Compositions, kits, and methods for detecting preclinical alzheimer's disease

Abstract

To provide compositions, kits and methods for detecting preclinical Alzheimer's disease.SOLUTION: Compositions and kits for diagnosing and prognosing Alzheimer's Disease (AD) in a human patient include a binding agent such as a monoclonal antibody for a biomarker conjugated to a detectable moiety such as a fluorophore, where the biomarker is chosen from CD163, CD91, CD59, MerTK and other phagocytosis-related molecules. Further, compositions and kits employ panels of fluorophore-conjugated monoclonal antibodies for biomarkers including scavenger receptors. Methods for determining the relative expression of biomarkers, diagnosing AD, and determining the efficacy of AD therapeutic candidates such as phagocytosis-promoting agents and scavenger receptor agonists also appear.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under PCT Chapter I, Article 8 of U.S. Provisional Patent Application No. 63 / 231,034, entitled "Compositions, Kits and Methods for Detecting Alzheimer's Disease," filed August 9, 2021, which is incorporated herein by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under APP 1048082 (2013-2015), APP 1061419 (2014-2016), and APP 1120095 (2017-2019) awarded by the National Health and Medical Research Council (Australia). The Australian Government may have certain rights in this invention. Further support was provided by the Bethlehem Griffiths Research Foundation.

[0003] FIELD OF THE INVENTION The present invention relates to the detection and prognosis of Alzheimer's disease, potentially before clinically significant symptoms appear. [Background technology]

[0004] Background of the Invention Although the causes of Alzheimer's disease (AD) are poorly understood, the disease correlates with a dramatic decline in quality of life and high care costs for patients in its most advanced stages. AD typically develops over several years. As it progresses from cognitively normal (CN) through mild cognitive impairment (MCI) to clinical AD dementia, patients experience declines in short-term memory, attention, and abstract thinking, along with increased emotional blunting, social withdrawal, and isolation. Accumulation of amyloid beta (Aβ) protein in the form of oligomers and plaques has been detected in both familial and sporadic cases of AD.

[0005] Using a novel hypothesis, applicants used flow cytometry technology to examine a series of cell surface leukocyte biomarkers for association with brain Aβ accumulation as potential biomarkers for AD. The study yielded surprising correlations that enabled the diagnosis and prognosis of AD for CN, MCI, and AD patient groups. Applicants' findings led to the compositions and methods of the present invention disclosed herein. Summary of the Invention

[0006] Summary of the Invention Certain embodiments of the present invention enable the use of individual biomarkers not previously associated with AD. Applicants unexpectedly identified several biomarkers suitable for the methods, compositions, and kits of the present invention, such as CD163, CD91, and MerTK. These biomarkers have been found to be decreased or increased (downregulated or upregulated) in AD and MCI patients compared with CN patients, demonstrating their utility, alone or in combination with one or more other biomarkers, in the diagnosis and prognosis of AD. Specifically, CD163 and MerTK are downregulated, while CD91 is upregulated in AD. See Table 1. Accordingly, some compositions of the present invention include binding agents, such as antibodies, directed against biomarkers such as CD163, CD91, and MerTK, which are coupled to a detectable moiety, such as a fluorophore. Furthermore, such compositions may include the use of specific binding agents, such as mouse anti-human IgG monoclonal antibodies, directed against these and other biomarkers. The compositions optionally include an anticoagulant and, optionally, human whole blood. Kits containing, for example, antibody-fluorophore conjugates to biomarkers are also described in embodiments of the present invention.

[0007] Further embodiments employ these antibody-fluorophore conjugates in useful methods. Thus, for example, Applicants have invented the use of binding agents for specific biomarkers in methods for diagnosing AD in human patients. Certain embodiments relate to methods for diagnosing AD in human patients, one such method comprising: Obtaining a blood sample from a human patient; storing the blood sample on ice, if necessary; If necessary, testing the blood sample within three hours of obtaining the blood sample; reacting the blood sample with an antibody against the biomarker conjugated with a fluorophore to obtain bound biomarker; measuring the concentration of the bound biomarker in the blood sample by fluorescence of the fluorophore; comparing the concentrations to the range of concentrations of the biomarker in healthy humans; observing changes in the concentration of biomarkers in the blood sample; and thereby diagnosing AD in a human patient.

[0008] Other embodiments relate to methods for determining the efficacy of a candidate AD treatment in a human patient, one such method comprising: (A) obtaining a first blood sample from a human patient; Optionally, storing the first blood sample on ice; optionally, testing the first blood sample within three hours of obtaining the first blood sample; reacting the first blood sample with an antibody against a biomarker conjugated to a fluorophore to obtain a first bound biomarker; measuring a first concentration of a first bound biomarker in the first blood sample by fluorescence of the fluorophore; (B) administering a candidate AD treatment to the human patient; and (C) obtaining a second blood sample from the human patient; Optionally, storing the second blood sample on ice; optionally, testing the second blood sample within three hours of obtaining the second blood sample; reacting the second blood sample with an antibody against the biomarker conjugated to a fluorophore to obtain a second bound biomarker; measuring a second concentration of the second bound biomarker in the second blood sample by fluorescence of the fluorophore; (D) observing a change from a first concentration of the first binding biomarker to a second concentration of the second binding biomarker; (E) thereby determining the efficacy of the candidate AD treatment in a human patient.

[0009] Further embodiments relate to panels of biomarkers for diagnosing AD and assessing the effectiveness of AD drugs. Accordingly, some additional embodiments include compositions and kits, such as compositions or kits comprising a first binding agent for a first biomarker conjugated to a first detectable moiety and a second binding agent for a second biomarker conjugated to a second detectable moiety. Other embodiments include a third binding agent for a third biomarker conjugated to a third detectable moiety. Still other embodiments contain more binding agents, each conjugated to a detectable moiety. Ideally, the emission spectra of several fluorophores do not significantly overlap or are otherwise distinguishable for quantitative assessment of the presence of several biomarkers. A panel of antibody-fluorophore conjugates may appear in a composition or kit. A composition represents one or more components in a single mixture, and a kit includes one or more compositions.

[0010] Suitable biomarkers for diagnosing AD and assessing drug efficacy for AD include, for example, one panel of CD11c, CD59, and CD163. Another example includes antibody-fluorophore conjugates against CD11b, CD11c, and CD18. Additional panels include, for example, antibody-fluorophore conjugates against CD91, CD59, and CD163. A further panel includes antibody-fluorophore conjugates against one or more scavenger receptors and their regulators. Another panel includes antibody-fluorophore conjugates against one or more scavenger receptors and their regulators, and one or more antibody-fluorophore conjugates against one or more leukocyte CD markers. Scavenger receptors and their regulators include, but are not limited to, CD163, CD91, P2X7, MerTK, CD59, CD11c, CD11b, and CD18. Additional scavenger receptors have been identified in the literature, for example, M. Prabhu Das et al., "Standardizing Scavenger Receptor Nomenclature," J. Immunol., 192(5), (2014) 1997-2006. Leukocyte CD markers include, but are not limited to, CD14, CD15, CD16, CD19, and CD3. Another panel includes antibody-fluorophore conjugates against one or more of CD33, CD35, CD36, CD59, CD91, P2X7, and RAGE, as well as one or more of CD11b, CD11c, CD18, CD163, and MerTK. Yet another panel includes antibody-fluorophore conjugates against one or more of CD11c, CD11b, CD18, CD59, and one or more of CD14, CD15, CD16, CD19, and CD3. Further panels include antibody-fluorophore conjugates against one or more of CD91, CD163, P2X7 and MerTK, and one or more of CD14, CD15, CD16, CD19 and CD3.Still other panels include antibody-fluorophore conjugates against three groups, for example, one or more of CD91, CD163, P2X7, and MerTK; one or more of CD11c, CD11b, CD18, CD59; and one or more of CD14, CD15, CD16, CD19, and CD3.

[0011] Yet another example of the invention relates to a method for determining the relative expression of biomarkers in a human patient, one such method comprising: Obtaining a sample of whole blood from a human patient; contacting the sample with a monoclonal antibody against the biomarker conjugated to a fluorophore to form a bound biomarker; measuring the fluorescence of the fluorophore; thereby determining the relative expression of the biomarkers.

[0012] Yet additional embodiments relate to methods for diagnosing AD in a human patient, one such method comprising: Obtaining a blood sample from a human patient; storing the blood sample on ice, if necessary; If necessary, testing the blood sample within three hours of obtaining the blood sample; The blood sample is reacting with a monoclonal antibody against the biomarker conjugated with a fluorophore to form a bound biomarker; measuring the concentration of the bound biomarker in the blood sample by fluorescence of the fluorophore; observing the change in concentration of each of the binding biomarkers over a range of concentrations of the biomarkers in healthy humans; and thereby diagnosing AD in a human patient.

