Isobaric peptide probes for biomarker detection

Abstract

Disclosed herein are reporter peptide-antibody conjugates and methods of using reporter peptide antibody conjugates to detect the presence of one or more antigens in a given sample. The detector peptide-antibody conjugates can be used in a sandwich immunoassay, and the reporter peptide may be liberated from the sandwich complex and detected using mass spectrometry.

Description

[0001] Attorney Docket No. 103362-047WO1

[0002] ISOBARIC PEPTIDE PROBES FOR BIOMARKER DETECTION

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of priority to United States Provisional Patent Application No. 63 / 745,893, filed January 16, 2025, the disclosure of which is incorporated herein by reference in its entirety.

[0005] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0006] This invention was made with government support under Grant / Contract No. R21 CA270727 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] REFERENCE TO SEQUENCE LISTING

[0008] A Sequence Listing conforming to the rules of WIPO Standard ST.26 is hereby incorporated by reference. Said Sequence Listing has been filed as an electronic document via PatentCenter in ASCII format encoded as XML. The electronic document, created on January 16, 2026, is entitled “iO3362-O47WOi_ST26.xml”, and is 17,571 bytes in size.

[0009] BACKGROUND

[0010] Biomarker detection from biofluids provides a faster, less invasive way of diagnosing a disease. A biomarker is a molecule found in biological serum, such as blood or other bodily fluids, that is used as a sign to detect a disease or a normal or abnormal process in the body. Clinical methods that detect biomarkers include enzyme-linked immunosorbent assay (ELISA), protein microarrays, flow cytometry, and biosensors. However, detecting a single biomarker is insufficient for diagnosing or treating a disease. This suggests that multiplex detection, the process of detecting several biomarkers in a single test, aids in better disease diagnosis and management. Screening various analytes simultaneously enables rapid prognosis, low-cost analysis, and reliable quantification. In point-of-care (POC) testing, multiplexing hasAttorney Docket No. 103362-047WO1

[0011] become more relevant since it can provide immediate and accurate results quickly, is relatively low-cost, and can be used by non-experts.

[0012] A device can be considered as multiplexing POC if the following requirements are met: (i) low sample consumption; (ii) simple or automated system operation; (iii) rapid turnaround times (10 minutes to 2 hours); (iv) prolonged reagent storage and shelf life; (v) accurate and quantitative results by clinical and central laboratory findings; and (vi) low-cost and portable readout devices. Immunoassay technologies in multiplexed POC include using enzymes or nanoparticles that need results within a specified time interval to ensure validity. Multiplex polymerase chain reaction (PCR) is one of the emerging technologies, but faces limitations on sensitivity and requires multi-step procedures to address challenges in method amplification. While these technologies continuously improve, tradeoffs are present; hence, a method that provides more accurate, high-throughput, and sensitive results is needed. While most clinical laboratories routinely use spectrophotometric or immunologic detection, mass spectrometry (MS) provides much greater specificity than these methods. However, complex MS instruments require large pumps and costly equipment that rely on stable electricity, which is challenging in resource-limited settings. To address this gap, a microfluidic paper-based analytical device (microPAD) that uses an ionic probe to detect disease biomarkers and is analyzed by a portable miniature mass spectrometer was developed. The ionic probe, smaller in molecular weight than the intact biomolecule, acts as a reporter tag in detecting the disease. Alongside this ionic probe, the 3D microPAD was designed to contain four spots to perform multiplex biomarker analysis by MS. Since the device requires a small amount of volume (< 60 qL), one limitation of the 3D microPAD is the insufficient sample that can flow through the four spots. Another challenge is the time required for the sample to flow through the layers to the spots. To improve the multiplex capability of the microfluidic device, a probe that can perform multiplex detection is ideal to address current challenges in this technology.

[0013] An immunoassay is a bioanalytical method based on the interaction of the antigen and antibody that has several applications in medicine and pharmaceutical analysis, including drug discovery, drug monitoring, and disease diagnosis. The strong interaction between antigen and antibody makes the immunoassay highly specific. Several immunoassay labels and detectors have been developed to provide opticalAttorney Docket No. 103362-047WO1

[0014] and electrical read-out systems. These immunoassay methods include radioisotope immunoassay, enzyme immunoassay, chemiluminescence immunoassay, and fluoroimmunoassay. Though they provide low detection limits (ng-pg / mL), multiplex analysis with a spectroscopic read-out system can suffer from overlapping signals. Multiplex detection studies are increasing since they offer more accurate results at a lower sample volume consumption. Hence, a sensitive, specific, and high-throughput detection method coupled with an immunoassay can help improve immunoassay analysis. Mass spectrometry is a high-throughput, sensitive instrument that has consistently undergone technical advancements to analyze various samples and can be coupled to another instrument in different applications. There remains a need for improved systems and methods for rapidly detecting the presence of multiple biomarkers in a single test sequence.

[0015] SUMMARY

[0016] In one aspect, methods of detecting an antigen in a composition are provided. In some aspects, the method can include contacting the composition with a capture antibody to form an antigen-capture antibody complex. In some aspects, the method can include contacting the antigen-capture antibody complex with a detector antibody to form a sandwich complex. In some aspects, the detector antibody includes at least one reporter peptide. In some aspects, the method can include separating the sandwich complex from unreacted detector antibody. In some aspects, the method can include separating the reporter peptide from the sandwich complex. In some aspects, the method can include detecting the reporter peptide using mass spectrometry.

[0017] In another aspect, antibody-reporter peptide conjugates are provided. In some aspects, the antibody-reporter peptide conjugate can include an antibody as described herein. In some aspects, the antibody-reporter peptide conjugate can include a reporter peptide having the structure:Attorney Docket No. 103362-047WO1

[0018]

[0019] wherein all variables are as defined herein.

[0020] In another aspect, assay devices are provided. In some aspects, the assay device can include a plurality of layers. In some aspects, the plurality of layers can include a detector layer having one or more antibody-reporter peptide conjugates. In some aspects, the plurality of layers can include a capture layer having one or more capture antibodies. In some aspects, the detector layer is in fluid communication with the capture layer.

[0021] The details of one or more aspects of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the disclosure will be apparent from the description, the drawings, and the claims.

[0022] DESCRIPTION OF DRAWINGS

[0023] FIG. 1 depicts a schematic of paper spray ionization.

[0024] FIGs. 2A-2D depict full MS and MS / MS of peptides (FIG. 2A) LRNPTIDPLN (SEQ ID NO2: 1) (FIG. 2B) LRNPDPYILN (SEQ ID NO2: 2) (FIG. 2C) LRNPTIDPLNR (SEQ ID NO2: 3) and (FIG. 2D) LRNPTIDPLNR (SEQ ID NO2: 3) in 1 mM NH4CH3COO.

[0025] FIGs. 3A-3B depict (FIG. 3A) full MS and (FIG. 3B) MS / MS of peptide LRNPTIDPLNR acetylated at N-terminus (SEQ ID NO2: 4).

[0026] FIGs. 4A-4B depict MS / MS spectra of (FIG. 4A) AcLRDPNPTLINR (SEQ ID NO2: 4) and (FIG. 4B) ACRLDPNPTLINR (SEQ ID NO2: 6) showing different intensities for 667 m / z.

[0027] FIGs. 5A-5B depict the structures of peptide (FIG. 5A) IRNPTIDPINR (SEQ ID NO2: 7) and internal standard (FIG.5B) IRNdsPTIDPINR (SEQ ID NO2: 8), both acetylated at the N-terminus.Attorney Docket No. 103362-047WO1

[0028] FIG. 6 depicts Solvent optimization for 1μg / ml AcIRNPTIDPINR (SEQ ID NO2: 7) at different solvent systems (1:1 MeOH H2O, 80:20 MeOH H2O, 1:1 ACN H2O, and 80:20 ACN: H2O with 0.1% FA.

[0029] FIGs. 7A-7D depict mass spectra of (FIG. 7A) MS / MS and (FIG. 7B) MS3 of peptide AcIRNPTIDPINR (SEQ ID NO2: 7). Cleavage at Asp-Pro produced (FIG. 7C) b7and (FIG. 7D) y4ions.

[0030] FIGs. 8A-8E depict mass spectra of Full MS and MS / MS of peptides (FIG. 8A) AcNLRPTDPLINR (SEQ ID NO2: 9), (FIG. 8B) AcNLRTDPIIPNR (SEQ ID NO2: 10), (FIG. 8C) AcRLDPNPTLINR (SEQ ID NO2: 6), (FIG. 8D) AcLNRPTLLDPNR (SEQ ID NO2: 11), and (FIG. 8E) AcNNITRDPPIIR (SEQ ID NO2: 12). Cleavage at Asp-Pro produced b- and y-type ions.

[0031] FIGs. 9A-9B depict mass spectra of (FIG. 9A) Full MS and (FIG. 9B) MS / MS of isobaric peptides in 80:20 ACN: H20 with 0.1% FA, pH = 3.00.

[0032] FIG. 10 depicts mass spectra of Full MS and MS / MS of peptide AcIRNdsPTIDPINR (SEQ ID NO2: 8). Cleavage at Asp-Pro produced b7and y4ions.

[0033] FIG. 11 depicts a calibration curve of Pep 1 (AcIRNPTIDPINR) (SEQ ID NO2: 7). Concentrations of 0, 0.1, 0.5, 1.0, 3.0, 5.0 ng / ml of Pep 1 were prepared and spiked with 1.0 ng / ml of IS in 80:20 ACN: H2O with 0.1% FA. Each datum point is an average of at least three replicates.

[0034] FIGs. 12A-12B depicts (FIG. 12A) Full MS and (FIG. 12B) MS / MS of peptide probe in 80:20 ACN: H2O with 0.1% FA, pH=3.00.

[0035] FIG. 13 depicts hydrolysis optimization of peptide probe at various pH conditions and different time intervals.

[0036] FIG. 14 depicts a hydrolysis experiment of peptide probe at pH 12 using 2M and 4M NH4OH.

[0037] FIG. 15 depicts a bioconjugation process of detector antibody and dendrimer-peptide probe.

[0038] FIG. 16 depicts a plot of positive and negative PfHRP2 human serum samples using the peptide probe as a mass reporter using PSMS.

[0039] FIG. 17 depicts a plot of positive and negative PfHRP2 human serum samples using the peptide probe as a mass reporter using nESI.Attorney Docket No. 103362-047WO1

[0040] DETAILED DESCRIPTION

[0041] Before the present methods and systems are disclosed and described, it is to be understood that the methods and systems are not limited to specific synthetic methods, specific components, or to particular compositions. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

[0042] 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. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0043] “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0044] Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps. “Exemplary” means “an example of’ and is not intended to convey an indication of a preferred or ideal aspect. “Such as” is not used in a restrictive sense, but for explanatory purposes.

[0045] Disclosed are components that can be used to perform the disclosed methods and systems. These and other components are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these components are disclosed that while specific reference of each various individual and collective combinations and permutation of these may not be explicitly disclosed, each is specifically contemplated and described herein, for all methods and systems. This applies to all aspects of this application including, but not limited to, steps in disclosed methods. Thus, if there are a variety of additional steps that can beAttorney Docket No. 103362-047WO1

[0046] performed it is understood that each of these additional steps can be performed with any specific aspect or combination of aspects of the disclosed methods.

[0047] Compounds are described using standard nomenclature. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this disclosure belongs.

[0048] The compounds described herein include enantiomers, mixtures of enantiomers, diastereomers, tautomers, racemates, and other isomers, such as rotamers, as if each is specifically described unless otherwise indicated or otherwise excluded by context. It is to be understood that the compounds provided herein may contain chiral centers. Such chiral centers may be of either the R) or (S) configuration. The compounds provided herein may either be enantiomerically pure or be diastereomeric or enantiomeric mixtures. It is to be understood that the chiral centers of the compounds provided herein may undergo epimerization in vivo. As such, one of ordinary skill in the art will recognize that administering a compound in its R) form is equivalent, for compounds that undergo epimerization in vivo, to administering the compound in its (S) form. Unless stated to the contrary, a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible isomer, e.g., each enantiomer, diastereomer, and meso compound, and a mixture of isomers, such as a racemic or scalemic mixture.

[0049] Compounds described herein may contain one or more double bonds and, thus, potentially give rise to cis / trans (E / Z) isomers, as well as other conformational isomers. Unless stated to the contrary, all such possible isomers are contemplated, as well as mixtures of such isomers.

[0050] Compounds described herein may also present as an equilibrium of tautomers. For example, ketones with an a-hydrogen can exist in an equilibrium of the keto form and the enol form. Likewise, amides with an N-hydrogen can exist in an equilibrium of the amide form and the imidic acid form. Unless stated to the contrary, all possible tautomers of the compounds described herein are contemplated.

[0051] A dash that is not between two letters or symbols is used to indicate a point of attachment for a substituent. For example, -(C=0)NH2is attached through the carbon of the keto (C=O) group.Attorney Docket No. 103362-047WO1

[0052] The term “substituted,” as used herein, means that any one or more hydrogens on the designated atom or group are replaced with a moiety selected from the indicated group, provided that the designated atom’s normal valence is not exceeded and the resulting compound is stable. For example, when the substituent is oxo (i.e., =0), two hydrogens on the atom are replaced. For example, a pyridyl group substituted by oxo is a pyridine. Combinations of substituents and / or variables are permissible only if such combinations result in stable compounds or useful synthetic intermediates. A stable active compound refers to a compound that can be isolated and / or can be formulated into a form with a shelflife of at least one month. A stable manufacturing intermediate or precursor to an active compound is stable if it does not degrade within the period needed for reaction or other use. A stable moiety or substituent group is one that does not degrade, react, or fall apart within the period necessary for use. Non-limiting examples of unstable moieties are those that combine heteroatoms in an unstable arrangement, as typically known and identifiable to those of skill in the art.

[0053] Any suitable group may be present on a “substituted” or “optionally substituted” position that forms a stable molecule and meets the desired purpose of the disclosure and includes, but is not limited to: halo, nitro, cyano, azido, oxo, Ci-Ce alkyl, Ci-Ce haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, (C3-C7 cycloalkyl) (C0-C3 alkyl)-, (3- to 8-membered monocyclic or bicyclic heterocycle)-(Co-C6 alkyl)-, (6- to 10-membered monocyclic or bicyclic aryl)-(Co-C6 alkyl)-, (5- to 10-membered monocyclic or bicyclic heteroaryl)-(Co-C6 alkyl)-, A O-(Co-C6 alkyl)-, A S-(Co-C6 alkyl)-, (A AyN)-(Co-C6 alkyl)-, AZC(O)-(CO-C6 alkyl)-, AZC(N)-(CO-C6 alkyl)-, and AzS(O)-(Co-C6 alkyl)-, and AZS(O)2-(CO-C6 alkyl)-, wherein Axand Av are independently selected at each occurrence from Aa, AZC(O)-, AZC(N)-, AZS(O)-, and AzS(0)2-, each of which maybe optionally substituted with one or more B groups as allowed by valency; wherein A is independently selected at each occurrence from hydrogen, halo, C₁-C₆alkyl, C₁-C₆haloalkyl, C2-C6alkenyl, C2-C6alkynyl, (C3-C7cycloalkyl)-(Co-C3alkyl)-, (4- to 6-membered heterocycle)-(Co-C3alkyl)-, (5- to 10-membered monocyclic or bicyclic aryl)-(Co-C3alkyl)-, (5- to 10-membered monocyclic or bicyclic heteroaryl)-(Co-C3alkyl)-, -0Aa, -SA“, and -NAaAb, each of which may be optionally substituted with one or more B groups as allowed by valency; wherein Aaand Abare independently selected at each occurrence from hydrogen, Ci-Cealkyl, Ci-Cehaloalkyl, C2-C6alkenyl, C2-C6alkynyl, (C3-C7 cycloalkyl)-(Co-C3alkyl)-, (4- to 6-membered heterocycle)-(Co-Attorney Docket No. 103362-047WO1

[0054] C3alkyl)-, (5- to 10-membered monocyclic or bicyclic aryl)-(Co-C3alkyl)-, (5- to 10-membered monocyclic or bicyclic heteroaryl)-(Co-C3alkyl)-, each of which may be optionally substituted by one or more B groups as allowed by valency; and wherein B is independently selected at each occurrence from hydrogen, halo, nitro, cyano, azido, oxo, Ci-Ce alkyl, Ci-Ce haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, (C3-C7cycloalkyl) (Co-C3alkyl)-, (3- to 8-membered monocyclic or bicyclic heterocycle)-(Co-Ce alkyl)-, (6- to 10-membered monocyclic or bicyclic aryl)-(Co-C6 alkyl)-, (5- to 10-membered monocyclic or bicyclic heteroaryl)-(Co-C6 alkyl)-, ApO-, ApS-, ApAqN-, AoC(O)-, AoC(O)-O-, AoC(O)-NAq-, AoS(O)2-, AoS(O)2-O-, and AoS(O)2-NAq-, wherein Aois independently selected at each occurrence from Ap, halo, ApO-, and ApAqN-, and wherein AP and AI are independently selected at each occurrence from hydrogen, Ci-Ce alkyl, Ci-Ce haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, (C3-C7cycloalkyl) (Co-C3alkyl)-, (3- to 8-membered monocyclic or bicyclic heterocycle)-(Co-Ce alkyl)-, (6- to 10-membered monocyclic or bicyclic aryl)-(Co-C6 alkyl)-, and (5- to 10-membered monocyclic or bicyclic heteroaryl)-(Co-C6 alkyl)-. In some aspects, a compound described herein maybe optionally substituted as described above.

