which the complex 100 can bind, such as polystyrene, polypropylene, nitrocellulose, nylon, polyvinylidene difluoride (PVDF), or the like. After the complex 100 is attached to the solid phase 102, the solid phase 102 can be analyzed by a microplate reader (e.g., a standard ELISA plate reader) or the like to determine the presence or absence of the complex 100. Thus, the presence or absence of the antibody- 104 in a solution can be determined and detected.

[0037] The antibody 104 can form or act as a bridge between two insulin and / or proinsulin antigen molecules (e.g., the capture antigen 106 and the detection antigen 108). In other words, the capture antigen 106 and the detection antigen 108 can be insulin and / or proinsulin molecules. Proinsulin can have an affinity for IAA such that the capture antigen 106 and the detection antigen 108 have an affinity for the antibody 104. A complex is formed between the capture antigen 106, the antibody 104, and the detection antigen 108. The complex includes a first antigen (capture antigen 106)-an autoantibody (antibody 104)-a second antigen (detection antigen 108).

[0038] Each of the capture antigen 106 and the detection antigen 108 can be labeled antigen molecules. For example, the capture antigen 106 can be labeled through the labeling molecule 112. The detection antigen 108 can be labeled through the labeling molecule 118. Labeling the capture antigen 106 and the detection antigen 108 through the labeling molecule 112 and the labeling molecule 118, respectively, can aid in detecting the antibody 104 and quantifying the antibody 104.

[0039] The capture antigen 106 can be attached to the solid phase 102 through the labeling molecule 112. The capture antigen 106 can be captured on a solid phase (e.g., on the solid phase 102) that matches its corresponding conjugate. The capture antigen 106 can be used to capture the antibody 104 on the solid phase 102 and is thus referred to as the capture antigen 106. The labeling molecule 112 can include dinitrophenol (DNP). The labeling molecule 112 can be attached to the solid phase 102 through a capture antibody 110. The capture antibody 110 can include a monoclonal antibody (mAb). The94916-3137-2662'1capture antibody 110 can be an antibody specific to the labeling molecule 112. For example, when the labeling molecule 112 includes dinitrophenol, the capture antibody 110 can be an anti-DNP antibody.

[0040] The detection antigen 108 can be bound to a substrate 1 14 and an enzyme 116 through a labeling molecule 118 and binding agent 120. The substrate 114 and the enzyme 116 can produce a color signal or other signal that can be detected to detect a presence and determine a quantity of the antibody 104 in a sample. The substrate 114 and the enzyme 116 can be attached to the solid phase 102 through the antibody 104. As such, in an example in which the antibody 104 is not present, the substrate 114 and the enzyme 116 will not be bound to the solid phase 102. The signal can be analyzed to determine the presence of the antibody 104 in a solution and / or the concentration of the antibody 104 in the solution. Thus, the detection antigen 108 is referred to as the detection antigen 108. The signal can be or include a color signal, such as a colorimetric, fluorescent, chemiluminescent, or other color-related signal. The substrate 114 can include a TMB (3,3', 5,5'-tetramethylbenzidine) peroxidase substrate. The enzyme 116 can include a horseradish peroxidase (HRP) enzyme. The substrate 114 can react with the enzyme 116 to produce a colored product, which can then be detected to detect a presence and / or concentration of the antibody 104 in a sample. In other words, the antigen-antibody complex present in the complex 100 can generate a measurable signal through enzyme-substrate reactions between the enzyme 116 and the substrate 114, which is detected using a plate reader, such as a standard ELISA plate reader. Other pairs of substrates 114 and enzymes 116 can be used in the complex 100, such as alkaline phosphatase (AP) for the substrate 114 and p-nitrophenyl-phosphate (pNPP) for the enzyme 116; 2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid] (ABTS), 3-amino-9- ethylcarbazole (AEC), for the substrate 114 and HRP for the enzyme 116; or the like.