[0013] Further embodiments relate to methods for determining the efficacy of a candidate AD treatment in a human patient, one such method comprising: (A) obtaining a first blood sample from a human patient; Optionally, storing the first blood sample on ice; optionally, testing the first blood sample within three hours of obtaining the first blood sample; reacting the first blood sample with a first aliquot of a monoclonal antibody against a biomarker conjugated to a fluorophore to obtain a first bound biomarker; measuring a first concentration of a first bound biomarker; (B) administering a candidate AD treatment to the human patient; and (C) obtaining a second blood sample from the human patient; Optionally, storing the second blood sample on ice; optionally, testing the second blood sample within three hours of obtaining the second blood sample; reacting the second blood sample with a second aliquot of a monoclonal antibody against the biomarker conjugated to a fluorophore to obtain a second bound biomarker; measuring a second concentration of the second bound biomarker by fluorescence of the fluorophore; (D) observing a change from a first concentration of the first binding biomarker to a second concentration of the second binding biomarker in the human patient; (E) thereby determining the efficacy of the candidate AD treatment in a human patient.

[0014] Another example of the invention relates to a method for diagnosing AD in a human patient, another such method comprising: Obtaining a blood sample from a human patient; storing the blood sample on ice, if necessary; If necessary, testing the blood sample within three hours of obtaining the blood sample; The blood sample is reacting with a monoclonal antibody against a scavenger receptor conjugated with a fluorophore to obtain a bound scavenger receptor; measuring the concentration of bound scavenger receptor in the blood sample by fluorescence of the fluorophore; Observing a decrease in the concentration of bound scavenger receptors compared to the concentration range of scavenger receptors in healthy humans; and thereby diagnosing AD in a human patient.

[0015] Yet further embodiments relate to methods for determining the efficacy of a candidate AD treatment in a human patient, one such method comprising: (A) obtaining a first blood sample from a human patient; Optionally, storing the first blood sample on ice; optionally, testing the first blood sample within three hours of obtaining the first blood sample; A first blood sample is reacting with a first aliquot of a plurality of antibody-fluorophore conjugates, each of the plurality of antibody-fluorophore conjugates comprising a monoclonal antibody against a scavenger receptor conjugated to a fluorophore; thereby forming a first bound scavenger receptor; measuring a first concentration of a first bound scavenger receptor in the first blood sample by fluorescence of the fluorophore; (B) administering a candidate AD treatment to the human patient; and (C) obtaining a second blood sample from the human patient; Optionally, storing the second blood sample on ice; optionally, testing the second blood sample within three hours of obtaining the second blood sample; A second blood sample is reacting with a second aliquot of the plurality of antibody-fluorophore conjugates; thereby forming a second bound scavenger receptor; measuring a second concentration of a second bound scavenger receptor in a second blood sample by fluorescence of the fluorophore; (D) observing an increase in the first concentration of the first bound scavenger receptor to a second concentration of the second bound scavenger receptor in the human patient; (E) thereby determining the efficacy of the candidate AD treatment in a human patient.

[0016] Further embodiments of the present invention relate to compositions, kits, and methods optimized for assessing a patient's risk of AD, the efficacy of AD medications, and other uses. In some cases, these embodiments include binding agents for specific biomarkers conjugated to a detectable moiety. For example, some of these embodiments include: a monoclonal antibody against CD11c conjugated to a first fluorophore; a monoclonal antibody against CD14 conjugated to a second fluorophore; a monoclonal antibody against CD59 conjugated to a third fluorophore; and and a monoclonal antibody against CD163 conjugated to a fourth fluorophore.

[0017] In another example, other embodiments of these embodiments include: a monoclonal antibody against CD91 conjugated to a first fluorophore; a monoclonal antibody against CD59 conjugated to a second fluorophore; a monoclonal antibody against CD163 conjugated to a third fluorophore.

[0018] A further example of the present invention is a monoclonal antibody against CD11c conjugated to a first fluorophore; a monoclonal antibody against CD14 conjugated to a second fluorophore; a monoclonal antibody against CD59 conjugated to a third fluorophore; and and a monoclonal antibody against CD163 conjugated to a fourth fluorophore.

[0019] Other examples include: a first binding agent to CD91 conjugated to a first detectable moiety; a second binding agent for CD59 conjugated to a second detectable moiety; a third binding agent to CD163 conjugated to a third detectable moiety; The present invention relates to a kit comprising:

[0020] Any suitable antibody may be used in various embodiments of the present invention, such as, for example, a mouse anti-human IgG monoclonal antibody.

[0021] Another aspect is a monoclonal antibody against CD91 conjugated to a first fluorophore; a monoclonal antibody against CD14 conjugated to a second fluorophore; a monoclonal antibody against CD59 conjugated to a third fluorophore; and and a monoclonal antibody against CD163 conjugated to a fourth fluorophore.

[0022] Additional aspects include: a monoclonal antibody against CD91 conjugated to a first fluorophore; a monoclonal antibody against CD59 conjugated to a second fluorophore; a monoclonal antibody against CD163 conjugated to a third fluorophore.

[0023] A further aspect is a monoclonal antibody against CD91 conjugated to a first fluorophore; a monoclonal antibody against CD14 conjugated to a second fluorophore; a monoclonal antibody against CD59 conjugated to a third fluorophore; and and a monoclonal antibody against CD163 conjugated to a fourth fluorophore.

[0024] In certain additional aspects, the kit comprises: a monoclonal antibody against CD91 conjugated to a first fluorophore; a monoclonal antibody against CD59 conjugated to a second fluorophore; and a monoclonal antibody against CD163 conjugated to a third fluorophore.

[0025] Still further aspects relate to the use of compositions and kits comprising binder-detectable moiety conjugates, such as antibody-fluorophore conjugates to CD11c, CD14, CD59, and CD163, for (1) determining the relative expression of these biomarkers in human patients, (2) diagnosing AD in human patients, and (3) determining the efficacy of candidate AD treatments in human patients, and the methods are described herein.

[0026] Although the present disclosure provides certain specific embodiments, the present invention is not limited to those embodiments. Those skilled in the art will understand from the description herein that modifications can be made to the described embodiments and that the present specification is therefore broader in scope than the described embodiments. Therefore, all examples are non-limiting. [Brief explanation of the drawings]

[0027] [Figure 1]Without binding the invention to any theory, FIG. 1 illustrates a mechanism that may explain the utility of some embodiments of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0028] Detailed Description Where necessary, detailed embodiments of the present invention are disclosed herein; however, it should be understood that the disclosed embodiments are merely exemplary of the invention, which may be embodied in various forms. The drawings are not necessarily to scale, and some features may be exaggerated to show details of particular components. Therefore, specific structural and functional details disclosed herein should not be construed as limiting, but merely as a basis for the claims and as a representative basis for teaching those skilled in the art how to use the present invention in various ways.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the event that there are a plurality of definitions for terms herein, those in the present disclosure prevail unless stated otherwise.

[0030] Whenever phrases such as "for example," "such as," "including," and the like are used herein, unless otherwise specified, they are understood to be followed by the phrase "without limitation." Similarly, "one example," "exemplary," and the like are understood to be non-limiting.

[0031] The term "substantially" allows for deviations from the descriptor that do not adversely affect the intended purpose. A descriptive term is understood to be modified by the term "substantially" even if the word "substantially" is not explicitly recited.

[0032] The term "about," when used in connection with a numerical value, refers to the actual given value and approximations to such a given value that would be reasonably estimated by one of ordinary skill in the art, including approximations based on experimental and / or measurement conditions to such a given value.

[0033] Terms such as "comprising," "including," "having," and "involving" (and similarly, "comprises," "includes," "has," and "involves") are used interchangeably and have the same meaning. Specifically, each term is defined consistent with the common U.S. patent law definition of "comprising" and, therefore, is interpreted as an open term meaning "at least" and not excluding additional features, limitations, embodiments, etc. Thus, for example, "an apparatus having components a, b, and c" means that the apparatus includes at least components a, b, and c. Similarly, the phrase "a method including steps a, b, and c" means that the method includes at least steps a, b, and c.

[0034] Biomarkers are specific chemical moieties expressed in the human body. Antibodies are immunological proteins adapted to bind to specific biomarkers. Antibodies can exist in monomeric or polymeric forms. "Fluorophore" refers to a chemical species that absorbs light at an excitation wavelength and emits fluorescence at an emission wavelength for the purpose of detection and quantification. As known in the art, fluorophores exhibit characteristic absorption and emission spectra. An antibody-fluorophore conjugate comprises an antibody and at least one fluorophore chemically linked to each other so that the antibody binds to a biomarker and can be detected or quantified by the fluorescence intensity of the linked fluorophore.

[0035] The kit comprises two or more compositions as described herein. Optionally, the kit includes other useful items such as sample preparation reagents, collection tubes, control samples, instructions for use, or a combination of such items.

[0036] Specific abbreviations and their definitions used herein include the following:

[0037] Aβ amyloid β peptide

[0038] AD Alzheimer's disease

[0039] AIBL Australian Imaging, Biomarker and Lifestyle Research

[0040] APC Allophycocyanin

[0041] APOE human apolipoprotein E (gene symbol is italicized, protein symbol is not italicized).