[0055] The terms for various functional groups as used herein are not intended to be limited to monovalent radicals and may include polyvalent radical groups as appropriate, such as divalent, trivalent, tetravalent, pentavalent, and hexavalent groups, and the like, based on the position and location of such groups in the compounds described herein as would be readily understood by the skilled person in the context in which said functional groups are recited.

[0056] _ i

[0057] As used herein, the symbol “ s ” (which hereinafter can be referred to as “a point of attachment bond”) denotes a bond that is a point of attachment between two chemical entities, one of which is depicted as being attached to the point of attachment bond and the other of which is not depicted as being attached to the point XY _ |

[0058] of attachment bond. For example, “ « ” indicates that the chemical entity “XY” is bonded to another chemical entity via the point of attachment bond. Furthermore, the specific point of attachment to the non-depicted chemical entity can be specified XY - 1 by inference. For example, the compound CH3-R3, wherein Rs is H or “? ” infers that when Rs is “XY”, the point of attachment bond is the same bond as the bond by which Rs is depicted as being bonded to CH3.Attorney Docket No. 103362-047WO1

[0059] Compounds disclosed herein may be provided in the form of acceptable salts, for example, pharmaceutically acceptable salts. Examples of such salts are acid addition salts formed with inorganic acids, for example, hydrochloric, hydrobromic, sulfuric, phosphoric, and nitric acids and the like; salts formed with organic acids such as acetic, oxalic, tartaric, succinic, maleic, fumaric, gluconic, citric, malic, methanesulfonic, p-toluenesulfonic, napthalenesulfonic, and polygalacturonic acids, and the like; salts formed from elemental anions such as chloride, bromide, and iodide; salts formed from metal hydroxides, for example, sodium hydroxide, potassium hydroxide, calcium hydroxide, lithium hydroxide, and magnesium hydroxide; salts formed from metal carbonates, for example, sodium carbonate, potassium carbonate, calcium carbonate, and magnesium carbonate; salts formed from metal bicarbonates, for example, sodium bicarbonate and potassium bicarbonate; salts formed from metal sulfates, for example, sodium sulfate and potassium sulfate; and salts formed from metal nitrates, for example, sodium nitrate and potassium nitrate.

[0060] Certain materials, compounds, compositions, and components disclosed herein can be obtained commercially or readily synthesized using techniques generally known to those of skill in the art. For example, the starting materials and reagents used in preparing the disclosed compounds and compositions are either available from commercial suppliers, such as Sigma-Aldrich (formerly MilliporeSigma, Burlington, MA) or Thermo Fisher Scientific Inc. (Waltham, MA), or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser's Reagents for Organic Synthesis (John Wiley and Sons, 2007); Organic Reactions (John Wiley and Sons, 2004); March's Advanced Organic Chemistry, (John Wiley and Sons, 8thEdition); and Larock's Comprehensive Organic Transformations (John Wiley and Sons, 3rdedition, 2017).

[0061] The present disclosure also includes compounds described herein with at least one desired isotopic substitution of an atom at an amount above the natural abundance of the isotope, i.e., enriched.

[0062] Examples of isotopes that can be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as2H,3H,11C,13C,15N,17O,18O,18F,31P,32P,35S,36Cl, and125I, respectively. Isotopically labeled compounds of this disclosure can generally beAttorney Docket No. 103362-047WO1

[0063] prepared by carrying out the procedures disclosed herein by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.

[0064] Byway of general example and without limitation, isotopes of hydrogen, for example, deuterium (2H) and tritium (3H), may optionally be used anywhere in the described structures that achieve the desired result. Alternatively, or in addition, isotopes of carbon, e.g.,13C and14C, maybe used. Isotopic substitutions, for example, deuterium substitutions, can be partial or complete. Partial deuterium substitution means that at least one hydrogen is substituted with deuterium. In certain aspects, the isotope is 80, 85, 90, 95, or 99% or more enriched in an isotope at any location of interest. In some aspects, deuterium is 80, 85, 90, 95, or 99% enriched at a desired location. Unless otherwise stated, enrichment at any point is above natural abundance and, in an aspect, is enough to alter a detectable property of the compounds.

[0065] The term "alkyl" refers to a radical of a straight-chain or branched hydrocarbon group having a specified range of carbon atoms (e.g., a " C1-16 alkyl" can have from 1 to 16 carbon atoms). An alkyl group can be a saturated alkyl group or an unsaturated alkyl group, i.e., an alkyl group having one or more carbon-carbon double / triple bonds, i.e., an alkenyl or alkynyl group. Unless specified to the contrary, an “alkyl” group includes both saturated alkyl groups and unsaturated alkyl groups.

[0066] The term "heteroalkyl" refers to an alkyl group, which further includes at least one heteroatom e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within (i.e., inserted between adjacent carbon atoms of) and / or placed at one or more terminal position(s) of the parent chain. By way of example, a heteroCi-ealkyl (which may also be designated a Ci-6heteroalkyl) group includes, but is not limited to, the following structures:

[0067]

[0068] The term “heteroalkyl,” preceded by a separate heteroatom, refers to a heteroalkyl group bonded through the specified heteroatom. By way of example, a OCi-oheteroalkyl group includes, but is not limited to, the following structures:Attorney Docket No. 103362-047WO1

[0069]

[0070] When a range of values is listed, it is intended to encompass each value and subrange within the range. For example, " C1-6 alkyl" is intended to encompass Ci, C2, C3, C4, C5, C6, C1-6, C1-5, C1-4, C1-3, C1-2, C2-6, C2-5, C2-4, C2-3, C3-6, C3-5, C3-4, C4-6, C4-5, and C5-6 alkyl.

[0071] Affixing the suffix "-ene" to a group indicates the group is a polyvalent moiety, e.g., boned to two or more groups. Alkylene is the polyvalent moiety of alkyl, alkenylene is the divalent moiety of alkenyl, alkynylene is the divalent moiety of alkynyl, heteroalkylene is the divalent moiety of heteroalkyl, heteroalkenylene is the divalent moiety of heteroalkenyl, heteroalkynylene is the divalent moiety of heteroalkynyl, carbocyclylene is the divalent moiety of carbocyclyl, heterocyclylene is the divalent moiety of heterocyclyl, arylene is the divalent moiety of aryl, and heteroarylene is the divalent moiety of heteroaryl (each of which parent groups as defined herein).

[0072] The term "alkoxy" refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom.

[0073] The term "aryl" refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system e.g., having 6, 10, or 1471 electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (" C6-14 aryl"). " Aryl" also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continues to designate the number of carbon atoms in the aryl ring system. Unless otherwise specified, each instance of an aryl group is independently unsubstituted (an "unsubstituted aryl") or substituted (a "substituted aryl") with one or more substituents.

[0074] " Aralkyl" is a subset of "alkyl" and refers to an alkyl group substituted by an aryl group, wherein the point of attachment is on the alkyl moiety.

[0075] The term "heteroaryl" refers to a radical of a 5-14 membered monocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 71 electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatomsAttorney Docket No. 103362-047WO1

[0076] provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-14 membered heteroaryl"). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl polycyclic ring systems can include one or more heteroatoms in one or both rings. " Heteroaryl" includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the point of attachment is on the heteroaryl ring, and in such instances, the number of ring members continues to designate the number of ring members in the heteroaryl ring system. " Heteroaryl" also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups, wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused polycyclic (aryl / heteroaryl) ring system. Polycyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like), the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl).

[0077] Exemplary heteroaryl and heterocyclyl rings include: benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromenyL cirrnolinyl, decahydroquinolinyl, 2H,6H~ 1,5, 2-di thiazinyl, dihydrofuro[2,3 b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl,Attorney Docket No. 103362-047WO1

[0078] tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl, and xanthenyl.

[0079] Unless specified to the contrary, the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups defined herein (and the “ene” versions of said groups) may be substituted or unsubstituted. A substituted group includes a non-hydrogen substituent at a position where, in the unsubstituted version, a hydrogen atom would be found. Substituents include, but are not limited to, halogen, hydroxy, alkyl, alkoxy, nitro, cyano, oxo, carbocyclyl, carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -NRaRb, -NRaC(=O)Rb, -NRaC(=O)NRaNRb, -NRaC(=O)ORb, - NRaS02Rb, -C(=O)Ra, -C(=O)ORa, -C(=O)NRaRb, -OC(=O)NRaRb, -ORa, -SRa, -SORa, - S(=0)2Ra, -0S(=0)2Raand -S(=0)20Ra. Raand Rbin this context can be the same or different and independently hydrogen, halogen, hydroxyl, alkyl, alkoxy, alkyl, amino, alkylamino, dialkylamino, carbocyclyl, carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl.

[0080] As used herein, the designation of a polyvalent moiety without specifying the specific order of attachment is intended to cover all possible arrangements. By way of example, a compound represented by the formula:

[0081] A-X-B,

[0082] wherein X is NHC(=O) embraces both:

[0083] 0 0

[0084] A B A JI

[0085] and

[0086] As used herein, a chemical bond depicted: ' I represents either a single, double, or triple bond, valency permitting. By way of example,

[0087]

[0088] Attorney Docket No. 103362-047WO1

[0089] Some compounds disclosed herein may exist as one or more tautomers. Tautomers are interconvertible structural isomers that differ in the position of one or more protons or other labile atoms. By way of example:

[0090]

[0091] The depiction of a compound in one tautomeric form is understood to include nondepicted tautomers as well.

[0092] Unless stated to the contrary, a substituent drawn without explicitly specifying the point of attachment indicates that the substituent may be attached at any possible atom. For example, in a benzofuran depicted:

[0093]

[0094] the substituent maybe present at any one of the six possible carbon atoms.

[0095] As used herein, the term “null,” when referring to a possible identity of a chemical moiety, indicates that the group is absent, and the two adjacent groups are directly bonded to one another. By way of example, for a genus of compounds having the formula CH3-X-CH3, if X is null, then the resulting compound has the formula CH3-CH3. A group having the subscript ‘o’ is understood to represent a null group as well. By way of example, in the compound CH3-(X)Z-CH3, if X is CH2and z is o, then the compound has the formula CH3-CH3.

[0096] A bracketed functional group with a subscripted variable should be understood to denote the number of repeated bracketed groups present. For example, a number that is selected from o or 1 should be interpreted as follows:

[0097] a = 0 = Ri-O-R2

[0098]

[0099] a = 1

[0100] In certain instances, two or more variable groups may together form a ring. It is understood that any depicted atoms separated from the identified groups will themselves form part of the ring:Attorney Docket No. 103362-047WO1

[0101] CH3

[0102]

[0103] When the variable groups are substituted on an aromatic system, the new ring will be a fused ring, and unless specified to the contrary, may be either aromatic or nonaromatic, carbocyclic or heterocyclic:

[0104]

[0105] The ring may further be defined by the number of carbon atoms in the specific ring formed by the variable groups, which includes the atoms separating the variable groups:

[0106]

[0107] Cecycloalkyl C6aryl C5heterocyclyl C5heteroaryl

[0108] Each of the above results occurs when R1and R2together form a six-membered (or six-atom) ring. Other rings, including 3, 4, 5, 7, and 8-member rings, may also be formed, and maybe further limited by a specified number of carbon atoms. Although the singular “a ring” maybe used to define the group, unless specified to the contrary, both monocyclic and polycyclic rings are possible:

[0109]

[0110]

[0111] monocyclic ring polycyclic ring

[0112] Unless stated to the contrary, a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible isomer, e.g., each enantiomer, diastereomer, and meso compound, and a mixture of isomers, such as a racemic or scalemic mixture. Unless stated to the contrary, a formula depicting one or more stereochemical features does not exclude the presence of other isomers. As used herein, “polypeptide” refers to a string of at least two amino acids attached to one another by a peptide bond. There is no upper limit to the number of amino acids that can be included in a polypeptide. Further, polypeptides may include non-Attorney Docket No. 103362-047WO1

[0113] natural amino acids, amino acid analogs, or other synthetic molecules that are capable of integrating into a polypeptide.

[0114] The polypeptides can be modified by either natural processes, such as post-translational processing, or by chemical modification techniques that are well-known in the art. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini. The same type of modification can be present in the same or varying degrees at several sites in the given polypeptide. Also, a given polypeptide can have many types of modifications. Modifications include, without limitation, acetylation, acylation, ADP-ribosylation, amidation, covalent cross-linking or cyclization, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleoside or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphatidylinositol, disulfide bond formation, demethylation, formation of cysteine or pyroglutamate, formylation, gammacarboxylation, glycosylation, GPI anchor formation, hydroxylating, iodination, methylation, myristoylation, oxidation, PEGylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer-RNA mediated addition of amino acids to proteins such as arginylation. Also included in the term “polypeptides” are cis- and trans-isomers, R- and S-enantiomers, D-isomers, L-isomers, diastereomers, conformers, and mixtures thereof.

[0115] The term “residue,” as used herein, refers to an amino acid that is incorporated into a polypeptide. The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.

[0116] Each amino acid can be a natural or non-natural amino acid. The term “non-natural amino acid” refers to an organic compound that is a congener of a natural amino acid in that it has a structure similar to a natural amino acid so that it mimics the structure and reactivity of a natural amino acid. The non-natural amino acid can be a modified amino acid, and / or amino acid analog, that is not one of the 20 common naturally occurring amino acids or the rare natural amino acids selenocysteine or pyrrolysine. Non-natural amino acids can also be the D-isomer of the natural amino acids. Thus, as used herein, the term “amino acid” refers to natural and non-natural amino acids, and analogs and derivatives thereof. Examples of suitable amino acids include, butAttorney Docket No. 103362-047WO1

[0117] are not limited to, alanine, allosoleucine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, napthylalanine, phenylalanine, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine, a derivative, or combinations thereof. Analogs of amino acids encompass those that have a structure similar, but not identical, to an amino acid, e.g., due to a modification to the side chain orbackbone of said amino acid. Such modifications may increase the hydrophobicity of the side chain, including elongation of the primary chain or side chain by one or more hydrocarbons, or increasing the solvent accessible surface area of an amino acid having an aromatic ring on its side chain, e.g., by conjugating a second aromatic ring or increasing the size of the aromatic ring. Derivatives of amino acids encompass natural and nonnatural amino acids that have been modified (e.g., by substitution) to include a hydrophobic group. For example, a derivative of lysine includes lysine whose side chain has been substituted with alkylcarboxamidyl.