[0041] The labeling molecule 118 can be attached or bound to the detection antigen 108 and the enzyme 116 can be attached or bound to the labeling molecule 118 through a binding agent 120. The labeling molecule 118 can include biotin. The binding agent 120 can include streptavidin (SA). The binding agent 120 can be a binding agent used to link the substrate 114 and the enzyme 116 to the biotinylated antibody including the labeling molecule 118 and the detection antigen 108. In one or all examples, dinitrophenol can be used for the labeling molecule 118 and biotin can be used for the labeling molecule 112.

[0042] In summary, the complex 100 can include the capture antibody 110 bound or attached to the solid phase 102. the labeling molecule 112 bound or attached to the104916-3137-2662'1capture antibody 110, the 106 bound or attached to the labeling molecule 112, the antibody 104 bound or attached to the capture antigen 106, the detection antigen 108 bound or attached to the antibody 104. the labeling molecule 118 bound or attached to the detection antigen 108, the binding agent 120 bound or attached to the labeling molecule 118, the enzyme 116 bound or attached to the binding agent 120. The substrate 114 can react with the enzyme 116 to produce a signal, which can be used to detect the presence and / or concentration of the antibody 104 in a sample.

[0043] The complex 100 can be formed by dissolving proinsulin (e.g., forming the capture antigen 106 and the detection antigen 108) in a double concentrated phosphate- buffered solution (2x PBS) with a pH of about 7.9. The proinsulin can be labeled with biotin and dinitrophenol (DNP). The biotin and the dinitrophenol can be added to the solution in a 1 :5 molar ratio and incubated at room temperature (RF) for about 2 hours (or 1 hour or greater) with shielding from light.

[0044] In one or all examples, the solution can be separated into a first portion and a second portion. The first portion can be labeled with biotin by supplying proinsulin and biotin in a molar ratio in a range from 1:3 to 1: 10 (such as about 1:5). The second portion can be labeled with DNP by supplying proinsulin and DNP in a molar ratio in a range from 1 :3 to 1 : 10 (such as about 1:5). After incubation, the products can be purified with desalting spin columns to remove any unbounded biotin or dinitro ph enol. The biotin can form the labeling molecule 118 and the dinitrophenol can form the labeling molecule 112. Thus, the capture antigen 106 can be labeled with the labeling molecule 112 (e.g., dinitrophenol) and the detection antigen 108 can be labeled with the labeling molecule 118 (e.g., biotin).

[0045] A protocol for forming an assay (e.g., an ELISA) can optimize concentrations and a ratio of biotinylated to DNP-labeled proinsulin (e.g., a ratio of the detection antigen 108 with the labeling molecule 118 to the capture antigen 106 with the labeling molecule 112). For example, a checkerboard assay can be performed to determine the optimal conditions.

[0046] To minimize assay background, a step of pre-heat treatment of serum can be introduced into the assay. The serum can be a serum to be tested for the presence of the antibody 104. As an example, about 5 pL of the serum can added into a 96-well polymerase chain reaction (PCR) plate and preheated at a temperature of about 56 °C for about 30 minutes. The serum can be diluted in a ratio of 1 :4 with phosphate-buffered solution (lx PBS). The solution can then be mixed with about 20 pL of antigen solution.114916-3137-2662'1The antigen solution can include about 100 ng / mL of biotinylated (e g., including the detection antigen 108 with the labeling molecule 118) and about 100 ng / mL of DNP- labeled proinsulin (e.g.. including the capture antigen 106 with the labeling molecule 112). The mixture can be incubated at room temperature (RT) for about 2 hours. This can be followed by an overnight incubation at about 4 °C. This solution can include any antibody 104 present in a solution, the detection antigen 108 attached to the labeling molecule 118, and the capture antigen 106 attached to the labeling molecule 112.