[0042] APP amyloid precursor protein

[0043] ATN Amyloid, tau, neurodegeneration

[0044] Area under the AUC curve

[0045] CDR Clinical Dementia Scale

[0046] CN Normal cognitive function

[0047] CSF cerebrospinal fluid

[0048] FACS Fluorescence-activated cell sorting

[0049] FCIP flow cytometry immunophenotyping

[0050] FITC Fluorescein isothiocyanate

[0051] GWAS Genome-Wide Association Study

[0052] IDI Integrated Discrimination Improvement

[0053] mAb monoclonal antibody

[0054] MAC membrane attack complex

[0055] MCI Mild Cognitive Impairment

[0056] MFI Mean Fluorescence Intensity

[0057] MRI Magnetic Resonance Imaging

[0058] MMSE Mini-Mental State Examination

[0059] PACC Preclinical Alzheimer's Disease Cognitive Complex

[0060] PE R-Phycoerythrin

[0061] PerCP peridinin-chlorophyll-protein complex

[0062] PET Positron Emission Tomography

[0063] RAGE Receptor for advanced glycation end products

[0064] ROC Receiver Operating Characteristic

[0065] Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprise", "comprising", and the like are to be construed in an inclusive sense rather than an exclusive or exhaustive sense, i.e., meaning "including, but not limited to".

[0066] Any discussion of prior art throughout this specification should in no way be taken as an admission that such prior art is widely known or forms part of the common general knowledge in the art.

[0067] It is an object of the present invention to overcome or ameliorate at least one disadvantage of the prior art, or to provide a useful alternative.

[0068] I. Individual Biomarkers As described above, the present applicants unexpectedly identified several biomarkers suitable for such methods and compositions, such as CD163, CD91, and MerTK. These biomarkers were found to be altered in AD and MCI patients compared to CN patients. Specifically, CD163 and MerTK are downregulated in AD, while CD91 is upregulated with a strong correlation to AD. See Table 1 below. They can be used individually or in combination with each other and / or other biomarkers to diagnose AD and other uses. Table 1 includes results obtained following the protocol described in the Examples. [Table 1-1] [Table 1-2]

[0069] Embodiments of the present invention use binding agents for biomarkers from, for example, single-chain variable fragments, antibody mimetics, antibody fragments, antibodies, monoclonal antibodies, and combinations thereof. The binding agent should exhibit sufficient selectivity and affinity for the desired biomarker. Any suitable antibody can be used in embodiments of the present invention. For example, the antibody can be monoclonal and can be derived from mouse, horse, rabbit, or pig, for example, via hybridoma. In some cases, the antibody is a mouse anti-human IgG monoclonal antibody against the biomarker. Care should be taken to avoid the presence of antibody inhibitors, such as heparin.

[0070] Further embodiments use detectable moieties, such as radioisotopes, stable isotopes, fluorophores, and combinations thereof. Detectable moieties should be selected to avoid overlapping signaling, which would confuse detection techniques. For example, the same radioisotope should not be conjugated to different binding agents in the same kit. Similarly, fluorophores should be selected to avoid overlapping emission spectra. Any suitable fluorophore can be used in embodiments of the present invention. Suitable fluorophores include, but are not limited to, those sold under the Alexa Fluor® trademark, such as Alexa Fluor® 488 and Alexa Fluor® 647; allophycocyanin; fluorescein compounds such as fluorescein isothiocyanate, fluorescein amidite, and 6-carboxyfluorescein; R-phycoerythrin; erythrosine; peridinin-chlorophyll-protein conjugates, such as those known as PerCP; rhodamine-based dyes, such as carboxytetramethylrhodamine, tetramethylrhodamine, and sulforhodamine; coumarins; lanthanide fluorophores; quantum dots; and other fluorophores known in the art. Care should be taken to avoid the use of two fluorophores with closely overlapping emission spectra. For example, Alexa Fluor® 647 should not be used with allophycocyanin. Alexa Fluor® 488 should not be used with fluorescein isothiocyanate. Also, in some cases, blocking / bleaching reagents, as known in the art, may be used to eliminate non-specific binding.

[0071] Further examples of fluorophores include, but are not limited to, the BD Horizon™ and BD Horizon Brilliant™ Ultraviolet families of dyes sold by Becton, Dickinson and Company (Franklin Lakes, New Jersey), e.g., the fluorophores known as BUV395; BUV563; BUV615; BUV661; BUV737; BUV805; BV421; BV480; BV510; BV605; BV650; BV711; BV750; and BV786. Additional fluorophores include BF Horizon™ tandem conjugate PE-CF594; tandem conjugate PerCP with a cyanine dye known as PerCP-Cy™ 5.5, offered by Becton, Dickinson and Company (Franklin Lakes, NJ); R-phycoerythrin (PE); and BD Horizon™ Red 718; or combinations of two or more thereof. Additional examples include NovaFluor™ brand fluorescent labels offered by Phitonex, Inc., now part of Thermo Fisher Scientific Inc. (Waltham, MA), such as NovaFluor™ Blue 510, NovaFluor™ Blue 530, NovaFluor™ Blue 555, NovaFluor™ Blue 610-30 S, and NovaFluor™ Blue 610-70. S, NovaFluor™ Blue 660-120 S, NovaFluor™ Blue 660-40 S, NovaFluor™ Yellow 570, NovaFluor™ Yellow 610, NovaFluor™ Yellow 660, NovaFluor™ Yellow 690, NovaFluor™ Yellow 700, NovaFluor™ Yellow 720, NovaFluor™ Red 660, NovaFluor™ Red 685, NovaFluor™ Red 700, or combinations thereof.

[0072] Additional fluorophores include GFP, YFP, RFP, eGFP, mCherry, tdtomato, FITC, Alexa Fluor 350, and Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 680, Alexa Fluor 750, Pacific Blue, Coumarin, BODIPY FL, Pacific Green, Oregon Green, Cy3, Cy5, Pacific Orange, TRITC, Texas Red, and combinations thereof.

[0073] Specific examples of antibody-fluorophore conjugates include mouse anti-human CD163 antigen conjugated to a fluorescent dye sold under the trademark Alexa Fluor® 647 (Molecular Probes, Inc., Eugene, Oregon); mouse anti-human CD91 antigen conjugated to fluorescein isothiocyanate; and mouse anti-human MerTK antigen conjugated to a fluorescent dye sold under the trademark BD Horizon Brilliant™ Blue 700 (Beckton, Dickinson and Company, Franklin Lakes, New Jersey).

[0074] Conjugation of the binding agent to a detectable moiety, such as an antibody to a fluorophore, can be accomplished by any suitable method. For example, known chemistries are well developed and commercially available. Fluorophore-conjugated antibodies are available, for example, from Becton, Dickinson and Company. 18 Radioactive isotopes such as F and 2 Stable isotopes such as H can be incorporated into the binding agent or into molecules that stably bind to the binding agent. For example, 18 Fluorodeoxyglucose or deuterated molecules containing F can be used. Quantum dots conjugated to binding agents such as antibodies can also be mentioned.

[0075] Compositions useful in the present invention are not limited. A specific example provides a composition comprising a mouse anti-human IgG monoclonal antibody against CD163 conjugated with a fluorophore and at least one anticoagulant. Any suitable anticoagulant can be used in various embodiments of the present invention, such as ethylenediaminetetraacetic acid, at least one salt thereof, citric acid, at least one salt thereof, or any combination thereof. Any suitable salt may be used, including alkali metal salts such as monosodium, disodium, trisodium, or tetrasodium salts. Another example relates to a composition comprising a mouse anti-human IgG monoclonal antibody against CD163 conjugated with a fluorophore, ethylenediaminetetraacetic acid, at least one salt thereof, citric acid, at least one salt thereof, or any combination thereof, and whole blood from a human patient. Any of these compositions can contain additional useful components, such as bovine serum albumin and sodium azide.

[0076] Various methods are described in embodiments of the present invention. Some methods relate to diagnosing AD in a human patient. Some of these methods involve obtaining a blood sample from the human patient, storing the blood sample on ice, which may include freezing, if desired; If necessary, testing the blood sample within three hours of obtaining the blood sample; reacting the blood sample with a monoclonal antibody against a biomarker, such as CD163, CD91, or MerTK, conjugated with a fluorophore to obtain a bound biomarker; measuring the concentration of the bound biomarker in the blood sample by fluorescence of the fluorophore; comparing the concentration to the range of concentrations of the biomarker in healthy humans; observing a change in the concentration of the biomarker in the blood sample; and thereby diagnosing AD in a human patient.