[0118] Amino Acid Abbreviations* Abbreviations* L-amino acid D-amino acid

[0119] Alanine Ala (A) ala (a)

[0120] Allosoleucine AIle Aile

[0121] Arginine Arg (R) arg (r)

[0122] Asparagine Asn (N) asn (n)

[0123] Aspartic acid Asp (D) asp (d)

[0124] Cysteine Cys (C) cys (c)

[0125] Cyclohexylalanine Cha Cha

[0126] 2,3-diaminopropionic acid Dap Dap

[0127] 4-fluorophenylalanine Fpa (Σ) Pfa

[0128] Glutamic acid Glu (E) glu (e)

[0129]

[0130] Attorney Docket No. 103362-047WO1

[0131] Amino Acid Abbreviations* Abbreviations*

[0132] L-amino acid D-amino acid

[0133] Glutamine Gin (Q) gin (q)

[0134] Glycine Gly(G) giy (g) Histidine His (H) his (h)

[0135] Homoproline (aka pipecolic acid) Pip (Θ) pip (θ) Isoleucine He (I) ile (i)

[0136] Leucine Leu (L) leu (1)

[0137] Lysine Lys (K) lys (k)

[0138] Methionine Met (M) met (m)

[0139] Naphthylalanine Nal (Φ) nal (φ)

[0140] Norleucine Nle (Ω) nle

[0141] Phenylalanine Phe (F) phe (f)

[0142] Phenylglycine Phg (Ψ) Phg

[0143] 4- F2Pmp (Λ) f2pmp (phosphonodifluoromethyl)phenylalani

[0144] ne

[0145] Proline Pro (P) pro (p)

[0146] Sarcosine Sar (Ξ) sar

[0147] Selenocysteine Sec (U) sec (u)

[0148] Serine Ser (S) ser (s)

[0149] Threonine Thr (T) thr (y)

[0150]

[0151] Attorney Docket No. 103362-047WO1

[0152] Amino Acid Abbreviations* Abbreviations* L-amino acid D-amino acid

[0153] Tyrosine Tyr (Y) tyr (y)

[0154] Tryptophan Trp (W) trp (w)

[0155] Valine Val (V) val (v)

[0156] 3-(3-benzothienyl)-alanine Bta Bta

[0157] 4-fluorophenylalanine Fpa or L-Fpa fpa or D-Fpa

[0158] 1-naphthylalanine L-i-Nal or 1-Nal D-i-Nal or 1-nal

[0159] 2 -naphthylalanine L-2-Nal or 2-Nal D-2-Nal or 2-nal

[0160] 2 -pyridylalanine L-2-Pya or 2-Pya D-2-Pya or 2-pya

[0161] 4-pyridylalanine L-4-Pya or 4-Pya D-4-Pya or 4-pya

[0162]

[0163] As used herein, an “amino acid-analog” or “analog” (e.g., “arginine-analog”, “lysine-analog” or “histidine-analog”) refers to a variant of an amino acid that retains at least one function of the amino acid. Such variants may have an elongated or shorter side chain (e.g., by one or more -CH2- groups. For example, an arginine analog may include an additional methylene or ethylene between the backbone and guanidine / guanidinium group. Other examples include amino acids with one or more additional substituents (e.g., Me, Et, halogen, thiol, methoxy, ethoxy, C1-haloalkyl, C2-haloalkyl, amine, guanidine, etc.). The amino acid analog can be monovalent, divalent, or trivalent. Similarly, an amino acid may have one or more -CH2- groups separating the amino and carboxy functional groups, e.g., P-alanine, y-alanine, etc.

[0164] Examples of arginine analogs include:Attorney Docket No. 103362-047WO1

[0165]

[0166] or any of the AAs that contain a guanidinium on the side chain group.

[0167] Throughout the present specification, peptides and amino acid monomers are depicted as charge-neutral species. It is to be understood that such species may bear a positive or negative charge depending on the conditions. For example, at pH 7, the N-terminus of an amino acid is protonated and bears a positive charge (-NH3+), and the C-terminus of an amino acid is deprotonated and bears a negative charge (-CO2- ). Similarly, the side chains of certain amino acids may bear a positive or negative charge.

[0168] Peptides described herein may include conservative substitutions of one or more amino acids. Conservative substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Exemplary conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine. Further exemplary conservative substitutions are provided in the table below, with others being known in the art:

[0169] Amino Acid Substitutions

[0170] Original Exemplary Conservative Residue SubstitutionsAttorney Docket No. 103362-047WO1

[0171] Ala Ser

[0172] Arg Lys; Gin

[0173] Asn Gin; His

[0174] Asp Glu

[0175] Cys Ser

[0176] Gin Asn, Lys

[0177] Glu Asp

[0178] Gly Pro

[0179] His Asn; Gin

[0180] Ile Leu; Val

[0181] Leu Ile; Val

[0182] Lys Arg; Gin

[0183] Met Leu; Ile

[0184] Phe Met; Leu; Tyr

[0185] Ser Thr

[0186] Thr Ser

[0187] Trp Tyr

[0188] Tyr Trp; Phe

[0189] Peptides can be prepared by any method, such as by synthesizing the peptide or by expressing a nucleic acid encoding an appropriate amino acid sequence in a cell and harvesting the peptide from the cell. Of course, a combination of such methods can also be used.Attorney Docket No. 103362-047WO1

[0190] Examples of chemical synthesis technologies are solid-phase synthesis and liquidphase synthesis. Solid phase synthesis methods are largely classified by the tBoc method and the Fmoc method, depending on the type of protective group used. Typically used protective groups include tBoc (t-butoxycarbonyl), Cl— Z (2-chlorobenzyloxycarbonl), Br— Z (2-bromobenzyloxycarbonyl), Bzl (benzyl), Fmoc (9-fluorenylmethoxycarbonyl), Mbh (4,4’-dimethoxydibenzyhydryl), Mtr (4-methoxy-2,3,6-trimethylbenzenesulfonyl), Trt (trityl), Tos (tosyl), Z (Benzyloxycarbonyl), and Clz-Bzl (2,6-dichlrobenzyl) for the amino groups; NO22 (nitro) and Pmc (2, 2, 5,7,8-pentamethylchromane-6-sulfonyl) for the guanidino groups; and t-Bu (t -butyl) for the hydroxyl groups. After synthesis of the desired peptide, it is subjected to one or more deprotection reactions and cut out from the solid support. Such peptide cutting reactions may be carried out with hydrogen fluoride or trifluoromethane sulfonic acid for the Boc method or with TFA for the Fmoc method. Methods of de novo synthesizing of peptides and peptidomimetics are described, for example, in Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2005; and Peptide and Protein Drug Analysis, ed. Redi., R., Marcel Dekker, Inc., 2000.

[0191] Alternatively, the peptide may be synthesized using recombinant techniques. In this case, a nucleic acid encoding the peptide is cloned into an expression vector under the control of expression control sequences (e.g., a promoter, a terminator, and / or an enhancer), allowing its expression. The expression vector is then transfected into a host cell (e.g., a human, CHO, mouse, monkey, fungal, or bacterial host cell), and the transfected host cell is cultivated under conditions suitable for the expression of the peptide. Standard recombinant DNA and molecular cloning techniques are described, for example, in Sambrook and Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (1989); Silhavy et al., Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (1984); and Ausubel et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc, and Wiley-Interscience (1987).

[0192] The method of producing the peptide may optionally include the steps of purifying said peptide, chemically modifying said peptide, and / or formulating said peptide into a composition.Attorney Docket No. 103362-047WO1

[0193] Disclosed herein are methods of detecting an antigen in a composition, including the steps:

[0194] (a) contacting the composition with a capture antibody to form an antigen-capture antibody complex;

[0195] (b) contacting the antigen-capture antibody complex with a detector antibody to form a sandwich complex, wherein the detector antibody includes at least one reporter peptide;

[0196] (c) separating the sandwich complex from unreacted detector antibody;

[0197] (d) separating the reporter peptide from the sandwich complex; and

[0198] (e) detecting the reporter peptide using mass spectrometry.

[0199] In some aspects, the reporter peptide can be detected using paper spray ionization mass spectrometry. A liquid composition including the reporter peptide can be dispensed onto a paper substrate, for example, having a triangular shape with a tip directed towards the inlet of the mass spectrometer.

[0200] In some aspects, the reporter peptide can be covalently conjugated to the detector antibody. In some aspects, the reporter peptide can be directly bonded to the detector antibody, while in other aspects, the reporter peptide can be bonded to the detector antibody through a linker. In certain aspects, the reporter peptide can be bonded to the detector antibody through a cleavable bond (e.g., an ester bond linking the reporter peptide directly to the detector antibody), or the linker can include a cleavable bond. The cleavable bond can be cleaved by exposure to acid or base (for example, a hydrolysable bond such as an ester, hydrazone, acetal, or oxime), by exposure to an oxidant (for example, a disulfide bond, thioacetal), by exposure to heat, or by exposure to actinic radiation (for example, an ortho-nitrobenzyl group). As used herein, an ortho-nitrobenzyl (or o-nitrobenzyl) linker has the following general formula:

[0201] NO2

[0202]

[0203] Attorney Docket No. 103362-047WO1

[0204] wherein one oxygen is bonded to the reporter peptide, optionally through an additional linker moiety, and the other oxygen is bonded to the detector antibody, optionally through additional linker moieties.

[0205] As used herein, “linker” refers to a moiety that covalently attaches two or more components of the compounds disclosed herein (e.g., a linker may covalently attach a reporter peptide and detector antibody). In some aspects, the linker may be an alkylene or heteroalkylene (e.g., poly(ethylene glycol)) moiety having two functional groups capable of forming covalent bonds with the reporter peptide and detector antibody. In some aspects, the linker is about 3 to about 100 (e.g., about 3 to about 20) atoms in linear length (not counting any branched atoms or substituents). In some aspects, the linker provides about 1 A to about 400 A in distance of the two groups to which it connects.

[0206] A linker as described herein can be used in either direction, i.e., either the left end is linked to the director antibody and the right end to the reporter peptide, or the left end is linked to the reporter peptide and the right end to the director antibody. In some aspects, the linker is a chain of 2 to 14, 15, 16, 17, 18, 19, or 20 or more carbon atoms, of which one or more carbons can be optionally replaced by a heteroatom such as O, N, S, or P as allowed by valency.

[0207] In some aspects, the chain has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 19, or 20 contiguous atoms. For example, the chain may include 1 or more ethylene glycol units that can be contiguous, partially contiguous, or non-contiguous (for example, 2, 3, 4, 5,, 7, 8, 9, 10, 11, or 12 ethylene glycol units).

[0208] In some aspects, the chain has at least 1, 2, 3, 4, 5, 6, 7, or 8 contiguous units which can be branched and which can be independently C1-C20 alkyl, 6- to 10-membered monocyclic or bicyclic aryl, 5- to 10-membered monocyclic or bicyclic heteroaryl, C2-C20 alkenyl, or C2-C20 alkynyl, C3-C7 cycloalkyl, or 3- to 8-membered monocyclic or bicyclic heterocycle substituents.

[0209] In some aspects, the linker can include one or more ethylene glycol, propylene glycol, lactic, and / or glycolic acid units. Block and random lactic acid-co-glycolic acid moieties, as well as ethylene glycol and propylene glycol, are known in the art and can be modified to obtain the desired half-life and hydrophilicity. In certain aspects, these units can be flanked or interspersed with other moieties, such as, for example,Attorney Docket No. 103362-047WO1

[0210] C1-C20 alkyl, 6- to 10-membered monocyclic or bicyclic aryl, 5- to 10-membered monocyclic or bicyclic heteroaryl, C3-C7 cycloalkyl, 3- to 8-membered monocyclic or bicyclic heterocycle, etc., each of which can be interspersed with optionally substituted O, N, S, P or Si atoms as allowed by valency, as desired to achieve the appropriate properties.

[0211] In some aspects, the linker is an optionally substituted (poly) ethylene glycol having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more, ethylene glycol units, or optionally substituted C1-C20 alkyl groups interspersed with optionally substituted O, N, S, P or Si atoms as allowed by valency.

[0212] In some aspects, the linker is flanked, substituted, or interspersed with a C1-C20 alkyl, C3-C7 cycloalkyl, 3- to 8-membered monocyclic or bicyclic heterocycle, 6- to 10-membered monocyclic or bicyclic aryl, or 5- to 10-membered monocyclic or bicyclic heteroaryl group.

[0213] In some aspects, the linker may be asymmetric or symmetric.

[0214] In some aspects, the linker can be a non-linear chain, and can be, or include, C3-C7cycloalkyl, 3- to 8-membered monocyclic or bicyclic heterocycle, 6- to 10-membered monocyclic or bicyclic aryl, or 5- to 10-membered monocyclic or bicyclic heteroaryl moieties.

[0215] In some aspects, the linker is selected from Li:

[0216]

[0217] In some aspects, the linker is selected from the group consisting of a moiety of Formula Li, Formula L2, Formula L3, Formula L4, Formula L5, Formula L6, Formula L7, Formula L8, Formula L9, or Formula Lio:

[0218] heteroaryl

[0219] \X102

[0220]

[0221] (L2)

[0222] heteroaryl

[0223] \R101

[0224] heteroaryl

[0225] \R10^

[0226]

[0227] Attorney Docket No. 103362-047WO1

[0228]

[0229] heterocycle

[0230] \R101

[0231] (L9)

[0232] heterocycle

[0233] \R1°^

[0234]

[0235] (LIO)

[0236] wherein:

[0237] X101and X102are independently at each occurrence selected from a bond, 6- to 10-membered monocyclic or bicyclic aryl, 5- to 10-membered monocyclic or bicyclic heteroaryl, C3-C7cycloalkyl, 3- to 8-membered monocyclic or bicyclic heterocycle, Ci-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, NR105, O, C(O), and S;

[0238] R100, R101, R102, R103, and R104 are independently at each occurrence selected from the group consisting of a bond, -C(O)-, -C(O)O-, -OC(O)-, -SO2-, -S(O)-, C(S)-, -C(O)NR105-, -NR105C(O)-, -O-, -S-, -NR105-, -P(O)(OR105))-, C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, 6- to 10-membered monocyclic or bicyclic aryl, 3- to 8-membered monocyclic or bicyclic heterocycle, C3-C7 cycloalkyl, 5- to 10-membered monocyclic or bicyclic heteroaryl, lactic acid, or glycolic acid, each of which may be optionally substituted with one or more (for example, 1, 2, 3, or 4) substituents independently selected from R'4°;

[0239] R105independently selected at each occurrence from R120, RlloC(O)-, R110C(N)-, RlloS(O)-, and R110S(0)2-, each of which may be optionally substituted with one or more R'4° groups as allowed by valency;

[0240] R110is independently selected at each occurrence from hydrogen, halo, C1-C6alkyl, C1-C6haloalkyl, C2-C6alkenyl, C2-C6alkynyl, (C3-C7cycloalkyl)-(Co-C6 alkyl)-, (4- to 6-Attorney Docket No. 103362-047WO1

[0241] membered heterocycle)-(Co-C6 alkyl)-, (5- to 10-membered monocyclic or bicyclic aryl)-(Co-C6 alkyl)-, (5- to 10-membered monocyclic or bicyclic heteroaryl)-(Co-C6 alkyl)-, -OR120, -SR120, and -NR120R130, each of which may be optionally substituted with one or more R'4° groups as allowed by valency; and

[0242] R120and R130are independently selected at each occurrence from hydrogen, C1-C6alkyl, C1-C6haloalkyl, C2-C6alkenyl, C2-C6alkynyl, (C3-C7 cycloalkyl)-(Co-C6 alkyl)-, (4- to 6-membered heterocycle)-(Co-C6 alkyl)-, (5- to 10-membered monocyclic or bicyclic aryl)-(Co-C6 alkyl)-, (5- to 10-membered monocyclic or bicyclic heteroaryl)-(Co-Ce alkyl)-, each of which may be optionally substituted by one or more R'4° groups as allowed by valency; and

[0243] R140is independently at each occurrence selected from the group consisting of hydrogen, halo, nitro, cyano, azido, oxo, C1-C6alkyl, C1-C6haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, (C3-C7 cycloalkyl)(Co-C6 alkyl)-, (3- to 8-membered monocyclic or bicyclic heterocycle)-(Co-C6 alkyl)-, (6- to 10-membered monocyclic or bicyclic aryl)-(Co-Ce alkyl)-, (5- to 10-membered monocyclic or bicyclic heteroaryl)-(Co-C6 alkyl)-, R'4'O-, R141S“, R 1R142N-, R143C(O)-, RX43C(O)-O-, R142C(0)-NR142-, R143S(O)2-, RX43S(O)2-O-, and R143S(O)2-NR142-;>wherein R143is independently selected at each occurrence from R141, halo, R141O-, and R141R142N-, and wherein R141and R142are independently selected at each occurrence from hydrogen, C1-C6alkyl, C1-C6haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, (C3-C7 cycloalkyl) (Co-Ce alkyl)-, (3- to 8-membered monocyclic or bicyclic heterocycle)-(Co-C6 alkyl)-, (6- to 10-membered monocyclic or bicyclic aryl)-(Co-C6 alkyl)-, and (5- to 10-membered monocyclic or bicyclic heteroaryl)-(Co-C6 alkyl)-.

[0244] The following are non-limiting examples of moieties that can be included in a linker in whole or in part that can be used in this disclosure. Based on this elaboration, those of skill in the art will understand how to use the full breadth of moieties that will accomplish the goal of the disclosure.