[0047] Separately, a 96-well ELISA plate can be pre-coated with about 150 pL of monoclonal anti-DNP antibody (e g., the capture antibody 110) at the concentration of about 125 ng / mL. This can be used to attach the capture antibody 110 to the solid phase 102. The pre-coated ELISA plate can be washed with ash buffer three times and blocked with the assay buffer at RT for about 1 hour.

[0048] About 40 pL of the overnight-incubated mixture (including the antibody 104, the capture antigen 106 with the labeling molecule 112 and the detection antigen 108 with the labeling molecule 118) can be transferred to the coated plate (including the capture antibody 110 attached to the solid phase 102) and incubated at RT for about 1 hour. The plate can be washed again with a final wash including 0.4 M sodium chloride (NaCl) to reduce any non-specific binding.

[0049] Horseradish peroxidase (HRP)-conjugated streptavidin can then be added to the solution and the solution can be incubated at RT for about 1 hour. The HRP-conjugated streptavidin includes the enzyme 116 conjugated with the binding agent 120.

[0050] This can be followed by another three washes, which can remove any unreacted reactants. A peroxidase substrate (3,3',5,5'-tetramethylbenzidine (TMB)) can then be added. The peroxidase substrate includes the substrate 114. The TMB can react with the HRP to produce a colored solution that indicates the presence of the antibody 104. Thus, the TMB can act as a reagent that interacts with the HRB (bound to the detection antigen 108) to produce a signal. If the solution does not change color, this shows a lack of the antibody 104. Absorbance of the solution can be measured at 450nm using a standard ELISA plate reader. The intra-assay coefficient variation was 4.3% (n=6) and inter-assay coefficient variation was 7.6% (n=6). A mouse monoclonal insulin antibody-125 was used as the assay internal standard positive control and the result was expressed as an index (index=[Signalsample - SignalNegativeControl] / [SignalPositiveControl- SignalNegativeControl]).124916-3137-2662'1

[0051] The complex 100 of the present disclosure can be used to detect IAA. This can be used to detect T1D risk and can be particularly useful in providing early diagnoses for children. The complex 100 of the present disclosure can be used with and included with other detection molecules in an ELISA or other assay to detect all four of the major islet antibodies (lAbs). This includes IAA, as well as glutamic acid decarboxylase-65 (GAD A), insulinoma-associated antigen-2 (IA-2A), and zinc transporter-8 (ZnT8A). Thus, a 4-panel ELISA can be provided.

[0052] The currently disclosed new “4-Screen” ELISA (including the complex 100 for detecting IAA) offers a complete panel, with enhanced disease-specificity and is an improved tool for the general population screening with high-throughput, low cost, and easy to use across laboratories. Follow-up testing using the individual ELISA assays for each lAb can be useful to confirm the specific lAbs. Nonetheless, the new multiplex assay will efficiently exclude over 95% of individuals who are negative for all lAbs in the general population screening, serving as an effective first-line screening tool.

[0053] In one or all examples, a complex the same as or similar to the complex 100 can be used in an electrochemiluminescence (ECL). The complex can use biotinylated and sulfo-tagged proinsulin to bind and detect IAA. The complex 100 can act in a similar way, using DNP-labeled and biotinylated proinsulin, which allows the interaction of antibody 104 with proinsulin (e.g., the capture antigen 106 and the detection antigen 108) completely in the fluid-phase. This is preferable to methods that do not interact completely in the fluid-phase. Both DNP-labeled and biotinylated proinsulin (capture antigen 106 and detection antigen 108, respectively) were tested as competitors in the standard RBA-IAA assay and demonstrated that both modified proteins (capture antigen 106 and detection antigen 108) were able to compete with I, 25-insulin for binding to patients’ IAA (antibody 104 in a sample). Monoclonal antibody (capture antibody 110) against DNP (labeling molecule 112) was coated onto the plate (solid phase 102) to capture DNP-labeled proinsulin (capture antigen 106). IAA (antibody 104) in the serum bridged the DNP-labeled and biotinylated proinsulin molecules (capture antigen 106 and detection antigen 108. respectively), then detection was achieved by streptavidin- conjugated HRP (including binding agent 120 and enzyme 116), which binds to the biotinylated proinsulin (detection antigen 108).