[0077] In some cases, blood samples are obtained from human patients and stored on ice or refrigerated as needed. As used herein, "storing on ice" includes any type of refrigeration, from a food-free refrigerator to an ice bath to dry ice, as long as the storage method does not harm the sample. Samples can be tested within any appropriate time, such as within three hours of being drawn. The sample is tested by reacting the blood sample with an antibody against the biomarker conjugated with a fluorophore to obtain bound biomarker, and measuring the concentration of bound biomarker in the blood sample by fluorescence of the fluorophore. Fluorescence can be measured by any appropriate technique, such as flow cytometry analysis.

[0078] Certain examples allow for a comparison of the concentration of a biomarker in a patient of interest with the range of biomarker concentrations in healthy individuals. This comparison can be accomplished in any suitable manner. Data on healthy individuals can be found in the literature or developed by testing a large number of otherwise healthy individuals who are not determined to have AD or MCI. These healthy individuals can exhibit any suitable indicator of health, such as having <25 CL as measured by amyloid PET imaging. Concentrations can be compared at any suitable level. In some cases, concentrations are compared in whole blood. In other cases, concentrations are compared in white blood cells. In still other cases, concentrations are compared in white blood cell subsets, such as white blood cells selected from monocytes, CD14+ monocytes, CD14+CD16- monocytes, neutrophils, CD14- neutrophils, CD14-CD16+ neutrophils, lymphocytes, B lymphocytes, T lymphocytes, natural killer cells, and combinations thereof. White blood cell subsets can be identified according to any suitable method. Cell sorting can be performed or cells can be gated according to known methods, for example in flow cytometry.

[0079] The concentration of detectable moiety can be measured by any suitable method.Scintillation counting, mass spectrometry and fluorescence can be mentioned.Measuring the concentration of bound biomarker includes, for example, measuring the mean fluorescence intensity of bound biomarker conjugated with fluorophore.In the case of CD163, observing a decrease in the concentration of CD163 in blood sample can mean measuring a decrease in mean fluorescence intensity of at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, or at least about 19%, compared to the mean fluorescence intensity of bound CD163 for a pool of human participants with <25CL measured by amyloid PET imaging.

[0080] Observing an increase in the concentration of CD91 in a blood sample can mean measuring an increase in mean fluorescence intensity of at least about 44%, at least about 50%, at least about 55%, or at least about 59% compared to the mean fluorescence intensity of bound CD91 for a pool of human patients with <25 CL as measured by amyloid PET imaging.

[0081] Observing a decrease in the concentration of MerTK in the blood sample includes measuring a decrease in mean fluorescence intensity of at least about 12% compared to the mean fluorescence intensity of bound MerTK for a pool of human patients with <25 CL as measured by amyloid PET imaging.

[0082] Other methods involve testing candidate AD therapeutics to determine whether they are effective as drugs to treat, prevent, or delay the onset of AD. (A) obtaining a first blood sample from a human patient; Optionally, storing the first blood sample on ice; optionally testing the first blood sample within three hours of obtaining the first blood sample; The test comprises: a first blood sample; testing the first blood sample within three hours of obtaining the first blood sample by reacting a first aliquot of a monoclonal antibody against a biomarker, such as CD163, CD91, or MerTK, conjugated with a fluorophore, to obtain a first bound biomarker; measuring a first concentration of a first bound biomarker in the first blood sample by fluorescence of the fluorophore; (B) administering a candidate AD treatment to the human patient; and (C) obtaining a second blood sample from the human patient; Optionally, storing the second blood sample on ice; optionally, testing the second blood sample within three hours of obtaining the second blood sample; reacting the second blood sample with a second aliquot of a monoclonal antibody against the biomarker conjugated to a fluorophore to obtain a second bound biomarker; measuring a second concentration of the second bound biomarker in the second blood sample by fluorescence of the fluorophore; (D) observing a change from a first concentration of the first binding biomarker to a second concentration of the second binding biomarker; (E) thereby determining the effectiveness of the AD treatment candidate in human patients. In the case of CD163 and MerTK, these biomarkers are downregulated in AD, so an effective AD treatment candidate would upregulate or increase the concentration of these biomarkers in human patients. In the case of CD91, this biomarker is upregulated in AD, so an effective AD treatment candidate would downregulate or decrease the concentration of this biomarker in human patients.

[0083] Any suitable AD therapeutic candidate can be used. In some cases, the AD therapeutic candidate is a phagocytosis promoter, a scavenger receptor agonist, or both. Thus, the candidate drug can act to promote phagocytosis. It can act as an agonist on a scavenger receptor, or both activities may be described. Of course, other mechanisms of action can also be tested by this protocol. Suitable AD therapeutic candidates can have any regulatory approval status, from investigational new drugs to approved therapeutics requiring post-marketing surveillance, for example, since the method investigates the efficacy of the candidate for a particular patient. The AD therapeutic candidate can be administered by any suitable route and in any suitable amount. In some cases, the amount is a therapeutically effective amount, or a suspected therapeutically effective amount. For example, the AD therapeutic candidate can be administered to a patient in an amount less than 1 g per kg of patient body weight per day. In another example, the AD therapeutic candidate can be administered to a patient in an amount greater than about 1 ng per kg of patient body weight per day. In other cases, the candidate AD treatment can be administered to a patient in an amount of about 0.1 μg / kg or less, about 1 μg / kg or less, about 10 μg / kg or less, or about 100 μg / kg or less per day. Any suitable period can be used, such as 1 day, 5 days, 10 days, 30 days, 60 days, 90 days, 180 days, 365 days, or 365 days or more. For example, oral administration, injection via intravenous or intramuscular routes, transdermal patch, implant, etc. can be used.

[0084] II. Biomarker combinations The present applicants have found that combinations of biomarkers hold predictive value in the diagnosis and prognosis of AD and in drug efficacy assessment. These biomarkers can represent indicators of a single mechanism, such as, for example, phagocytic regulatory molecules. Alternatively, the biomarkers can indicate more than two mechanisms or aspects of a patient's health, such as, for example, one or more phagocytic regulatory molecules combined with one or more leukocyte CD markers, including, for example, CD14 (monocytes), CD16 (monocytes, NK cells, neutrophils), CD15 (neutrophils), CD19 (B lymphocytes), and CD3 (T lymphocytes).

[0085] Some embodiments of the present invention probe the expression of one or more scavenger receptors to diagnose or prognose AD. Table 1 above lists the expected activity of certain biomarkers, including scavenger receptors. Specific panels of scavenger receptors and other biomarkers include, for example: CD11c, CD59 and CD163; CD11b, CD11c and CD18; CD91, CD59 and CD163; one or more of CD33, CD35, CD36, CD59, CD91, P2X7 and RAGE and one or more of CD11b, CD11c, CD18, CD163 and MerTK; one or more of CD163, CD91, P2X7, MerTK CD59, CD11c, CD11b and CD18 and one or more of CD14, CD15, CD16, CD19 and CD3; one or more of CD11c, CD11b, CD18, and CD59 and one or more of CD14, CD15, CD16, CD19, and CD3; one or more of CD11c, CD11b, CD18, and CD59 and one or more of CD14, CD15, CD16, CD19, and CD3; one or more of CD91, CD163, P2X7 and MerTK and one or more of CD14, CD15, CD16, CD19 and CD3; One or more of CD91, CD163, P2X7 and MerTK, one or more of CD11c, CD11b, CD18 and CD59, and one or more of CD14, CD15, CD16, CD19 and CD3.

[0086] These panels are represented by the presence of binding agents, such as monoclonal antibodies, for specific biomarkers conjugated with appropriate detectable moieties, such as fluorophores. It should be understood that in any combination of antibody-fluorophore conjugates, the fluorophores should be selected so that their emission spectra do not have significant or irresolvable overlap, or so that the relative intensities of overlapping spectra cannot be easily determined. In some cases, attention should also be paid to excitation parameters so that qualitative information can be obtained. All fluorophores should be sufficiently illuminated under the excitation intensity used, so that quantitative data can be described. Some embodiments of the present invention rely on the relative concentrations of biomarkers, or the concentration of one or more biomarkers in a patient sample relative to that of a healthy individual.

[0087] Particular examples relate to compositions and kits. For example, one example relates to a composition or kit comprising a monoclonal antibody against CD11c conjugated with a first fluorophore, a monoclonal antibody against CD59 conjugated with a second fluorophore, and a monoclonal antibody against CD163 conjugated with a third fluorophore. Another example relates to a composition or kit comprising a mouse anti-human IgG monoclonal antibody against CD11c conjugated with a first fluorophore, a mouse anti-human IgG monoclonal antibody against CD59 conjugated with a second fluorophore, and a mouse anti-human IgG monoclonal antibody against CD163 conjugated with a third fluorophore. A further example relates to a composition or kit comprising a mouse anti-human IgG monoclonal antibody against CD91 conjugated to a first fluorophore, a mouse anti-human IgG monoclonal antibody against CD59 conjugated to a second fluorophore, and a mouse anti-human IgG monoclonal antibody against CD163 conjugated to a third fluorophore. Suitable fluorophores include those described above. In some cases, the first fluorophore is allophycocyanin or fluorescein isothiocyanate, the second fluorophore is R-phycoerythrin, and the third fluorophore is a fluorescent dye sold under the trademark Alexa Fluor® 647 (Molecular Probes, Inc., Eugene, Oregon).