[0245] Non-limiting examples of moieties which may be included in the linker, either in whole or in part, include, but are not limited to:

[0246] a bond; -C(=O)-; -C=C-; -NH-; -N(CH3)-; -O-; -CH2-; -(CH2)2-; -(CH2)3-; -(CH2)4-; -(CH2)5-; -(CH2)6-; -(CH2)7-; -(CH2)8-; -(CH2)9-; -(CH2)10-; -NH(C=O)-; -C(=O)NH-; -C(=O)CH2-; -C(=O)(CH2)2-; -C(=O)(CH2)3-; -C(=O)(CH2)4-; -C(=O)(CH2)5-;Attorney Docket No. 103362-047WO1

[0247] -C(=O)(CH2)6-; -CH2C(=0)-; -(CH2)2C(=0)-; -(CH2)3C(=O)-; -(CH2)4C(=O)-; -(CH2)5C(=O)-; -(CH2)6C(=O)-; -CH2NH-; -(CH2)2NH-; -(CH2)3NH-; -(CH2)4NH-; -(CH2)5NH-; -(CH2)6NH-; -NHCH2-; -NH(CH2)2-; -NH(CH2)3-; -NH(CH2)4-; -NH(CH2)5-; -NH(CH2)6-; -CH20-; -(CH2)20-; -(CH2)3O-; -(CH2)4O-; -(CH2)5O-; -(CH2)6O-; -0CH2-; -0(CH2)2-; -O(CH2)3-; -O(CH2)4-; -O(CH2)5-; -O(CH2)6-;

[0248] O

[0249]

[0250] Further non-limiting examples of moieties which may be included in the linker, either in whole or in part, include, but are not limited to:

[0251] O 0 0

[0252] O O

[0253]

[0254] Attorney Docket No. 103362-047WO1

[0255]

[0256] In certain aspects, the reporter peptide can be separated from the sandwich complex by washing the complex with an acidic or alkaline solution, for example, aqueous hydrochloric acid or aqueous ammonium hydroxide. In some aspects, the sandwich complex may be washed with the solution having a pH from 1-13, from 1-7, from 1-5, from 2-6, from 3-6, from 4-6, from 7-13, from 9-13, or from 10-13. The reporter peptides are cleaved from the sandwich complex and may be analyzed in the wash solution.

[0257] In some aspects, the reporter peptide can include glutamic acid or aspartic acid residues, and the reporter peptide is linked to the detector antibody via the carboxy side chain in the glutamic acid or aspartic acid residue, for example, through an ester or thioester bond. In some aspects, the reporter peptide can include a cysteine, serine, or threonine residue, and the reporter peptide is linked to the detectorAttorney Docket No. 103362-047WO1

[0258] antibody via the alcohol / thiol side chain in the cysteine, serine, or threonine residue, for example, through a thioester, disulfide, thioacetal, ester, or acetal.

[0259] In some aspects, the detector antibody may be linked to the reporter peptide via a non-hydrolyzable bond. In some aspects, the detector antibody may be reacted with a linker compound having an isothiocyanate group to form a thiourea bond to the linker via a lysine residue in the detector antibody. In some aspects the linker may have the formula:

[0260] N

[0261]

[0262] H H

[0263] wherein the thiourea is bonded directly to the detector antibody, for example, via a lysine residue, or the thiourea is bonded to an additional linker moiety.

[0264] The reporter peptide will generally include an aspartic acid / proline motif. In some aspects, the reporter peptide includes an aspartic acid / proline motif and exactly two arginine residues. In some aspects, the reporter peptide includes an aspartic acid / proline motif, exactly two arginine residues, and no other basic amino acid residues, e.g., no lysine or histidine residues. In some aspects, the reporter peptide includes an aspartic acid / proline motif, exactly two arginine residues, no other basic amino acid residues, e.g., no lysine or histidine residues, wherein one arginine residue is at the C-terminus and the other arginine residue is at the N-terminus or within two amino acid residues of the N-terminus. In some aspects, the reporter peptide includes an aspartic acid / proline motif, exactly two arginine residues, no other basic amino acid residues, e.g., no lysine or histidine residues, wherein the N-terminus is acylated, and one arginine residue is at the C-terminus and the other arginine residue is at the N-terminus or within two amino acid residues of the N-terminus.

[0265] In some aspects, the reporter peptide has the following formula:Attorney Docket No. 103362-047WO1

[0266]

[0267] wherein each X group is independently selected from null or an amino acid residue other than arginine, lysine, or histidine.

[0268] wherein one of Y1, Y2, or Y³ is arginine or an arginine derivative, and the other two are selected from null or an amino acid residue other than arginine, arginine derivative, lysine, or histidine. It should be understood that the reporter peptide may be conjugated to the detector antibody via the carboxylate in the depicted aspartic acid residue or the side chain in one of the X or Y residues.

[0269] In some aspects, the reporter peptide can include from 8-18 amino acid residues, e.g., from 3 to 14 of X and Y are chosen to be null. In some aspects, the reporter peptide can include from 8-16 amino acid residues, from 8-14 amino acid residues, from 8- 12 amino acid residues, or from 10-12 amino acid residues.

[0270] In certain aspects, from 4-16 of X and Y are null. In some aspects, from 6-16, from 8- 14, from 10-14, from 10-12, or 11 of X and Y are null.

[0271] In some aspects, Y1is arginine.

[0272] In some aspects, Y2is arginine, and Y1is an amino acid other than arginine, lysine, or histidine. For example, Y1can be alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0273] In some aspects, Y³ is arginine and Y1and Y2are amino acids other than arginine, lysine, or histidine, for example Y1can be alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine, and Y2can be alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.Attorney Docket No. 103362-047WO1

[0274] In some aspects, X1is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0275] In some aspects, X2is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0276] In some aspects, X3 is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0277] In some aspects, X4 is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0278] In some aspects, X⁵ is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0279] In some aspects, X6is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0280] In some aspects, X⁷ is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0281] In some aspects, X8is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0282] In some aspects, X⁹ is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0283] In some aspects, X10is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.Attorney Docket No. 103362-047WO1

[0284] In some aspects, X11is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0285] In some aspects, X12is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0286] In some aspects, X¹³ is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0287] In some aspects, X¹⁴ is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0288] In some aspects, X1 r> is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0289] In some aspects, X16is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0290] In some aspects, the detector antibody can be conjugated to a plurality of reporter peptides in order to enhance the reporting signal of a particular sandwich complex. In such aspects, the plurality of reporter peptides conjugated to the detector antibody will be the same, conjugated to the reporter peptide in the same way. In some aspects, the detector antibody can include from 1-500 reporter peptides, from 1-250 reporter peptides, from 1-125 reporter peptides, from 1-100 reporter peptides, from 1-75 reporter peptides, from 1-50 reporter peptides, from 1-25 reporter peptides, from 25-250 reporter peptides, from 100-250 reporter peptides, from 10-50 reporter peptides, from 25-100 reporter peptides, or from 50-150 reporter peptides.

[0291] In some aspects, the reporter peptides may be conjugated to the detector antibody through a dendrimer, for example, a polyamidoamine dendrimer, such as G1, G2, G3, G4, G5, G6, G7, or G8 PAMAM. The polyamidoamine dendrimer maybe conjugated to a plurality of reporter peptides via the amine groups on the surface of the dendrimer. Unreacted amine groups may then be used to conjugate the reporter-Attorney Docket No. 103362-047WO1

[0292] dendrimer complex to the detector antibody. In some aspects, the unreacted amine groups may be treated with glutaraldehyde or a similar bis-aldehyde compound and then condensed with a lysine residue on the detector antibody.

[0293] Also disclosed herein are methods of detecting a plurality of different antigens within a single composition, including

[0294] (a) contacting the composition with a detector array, wherein the detector array comprises a plurality of detector antibodies having affinity for different antigens, wherein each detector antibody having affinity for a different antigen is conjugated to a distinct reporter peptide;

[0295] (b) contacting the plurality of antigen-detector antibody complexes with a plurality of capture antibodies to form a plurality of sandwich complexes; (c) separating the plurality of sandwich complexes from the unreacted detector antibodies;

[0296] (d) separating the reporter peptides from the plurality of sandwich complexes;

[0297] and

[0298] (e) detecting the plurality of reporter peptides using mass spectrometry. By pairing a unique reporter peptide with different detector antibodies, a plurality of sandwich complexes can be identified in a single mass spectrometry experiment. Each reporter peptide will fall within the general description given above, but each will be distinct (e.g., a distinct selection of the X and Y residues) so as to generate a unique MS fingerprint for each antigen.

[0299] The types of antigens that can be detected using the disclosed methods are not particularly limited, so long as a relevant detector antibody specific to the antigen is available. In some aspects, the methods may be used to detect cancer antigens (including tumor antigens), viral antigens, bacterial antigens, fungal antigens, parasitic antigens, neuronal antigens, and others. In certain aspects, the antigen is a marker for HIV, malaria, dengue, Chagas’ disease, Leishmania, Trypanosoma, Plasmodium, Toxoplasma, adenovirus, Campylobacter, rotavirus, norovirus, E. coli, Salmonella, influenza, anthrax, Legionella, chlamydia, trachomatis, herpes simplex, gonorrhoeae, hepatitis (including A, B, C and other strains), measles, pneumonia, or tuberculosis.Attorney Docket No. 103362-047WO1

[0300] Antigens may be detected in a variety of biological fluids, including urine, whole blood, blood serum, plasma, lymph, saliva, sweat, tears, cerebrospinal fluid, ocular fluid, joint fluid, gastrointestinal fluid, stomach acid, pancreatic fluid, serous fluid, synovial fluid, aqueous humor of the eye, perilymph, or endolymph.

[0301] In some aspects, the antigen can be a cancer antigen. Representative examples of cancer antigens that may be detected include, but are not limited to, Alpha-phetoprotein (AFP), Carcinoemobryonic antigen (CEA), prostate-specific antigen (PSA), CA 19-9, CA 125, CA 15-3, CA 27-29, Beta-hCG, Lactate dehydrogenase-1 (LDH-1), Lactate dehydrogenase-2 (LDH-2), Lactate dehydrogenase-3 (LDH-3), Lactate dehydrogenase-4 (LDH-4), Lactate dehydrogenase-5 (LDH-5), Beta-2-microglobulin, neuron-specific enolase (NSE), 5-hydroxyindoleacetic acid (5-HIAA), Estrogen receptora (ESR1), Estrogen receptorP (ESR2), GPER, Estrogen receptora 66 (ERa66), Estrogen receptora 46 (ERa46), Androgen receptor-V7 (ARv7), Androgen receptor-v567es (AR-v567es), full-length HER2, P95HER2, Δ16-HER2, EGFR, full-length ALK, NMP1-ALK, EML4-ALK, Full-length ROS1, PD-1 / PD-L1, SSTR1, SSTR2A, SSTR2B, SSTR3, SSTR4, SSTR5, and the like. In some aspects, the capture antibody and / or the detection antibody is capable of binding one or more of the above cancer antigens.

[0302] In some aspects, the antigen can be a viral antigen. Representative examples of viral antigens that may be detected include, but are not limited to, EBV-encoded latent membrane proteins (LMP1), HPV E6 / E7 oncoproteins, SARS-CoV-2 Nucleocapsid (N) protein, SARS-CoV-2 Spike (S) protein, Respiratory syncytial virus (RSV) fusion (F) protein, Myxovirus resistance protein A (MxA), TNF-related apoptosis-inducing ligand (TRAIL), interferon gamma-induced protein 10 (IP-10), HPV E6 / E7 gene, CMV UL54 genes, CMV UL83 genes, EBV LMP1 / EBNA, and the like. In some aspects, the capture antibody and / or the detection antibody is capable of binding to one or more of the above viral antigens.

[0303] In some aspects, the antigen can be a bacterial antigen. Representative examples of bacterial antigens that maybe detected include, but are not limited to, Streptococcus group A antigen, Staphylococcus protein A, Clostridium difficile toxins A / B, Lipopolysaccharide, C-reactive protein (CRP), Procalcitonin (PCT), Presepsin (SCD14-ST), and the like. In some aspects, the capture antibody and / or the detection antibody is capable of binding to one or more of the above viral antigens.Attorney Docket No. 103362-047WO1

[0304] In some aspects, the antigen can be a fungal antigen. Representative examples of fungal antigens that may be detected include, but are not limited to, Galactomannan (GM), Mannan, (i^3)-P-D-glucan (BDG), and the like. In some aspects, the capture antibody and / or the detection antibody is capable of binding to one or more of the above fungal antigens.

[0305] In some aspects, the antigen can be a parasitic antigen. Representative examples of parasitic antigens include, but are not limited to, BmRi, circulating cathodic antigen (CCA), circulating anodic antigen (CAA), and the like. In some aspects, the capture antibody and / or the detection antibody is capable of binding to one or more of the above parasitic antigens.

[0306] In some aspects, the antigen can be a neuronal antigen. Representative examples of neuronal antigens include, but are not limited to, N-methyl-D-aspartate receptor (NMDAR), leucine-rich glioma-inactivated 1 (LGI1), contactin-associated proteinlike 2 (CASPR2), a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), GABA B receptor, and the like. In some aspects, the capture antibody and / or the detection antibody is capable of binding to one or more of the above parasitic antigens.

[0307] In some aspects, the antigen can be a HIV antigen (such as p24 capsid antigen, gp4i transmembrane glycoprotein, gpi2o / gpi6o envelop glycoprotein, and HIV gag / pol genes), dengue virus antigens (such as Dengue NS1 glycoprotein, Dengue envelope (E) protein, Dengue pre-membrane (PrM) protein, IFN-inducible protein 44-like (IFI44L), IFNα-inducible protein 6 (IFI6), VCAM1, FGL1, MFAP4, GLUL, PLAT, LAMB2, F9, and Dengue NS5 or 3' UTR gene), Trypanosoma cruzi antigens (such as calreticulin-like protein (Tc24), trypomastigote excreted-secreted antigens (TESA), Ag3, Tc85, SAPA, and Tc24), Leishmania antigens (such as recombinant kinesin-related protein 39 (rK39), L. infantum chagasin (LiCHN1), rk28 fusion protein, and Ld-rBP23 / Ld-rBP29), Trypanosoma antigens (such as Variant surface glycoprotein 1 (VSG1), Variant surface glycoprotein 2 VSG2, ILTati.24, Variant surface glycoprotein 13 VSG13, VSGsur, Variant surface glycoprotein 3 VSG3, Variant surface glycoprotein 11 VSG11, Variant surface glycoprotein 615 VSG615, glycosylphosphatidylinositol (GPI) anchors, Native LiTat 1.3 antigen, Native LiTat 1.5 antigen, Trypanosoma brucei enolase (TbrENO2), and Tc_5171 (TESA antigen)), plasmodium antigens (such as PfLDH (P. falciparum specific lactate dehydrogenase),Attorney Docket No. 103362-047WO1

[0308] Plasmodium histidine-rich protein 2 (HRP2), PvLDH (P. vivax specific lactate dehydrogenase), PanLDH (Pan-plasmodium lactate dehydrogenase), PoLDH (P. ovale specific lactate dehydrogenase), PmLDH (P. malariae specific lactate dehydrogenase), merozoite surface protein 1 (MSP-1), Hemozoin, aldolase, and pGDH (plasmodium glutamate dehydrogenase)), toxoplasma antigens (such as p30 surface antigen 1 (SAG1), p22 surface antigen (SAG2), dense granule antigen 1 (GRA1), dense granule antigen 6 (GRA6), dense granule antigen 7 (GRA7), bradyzoite antigen 1 (BAG1), IgM antigen GAP50, IgM antigen EF1γ, and IgM antigen PGKI), adenovirus antigens (such as Hadv-B3, B7, B21 hexon, HAdV-D26, D48 hexon, HAdV-C5 hexon, Hexon capsid proteins, HAdV-F40 short fiber knobs (SFK), HAdV-F41 short fiber knobs (SFK), coxsackievirus-adenovirus receptor (CAR) protein, HAdV-C2 / C5 penton base, HAdV-D8 penton base, and Species B penton base), Campylobacter antigens (such as Flagellin protein (FlaA), Flagellin protein (FlaB), Campylobacter jejuni antigen, and Campylobacter coli antigen), rotavirus antigens (such as inner capsid protein (VP6), outer capsid glycoprotein, G serotype (VP7), and outer capsid protein, P serotype (VP4)), norovirus antigens (such as major capsid protein (VP1), minor capsid protein (VP2), and Norovirus GI / GII ORF1-ORF2 junction genes), E. coli antigens (such as E. coli O157: H7 LPS and E. coli O111 / O55), Salmonella antigens (such as Salmonella O-antigens, H-antigens (flagellin), Vi antigen, and Hemolysin E (HlyE)), Influenza antigens (such as Influenza A / B nucleoprotein (NP), Hemagglutinin (HA), Neuraminidase (NA), Matrix protein (Ml), Influenza A / B matrix (M) genes, and Influenza hemaglutinin genes), Anthrax antigens (such as protective antigen (PA), protective antigen 83 (PA83), protective antigen 63 (PA63), Lethal factor (LF), edema factor (EF), anthrose-containing tetrasaccharide, and poly-y-D-glutamic acid (PDGA / PGA)), Legionella antigens (such as Pan-L. pneumophila antigen, L. pneumophila serogroup 1 (LPS) antigen, Ribosomal L7 / L12 antigen, major outer membrane protein (MOMP), and DotC / DotD outer membrane lipoproteins), trachomatis antigens (such as major outer membrane porin protein (OmpA), chlamydia lipopolysaccharide antigen (LPS), and CT443 / omcB, CT381 arginine binding protein), Herpes simplex antigens (such as glycoprotein G, HSV-1 specific (gG-1), HSV-1 / 2 glycoprotein B (gB) gene, glycoprotein B (gB), glycoprotein D (gD), and glycoprotein G, HSV-2 specific (gG-2)), Neisseria gonorroeae antigens (such as Porin IA (PIA), Porin IB (PIB), major pilin subunit (PilE), PilinAV, Lacto-N-neotetraose,Attorney Docket No. 103362-047WO1

[0309] phenotype Li (LNT), and trisaccharide to pentasaccharide a-chains (L2-L8)), hepatitis antigens (such as Hepatitis B surface antigen (HBsAg), Hepatitis B e antigen (HBeAg), HCV core antigen, Hepatitis B core-related antigen (HBcrAg), HBV core antigen (HBeAg), HBV polymerase genes, and HBV precore / core genes, HCV 5' UTR), measles antigens (such as Measles Nucleoprotein (N protein), Measles hemagglutinin (H protein), Measles C protein, Measles fusion protein (F protein), and Measles large protein (L protein)), pneumonia bacterial antigens (such as Streptococcus pneumoniae urinary antigen, Klebsiella pneumoniae O1-12, and Streptococcus pneumoniae C-polysaccharide), and tuberculosis antigens (such as lipoarabinomannan (LAM), ESTAT-6 / CFP-10, MPT64, and interferon-y-inducible protein 10 (IP-10)). In some aspects, the capture antibody and / or the detection antibody is capable of binding to one or more of the above-listed antigens.