[0054] RBA assays and ECL assays for IAA, GAD A, IA-2A, and ZnT8A are discussed throughout the present disclosure. The RBA assays can have sensitivities and specificities of 56% and 99% for IAA. 90% and 99% for GADA, 76% and 100% for IA-134916-3137-2662'12A, and 80% and 100% for ZnT8A, respectively. To determine IAA affinity by RBA, standard IAA RBA was performed in which IAA in sera were absorbed with a serial dilution of unlabeled insulin antigen protein in 6 different concentrations: (5.7 / 106- 5.7 / 101 1M). Inhibitions for 50% of signals at the concentrations of unlabeled IAA were counted and the relative affinities were compared between antibodies. The ECL assays can have sensitivities and specificities of 58% and 99% for IAA. respectively.

[0055] All statistical analyses were performed using GraphPad Prism software (version 8.0; GraphPad Software Institute). For continuous variables, the mean, median, range, and SD were presented. Frequency and its percentage were generated for categorical variables. Between-group comparisons w ere performed with the t test for continuous variables and with the chi-square or Fisher exact test for categorical variables as appropriate. Pearson correlation analysis was performed to assess the strength of correlation betw een levels of IAA in different assays. A P value of less than 0.05 on the 2-sided test was considered statistically significant.

[0056] FIG. 2 illustrates a graph 200 of positivity rates for IAA detected by the complex 100 of FIG. 1 in an ELISA. FIG. 2 compares results for a control group 202 and a group of type 1 diabetes (T1D) patients (T1D positive group 204). The T1D positive group 204 included serum samples from 227 T1D patients that tested positive for at least one islet autoantibody by radio-binding assay (RBA). The T1D patents had a median age of 9.9 years and were 46.7% female. The serum samples were obtained within two weeks of diagnosis. The control group 202 includes serum samples obtained from 108 age-matched healthy children negative for all islet autoantibodies by RBA. The y-axis indicates the log 10 of the reactivity index for IAA detected in samples using the ELISA. Samples with reactivity indexes greater than a cut-off 206 were determined to be positive for IAA, while samples with reactivity indexes less than the cut-off 206 were determined to be negative for IAA.

[0057] The specificity of the complex 100 in the ELISA was set to 99.4%. An upper limit of a normal range was set at the 99.4th percentile (assay specificity) of index values in the control group 202 (index = 0.083). In one or all examples, the specificity can be set to 98% or higher or the 98th percentile or higher. Thus, the complex 100 and a composition including portions thereof can be operable to detect IAA with a specificity of 98% or greater. In the 108 control subjects of the control group 202, the bridging ELISA (using the complex 100) detected IAA in two subjects. In the 227 T1D patients, IAA was detected by the complex 100 (also referred to as the bridging ELISA due to the144916-3137-2662'1detection of bridging by the antibody 104 between the capture antigen 106 and the detection antigen 108) in 148 subjects. The same T1D positive group 204 was analyzed by RBA and IAA was only detected in 106 subjects, resulting in a sensitivity of 60.8%. Thus, the configurations and methods of the present disclosure can provide an increased sensitivity to IAA relative to conventional RBA tests.

[0058] FIG. 3 illustrates a graph 300 of positivity rates for IAA detected by the complex 100 of FIG. 1 in an ELISA as compared to positivity rates for IAA detected by RBA. The y-axis indicates the loglO of the reactivity index for IAA detected in samples using the ELISA. The x-axis indicates the loglO of the reactivity index for IAA detected in samples using the RBA. Samples with reactivity indexes greater than a cut-off 302 were determined to be positive for IAA by ELISA. Samples with reactivity indexes less than the cut-off 302 were determined to be negative for IAA by ELISA. Samples with reactivity’ indexes greater than a cut-off 304 were determined to be positive for IAA by RBA. Samples with reactivity indexes less than the cut-off 304 were determined to be negative for IAA by RBA.