[0088] Optionally, the composition or kit includes at least one anticoagulant. As mentioned above, any suitable anticoagulant can be used. Any suitable additional component can also be included, such as bovine serum albumin and sodium azide.

[0089] Compositions according to the invention may comprise whole blood from a human patient. Such compositions are described where a sample from the patient is being tested according to the invention.

[0090] Various methods are described in some embodiments of the present invention. Some methods involve measuring the relative expression of specific biomarkers in patients. It has been found that certain biomarkers are increased in MCI and AD patients, while other biomarkers are decreased, compared with CN patients. For example, one such method involves measuring the relative expression of CD11c, CD59, and CD163 in human patients, which method includes: Obtaining a sample of whole blood from a human patient; The sample a monoclonal antibody against CD11c conjugated to a first fluorophore; a monoclonal antibody against CD59 conjugated to a second fluorophore; contacting the cells with a monoclonal antibody against CD163 conjugated to a third fluorophore to form bound CD11c, bound CD59, and bound CD163; and determining the relative expression of CD11c, CD59, and CD163 by measuring the fluorescence of the first, second, and third fluorophores. As described above, any suitable antibody and fluorophore can be used. Referring to Table 1, if CD11c and CD163 appear to be downregulated and CD59 appears to be upregulated, the patient can be evaluated for MCI or AD, and further treatment or preventative measures can be taken.

[0091] Another such method involves measuring the relative expression of CD91, CD59 and CD163 in a human patient, the method comprising: Obtaining a sample of whole blood from a human patient; The sample a monoclonal antibody against CD91 conjugated to a first fluorophore; a monoclonal antibody against CD59 conjugated to a second fluorophore; contacting the cells with a monoclonal antibody against CD163 conjugated to a third fluorophore to form bound CD91, bound CD59, and bound CD163; and determining the relative expression of CD91, CD59, and CD163 by measuring the fluorescence of the first, second, and third fluorophores. As described above, any suitable antibody and fluorophore can be used. Referring to Table 1, if CD91 and CD59 appear to be upregulated and CD163 appears to be downregulated, the patient can be evaluated for MCI or AD, and further treatment or preventative measures can be taken.

[0092] Methods for diagnosing AD in a human patient, which may include assessing whether the patient meets a CN diagnosis, an MCI diagnosis, or an AD diagnosis, may also use a combination of biomarkers. reacting a blood sample from the patient with a plurality of antibody-fluorophore conjugates, each of the plurality of antibody-fluorophore conjugates comprising a mouse anti-human IgG monoclonal antibody against a scavenger receptor conjugated to a fluorophore, thereby forming bound scavenger receptors; measuring the concentration of bound scavenger receptor in the blood sample by fluorescence of the fluorophore; and observing a decrease in the concentration of at least one bound scavenger receptor relative to a concentration range of the corresponding scavenger receptor in a healthy human, thereby diagnosing AD in the human patient. In some cases, the scavenger receptor is selected from CD163, MerTK, CD11b, CD11c, CD18, and combinations thereof.

[0093] Measuring the concentration of the bound biomarker can proceed in any suitable manner. Whole blood, white blood cells, or a white blood cell subset can be tested. For example, one biomarker can be tested in one white blood cell subset, and another biomarker can be tested in a different white blood cell subset. In some cases, for example, measuring the concentration of the bound biomarker includes measuring the mean fluorescence intensity of the bound biomarker. Similarly, observing a change (increase or decrease) in the concentration of the bound biomarker can include comparing the mean fluorescence intensity of the bound biomarker in a human patient with the mean fluorescence intensity of the bound biomarker for a pool of human participants with <25 CLs as measured by amyloid PET imaging. For example, observing a decrease in the concentration of bound CD11c can include measuring a decrease in mean fluorescence intensity of at least about 15%, at least about 19%, at least about 22%, at least about 25%, at least about 28%, at least about 30%, or at least about 34% compared to the mean fluorescence intensity of bound CD11c for a pool of human participants with <25 CLs as measured by amyloid PET imaging. In another example, observing an increase in the concentration of bound CD59 comprises measuring an increase in mean fluorescence intensity of at least about 27%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 78% compared to the mean fluorescence intensity of bound CD59 for a pool of human participants with <25 CL as measured by amyloid PET imaging.

[0094] In certain cases, measuring the concentration of a biomarker correlates with specific cells. For example, the concentration of a biomarker may show a significant difference from that of a healthy individual when measured in specific cells. Cell population subsets can be identified by any suitable method and correlated with other biomarkers. For example, leukocytes can be isolated from whole blood, and the concentration of CD163 can be measured only in leukocytes. Alternatively, leukocytes or their subsets can be gated according to known methods useful in flow cytometry. For example, natural killer (NK) cells, B lymphocytes, T lymphocytes, classical monocytes, intermediate monocytes, non-classical monocytes, and mature neutrophils can be gated and probed for the expression of one or more biomarkers, such as CD163, CD91, and MerTK. Such gating strategies typically include forward and side scatter of lymphocytes, monocytes, and neutrophils, along with surface expression of cell-specific CD markers, including, for example, monocytes (CD14+), neutrophils (CD15+ or CD16+), B lymphocytes (CD19+), T lymphocytes (CD3+), natural killer cells (CD16+), and combinations thereof. As used herein, classical monocytes can be described as CD14+CD16-, non-classical monocytes are CD14dimCD16+, and intermediate monocytes are CD14+CD16+.

[0095] Certain further embodiments relate to using a combination of biomarkers to determine the effectiveness of a candidate AD treatment in a human patient. One such method comprises: (A) obtaining a first blood sample from a human patient; reacting the first blood sample with a first aliquot of a plurality of antibody-fluorophore conjugates, each of the plurality of antibody-fluorophore conjugates comprising a mouse anti-human IgG monoclonal antibody against a scavenger receptor conjugated to a fluorophore, thereby forming a first bound scavenger receptor; and measuring a first concentration of the first bound scavenger receptor in the first blood sample by fluorescence of the fluorophore; (B) administering a candidate AD treatment to the human patient; and (C) obtaining a second blood sample from the human patient; reacting the second blood sample with a second aliquot of a plurality of antibody-fluorophore conjugates, thereby forming second bound scavenger receptors; measuring a second concentration of a second bound scavenger receptor in the second blood sample by fluorescence of the fluorophore; (D) observing an increase in the first concentration of the first bound scavenger receptor to a second concentration of the second bound scavenger receptor in the human patient; (E) thereby determining the efficacy of the candidate AD treatment in human patients. Any suitable candidate AD treatment can be tested, for example, a phagocytosis promoter, a scavenger receptor agonist, or a candidate that exhibits both mechanisms.

[0096] III. Two Panels The applicants have discovered two combinations of biomarkers that are useful in certain of the compositions, kits, and methods of the present invention. One combination involves the biomarkers CD163, CD59, CD11c, and CD14. The second combination involves the biomarkers CD163, CD59, CD91, and CD14. These combinations can be represented by a binding agent for the biomarkers, such as a monoclonal antibody, conjugated to a unique detectable moiety, such as a fluorophore. Thus, one embodiment includes: a monoclonal antibody against CD11c conjugated to a first fluorophore; a monoclonal antibody against CD14 conjugated to a second fluorophore; a monoclonal antibody against CD59 conjugated to a third fluorophore; and and a monoclonal antibody against CD163 conjugated to a fourth fluorophore. a monoclonal antibody against CD91 conjugated to a first fluorophore; a monoclonal antibody against CD14 conjugated to a second fluorophore; a monoclonal antibody against CD59 conjugated to a third fluorophore; and The present invention relates to compositions comprising a monoclonal antibody against CD163 conjugated with a fourth fluorophore. These monoclonal antibodies can include any suitable antibody, such as a mouse anti-human IgG monoclonal antibody. As described above, monoclonal antibodies derived from mice, horses, rabbits, or pigs, for example, via hybridomas, can be used. At least one anticoagulant can also be included in these compositions, such as ethylenediaminetetraacetic acid, at least one salt thereof, citric acid, at least one salt thereof, or any combination of the foregoing. Suitable additional components can be present, such as bovine serum albumin and sodium azide, or, in another example, phosphate-buffered saline, gelatin, and sodium azide.

[0097] Detectable moieties include radioisotopes, stable isotopes, fluorophores, and combinations thereof. Any suitable fluorophores can be conjugated with their binding agents, taking care to avoid the use of two fluorophores with closely overlapping emission spectra. In some cases, the first fluorophore is fluorescein isothiocyanate, allophycocyanin, or a combination thereof. In further cases, the second fluorophore is a peridinin-chlorophyll-protein complex. In yet other cases, the third fluorophore is R-phycoerythrin. In additional cases, the fourth fluorophore is a fluorescent dye sold under the trademark Alexa Fluor® 647 (Molecular Probes, Inc., Eugene, Oregon). In yet other cases, the composition does not include a fluorescent dye sold under the trademark Alexa Fluor® 488 (Molecular Probes, Inc., Eugene, Oregon). The composition can include, for example, whole blood from a human patient when the patient is being evaluated for AD.