[0310] Representative examples of antibodies than can be used for the capture and / or detection antibody include, but are not limited to, Depemokimab, Apitegromab, Telisotuzumab vedotin, Clesrovimab, Sipavibart, Nipocalimab, Bentracimab, Datopotamab deruxtecan, Zenocutuzumab, Nemolizumab, Zanidatamab, Linvoseltamab, Axatilimab, Patritumab deruxtecan, Tarlatamab, Marstacimab, Garadacimab, Vilobelimab, Zolbetuximab, Odronextamab, Crovalimab, Camrelizumab, Serplulimab, Sugemalimab, Concizumab, Cosibelimab, Trastuzumab duocarmazine, Donanemab, Narsoplimab, Pozelimab, Elranatamab, Rozanolixizumab, Talquetamab, Epcoritamab, Lebrikizumab, Glofitamab, Mirikizumab, Tislelizumab, Toripalimab, Retifanlimab, Lecanemab, Teplizumab, Ublituximab, Mirvetuximab soravtansine, Nirsevimab, Tremelimumab, Spesolimab, Teclistamab, Mosunetuzumab, Tixagevimab, Relatlimab, Tebentafusp, Faricimab, Sutimlimab, Sotrovimab, Regdanvimab, Tezepelumab, Tisotumab vedotin, Amivantamab, Anifrolumab, Loncastuximab tesirine, Bimekizumab, Tralokinumab, Evinacumab, Aducanumab, Dostarlimab, Ansuvimab, Margetuximab, Naxitamab, Atoltivimab, Belantamab mafodotin, Tafasitamab, Satralizumab, Inebilizumab, Sacituzumab govitecan, Teprotumumab, Isatuximab, Eptinezumab, [fam]-trastuzumab deruxtecan, Enfortumab vedotin, Crizanlizumab, Brolucizumab, Polatuzumab vedotin, Risankizumab, Romosozumab, Caplacizumab, Ravulizumab, Emapalumab, Cemiplimab, Fremanezumab, Moxetumomab pasudotox, Galcanezumab, Lanadelumab, Mogamulizumab, Erenumab, Tildrakizumab, Ibalizumab, Burosumab, Durvalumab, Emicizumab, Benralizumab, Ocrelizumab,Attorney Docket No. 103362-047WO1

[0311] Guselkumab, Inotuzumab ozogamicin, Sarilumab, Dupilumab, Avelumab, Brodalumab, Atezolizumab, Bezlotoxumab, Olaratumab, Reslizumab Obiltoxaximab, Ixekizumab, Daratumumab, Elotuzumab, Necitumumab Idarucizumab Alirocumab, Mepolizumab, Evolocumab, Dinutuximab Secukinumab, Nivolumab, Blinatumomab, Pembrolizumab, Ramucirumab, Maftivimab, Odesivimab-ebgn, cilgavimab, and the like.

[0312] Further representative examples of antibodies which can be used for the capture and / or detection antibody include, but are not limited to, anti-HIV-i p24 antibodies, anti-HIV-i gp4i antibodies, anti-SARS-CoV-2 spike or nucleocapsid antibodies, anti-hepatitis B surface antigen (HBsAg) and anti-HBc antibodies, anti-HCV core / NS3 / NS5 antibodies, anti-influenza A / B nucleoprotein or hemagglutinin antibodies, anti-bacterial toxin / antigen antibodies (such as anti-Clostridioides difficile toxins, anti-Streptococcus pneumoniae polysaccharide, or anti-Haemophilus influenzae polysaccharide), anti-human chorionic gonadotropin (hCG) antibodies, anti-thyroid stimulating hormone (TSH) antibodies, anti-free and total thyroxine (T4) and triiodothyronine (T3) antibodies, anti-insulin antibodies, anti-cortisol, anti-estradiol, anti-testosterone, anti-progesterone antibodies, anti-prostate-specific antigen (PSA) antibodies, anti-carcinoembryonic antigen (CEA) antibodies, anti-alpha-fetoprotein (AFP) antibodies, anti-CA-125 antibodies, anti-HER2 / neu, anti-ER, and anti-PR antibodies, anti-CD20, anti-CD3, anti-CD4, anti-CD8, and anti-CD19 antibodies, anti-cardiac troponin I and troponin T antibodies, anti-creatine kinase-MB (CK-MB) antibodies, anti-B-type natriuretic peptide (BNP) or NT-proBNP antibodies, anti-interleukin-6 (IL-6) antibodies, anti-tumor necrosis factor alpha (TNF-a), anti-IL-iP, anti-IL-10, and anti-IFN-y antibodies, anti-C-reactive protein (CRP) antibodies, anti-double-stranded DNA (dsDNA) antibodies, anti-nuclear antigen (ANA) profile antibodies (such as, anti-Sm, anti-RNP, anti-Ro / SSA, and anti-La / SSB), anti-cyclic citrullinated peptide (CCP) antibodies, anti-IgE antibodies, anti-human IgG, IgM, IgA, IgD, and IgE antibodies, anti-IgG subclass-specific antibodies (such as anti-IgG1, anti-IgG2, anti-IgG3, and anti-IgG4), anti-light-chain antibodies (such as anti-kappa and anti-lambda), and the like.

[0313] Also disclosed are reporter peptide-detector antibody complexes. In some aspects, the reporter peptide can be covalently conjugated to the detector antibody. In someAttorney Docket No. 103362-047WO1

[0314] aspects, the reporter peptide can be directly bonded to the detector antibody, while in other aspects, the reporter peptide can be bonded to the detector antibody through a linker. In certain aspects, the reporter peptide can be bonded to the detector antibody through a cleavable bond (e.g., an ester bond linking the reporter peptide directly to the detector antibody), or the linker can include a cleavable bond. The cleavable bond can be cleaved by exposure to acid or base (for example, a hydrolysable bond such as an ester, hydrazone, acetal, or oxime), by exposure to an oxidant (for example, a disulfide bond, thioacetal), by exposure to heat, or by exposure to actinic radiation (for example, an ortho-nitrobenzyl group). As used herein, an ortho-nitrobenzyl (or o-nitrobenzyl) linker has the following general formula:

[0315] NO2

[0316]

[0317] wherein one oxygen is bonded to the reporter peptide, optionally through an additional linker moiety, and the other oxygen is bonded to the detector antibody, optionally through additional linker moieties.

[0318] In some aspects, the reporter peptide can include glutamic acid or aspartic acid residues, and the reporter peptide is linked to the detector antibody via the carboxy side chain in the glutamic acid or aspartic acid residue, for example, through an ester or thioester bond. In some aspects, the reporter peptide can include a cysteine, serine, or threonine residue, and the reporter peptide is linked to the detector antibody via the alcohol / thiol side chain in the cysteine, serine, or threonine residue, for example, through a thioester, disulfide, thioacetal, ester, or acetal.

[0319] In some aspects, the detector antibody may be linked to the reporter peptide via a non-hydrolyzable bond. In some aspects, the detector antibody may be reacted with a linker compound having an isothiocyanate group to form a thiourea bond to the linker via a lysine residue in the detector antibody. In some aspects, the linker may have the formula:Attorney Docket No. 103362-047WO1

[0320] 'reporter'

[0321] peptide

[0322]

[0323] wherein the thiourea is bonded directly to the detector antibody, for example, via a lysine residue, or the thiourea is bonded to an additional linker moiety.

[0324] The reporter peptide will generally include an aspartic acid / proline motif. In some aspects, the reporter peptide includes an aspartic acid / proline motif and exactly two arginine residues. In some aspects, the reporter peptide includes an aspartic acid / proline motif, exactly two arginine residues, and no other basic amino acid residues, e.g., no lysine or histidine residues. In some aspects, the reporter peptide includes an aspartic acid / proline motif, exactly two arginine residues, no other basic amino acid residues, e.g., no lysine or histidine residues, wherein one arginine residue is at the C-terminus and the other arginine residue is at the N-terminus or within two amino acid residues of the N-terminus. In some aspects, the reporter peptide includes an aspartic acid / proline motif, exactly two arginine residues, no other basic amino acid residues, e.g., no lysine or histidine residues, wherein the N- terminus is acylated, and one arginine residue is at the C-terminus and the other arginine residue is at the N-terminus or within two amino acid residues of the N- terminus.

[0325] In some aspects, the reported peptide has the following formula:

[0326]

[0327] wherein each X group is independently selected from null or an amino acid residue other than arginine, lysine, or histidine.

[0328] wherein one of Y1, Y2, or Y3is arginine, and the other two are selected from null or an amino acid residue other than arginine, lysine, or histidine. It should be understoodAttorney Docket No. 103362-047WO1

[0329] that the reporter peptide is conjugated to the detector antibody via the carboxylate in the depicted aspartic acid residue or the side chain of one of the X or Y residues. In some aspects, the reporter peptide can include from 8-18 amino acid residues, e.g., from 3 to 14 of X and Y are chosen to be null. In some aspects, the reporter peptide can include from 8-16 amino acid residues, from 8-14 amino acid residues, from 8-12 amino acid residues, or from 10-12 amino acid residues.

[0330] In certain aspects, from 4-16 of X and Y are null. In some aspects, from 6-16, from 8-14, from 10-14, from 10-12, or 11 of X and Y are null.

[0331] In some aspects, Y1is arginine.

[0332] In some aspects, Y2is arginine, and Y1is an amino acid other than arginine, lysine, or histidine. For example, Y1can be alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0333] In some aspects, Y3is arginine and Y1and Y2are amino acids other than arginine, lysine, or histidine, for example Y1can be alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine, and Y2can be alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0334] In some aspects, X1is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0335] In some aspects, X2is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0336] In some aspects, X3 is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0337] In some aspects, X4 is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.Attorney Docket No. 103362-047WO1

[0338] In some aspects, X⁵ is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0339] In some aspects, X6is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0340] In some aspects, X⁷ is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0341] In some aspects, X8is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0342] In some aspects, X⁹ is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0343] In some aspects, X10is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0344] In some aspects, X11is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0345] In some aspects, X12is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0346] In some aspects, X¹³ is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0347] In some aspects, X¹⁴ is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.Attorney Docket No. 103362-047WO1

[0348] In some aspects, X1 r> is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0349] In some aspects, X16is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0350] In some aspects, the detector antibody can be conjugated to a plurality of reporter peptides in order to enhance the reporting signal of a particular sandwich complex. In such aspects, the plurality of reporter peptides conjugated to the detector antibody will be the same, conjugated to the reporter peptide in the same way. In some aspects, the detector antibody can include from 1-500 reporter peptides, from 1-250 reporter peptides, from 1-125 reporter peptides, from 1-100 reporter peptides, from 1-75 reporter peptides, from 1-50 reporter peptides, from 1-25 reporter peptides, from 25-250 reporter peptides, from 100-250 reporter peptides, from 10-50 reporter peptides, from 25-100 reporter peptides, or from 50-150 reporter peptides.

[0351] In some aspects, the reporter peptides may be conjugated to the detector antibody through a dendrimer, for example, a polyamidoamine dendrimer, such as G1, G2, G3, G4, G5, G6, G7, or G8 PAMAM. The polyamidoamine dendrimer maybe conjugated to a plurality of reporter peptides via the amine groups on the surface of the dendrimer. Unreacted amine groups may then be used to conjugate the reporterdendrimer complex to the detector antibody. In some aspects, the unreacted amine groups may be treated with glutaraldehyde or a similar bis-aldehyde compound and then condensed with a lysine residue on the detector antibody.

[0352] Also disclosed herein are assay devices incorporating the disclosed reporter peptide / detector antibody complexes. In certain aspects, the assay device can include a plurality of layers, including a detector layer having the reporter peptide / detector antibody complex and a capture layer having the capture antibody immobilized to the capture layer. The individual layers maybe composed of a cellulosic material, for example, a paper. The cellulosic material may be oxidatively functionalized with aldehydes, which may be condensed with lysine residues in the capture antibody to immobilize it. The reporter peptide / detector antibody complex may simply be dispersed (i.e., non-covalently conjugated) to the capture layer (i.e., paper), such that an aqueous composition containing an analyte is contacted with the detector layer.Attorney Docket No. 103362-047WO1

[0353] The aqueous composition extracts the reporter peptide / detector antibody complex from the paper, and an antigen within the aqueous composition binds the detector antibody. The aqueous composition (including the reporter peptide / detector antibody / antigen complex flows to the capture layer, where the complex can form a sandwich complex with the capture antibody. The capture layer can then be washed to remove unreacted reporter peptide / detector antibody. The kit may further include a reservoir layer, which may then be placed in fluid communication with the capture layer. The capture layer is then treated to liberate the reporter peptide (for example, by washing with an aqueous base, acid, or oxidant, or irradiation) and then collecting the liberated reporter peptides in the reservoir layer. The reservoir layer may itself be a paper substrate that can be directly used in paper spray ionization as described herein.

[0354] In some aspects, the assay device can include a plurality of reporter peptide / detector antibody complexes, each specific to a particular antigen and having a distinct reporter peptide. The detection of different reporter peptides in the reservoir confirms the presence of different antigens.

[0355] In some aspects, the detector layer can include two or more regions, each region containing a distinct reporter peptide / detector antibody complex. In some aspects, the regions are not in fluid communication with one another, for example, by treating the paper spaces between the regions with wax. Such a device can also include two or more regions in the capture layer, each region in fluid communication with a single region in the detector layer. The device can include a reservoir layer configured to be in fluid communication with each capture region, and the aqueous solution used to liberate the reporter peptides is directed to a single portion, thereby combining the reporter peptides for analysis.

[0356] The assay device can further include a dispensing layer having a single portion for receiving a biological fluid or other composition for analysis. The dispensing layer is in fluid communication with each region of the detector layer, such that the composition is directed into each region of the detector layer for analysis.

[0357] In view of the described compositions, articles, devices, and methods, certain more particular aspects of the disclosure are described below. These particularly recited aspects should not, however, be interpreted to have any limiting effect on any different claims containing different or more general teachings described herein, orAttorney Docket No. 103362-047WO1

[0358] that the “particular” aspects are somehow limited in some way other than the inherent meanings of the language and formulae literally used therein.

[0359] Aspect 1. A method of detecting an antigen in a composition, comprising: a) contacting the composition with a capture antibody to form an antigen-capture antibody complex;

[0360] b) contacting the antigen-capture antibody complex with a detector antibody to form a sandwich complex, wherein the detector antibody includes at least one reporter peptide;

[0361] c) separating the sandwich complex from unreacted detector antibody;

[0362] d) separating the reporter peptide from the sandwich complex; and detecting the reporter peptide using mass spectrometry.