[0059] The positivity rates and antibody levels measured by the ELISA-IAA and the standard RBA (mlAA) are compared in the graph 300. With the 99th percentile of specificity set for both assays, 65.2% (148 / 227) of the new- onset T1D patients tested positive for ELISA-IAA, compared to 60.8% (138 / 227) by RBA. Graph 300 compares the same T1D positive group 204 as the graph 200. The IAA levels of the two assays were well correlated (r = 0.467, p < 0.0001). Of the 138 patients positive for IAA by RBA, 136 were also positive for IAA by ELISA. The two remaining patients that tested positive for IAA by RBA but not by ELISA had very low IAA levels by RBA. The ELISA test for IAA identified IAA in 12 additional patients who were negative by RBA. Thus, the complex 100 and the configurations and methods of the present disclosure can detect IAA by ELISA with higher sensitivity relative to RBA.

[0060] FIG. 4 illustrates a graph 400 of positivity rates for IAA detected by the complex 100 of FIG. 1 in an ELISA as compared to positivity rates for IAA detected by the complex 100 of FIG. 1 or a similar complex in an electrochemiluminescence (ECL) assay. The y-axis indicates the loglO of the reactivity index for IAA detected in samples using the ELISA. The x-axis indicates the loglO of the reactivity’ index for IAA detected in samples using the ECL. Samples with reactivity indexes greater than a cut-off 402 were determined to be positive for IAA by ELISA. Samples with reactivity indexes less than the cut-off 402 were determined to be negative for IAA by ELISA. Samples with154916-3137-2662'1reactivity indexes greater than a cut-off 404 were determined to be positive for IAA by ECL. Samples with reactivity indexes less than the cut-off 404 were determined to be negative for IAA by ECL.

[0061] The positivity rates and antibody levels measured by the ELISA-IAA and the ECL-ISA are compared in the graph 400. With a 98th or 99th percentile of specificity set for both assays, 81.1% (128 / 158) of the new onset T1D patients tested positive for ELISA-IAA. compared to 73.4% (116 / 158) by ECL. Of the 116 patients positive for IAA by ECL, 111 were also positive for IAA by ELISA. The ELISA test for IAA identified IAA in 18 additional patients who were negative for IAA by ECL. Thus, the complex 100 and the configurations and methods of the present disclosure can detect IAA by ELISA with higher sensitivity relative to ECL.

[0062] FIGS. 5A and 5B illustrate a graph 500 and a graph 502, respectively, comparing positivity' rates for IAA detected by the complex 100 of FIG. 1 in an ELISA with positivity' rates for IAA detected by RBA and positivity rates for IAA detected by an ECL assay. The y-axis in graphs 500, 502 indicates the loglO of the reactivity index for IAA detected in samples using the RBA. The x-axis in graphs 500, 502 indicates the log 10 of the reactivity index for IAA detected in samples using the ELISA. Samples with reactivity indexes greater than a cut-off 504 in graph 500 were determined to be positive for IAA by ELISA. Samples with reactivity indexes less than a cut-off 504 in graph 500 were determined to be negative for IAA by ELISA. Samples with reactivity indexes greater than a cut-off 506 in graph 502 were determined to be positive for IAA by ELISA. Samples with reactivity indexes less than a cut-off 506 in graph 502 were determined to be negative for IAA by ELISA. Samples 508 depicted by circles in graphs 500, 502 indicate positive results for IAA by ECL. Samples 510 depicted by squares in graphs 500, 502 indicate negative results for IAA by ECL.