[0098] Kits using these combinations are also described in the present invention. a monoclonal antibody against CD11c conjugated to a first fluorophore; a monoclonal antibody against CD14 conjugated to a second fluorophore; a monoclonal antibody against CD59 conjugated to a third fluorophore; and and a monoclonal antibody against CD163 conjugated to a fourth fluorophore. a monoclonal antibody against CD91 conjugated to a first fluorophore; a monoclonal antibody against CD14 conjugated to a second fluorophore; a monoclonal antibody against CD59 conjugated to a third fluorophore; and and a monoclonal antibody against CD163 conjugated to a fourth fluorophore.

[0099] These kits can include binding agents such as antibodies, detectable moieties such as fluorophores, anticoagulants, and the additional components described above. In some examples, the compositions and kits can include the use of specific antibodies, such as mouse anti-human IgG monoclonal antibodies, directed against these and other biomarkers. The compositions optionally include an anticoagulant and, optionally, human whole blood. Suitable anticoagulants include, but are not limited to, ethylenediaminetetraacetic acid, at least one salt thereof, citric acid, at least one salt thereof, or any combination of the foregoing. The compositions and kits can further include any suitable additional components, such as bovine serum albumin and sodium azide, and, as another example, phosphate-buffered saline, gelatin, and sodium azide.

[0100] Suitable fluorophores include those described above. In some cases, the first fluorophore is fluorescein isothiocyanate, allophycocyanin, or a combination thereof. In further cases, the second fluorophore is a peridinin-chlorophyll-protein complex. In additional cases, R-phycoerythrin is provided as the third fluorophore. In still other cases, the fourth fluorophore is a fluorescent dye sold under the trademark Alexa Fluor® 647 (Molecular Probes, Inc., Eugene, Oregon).

[0101] Methods of using these combinations of biomarkers are also described. The methods described herein can be used to determine relative expression and to diagnose AD in human patients and to determine the efficacy of candidate AD treatments.

[0102] Detailed Description of the Drawings Further embodiments of the present invention can be understood by reference to the accompanying drawings.

[0103] Figure 1 illustrates the proposed mechanism. Without wishing to be bound by theory, the proposed mechanism may explain the utility of certain embodiments of the present invention. Further explanation is provided in the discussion below. [Example]

[0104] Testing biomarkers as indicators of AD We used flow cytometry techniques to examine a set of common cell surface leukocyte biomarkers for association with cerebral amyloid-β (Aβ) accumulation as potential biomarkers of AD in a total of 418 human patients, including 241 cognitively normal (CN), 106 with mild cognitive impairment (MCI), and 71 with clinical AD dementia.

[0105] 2. Method:

[0106] 2.1. Subjects and ethical approval

[0107] Participants for Phases I, II, and III were recruited from the Australian Imaging, Biomarker, and Lifestyle Study (AIBL). This study integrates data from neuroimaging, biomarker, lifestyle, clinical, and neuropsychological analyses. Ninety-nine percent of participants recruited by the AIBL study were of genetic Northern European descent and fluent in English. Classification of AIBL participants was based on both clinical and neuroimaging / CSF evidence of Aβ accumulation. This study was approved by the Human Research Ethics Committee of the Research Governance Unit at St. Vincent's Hospital, Melbourne, Australia (reference number: HREC-A 028 / 06). All participants and patient caregivers completed written informed consent prior to participation. All clinical and demographic information of AIBL participants was masked until the collection of all biological measurements. For Phase IV, samples were randomly collected from subjects participating in the Anti-Amyloid Treatment in Asymptomatic Alzheimer's Disease (A4) study. In this phase, whole blood was obtained from 112 participants blindly and randomly selected from a cohort of subjects admitted at US centers as part of the anti-amyloid screening process in the Asymptomatic AD (A4) clinical trial (e.g., A4 Biobank Addendum, PI Rissman)

[52] .

[0108] 2.2. Aβ-PET imaging and centiloid (CL) scale

[0109] Participants underwent Aβ-PET imaging as previously described [17, 18]. 3D T1-weighted MRI was obtained for screening and co-registration with Aβ-PET images. Researchers involved in blood assessments were blinded to all Aβ-PET and clinical data until statistical analysis was reported. Aβ-PET status was based on centiroids (CL): negative <25 CL (including borderline >15 CL and <25 CL) and positive ≥25 CL. The threshold was selected according to its association with risk of disease progression.

[0110] 2.3. Episodic Memory and Preclinical Alzheimer's Cognitive Composite (PACC) Scores

[0111] The methods used to develop the episodic memory composite score and PACC score have been published elsewhere [ 19 , 20 ].

[0112] 2.4.Blood collection

[0113] For Phases I-III, peripheral whole blood was collected by venipuncture between 8:00 AM and 10:30 AM after an overnight fast. Whole blood was kept in EDTA anticoagulant Vacutainer® tubes (Becton Dickinson Biosciences) and kept on ice during transport. Whole blood processing was completed within 3 hours of collection. For Phase IV, whole blood was collected by venipuncture from participants who had fasted overnight. Collected blood was transported overnight in gel packs at ambient temperature and processed the next morning for analysis.

[0114] 2.5. Cell flow cytometry staining

[0115] The cell surface staining procedure was performed using BD standard protocols: 100 μL aliquots of fresh blood were added to fluorescence-activated cell sorting (FACS®) tubes containing premixed antibody cocktails. Mouse anti-human IgG monoclonal antibodies (mAbs) conjugated with fluorophores excited in the FITC, PE, PerCP, and APC channels were used to stain leukocytes (Table A.1 below). An autofluorescence tube containing only whole blood and an IgG isotype control (BD Australia) tube were prepared for each AIBL sample. The optimal antibody concentration was determined by titration testing. For intracellular staining, cells were fixed, permeabilized, and then mixed with the antibody cocktail according to BD standard protocols. Immunophenotyping was performed by flow cytometry using a FACSCalibre (BD Biosciences). All flow data was digitally stored.

[0116] 2.6. Definitions of Phase I, II, III, and IV

[0117] Phase I consisted of a three-color cytometry panel consisting of 16 surface markers and two intracellular markers. Phase II consisted of a four-color cytometry panel including 18 surface markers. Phases I and II covered several key leukocyte CD markers. Phase III consisted of a four-color cytometry panel with 19 surface markers, enriched to reflect emerging evidence that innate immune pathways may play an important role in the pathogenesis of AD. A total of 34 leukocyte markers were tested. The antibody-fluorophore conjugate panel, along with the catalog numbers of the products used, is listed in Table A.1.

[0118] [Table A.1.1]

[0119] [Table A.1.2]

[0120] [Table A.1.3]

[0121] [Table A.1.4]

[0122] Flow data were analyzed using FlowJo software (V10, FlowJo, LLC). Applicants used forward and side scatter to gate lymphocytes, monocytes, and neutrophils. Subpopulations were then gated based on the fluorescence intensity of specific markers. In Phase III, applicants used the fluorescence intensity of anti-CD14 and anti-CD16 mAbs to gate natural killer (NK) cells, B and T lymphocytes, classical monocytes, intermediate monocytes, non-classical monocytes, mature neutrophils, and immature neutrophils. Cell counts and frequencies of subpopulations were calculated. The mean fluorescence intensity (MFI) of different markers on different cell groups was also measured.

[0123] 2.7.Statistical analysis

[0124] Statistical analyses were performed using GraphPad Prism for Windows® (San Diego, CA, USA, version 8.4.2) and IBM SPSS® Statistics Software (version 25.0, Armonk, NY: IBM Corp.). mAb fluorescence intensity was compared between the CN, MCI, and AD groups using one-way ANOVA. Pearson product-moment correlation analysis was performed to examine the strength of the relationship between mAb fluorescence intensity and PET Aβ centimeters, episodic memory, and PACC scores. For predictive analysis, receiver operating characteristic (ROC) analysis was used to define sensitivity, specificity, and accuracy values. Thresholds were selected via the Youden index. To predict outcomes after logistic regression, models were calculated for individual biomarkers, individual biomarkers adjusted for covariates, and combined sets of biomarkers. Figures were created using GraphPad Prism for Windows® (San Diego, CA, USA, version 8.4.2), and statistical analyses were performed using R (version 4.0).