[0363] Aspect 2. The method of any aspect herein, such as aspect 1, wherein the reporter peptide is detected using paper spray mass spectrometry.

[0364] Aspect 3. The method of any aspect herein, such as aspect 2, wherein a liquid composition comprising the reporter peptide is contacted with a paper substrate, and a electrical current is applied to the paper substrate to ionize the reporter peptide. Aspect 4. The method of any aspect herein, such as aspect 3, wherein the paper substrate has a triangular shape, having a tip disposed towards an inlet of a mass spectrometer.

[0365] Aspects. The method of any aspect herein, such as any one of aspects 1-4, wherein the reporter peptide is covalently conjugated to the detector antibody.

[0366] Aspect 6. The method of any aspect herein, such as any one of aspects 1-5, wherein the reporter peptide is covalently conjugated to the detector antibody through a linker.

[0367] Aspect 7. The method of any aspect herein, such as any one of aspects 1-6, wherein the reporter peptide is covalently conjugated to the detector antibody through a linker having at least one bond cleavable by exposure to acid, base, oxidant, heat, or actinic radiation.

[0368] Aspect 8. The method of any aspect herein, such as any one of aspects 1-6, wherein the reporter peptide is covalently conjugated to the detector antibodyAttorney Docket No. 103362-047WO1

[0369] through a linker, wherein the reporter peptide is covalently conjugated to the linker via an ester bond.

[0370] Aspect 9. The method of any aspect herein, such as any one of aspects 1-6, wherein the reporter peptide is covalently conjugated to the detector antibody through a linker, wherein the reporter peptide is covalently conjugated to the linker via a thiourea bond or urea bond.

[0371] Aspect 10. The method of any aspect herein, such as any one of aspects 1-9, wherein the reporter peptide is separated from the sandwich complex by washing the sandwich complex with an aqueous acid or aqueous base.

[0372] Aspect 11. The method of any aspect herein, such as any one of aspects 1-10, wherein the reporter peptide is separated from the sandwich complex by washing the sandwich complex with solution having a pH from 1-13, from 1-7, from 1-5, from 2-6, from 3-6, from 4-6, from 7-13, from 9-13, or from 10-13.

[0373] Aspect 12. The method of any aspect herein, such as any one of aspects 1-11, wherein the sandwich complex is washed with a solution comprising a reagent effective to separate the reporter peptide from the sandwich complex to generate a solution comprising the reporter peptide, and the solution comprising the reporter peptide is dispensed onto a paper substrate.

[0374] Aspect 13. The method of any aspect herein, such as any one of aspects 6-12, wherein the linker is conjugated to the reporter peptide via an ester with a glutamic acid or aspartic acid residue in the reporter peptide.

[0375] Aspect 14. The method of any aspect herein, such as any one of aspects 1-13, wherein the reporter peptide comprises an aspartic acid / proline motif.

[0376] Aspect 15. The method of any aspect herein, such as any one of aspects 1-14, wherein the reporter peptide comprises exactly two arginine or arginine derivative residues.

[0377] Aspect 16. The method of any aspect herein, such as any one of aspects 1-15, wherein the reporter peptide comprises exactly two arginine or arginine derivative residues, and no other basic amino acid residues.Attorney Docket No. 103362-047WO1

[0378] Aspect 17. The method of any aspect herein, such as any one of aspects 1-16, wherein the reporter peptide comprises exactly two arginine or arginine derivative residues, and no lysine or histidine residues.

[0379] Aspect 18. The method of any aspect herein, such as any one of aspects 1-17, wherein the reporter peptide comprises exactly two arginine or arginine derivative residues, the first arginine or arginine derivative located at the C-terminus of the reporter peptide, and the other arginine or arginine derivative located at the N- terminus, or within two residues of the N-terminus.

[0380] Aspect 19. The method of any aspect herein, such as any one of aspects 1-18, wherein the reporter peptide has the formula:

[0381]

[0382] wherein each X group is independently selected from null or an amino acid residue other than arginine, lysine, or histidine;

[0383] wherein one of Y1, Y2, or Y3, is arginine, and the other two are selected from null or an amino acid residue other than arginine, lysine, or histidine.

[0384] Aspect 20. The method of any aspect herein, such as aspect 19, wherein from 3 to 14 of X and Y are chosen to be null.

[0385] Aspect 21. The method of any aspect herein, such as aspect 19 or 20, wherein the reporter peptide comprises from 8-16 amino acid residues from 8-14 amino acid residues from 8-12 amino acid residues, or from 10-12 amino acid residues.

[0386] Aspect 22. The method of any aspect herein, such as any one of aspects 19-21, wherein from 4-16, from 6-16, from 8-14, from 10-14, from 10-12, or 11 of X and Y are null.

[0387] Aspect 23. The method of any aspect herein, such as any one of aspects 19-22, wherein Y1is arginine or an arginine derivative.Attorney Docket No. 103362-047WO1

[0388] Aspect 24. The method of any aspect herein, such as any one of aspects 19-22, wherein Y1is arginine.

[0389] Aspect 25. The method of any aspect herein, such as any one of aspects 19-22, wherein Y2is arginine or an arginine derivative, and Y1is an amino acid other than arginine, arginine derivative, lysine, or histidine.

[0390] Aspect 26. The method of any aspect herein, such as any one of aspects 19-22, wherein Y2is arginine.

[0391] Aspect 27. The method of any aspect herein, such as aspect 25 or 26, wherein Y1is alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0392] Aspect 28. The method of any aspect herein, such as any one of aspects 19-22, wherein Y³ is arginine or an arginine derivative, and Y1and Y2are amino acids other than arginine, arginine derivative, lysine, or histidine.

[0393] Aspect 29. The method of any aspect herein, such as any one of aspects 19-22, wherein Ys is arginine.

[0394] Aspect 30. The method of any aspect herein, such as aspect 28 or 29, wherein Y1is alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine, and Y2is alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0395] Aspect 31. The method of any aspect herein, such as any one of aspects 19-30, wherein X1is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0396] Aspect 32. The method of any aspect herein, such as any one of aspects 19-31, wherein X2is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.Attorney Docket No. 103362-047WO1

[0397] Aspect 33. The method of any aspect herein, such as any one of aspects 19-32, wherein X3 is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0398] Aspect 34. The method of any aspect herein, such as any one of aspects 19-33, wherein X4 is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0399] Aspect 35. The method of any aspect herein, such as any one of aspects 19-34, wherein Xs is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0400] Aspect 36. The method of any aspect herein, such as any one of aspects 19-35, wherein X6is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0401] Aspect 37. The method of any aspect herein, such as any one of aspects 19-36, wherein X8is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0402] Aspect 38. The method of any aspect herein, such as any one of aspects 19-37, wherein X9 is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0403] Aspect 39. The method of any aspect herein, such as any one of aspects 19-38, wherein X10is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0404] Aspect 40. The method of any aspect herein, such as any one of aspects 19-39, wherein X11is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.Attorney Docket No. 103362-047WO1

[0405] Aspect 41. The method of any aspect herein, such as any one of aspects 19-40, wherein X12is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0406] Aspect 42. The method of any aspect herein, such as any one of aspects 19-41, wherein X' is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0407] Aspect 43. The method of any aspect herein, such as any one of aspects 19-42, wherein X'4 is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0408] Aspect 44. The method of any aspect herein, such as any one of aspects 19-43, wherein X|r> is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0409] Aspect 45. The method of any aspect herein, such as any one of aspects 19-44, wherein X16is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0410] Aspect 46. The method of any aspect herein, such as any one of aspects 1-45, wherein the detector antibody is conjugated to a plurality of reporter peptides.

[0411] Aspect 47. The method of any aspect herein, such as aspect 46, wherein the plurality of reporter peptides is conjugated to the detector antibody through a dendrimer.

[0412] Aspect 48. The method of any aspect herein, such as aspect 47, wherein the dendrimer is a polyamidoamide dendrimer, preferably a G1, G2, G3, G4, G5, G6, G7, or G8 PAMAM.

[0413] Aspect 49. The method of any aspect herein, such as any one of aspects 1-48, comprising detecting a plurality of different antigens in the composition, comprising contacting the composition with a plurality of different detector antibodies, wherein each different detector antibody is conjugated to a distinct reporter peptide.Attorney Docket No. 103362-047WO1

[0414] Aspect 50. An antibody-reporter peptide conjugate, comprising an antibody and a reporter peptide having the structure:

[0415]

[0416] wherein each X group is independently selected from null or an amino acid residue other than arginine, lysine, or histidine;

[0417] wherein one of Y1, Y2, or Y3, is arginine, and the other two are selected from null or an amino acid residue other than arginine, lysine, or histidine.

[0418] Aspect 51. The conjugate of any aspect herein, such as aspect 50, wherein from 3 to 14 of X and Y are chosen to be null.

[0419] Aspect 52. The conjugate of any aspect herein, such as aspect 50 or 51, wherein the reporter peptide comprises from 8-16 amino acid residues from 8-14 amino acid residues from 8-12 amino acid residues, or from 10-12 amino acid residues.

[0420] Aspect 53. The conjugate of any aspect herein, such as any one of aspects 50-52, wherein from 4-16, from 6-16, from 8-14, from 10-14, from 10-12, or 11 of X and Y are null.

[0421] Aspect 54. The conjugate of any aspect herein, such as any one of aspects 50-52, wherein Y1is arginine or an arginine derivative.

[0422] Aspect 55. The conjugate of any aspect herein, such as any one of aspects 50-52, wherein Y1is arginine.

[0423] Aspect 56. The conjugate of any aspect herein, such as any one of aspects 50-52, wherein Y2is arginine or an arginine derivative, and Y1is an amino acid other than arginine, arginine derivative, lysine, or histidine.

[0424] Aspect 57. The conjugate of any aspect herein, such as any one of aspects 50-52, wherein Y2is arginine.

[0425] Aspect 58. The conjugate of any aspect herein, such as aspect 56 or 57, wherein Y1is alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine,Attorney Docket No. 103362-047WO1

[0426] isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0427] Aspect 59. The conjugate of any aspect herein, such as any one of aspects 50-52, wherein Y3 is arginine or an arginine derivative, and Y1and Y2are amino acids other than arginine, arginine derivative, lysine, or histidine.

[0428] Aspect 60. The conjugate of any aspect herein, such as any one of aspects 50-52, wherein Ys is arginine.

[0429] Aspect 61. The conjugate of any aspect herein, such as aspect 59 or 60, wherein Y1is alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine, and Y2is alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0430] Aspect 62. The conjugate of any aspect herein, such as any one of aspects 50-61, wherein X1is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0431] Aspect 63. The conjugate of any aspect herein, such as any one of aspects 50-62, wherein X2is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0432] Aspect 64. The conjugate of any aspect herein, such as any one of aspects 50-63, wherein X3 is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0433] Aspect 65. The conjugate of any aspect herein, such as any one of aspects 50-64, wherein X4 is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0434] Aspect 66. The conjugate of any aspect herein, such as any one of aspects 50-65, wherein Xs is selected from null, alanine, asparagine, aspartic acid, cysteine,Attorney Docket No. 103362-047WO1

[0435] glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0436] Aspect 67. The conjugate of any aspect herein, such as any one of aspects 50-66, wherein X6is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0437] Aspect 68. The conjugate of any aspect herein, such as any one of aspects 50-67, wherein X8is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0438] Aspect 69. The conjugate of any aspect herein, such as any one of aspects 50-68, wherein X9 is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0439] Aspect 70. The conjugate of any aspect herein, such as any one of aspects 50-69, wherein X10is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0440] Aspect 71. The conjugate of any aspect herein, such as any one of aspects 50-70, wherein X11is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0441] Aspect 72. The conjugate of any aspect herein, such as any one of aspects 50-71, wherein X12is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0442] Aspect 73. The conjugate of any aspect herein, such as any one of aspects 50-72, wherein X' is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0443] Aspect 74. The conjugate of any aspect herein, such as any one of aspects 50-73, wherein X'4 is selected from null, alanine, asparagine, aspartic acid, cysteine,Attorney Docket No. 103362-047WO1

[0444] glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0445] Aspect 75. The conjugate of any aspect herein, such as any one of aspects 50-74, wherein X|r> is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0446] Aspect 76. The conjugate of any aspect herein, such as any one of aspects 50-75, wherein X16is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0447] Aspect 77. The conjugate of any aspect herein, such as any one of aspects 50-76, wherein the antibody is conjugated to a plurality of reporter peptides.

[0448] Aspect 78. The conjugate of any aspect herein, such as aspect 77, wherein the plurality of reporter peptides is conjugated to the antibody through a dendrimer. Aspect 79. The conjugate of any aspect herein, such as aspect 78, wherein the dendrimer is a polyamidoamide dendrimer, preferably a G1, G2, G3, G4, G5, G6, G7, or G8 PAMAM.

[0449] Aspect 80. An assay device comprising a plurality of layers, wherein the plurality of layers comprises a detector layer comprising one or more antibody-reporter peptide conjugates and a capture layer comprising one or more capture antibodies, wherein the detector layer is in fluid communication with the capture layer.

[0450] A number of aspects of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the disclosure. Accordingly, other aspects are within the scope of the following claims.

[0451] By way of non-limiting illustration, examples of certain aspects of the present disclosure are given below.

[0452] EXAMPLES

[0453] The following examples are set forth below to illustrate the compositions, articles, devices, and methods claimed herein, along with associated methods and resultsAttorney Docket No. 103362-047WO1

[0454] according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present disclosure, which are apparent to one skilled in the art.

[0455] Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of reaction conditions, e.g., component concentrations, temperatures, pressures, and other reaction ranges and conditions that can be used to optimize the product purity and yield obtained from the described process. Only reasonable and routine experimentation will be required to optimize such process conditions.

[0456] Acetylated N-terminal peptides (AcIRNPTIDPINR (SEQ ID NO2: 7), AcNLRPTDPLINR (SEQ ID NO2: 9), AcNLRTDPIIPNR (SEQ ID NO2: 10), AcLRDPNPTLINR (SEQ ID NO2: 5), AcLNRPTLLDPNR (SEQ ID NO2: 11) and deuterated peptide (AcIRN-(Proline-2,5,5-d3)-TIDPINR (SEQ ID NO2: 8) was purchased from LifeTein® LLC (Somerset, NJ, USA). Anhydrous dimethylformamide (DMF), 1-Butanol, Methanol, dichloromethane (DCM), hydrochloric acid (HC1), ammonium hydroxide (NH4OH), ammonium acetate (NH4CH3COO), formic acid and potassium periodate (KIO4) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile (ACN) was purchased from Fisher Chemical (Pittsburgh, PA, USA), i-ethyl-3-(3-(dimethylamino) propyl) carbodiimide (EDC) from Thermo Scientific (Waltham, MA, USA), 4-dimethylaminopyridine (DMAP) from Acros Organics (Geel, Belgium), 4-(2-hydroxyethyl)phenyl isothiocyanate from Organix, Inc. (Woburn, MA, USA), and 2,4-dinitrophenyl hydrazine (2,4-DNP) from Spectrum Chemical MFG. Corp. (Gardena, CA. The deionized water used to prepare reagents and stock solutions of peptides was obtained from a Milli-Q nano pure water filtration system purchased from Merck Millipore (Burlington, MA, USA).

[0457] A set of peptide sequences was initially characterized and analyzed in MS to check the fragmentation pattern. The final peptides of six unique sequences were designedAttorney Docket No. 103362-047WO1

[0458] to contain the same set of amino acids but different Asp and Pro amino acid locations. The N-terminal was acetylated to protect the primary amine from reacting with the isothiocyanate functional group of the conjugate unit during peptide probe synthesis. These peptides act as mass labels in the immunoassay analysis; hence, to avoid any false positives, they must not be naturally occurring peptides. A database search in UniProt Blast (https: / / www.uniprot.org / blast) was done for each peptide to ensure that the peptides are unique and not present in any protein.

[0459] The isobaric and deuterated peptides were characterized by analyzing their sensitivity in nESI and fragmentation patterns in MS / MS. First, solvent optimization was performed using the following solvents with 0.1% Formic Acid (FA): 1:1 Methanol / Water (Me0H: H20), 80:20 Me0H: H20, 1:1 Acetonitrile / Water (ACN: H20), and 80:20 ACN: H2O. After solvent optimization, peptide and deuterated peptides were analyzed in MS, and diagnostic peaks were identified. Synthesis of the peptide probe was accomplished by Steglich esterification. The peptide (1 mol) was dissolved in DMF in a round bottom flask suspended in an ice bath to maintain a temperature of o°C, followed by the addition of EDC (1.2 mol). The mixture was allowed to react for one hour. A 10 mol equivalent of 4-(2-hydroxyethyl)phenyl isothiocyanate was added to the solution, followed by DMAP (6-7 mol %). The reaction mixture was stirred for 1.5 hours at o°C. The flask was removed from the ice bath and continuously stirred for 20 hours at room temperature. After reacting, the solvent was evaporated under a high vacuum. The residue was in dissolved deionized distilled water. The peptide probe was purified by manual separation and preparative HPLC. The purified peptide probe was reconstituted with deionized distilled water and stored at -20°C until use.