[0063] The subjects analyzed in FIGS. 5A and 5B included 202 at-risk children who tested positive for IAA by RBA from an Autoimmunity Screening for Kids (ASK) study. The ASK is a screening study for T1D and celiac disease in the general population children aged 1-17 years. All individuals are screened for all 4 major lAbs and transglutaminase autoantibodies for celiac disease by two assay methods including RBA and ECL assays. The ASK samples recruited in the present study include 71 children positive for multiple lAbs (IAA plus other lAbs) and 131 positive for single IAA by RBA. 52 patients were positive by high-affinity ECL assay.164916-3137-2662'1

[0064] As illustrated in graph 500 of FIG. 5A, among the 71 children with multiple lAbs, 80.3% (57 / 71) were ELISA-IAA positive. Of the 14 ELISA-IAA negative cases. 7 were also negative by high-affinity ECL-IAA assay. As illustrated in graph 502 of FIG. 5B, in contrast, among the 131 children with single IAA by RBA, only 48.1 % (63 / 131 , p < 0.0001) were ELISA-IAA positive. When stratified by ECL-IAA results, ELISA-IAA positivity was 78.9% (41 / 52) in those positive for ECL-IAA (indicating high-affinity and predictive antibodies), and only 27.9% (22 / 79, p < 0.0001) in those negative for ECL- IAA (suggesting low-affinity and less predictive antibodies). Thus, the complex 100 and the configurations and methods of the present disclosure can detect high-affinity antibody binding for IAA by ELISA and can discriminate between high- and low-affinity autoantibodies. The high-affinity antibodies can be particularly relevant in diagnosing T1D and the ELISA-IAA can be used to identify subjects with high-affinity IAA.

[0065] FIG. 6 illustrates a graph 600 of an IAA affinity analysis of 10 subjects with single IAA positivity. The graph 600 focuses on 10 samples from the ASK study including 5 samples that were positive for IAA by both RBA and ELISA and 5 samples that were positive for IAA by RBA but negative for IAA by ELISA. The samples selected for IAA affinity analysis include comparable levels of IAA between the two subgroups. The y-axis in graph 600 indicates the percent of insulin not absorbed or bound to IAA. The x-axis indicates the concentration of insulin added. Six different concentrations of unlabeled insulin were used to compete with I125-labeled insulin to compare the speed of IAA absorption. Table 1, below, provides the data illustrated in graph 600 where IC50 is the concentration used to inhibit 50% of the signal.

[0066] A competitive RBA analysis was performed using varying concentrations of unlabeled insulin to compete with I, 25-labeled insulin. Dashed lines with circles represent samples 602 positive for IAA by both RBA and ELISA. Solid lines with triangles represent samples 604 positive by RBA only. The lAA-positive samples detected by both RBA and ELISA used lower concentrations of unlabeled insulin to reach 50% inhibition of binding. The RBA-only positive samples used higher concentrations of insulin to reach 50% inhibition of binding. These findings suggest that IAA detected by both RBA and ELISA are high-affinity antibodies, while those not identified by ELISA are low- affinity and potentially less predictive.

[0067] Table 1: Competitive RBA in samples with RBA-IAA positive174916-3137-2662'1

[0068] While multiple embodiments are disclosed, still other embodiments of the present invention will become apparent to those skilled in the art from the following detailed description. As will be apparent, the invention is capable of modifications in various obvious aspects, all without departing from the spirit and scope of the present invention. Accordingly, the detailed description is to be regarded as illustrative in nature and not restrictive.

[0069] All references disclosed herein, whether patent or non-patent, are hereby incorporated by reference as if each was included at its citation, in its entirety. In case of conflict between reference and specification, the present specification, including definitions, will control.

[0070] Although the present disclosure has been described with a certain degree of particularity, it is understood the disclosure has been made by way of example, and changes in detail or structure may be made without departing from the spirit of the disclosure as defined in the appended claims.184916-3137-2662'1

Claims

CLAIMSWhat is claimed is:1 . A composition for use with an enzyme-linked immunosorbent assay (ELISA), the composition comprising: a first antigen with a first label molecule; and a second antigen with a second label molecule, wherein the first antigen and the second antigen have affinity for insulin autoantibody (IAA).