[0125] 2.8 Biomarker Evaluation

[0126] Here, we seek to discover whether adding new biomarkers can better predict disease risk. We evaluated biomarker performance in a logistic regression model created using the rms R package and evaluated using the rap R package. R version 4.0.2 was used. The value of adding biomarkers to a baseline model was assessed using the change in the area under the receiver operating characteristic curve (AUC), the integrated discrimination improvement (IDI) metric, and the Brier skill score. The change in AUC reflects the change in the probability ranking of the baseline model and the new model. IDIs are presented separately for those with and without the event of interest. They represent the net average risk increase for those with an event (IDI event) and the average risk decrease for those without an event (IDI non-event). Brier improvement is the relative improvement in Brier score from the baseline model. The Brier score is the mean squared error of the model, and Brier improvement gives the percentage improvement in accuracy due to the addition of a biomarker.

[0127] The model creation process, briefly, consisted of constructing an artificial baseline logistic regression model to predict a subject's brain Aβ-PET status. First, variables were selected using principal component regression, a variable reduction technique that reduces the number of variables by collapsing neuropsychological diagnoses along with demographics (i.e., age, sex, and years of education). Second, we added each biomarker to the baseline model one by one to evaluate whether the new biomarker improved the baseline model's diagnostic performance. These were the "new models." Finally, we combined the best-performing biomarkers into a panel of biomarkers to achieve optimal performance. Here, for Phase III Model 1, we defined disease status solely by centiroid >25CL. In Phase III Model 2, we sought to explore whether specific biomarkers have physiological value in differentiating early stages of disease (i.e., borderline). However, the borderline ranges of Aβ, >15CL and <25CL, were too narrow to include enough cases. Therefore, we removed the arbitrarily defined centimeters ≥ 25 CL and < 100 CL, as well as the early stage of AD due to individuals with centimeters > 100 CL. Model 3 further considered the patient's clinical status along with Aβ status to stratify the study cohort into healthy control (CN & < 25 CL), preclinical (CN & ≥ 25 CL), or prodromal (MCI and CL not considered) and AD (CL not considered) stages. We then arbitrarily defined the disease state in Model 3 (removing the AD stage) by combining the preclinical and prodromal stages. Data skewness was tested using the Kolmogorov-Smirnova and Shapiro-Wilk methods.

[0128] 3.Results

[0129] 3.1. Demographics

[0130] This cross-sectional study included four phases: Phases I and II served as discovery datasets, and Phases III and IV served as validation datasets. Applicants recruited 76,142 and 200 participants from the AIBL trial in Phases I, II, and III, respectively, and 112 participants from the A4 trial in Phase IV. All datasets contained balanced numbers of individuals clinically classified as CN, MCI, and AD dementia. All samples had corresponding Aβ-PET and cognitive data. Study demographics were simply stratified by high or low Aβ burden, except for Phase III models 2 and 3 (see Methods 2.8).

[0131] 3.2. Phase I and Phase II Candidate Biomarkers

[0132] Phase I identified CD11c and P2X7 as candidate biomarkers (Table A.2.1 below). Compared with the CN group, the MFI of CD11c was reduced by 19–34% (P<0.05) and P2X7 by 20–42% (P<0.01) in the MCI and AD groups. They also showed a negative correlation with Aβ CL, such that subjects with higher Aβ burden had lower CD11c and P2X7 expression levels. Phase II identified CD11c, CD11b, and CD33, but not P2X7, as candidate biomarkers (Table A.2.2 below). Compared with the CN group, the MFI of CD11c, CD11b, and CD33 was reduced by 14–23% (P<0.05), 14–21% (P<0.05), and 16–18% (P<0.05) in the MCI and AD groups, respectively. However, only CD11c on NK cells correlated with the Aβ-PET data.

[0133] 3.3 Phase III Candidate Biomarkers

[0134] In Phase III, we identified 10 candidate markers related to MFI of CD11c, CD59, CD91, and CD163 that differed between CN, MCI, and AD groups (top 10 in Table A.2.3 below) and correlated with Aβ burden. The MFI of CD11c and CD163 decreased by 15-19% (P<0.01) and 11-16% (P<0.01) in MCI and AD groups, respectively, compared with the CN group, whereas the MFI of CD59 and CD91 increased by 27-78% (P<0.0001) and 44-59% (P<0.001) in MCI and AD groups, respectively. The bottom four in Table A.2.3, related to MerTK, CD18, and RAGE, were excluded due to small sample sizes. Nevertheless, compared with the CN group, the MCI and AD groups showed a 20-26% (P<0.05) and 12% (P<0.05) decrease in MFI of CD18 and MerTK, respectively, and a 31% (P<0.05) increase in MFI of RAGE on monocytes. All potential biomarker changes and their relevance to AD are listed in Table 1 above.

[0135] The absolute percentage of CD14- lymphocytes in whole blood was reduced by 11% (P<0.01) in the MCI and AD groups compared to the CN group (Table A.2.4 below) and correlated with CL, episodic memory, and PACC scores. Because the differences in the percentage of monocytes in whole blood between the CN, MCI, and AD groups were minimal, Applicant divided the total monocyte population into CD14+CD16+, CD14+CD16-, and CD14-CD16+ subpopulations and assessed their relative percentages from the total monocyte population. The relative percentage of CD14+CD16- monocytes was increased by 13% (P<0.01) in the AD and MCI groups compared to the CN group (Table A.2.4). Interestingly, the relative percentage of CD14+CD16+ monocytes was reduced by 30% in the MCI group compared with the CN group (P = 0.02), whereas AD and CN showed no group difference. A lower relative percentage of CD14-CD16+ monocytes in the MCI group (-29% P = 0.02) was also observed in a separate tube with a smaller sample size. Subsequently, the percentages of CD14- lymphocytes and CD14+CD16- monocytes were added to the above 10 to create 12 biomarkers for further evaluation. [Table A.2.1] [Table A.2.2] [Table A.2.3]

[0136] [Table A.2.4]

[0137] 3.5. Biomarker evaluation

[0138] In Table 3, the 12 biomarkers selected in Phase III are named Biomarker 1 (BM1), Biomarker 2 (BM2), ..., and Biomarker 12 (BM12). Distribution tests found that Biomarker 7 and Biomarker 8 followed approximately normal distributions (Table A.6). The others were transformed using both natural logarithm (lnBM) and square root (sqrtBM). Collinearity tests were used to select only independent biomarkers for combination into panels (Table A.7).

[0139] [Table 3.1-1] [Table 3.1-2] [Table 3.1-3]

[0140] [Table 3.2-1] [Table 3.2-2] [Table 3.2-3]

[0141] [Table 3.3-1] [Table 3.3-2] [Table 3.3-3]

[0142] [Table 3.4-1] [Table 3.4-2] Table 3.4-3

[0143] Table 3.5-1 Table 3.5-2

[0144] Table A.6.1

[0145] Table A.6.2 Table A.6.3

[0146] Table A.7.1

[0147] Table A.7.2

[0148] In Model 1 (Table 3.1), Baseline 1 distinguished between <25CL and >25CL with an AUC of 0.85 (0.79-0.91). The MFI of CD163 on neutrophils (lnBM8) had the best improved AUC of 0.89 (0.84-0.94), a 16% Brier improvement, and an IDI with events of 0.06 (0.024-0.096) and an IDI without events of 0.041 (0.015-0.067). Two other running biomarkers, the MFI of CD59 on CD14-CD16+ monocytes (lnBM5) and the MFI of CD11c on total neutrophils (lnBM3), formed a panel of biomarkers: Novel 1. Novel 1 had an area under the curve (AUC) of 0.91 (0.86-0.95), a Brier improvement of 17.8%, and an IDI of 0.085 (0.049-0.12) and 0.050 (0.019-0.081) for those with and without events, respectively. Novel 1 had a sensitivity of 89.9% (95% CI 80.2%-95.8%), a specificity of 76.9% (66.0%-85.7%), and an overall accuracy of 83.0% at the Youden Index cutoff point (Table A.8.1).

[0149] [Table A.8.1]

[0150] In Model 2 (Table 3.2 above), Baseline 2 distinguished between <25CL and >25CL and <100CL with an AUC of 0.84 (0.77 to 0.91). MFI of CD163 on neutrophils (lnBM8) had the best improved AUC of 0.90 (0.84 to 0.96), a 20% Brier improvement, and an IDI with events of 0.106 (0.050 to 0.162) and an IDI without events of 0.051 (0.024 to 0.077). Notably, Model 2 only had 52 cases, and only one biomarker was added to fit the model, forming Novel 2. Novel 2 had a sensitivity of 90.0% (76.3%-97.2%), a specificity of 76.5% (65.8%-85.3%), and an overall accuracy of 81.0% at the Youden index cutoff point (Table A.8.1).

[0151] In Model 3 (Table 3.3 above), all MCI subjects were classified as cases, resulting in over-aggregation of Baseline 3. The MFI of CD163 on neutrophils (lnBM8) had the best improved AUC of 0.96 (0.93–0.99), a significant improvement of 24.4%, with an IDI of 0.049 (0.013–0.085) with events and an IDI of 0.035 (0.009–0.062) without events. Two other running biomarkers, the MFI of CD59 on CD14-CD16- lymphocytes (sqrtBM4) and the absolute percentage of CD14- lymphocytes in whole blood (sqrtBM11), were also included to form a panel of biomarkers: New3. New3 improved AUC by 6% from baseline. Novel 3 had a sensitivity of 91.2% (80.7%–97.1%), a specificity of 93.9% (85.2%–98.3%), and an overall accuracy of 92.7% at the Youden index cutoff point (Table A.8.1).