[0460] The synthesized peptide probe was subjected to hydrolysis optimization studies to identify the optimum pH and time to cleave the ester bond. Various solvents ranging from 3-12 pH were prepared using HC1 and NH40H solutions. The peptide probe was subjected to different pH at different time intervals.

[0461] Paper spray mass spectrometry was used to analyze immunoassay performed in wax-printed functionalized 2D pPAD. Paper functionalization was done by soaking Whatman® cellulose chromatography papers in 0.03 M KI solution for four hours at 8o°C. After soaking, the papers were washed thrice with deionized distilled water and air-dried in blotting paper for 12 hours. To ensure the conversion of alcoholAttorney Docket No. 103362-047WO1

[0462] functional groups in cellulose to aldehyde groups, a 2,4-dinitrophenyl hydrazine test was used. The paper was then wax-printed to convert portions of the paper to hydrophobic zones to ensure that immunoassay reactions were only occurring in the hydrophilic test zones. The wax-printed paper was designed using Adobe Illustrator (Al) and printed using a Xerox ColorQube 8870 wax printer (Norwalk, CT, USA). Wax-printed papers were then placed in the oven at 13O°C for 20 seconds to allow the wax to permeate both sides of the paper. The papers were then cut according to their design and kept in the desiccator until use.

[0463] In this example, an immunoassay for detecting P. falciparum histidine-rich protein II (HRP-2) was done. The peptide probe was conjugated to the dAb in bioconjugation and served as reporter tags for detecting the biomarker. A sandwich immunoassay was performed by first reacting the aldehyde functional groups on the hydrophilic test zones of the functionalized paper to the capture antibody (cAb) ABMAL-0444 monoclonal antibody (mAb) anti-malaria HRP2 (Arista Biologicals, Inc. AIlentown, PA, USA). After blocking, incubation, and washing steps, 100 nM of the antigen spiked in human serum was added to each test zone, followed by another round of incubation and washings. To complete the sandwich immunoassay, the dAb conjugated to the dendrimer probe was added to each test spot, followed by final washings. Malaria jyHRP2 antigen was detected by analyzing negative and positive samples using paper spray ionization (PSI).

[0464] PSI is a substrate spray technique of liquid extraction, a type of ambient ionization. The spray solvent used was 1:1 Acetonitrile: water (ACN: H20) with 1% formic acid (FA) and a spray voltage of 4.5 kV. The distance of the paper triangle tip to the MS inlet was 5mm.

[0465] The first part of the peptide probe development is designing peptide sequences that will be used as mass tags in multiplexed immunoassays. The most critical factors for the peptide fragmentation process are the mobility of the proton, the position and basicity of the most basic residue, and the presence of proline (Pro). Specific cleavages forming b and / or y ions can occur depending on the localization of proton(s) and the availability and relative position of basic residue(s). Three 11-amino acid peptides were studied in low-energy CID fragmentation: LRNPTIDPLN (SEQ ID NO2: 1) (Exact Mass = 1151.63 Da), LRNPDPYILN (SEQ ID NO2: 2) (Exact Mass = 1213.65 Da), and LRNPTIDPLNR (SEQ ID NO2: 3) (Exact Mass = 1307.73 Da).Attorney Docket No. 103362-047WO1

[0466] The full MS and MS / MS (CID at 20) are shown below in FIGs. 2A-2D. Peptides 1 and 2 contain Asp, Pro, and one Arg, while Peptide 3 contains two Arg, having one Arg close to the N-terminal and another Arg at the N-terminal of the peptide. Upon protonation of the peptides (10 pM peptides dissolved in 1:1 MeOH-. H2O with 0.1% FA, pH=3.oo), Peptides 1 and 2 were protonated of up to +2 charge state, while Peptide 3 was up to +3. The results of MS / MS show that in Peptides 1 and 2, more fragments were generated since only one basic amino acid is present in the sequence that can sequester a proton.

[0467] The available proton can be immediately mobilized upon the addition of energy during CID, which results in several fragmentations. The results are consistent with the notion that selective cleavage is observed in peptides where the number of protons is less than or equal to the number of Arg. If the added proton(s) is sequestered by basic residue(s), selective cleavage occurs at Asp-Xxx (Xxx is C-terminus residue) as initiated by the acidic hydrogen of the Asp side chain. The MS / MS spectrum of Peptide 3 (see FIG. 2C) shows selective cleavage for the +2-charge state of the peptide (m / z 655) forming +1 charge states for b (m / z 810) and y (m / z 499) ions. The two added protons are sequestered by the guanidino side chains of the two Arg residues, allowing the acidic hydrogen of Asp to initiate cleavage. It is also worth mentioning the proline effect in enhancing cleavage N-terminal to proline (Zzz-Pro, Zzz is N-terminal residue), which is further amplified by the presence of Asp. MS / MS for the +3-charge state (m / z 437) resulted in the formation of several fragments since an available proton can initiate cleavage in different protonation sites at the peptide backbone. An attempt to reduce the +3-charge state of Peptide 3 was conducted by dissolving 10 pM of peptide in a series of ammonium acetate NH4CH2COO) solutions. The imM concentration of NH4CH2COO gave the highest intensity for 655 m / z of Peptide 3 and reduced the +3-charge state (see FIG. 2D). This also paved the way to acetylate the N-terminal of the peptide to reduce the charge state, and to avoid side reactions of the reactive N-terminal to the isothiocyanate functional group of the conjugate unit during peptide probe synthesis (see next section). Acetylated Peptide 3 (10 pM in 1:1 MeOH H2O with 0.1% FA) provided a dominant +2-charge state in the mass spectrum (see FIGs. 3A-3B).

[0468] From the preliminary studies on what factors to consider in peptide design, the following rules have been established so far: (1) the sequence must contain two Arg,Attorney Docket No. 103362-047WO1

[0469] one at the C-terminal and another close to the N-terminus; (2) the sequence must have Asp-Pro residues as cleavage site, and (3) the number of proton must not exceed the number of Arg residues. One more important factor to consider in designing peptide sequences is its unique identity. To avoid any false positives, the peptide must not be present in any protein that can be confirmed in the UniProt Blast database. Peptide 3 LRNPTIDPLNR (SEQ ID NO2: 3) was confirmed to have two hits in the database. Thus, the sequence cannot be used. Five unique peptide sequences were generated by rearranging the amino acids and interchanging leucine (Leu) and Isoleucine (He), as listed in Table 1.

[0470] Table 1.

[0471] Pep Sequence

[0472] 1 AcIRNPTIDPINR (SEQ ID NO2: 7)

[0473] 2 AcNLRPTDPLINR (SEQ ID NO2: 9)

[0474] 3 AcNLRTDPIIPNR (SEQ ID NO2: 10)

[0475] 4 AcRLDPNPTLINR (SEQ ID NO2: 6)

[0476] 5 AcLNRPTLLDPNR (SEQ ID NO2: 11)

[0477] 6 AcNNITRDPPIIR (SEQ ID NO2. 12)

[0478]

[0479] For peptide 4, the initial sequence was AcLRDPNPTLINR (SEQ ID NO2: 5). However, upon MS / MS, it provides a third fragment at 667 m / z, which is a +2-charge state of the peptide with ammonia loss (-NH3). My hypothesis for this is that the guanidino group of Arg (R) interacts with the proton in the side chain of Asp (D). The acidic hydrogen cannot initiate charge-remote fragmentation due to strong hydrogen bonding. Instead, an ammonia loss is the next most labile bond that breaks upon MS / MS. Upon changing the position of L and R at the N-terminus, the third fragment, 667 m / z, significantly reduced relative to the b- and y-ions, see FIGs. 4A-4B. All other peptides also produce a third fragment at 667 m / z but at a relatively lower frequency (see FIGs. 8A-8E). All the peptides were acetylated at the N-Attorney Docket No. 103362-047WO1

[0480] terminus. For optimization studies, only AcIRNPTIDPINR (Pep 1) (SEQ ID NO2: 7) was analyzed.

[0481] Acetylated peptides listed in Table 1 and an internal standard with the same sequence as Pep 1 (AcIRNPTIDPINR) (SEQ ID NO2: 7) but deuterated at Pro near the N-terminal (AcIRNdsPTIDPINR (SEQ ID NO2: 8)), were characterized based on their sensitivity in nano-ESI and tandem mass spectrometry.

[0482] First, the nano-ESI solvent was optimized, and for this example, only Pep 1 was used. The following solvents with 0.1% FA were used for solvent optimization studies: 1:1 MeOH H2O, 80:20 MeOH H2O, 1:1 ACN H2O, and 80:20 ACN H2O. A peptide concentration of lqg / ml was analyzed. Based on the results solvent of 80:20 ACN-. H2O gave the highest intensity as shown in the FIG. 6.

[0483] A 30pg / ml of Pep 1 was prepared in 80:20 ACN-. H2O with 0.1% FA and was analyzed using nESI. The +2-charge state of the peptide was detected at 676 m / z. This was then subjected to MS / MS, which detected two dominant ions of b7(m / z 852.50) and y4(m / z 499.33) cleaving at Asp-Pro residues giving an anhydride b ion. Formation of an anhydride product can be confirmed by MSs with the loss of CO2 and CO (total of 72 Da) and maleic anhydride (98 Da)5?. It is also confirmed by MSs applying CID 25 to m / z 852, forming m / z 780 (loss of 72 Da) and m / z 754 (loss of 98 Da), as shown in FIGs. 7A-7D. In terms of mass tag characteristics for Pep 1, 852 m / z and 499 m / z are selected as diagnostic peaks. Mass Spectra of full MS and MS / MS of other peptides are shown in FIGs. 8A-8E, and their corresponding diagnostic peaks are listed in Table 2. A mass spectrum of all the peptides in one solution is shown in FIGs.

[0484] 9A-9B.

[0485] Table 2: Diagnostic b and y ion peaks of the peptides.

[0486] Peptide Code Sequence* Diagnostic Peaks

[0487] b ion yion

[0488] Pep 1 AcIRNPTIDPINR 852 499

[0489] (SEQ ID NO2: 7)

[0490]

[0491] Attorney Docket No. 103362-047WO1

[0492] Pep 2 AcNLRPTDPLINR 739 612

[0493] (SEQ ID NO2: 9)

[0494] Pep 3 AcNLRTDPIIPNR 642 709

[0495] (SEQ ID NO2: 10)

[0496] Pep 4 AcRLDPNPTLINR 427 924

[0497] (SEQ ID NO2: 6)

[0498] Pep 5 AcLNRPTLLDPNR 965 386

[0499] (SEQ ID NO2: 11)

[0500] Pep 6 AcNNITRDPPIIR 756 595

[0501] (SEQ ID NO2: 12)

[0502]

[0503] *All peptides are acetylated at the N-terminus

[0504] The internal standard (IS) was designed to contain deuterium at the first Pro (see FIG. 10). A 30,g / ml of the IS was prepared by dissolving appropriate amount of peptide stock solution in 80:20 ACN-. H2O with 01.% FA. The protonated form at +2-charge state gives a peak at 678 m / z and a +i-charge state of 1353 m / z. MS / MS of 678 m / z gives b ion at 856 m / z and y4at 499 m / z (see FIG. 10). The diagnostic peak for the IS is 856 m / z upon MS / MS.

[0505] A calibration curve (see FIG. 11) was constructed for Pep 1, wherein quantification was done by obtaining the relative intensity of the analyte to the internal standard. An internal standard (IS) is a compound that shares similar physical and chemical characteristics to the target analyte, allowing variations due to inefficiencies in sample preparation and ionization and instrumentation to be corrected since it is anticipated that the initial ratio of analyte to internal standard does not change. In this example, the same peptide sequence as with Pep 1 but deuterated at proline residue located near the N-terminal is used as the internal standard. Quantification analysis was based on the ratio of intensities of the analyte signal (m / z 676 -> m / z 852) to the internal standard signal m / z 678 m / z 856).Attorney Docket No. 103362-047WO1

[0506] The limit of detection (LOD) and limit of quantification (LOQ) were also calculated from the calibration curve.

[0507] ,n n_ Ublk + 3sbik) — b

[0508] m

[0509] . > (Jblk K^blk) ~ b

[0510]

[0511] m

[0512] where:

[0513] Ibik = average ratio of blank / IS

[0514] sbik = standard deviation of the blank / IS b = y — intercept in the calibration curve

[0515] m = slope of the calibration curve

[0516] Based on the calibration curve, Pep 1 LOD is 0.35 ng / ml (0.26 nM) and LOQ 1.03 ng / ml (0.77 nM).

[0517] The synthesis of the peptide probe was accomplished following the Steglich esterification procedure, using EDC as coupling reagent instead of dicyclohexylcarbodiimide (DCC). DCC is toxic to human health and produces byproduct N, N’-dicyclohexylurea (DCU), which is insoluble in water and partially soluble in many organic solvents. EDC produces a by-product EDU that is more soluble than DCU. Steglich esterification is catalyzed by DMAP, which possesses a higher nucleophilicity than alcohol. DMAP reacts with the O-acylisourea intermediate, forming a highly activated electrophilic acylated pyridinium intermediate, which will subsequently be attacked by the nucleophilic oxygen of the alcohol. Initially, a 0.1 mol equivalent of DMAP was added to the reaction, which accounts for only ~ 2 mol%. Upon increasing DMAP from 2mol% to 6-7 mol%, yield increased from 23-24% to 47-50%. It was noted that increasing the mol equivalents of (4-hydroxymethyl)phenylisothiocyanate, the alcohol functional group (-OH) source, also helped in increasing the yield. Initially, 2.4 mol equivalents were used then eventually doubling it to 4.8 mol equivalents. Though the yield was around 50%, separating the peptide from the peptide probe is difficult since they have similar polarity. Increasing the -OH source to up to 10 mol equivalents increased the yield to 88% but also produced a side product of 10% yield. With this, two separation techniques were employed: (1) liquid-liquid extraction and (2) purification byAttorney Docket No. 103362-047WO1

[0518] preparative HPLC. Liquid-liquid extraction aided in the removal of by-products insoluble in water, and HPLC aided in separating peptide probes, peptides, and side products of the same polarity. Collected samples in HPLC were subjected to solvent evaporation and then reconstituted with dH20. Samples were analyzed in MS to confirm the presence of the peptide probe product providing [M+H]+(1511 m / z) and [M+2H]2+(m / z 756.5 ) in full MS, and fragment ions of m / z 852.42 and m / z 660.42 in MS / MS. Ester bond was confirmed to form at the C-terminal by subjecting the precursor ion to MS / MS, forming fragments of m / z 852.5 and m / z 660. The fragment ion m / z 852.5 is the anhydride fragment ion which is the same as the b-ion forms when the Pep 1 is subjected to MS / MS. The m / z 660 fragment ion is similar to the y-ion with the loss of a methylene residue (-CH2) but includes the mass of the conjugate unit.

[0519] Several studies have demonstrated the labile ester bond in ionic probes is cleaved via basic hydrolysis. In this example, hydrolysis of the ester bond connecting the peptide and the conjugate unit (4-hydroxymethyl)phenylisothiocyanate was investigated in different pH solvents ranging from 3-12 using solutions of HC1 and NH40H. Optimized time and pH conditions will be used in subsequent immunoassay studies. Hydrolysis of esters at neutral pH is a kinetically slow process, ideal in immunoassay analysis, wherein pH is around 7. Reporter mass tags are expected to be liberated after sandwich immunoassay and then will be analyzed by MS. In this optimization analysis results show that peptides have been liberated to 40% for the first 10 mins at pH 12. The % hydrolysis is computed as follows:

[0520] ..,.. Ipeptide.

[0521] % Hydrolysis = - - - - x 100

[0522] 'peptide + 'probe

[0523] where:

[0524] Ipeptide = Signal Intentslty of peptide probe

[0525] Iprobe = signal intensity of probe

[0526] Results show that after 60 mins at pH 12, 80% of the peptides had been liberated, and not much change was seen after 100 mins. It can also be noted that at 40 mins, more than 70% of peptides are hydrolyzed. For this example, and considering the time in immunoassay, 40 mins at pH 12 can be used as the optimized condition in liberating peptides.Attorney Docket No. 103362-047WO1

[0527] To hasten the reaction, the hydrolysis experiment was repeated using 2M and 4M NH4OH, both at pH 12. Results show that at 10 minutes, 97% of the peptides are liberated from the probe using 2M NH4OH. The difference in the results may be due to the reagents used in the first hydrolysis experiment, which were 7 days older than the freshly prepared reagents in the second hydrolysis experiment.