2. The composition of claim 1, wherein: at least one of the first label molecule or the second label molecule is selected from biotin or dinitrophenol (DNP); and at least one of the first antigen or the second antigen is proinsulin.

3. The composition for use of claim 1, wherein: the first antigen comprises proinsulin; the first label molecule comprises biotin; the second antigen comprises proinsulin; and the second label molecule comprises DNP.

4. The composition for use of claim 1, wherein: the second label molecule is different from the first label molecule; and the composition is configured to form a bridge between the first antigen with the first label molecule, the IAA, and the second antigen with the second label molecule.

5. The composition for use of claim 1, wherein the composition is operable to detect ty pe 1 diabetes (T1D) with a specificity of at least 98%.

6. The composition of claim 1, further comprising a plurality of molecules to detect one or more of glutamic acid decarboxylase-65 (GADA), insulinoma-associated antigen- 2 (IA-2A), or zinc transporter-8 (ZnT8A).

7. A method of detecting an insulin autoantibody (IAA). the method comprising:194916-3137-2662'1forming a fluid by combining a biological fluid sample with a first antigen and a second antigen, the first antigen comprising a first labeling molecule, the second antigen comprising a second labeling molecule, the first antigen and the second antigen having an affinity for the 1AA; adding the fluid to a well comprising a plate with a molecule with an affinity for at least one of the first antigen or the second antigen; adding a reagent to the well such that the reagent interacts with at least one of the first antigen or the second antigen to produce a signal; and detecting the signal.

8. The method of claim 7, wherein the first antigen and the second antigen form a complex with the IAA in the fluid.

9. The method of claim 7, wherein at least one of the first antigen or the second antigen comprises proinsulin.

10. The method of claim 7, wherein: one of the first labeling molecule or the second labeling molecule comprises biotin; and the other of the first labeling molecule or the second labeling molecule comprises dinitro phenol (DNP).

11. The method of claim 7, wherein: the molecule is attached to the plate; and the molecule comprises an anti-DNP antibody.

12. The method of claim 7, further comprising adding horseradish peroxidase (HRP)- conjugated streptavidin to the well before adding the reagent to the well.

13. The method of claim 7, wherein the reagent comprises a 3, 3', 5,5'- tetramethylbenzidine (TMB) substrate.

14. The method of claim 7, wherein the signal comprises a color signal and the signal is detected through an enzyme-linked immunosorbent assay (ELISA) plate reader.204916-3137-2662'115. The method of claim 7, wherein the signal is operable to detect type 1 diabetes (T1D) with a specificity of at least 98%.

16. The method of claim 7, further comprising detecting at least one of glutamic acid decarboxylase-65 (GAD A), insulinoma-associated antigen-2 (IA-2A), or zinc transporter- 8 (ZnT8A) using an additional molecule provided in the well.

17. A method of forming a composition for detecting an insulin autoantibody (IAA), the method comprising: providing a first solution comprising dissolved proinsulin; labeling a first portion of the first solution with biotin; labeling a second portion of the first solution with dinitrophenol (DNP); and mixing the first portion of the solution with the second portion of the solution to form a second solution.

18. The method of claim 17, wherein: the first portion of the first solution is labeled using a molar ratio of proinsulin to biotin in a range from 1 : 3 to 1: 10; and the second portion of the first solution is labeled using a molar ratio of proinsulin to DNP in a range from 1 : 3 to 1: 10.

19. The method of claim 17, further comprising: incubating the first portion and the second portion of the first solution at room temperature with shielding from light for a period of greater than 1 hour; and purifying the first portion and the second portion of the first solution to remove unbounded biotin and DNP.

20. The method of claim 17, further comprising mixing the second solution with a sample of blood or serum comprising an IAA such that the IAA binds to biotin labeled proinsulin and DNP-labeled proinsulin.214916-3137-2662'1

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