[0528] Bioconjugation involves three steps: (1) conjugating dendrimer (MW = 14 kDa) and peptide probe (MW =1.5 kDa) via reaction of isothiocyanate group (-N = C = S), (2) conjugating detection antibody (dAb), ABMAL-0445 monoclonal antibody (mAb) anti-malaria HRP2 (Arista Biologicals, Inc. AIlentown, PA, USA) to the dendrimerprobe via Schiff base chemistry, and (3) dilution and purification of conjugated dAb and peptide probe. For step 1, 10% vol / vol G4 PAMAM dendrimer with 64 primary amines on the surface (-NH2,pKa = 6.85) was prepared in phosphate buffer saline (PBS, pH 7.4), and then the peptide probe was added to 5pL of dendrimer in PBS to make up a ratio of 1 dendrimer: 10 probes. The use of PBS (pH 7.4) is to ensure that the primary amines at the surface of the dendrimer are deprotonated, making the isothiocyanate functional group of the probe readily react with the amines of the dendrimer. The solution was then incubated for 12 hours at 4°C. In step 2, the incubated solution was diluted to 500 pL using PBS and then purified by filtration using Amicon® Ultra Centrifugal Filters 10 kDa MWCO (Sigma Aldrich, MO, USA). A 0.02% w / w glutaraldehyde was reacted with the dendrimer solution for 10 minutes at room temperature (RT) to form a CHO-G4-peptide probe (molecule A). Before conjugating the dAb to molecule A, a buffer exchange was done to replace PBS with sodium buffer (Na2CO3— NaHCO3~) to ensure pH is 9-11. Purification of dAb in sodium buffer was done using Amicon® Ultra Centrifugal Filters 30 kDa MWCO (Sigma Aldrich, MO, USA) The lysine (pKa = 10.79) primary amine of the dAb forms a Schiff base upon reacting with the aldehyde group of molecule A. A 1 dAb: 100 molecule A ratio is prepared and then incubated for 12 hours at 4°C. The last step was the purification of dAb-dendrimer-probe using Amicon® Ultra Centrifugal Filters 100 kDa MWCO (Sigma Aldrich, MO, USA). The process is described in FIG.

[0529] 15.

[0530] To test the peptide probe's capability for disease detection, the malaria jyHRP2 antigen was used for biomarker detection. The P / HRP2 is a protein associated with malaria infection which is produced by the asexual stages and gametocytes of P.Attorney Docket No. 103362-047WO1

[0531] falciparum. This is a water-soluble protein and can be easily found in human serum, plasma, cerebrospinal fluid and urine of infected person. For a proof of concept, the positive sample was prepared by spiking / yHRP2 into human serum, while the negative sample did not contain any jyHRP2. After the immunoassay, both the samples were analyzed using nESI and PSI. For nESI, liquid samples were extracted from the microfluidic device, then a spray solvent was added and analyzed in MS. For PSI, each spot was directly analyzed in the MS. The positive samples are expected to give higher signal intensity of cleaved peptides than the negative sample. The analysis proved that mass-spectrometry-based immunoassay using peptide probes as mass reporters was able to distinguish the positive from the negative, as shown in FIGs. 16 and 17.

[0532] This example developed isobaric peptide probes intended to provide peptide mass tags for the simultaneous detection of disease biomarkers. This was accomplished by designing peptide sequences, characterizing reporter mass tags in nano-ESI, synthesizing the peptide probe via Steglich esterification, and optimizing system conditions to efficiently liberate the peptide from the probe. Bioconjugation of the peptide probe to the dAb and performing immunoassay were also done in this example to prove that the probes can be used as reporter tags for biomarker detection. For the design of the peptides, the same set of amino acids is designed since isobaric peptides are ideal for simplicity in mass spectra. Given that the peptide is in a +2-charge state, the sequences need to contain two Arg, one at the C-terminus and the other near the N-terminal, to sequester the protons available. This way, charge remote fragmentation is initiated by the acidic proton from the Asp side chain, thus cleaving the Asp-Pro amide bond. In this example, six isobaric peptides that are not present in any protein, as confirmed by a search in Uniprot BLAST, were developed. These peptides were then characterized by investigating their fragmentation behavior in MS / MS using a 2D linear ion trap mass spectrometer. A calibration curve was also constructed to determine the sensitivity of peptides in nESI, and the results provided LOD is 0.35 ng / ml (0.26 nM) and LOQ 1.03 ng / ml (0.77 nM) for Pep 1 (AcIRNPTIDPINR). After characterizing and analyzing the sensitivity of the peptide mass reporters, the peptide probe was synthesized by Steglich esterification. In this reaction, an ester bond is formed by reacting the carboxylic terminal of the peptide (-COOH) to the alcohol (-OH) functional group of the conjugate unit. The formation of an ester bond was confirmed by determining theAttorney Docket No. 103362-047WO1

[0533] mass of the peptide probe at 1511 m / z [M+H]+and m / z 756.5 [M+2H]2+. Ester bond was confirmed to form at the C-terminal by subjecting the precursor ion to MS / MS, forming fragments of m / z 852.5 and m / z 660. The fragment ion m / z 852.5 is the anhydride fragment ion which is the same as the b-ion forms when the Pep 1 is subjected to MS / MS. The m / z 660 fragment ion is similar to the y-ion with the loss of a methylene residue (-CH2) but includes the mass of the conjugate unit. Finally, this example investigated the hydrolysis of the ester bond in the peptide probe by determining the percentage of peptides liberated from the probe. This has to be done since, after performing an immunoassay, peptides as mass tags need to be detected by the mass spectrometer for the indirect detection of the protein. Based on the results, 97% of the peptides can be hydrolyzed from the probe at 10 minutes. After hydrolysis experiments, bioconjugation and sandwich immunoassay were conducted using the peptide probe. Results showed that peptide probe has successfully distinguished a malaria-positive P / HRP2 human serum sample and a negative sample.

[0534] The references cited herein are hereby incorporated by reference to disclose and describe the methods or materials in connection with which the publications are cited or to provide background for the present disclosure. Any incorporation by reference of documents herein is limited such that no subject matter is incorporated by reference that is contrary to the explicit disclosure herein. In the event of inconsistent usages between this document and those documents so incorporated by reference herein, the use in the incorporated references should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0535] The compositions and methods of the appended claims are not limited in scope by the specific compositions and methods described herein, which are intended as illustrations of a few aspects of the claims and any compositions and methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the compositions and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative compositions and method steps disclosed herein are specifically described, other combinations of the compositions and method steps also are intended to fall within the scope of the appended claims, evenAttorney Docket No. 103362-047WO1

[0536] if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein; however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated.

Claims

1. Attorney Docket No. 103362-047WO1WHAT IS CLAIMED IS:

1. A method of detecting an antigen in a composition, comprising:a) contacting the composition with a capture antibody to form an antigen-capture antibody complex;b) contacting the antigen-capture antibody complex with a detector antibody to form a sandwich complex, wherein the detector antibody includes at least one reporter peptide;c) separating the sandwich complex from unreacted detector antibody;d) separating the reporter peptide from the sandwich complex; and detecting the reporter peptide using mass spectrometry.

2. The method of claim 1, wherein the reporter peptide is detected using paper spray mass spectrometry.

3. The method of claim 2, wherein a liquid composition comprising the reporter peptide is contacted with a paper substrate, and a electrical current is applied to the paper substrate to ionize the reporter peptide.

4. The method of claim 3, wherein the paper substrate has a triangular shape, having a tip disposed towards an inlet of a mass spectrometer.

5. The method of any one of claims 1-4, wherein the reporter peptide is covalently conjugated to the detector antibody.

6. The method of any one of claims 1-5, wherein the reporter peptide is covalently conjugated to the detector antibody through a linker.

7. The method of any one of claims 1-6, wherein the reporter peptide is covalently conjugated to the detector antibody through a linker having at least one bond cleavable by exposure to acid, base, oxidant, heat, or actinic radiation.

8. The method of any one of claims 1-6, wherein the reporter peptide is covalently conjugated to the detector antibody through a linker, wherein the reporter peptide is covalently conjugated to the linker via an ester bond.Attorney Docket No. 103362-047WO19. The method of any one of claims 1-6, wherein the reporter peptide is covalently conjugated to the detector antibody through a linker, wherein the reporter peptide is covalently conjugated to the linker via a thiourea bond or urea bond.

10. The method of any one of claims 1-9, wherein the reporter peptide is separated from the sandwich complex by washing the sandwich complex with an aqueous acid or aqueous base.

11. The method of any one of claims 1-10, wherein the reporter peptide is separated from the sandwich complex by washing the sandwich complex with solution having a pH from 1-13, from 1-7, from 1-5, from 2-6, from 3-6, from 4-6, from 7-13, from 9-13, or from 10-13.

12. The method of any one of claims 1-11, wherein the sandwich complex is washed with a solution comprising a reagent effective to separate the reporter peptide from the sandwich complex to generate a solution comprising the reporter peptide, and the solution comprising the reporter peptide is dispensed onto a paper substrate.

13. The method of any one of claims 6-12, wherein the linker is conjugated to the reporter peptide via an ester with a glutamic acid or aspartic acid residue in the reporter peptide.

14. The method of any one of claims 1-13, wherein the reporter peptide comprises an aspartic acid / proline motif.

15. The method of any one of claims 1-14, wherein the reporter peptide comprises exactly two arginine or arginine derivative residues.

16. The method of any one of claims 1-15, wherein the reporter peptide comprises exactly two arginine or arginine derivative residues, and no other basic amino acid residues.

17. The method of any one of claims 1-16, wherein the reporter peptide comprises exactly two arginine or arginine derivative residues, and no lysine or histidine residues.

18. The method of any one of claims 1-17, wherein the reporter peptide comprises exactly two arginine or arginine derivative residues, the first arginine or arginine derivative located at the C-terminus of the reporter peptide, and the other arginineAttorney Docket No. 103362-047WO1or arginine derivative located at the N-terminus, or within two residues of the N- terminus.

19. The method of any one of claims 1-18, wherein the reporter peptide has the formula:wherein each X group is independently selected from null or an amino acid residue other than arginine, lysine, or histidine;wherein one of Y1, Y2, or Y3, is arginine, and the other two are selected from null or an amino acid residue other than arginine, lysine, or histidine.

20. The method of claim 19, wherein from 3 to 14 of X and Y are chosen to be null.

21. The method of claim 19 or 20, wherein the reporter peptide comprises from 8-16 amino acid residues from 8-14 amino acid residues from 8-12 amino acid residues, or from 10-12 amino acid residues.

22. The method of any one of claims 19-21, wherein from 4-16, from 6-16, from 8- 14, from 10-14, from 10-12, or 11 of X and Y are null.

23. The method of any one of claims 19-22, wherein Y1is arginine or an arginine derivative.

24. The method of any one of claims 19-22, wherein Y1is arginine.

25. The method of any one of claims 19-22, wherein Y2is arginine or an arginine derivative, and Y1is an amino acid other than arginine, arginine derivative, lysine, or histidine.

26. The method of any one of claims 19-22, wherein Y2is arginine.

27. The method of claim 25 or 26, wherein Y1is alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.Attorney Docket No. 103362-047WO128. The method of any one of claims 19-22, wherein Y3 is arginine or an arginine derivative, and Y1and Y2are amino acids other than arginine, arginine derivative, lysine, or histidine.

29. The method of any one of claims 19-22, wherein Y3 is arginine.

30. The method of claim 28 or 29, wherein Y1is alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine, and Y2is alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

31. The method of any one of claims 19-30, wherein X1is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

32. The method of any one of claims 19-31, wherein X2is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

33. The method of any one of claims 19-32, wherein X3 is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

34. The method of any one of claims 19-33, wherein X4 is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

35. The method of any one of claims 19-34, wherein Xs is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.Attorney Docket No. 103362-047WO136. The method of any one of claims 19-35, wherein X6is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

37. The method of any one of claims 19-36, wherein X8is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

38. The method of any one of claims 19-37, wherein X9 is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

39. The method of any one of claims 19-38, wherein X10is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

40. The method of any one of claims 19-39, wherein X11is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

41. The method of any one of claims 19-40, wherein X12is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

42. The method of any one of claims 19-41, wherein X' is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

43. The method of any one of claims 19-42, wherein X'4 is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.Attorney Docket No. 103362-047WO144- The method of any one of claims 19-43, wherein X|r> is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

45. The method of any one of claims 19-44, wherein X16is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

46. The method of any one of claims 1-45, wherein the detector antibody is conjugated to a plurality of reporter peptides.

47. The method of claim 46, wherein the plurality of reporter peptides is conjugated to the detector antibody through a dendrimer.

48. The method of claim 47, wherein the dendrimer is a polyamidoamide dendrimer, preferably a G1, G2, G3, G4, G5, G6, G7, or G8 PAMAM.

49. The method of any one of claims 1-48, comprising detecting a plurality of different antigens in the composition, comprising contacting the composition with a plurality of different detector antibodies, wherein each different detector antibody is conjugated to a distinct reporter peptide.

50. An antibody-reporter peptide conjugate, comprising an antibody and a reporter peptide having the structure:wherein each X group is independently selected from null or an amino acid residue other than arginine, lysine, or histidine;wherein one of Y1, Y2, or Y3, is arginine, and the other two are selected from null or an amino acid residue other than arginine, lysine, or histidine.Attorney Docket No. 103362-047WO151. The conjugate of claim 50, wherein from 3 to 14 of X and Y are chosen to be null.

52. The conjugate of claim 50 or 51, wherein the reporter peptide comprises from 8-16 amino acid residues from 8-14 amino acid residues from 8-12 amino acid residues, or from 10-12 amino acid residues.

53. The conjugate of any one of claims 50-52, wherein from 4-16, from 6-16, from 8-14, from 10-14, from 10-12, or 11 of X and Y are null.

54. The conjugate of any one of claims 50-52, wherein Y1is arginine or an arginine derivative.

55. The conjugate of any one of claims 50-52, wherein Y1is arginine.

56. The conjugate of any one of claims 50-52, wherein Y2is arginine or an arginine derivative, and Y1is an amino acid other than arginine, arginine derivative, lysine, or histidine.

57. The conjugate of any one of claims 50-52, wherein Y2is arginine.

58. The conjugate of claim 56 or 57, wherein Y1is alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

59. The conjugate of any one of claims 50-52, wherein Y³ is arginine or an arginine derivative, and Y1and Y2are amino acids other than arginine, arginine derivative, lysine, or histidine.

60. The conjugate of any one of claims 50-52, wherein Y3 is arginine.

61. The conjugate of claim 59 or 60, wherein Y1is alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine, and Y2is alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

62. The conjugate of any one of claims 50-61, wherein X1is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.Attorney Docket No. 103362-047WO163. The conjugate of any one of claims 50-62, wherein X2is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

64. The conjugate of any one of claims 50-63, wherein X3 is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

65. The conjugate of any one of claims 50-64, wherein X4 is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

66. The conjugate of any one of claims 50-65, wherein Xs is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

67. The conjugate of any one of claims 50-66, wherein X6is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

68. The conjugate of any one of claims 50-67, wherein X8is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

69. The conjugate of any one of claims 50-68, wherein X9 is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

70. The conjugate of any one of claims 50-69, wherein X10is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.Attorney Docket No. 103362-047WO171. The conjugate of any one of claims 50-70, wherein X11is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

72. The conjugate of any one of claims 50-71, wherein X12is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

73. The conjugate of any one of claims 50-72, wherein X' is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

74. The conjugate of any one of claims 50-73, wherein X'4 is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

75. The conjugate of any one of claims 50-74, wherein X|r> is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

76. The conjugate of any one of claims 50-75, wherein X16is selected from null, alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

77. The conjugate of any one of claims 50-76, wherein the antibody is conjugated to a plurality of reporter peptides.

78. The conjugate of claim 77, wherein the plurality of reporter peptides is conjugated to the antibody through a dendrimer.

79. The conjugate of claim 78, wherein the dendrimer is a polyamidoamide dendrimer, preferably a G1, G2, G3, G4, G5, G6, G7, or G8 PAMAM.Attorney Docket No. 103362-047WO180. An assay device comprising a plurality of layers, wherein the plurality of layers comprises a detector layer comprising one or more antibody-reporter peptide conjugates and a capture layer comprising one or more capture antibodies, wherein the detector layer is in fluid communication with the capture layer.

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