[0152] Overall, different models proposed different biomarkers, complexes, or panels. Three candidate biomarkers: CD11c, CD59, and CD163 performed very well, with CD163 consistently performing the best. The model cutoff values, sensitivity, specificity, and accuracy for each biomarker can be found in Tables A.8.2-4.

[0153] [Table A.8.2] [Table A.8.3] [Table A.8.4]

[0154] 3.6. Phase IV Results

[0155] Phase III identified CD11c, CD59, CD91, and CD163 as potential biomarkers for distinguishing between PET Aβ-positive (Cl>25) and PET Aβ-negative (CL<25) individuals after adjusting for age, sex, education, and APOE ε4 allele status (Model 1, P=0.021). We then validated them in an independent cohort (Phase IV, N=112). Several leukocyte markers showed associations with clinical diagnosis or with phakicosis, consistent with Phase III. A multivariate model of the validation cohort found that the combination of CD59 and CD91 discriminated between Aβ status, CL>25, or CL<25, with an AUC of 0.91 (CI: 0.81-0.99). The AUC was 0.81 (CI: 0.72-0.9) and 0.1 higher than the average base model AUC (p=0.072) after adjusting for age, sex, education, and APOE ε4 allele status. When combining CD11c, CD59, and CD91, the adjusted AUC was 0.95 (CI: 0.86-1), 0.14 higher than the base model, p=0.05, although this value may reflect overfitting due to the relatively small sample size (details in Table 3.5).

[0156] 4. Consideration:

[0157] Leukocyte surface markers showed differential expression when compared between CN, MCI, and AD groups and / or in conjunction with Aβ-PET data. The Phase I and II designs were limited in their screening capabilities due to the large number of key CD markers. The new range of leukocyte surface markers in Phase III was adjusted to reflect GWAS and other evidence that phagocytosis / endocytosis in the microglia / monocyte pathway may play an important role in AD pathogenesis [7, 21]. Although the precise molecular mechanisms remain unclear, the decreased expression of phagocytic receptors, MerTK and P2X7; scavenger receptors, CD36 and CD163; and integrins, CD11b, CD11c, and CD18, closely aligns with some of our previous studies [7] inferring impaired monocyte phagocytic function in AD patients (Table 1; Figure 1, left). It is also worth noting that CD18 is a common subunit of CD11b / CD18 and CD11c / CD18, also known as complement receptors 3 and 4 (CR3, CR4). This study found that both CR3 and CR4 are downregulated in AD. CD59 is a major inhibitor of the membrane attack complex (MAC)

[22] . The present applicants found that upregulation of CD59 in AD can interfere with cell lysis and phagocytic clearance. See the right side of Figure 1. Coordinated changes in two or more pathways represent functional inhibition in AD related to opsonization, phagocytosis, and removal of pathogenic factors.

[0158] In this study, three markers, CD33, CD35 / CR1, and CD91, are protein products of AD risk genes reported by GWAS meta-analyses [23, 24] or other genetic association studies [25-27]. Both CD35 and CD91 were found to be upregulated in AD in this study. Bradshaw et al.

[28] found that the CD33 risk allele rs3865444C was associated with greater cell surface expression of CD33 on monocytes from young and older individuals. However, it was associated with reduced Aβ42 internalization, accumulation of neuritic tau-positive tangles, and increased numbers of activated human microglia. Overexpression of CD33 on monocytes in individuals with the rs3865444C risk genotype may be a mechanism to compensate for its loss of function. CD35 / CR1 is a receptor for complement factors C3b and C4b

[29] , but its relevance to AD is unclear. CD91 is a receptor for ApoE and the endocytosis and degradation of secreted amyloid precursor protein (APP)

[30] , suggesting that a single pathway links two molecules involved in the pathophysiology of AD.

[0159] In addition to CD91, RAGE is also a receptor for Aβ. Yan et al.

[31] reported that RAGE expression was increased in AD, particularly in neurons near Aβ deposits and neurofibrillary tangles. In mice, Deane et al.

[32] demonstrated that RAGE mediated the transport of human Aβ40 and Aβ42 across the blood-brain barrier, resulting in the expression of pro-inflammatory cytokines and endothelin-1. Arancio et al.

[33] demonstrated that RAGE is a cofactor for Aβ-induced neuronal perturbations and is involved in microglial cell activation in transgenic mice. In this study, RAGE expression was increased in AD, consistent with Yan et al.

[31] , and may provide a mirror image of what occurs in brain-resident microglia. CD36 was found to be downregulated in AD in this study. CD36 is a receptor for oxidized low-density lipoprotein (LDL)

[34] and Aβ

[35] . Immobilized Aβ oligomer-binding CD36-Fc protein showed affinity similar to that of RAGE-Fc and TREM2-Fc

[36] .

[0160] Our data from blood typing suggest that there may be abnormalities in the percentages of lymphocyte and monocyte subsets. Hematopoietic aging is characterized by dysfunctional hematopoietic stem cell (HSC) proliferation, resulting in the generation of more myeloid and fewer lymphoid daughter cells, a phenomenon called myeloid-biased hematopoiesis [37, 38]. The percentage of each type of leukocyte in the blood is primarily determined by differences in stem cell activity; therefore, the decrease in lymphocytes may be attributable to this myeloid hematopoiesis

[39] . However, such differences do not explain the observed pattern of changes in other monocyte subsets in the opposite direction. CD14+CD16+ monocytes (highly phagocytic cells similar to tissue macrophages) may infiltrate into the brain at sites of Aβ plaques, resulting in a transient decline in MCI and incomplete recovery from AD, possibly due to impaired chemotactic function or obstructive passage, representing a U-shaped response. The pattern of CD14-CD16+ monocytes reveals that these two small monocyte subsets had similar responses, but CD14-CD16+ monocytes were less likely to reverse AD. Neither the percentage of CD14+CD16+ nor the percentage of CD14-CD16+ correlated with CL, EM, or PACC due to this U-shaped response. It is interesting to note that the percentage of monocytes in the blood did not change, likely due to this subset discrepancy.

[0161] Applicants have demonstrated that a significant percentage (30%) of CN individuals have high Aβ levels in the CSF. We found that individuals with AD exhibited high PET and low Aβ42 levels, and these individuals are now classified as having preclinical AD. Mild cognitive impairment (MCI) is considered a prodromal stage of AD, with 40–60% of individuals meeting criteria for MCI and eventually progressing to clinical AD, or approximately 5–25% per year progressing to clinical AD

[40] . Because diagnostic decision-making is a two-class prediction problem, we established a binary classifier, i.e., healthy and disease, and determined the classifier boundary using a logistic model. Unfortunately, AD diagnosis is based on multidimensional criteria including clinical considerations, cognition, behavior, advanced imaging, and CSF biomarkers. Furthermore, there are no specific criteria for early detection or clear definitions of preclinical stages. Therefore, we used CL as a simple proxy for identifying stages in Models 1 and 2. However, we added a clinical classification in Model 3 to reflect the latest developments made by the NIA-AA

[13] and IWG

[41] .

[0162] Our findings may have implications for the early diagnosis of individuals at high risk for AD due to genetic factors (e.g., APOE) to monitor preclinical signs and initiate early intervention without obvious changes in neuroimaging or CSF biomarkers. The use of plasma biomarkers for AD has recently been demonstrated by Nakamura et al. and Fossati et al. [15, 42], justifying the use of blood-based biomarkers for AD diagnosis. Our novel findings, if relevant, may be attributable to AD proteinopathy in the blood. The role of altered leukocyte biomarkers, such as PU.1, or blood grouping or transcriptional networks [21, 43] in regulating these processes remains to be determined but may contribute to the molecular pathology of AD.

[0163] 4.7. In conclusion, our results suggest that leukocyte biomarkers may be objective, measurable indicators of AD progression and useful for early diagnosis. Furthermore, leukocyte biomarkers are cost-effective and simple to use, allowing them to be readily employed in the clinic, confirming their importance for the development of novel, effective drugs and as outcome measures in all phases of clinical trials.

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[0165] Terms Item 1. A composition comprising: A composition comprising a binding agent to CD163 conjugated to a detectable moiety.

[0166] Clause 2. The composition of clause 1, wherein the binding agent to CD163 is selected from a single chain variable fragment, an antibody mimetic, an antibody fragment, an antibody, a monoclonal antibody, and combinations thereof.

[0167] Item 3. The composition according to any one of Items 1 to 2, wherein the detectable moiety is selected from a radioisotope, a stable isotope, a fluorophore, and combinations thereof.

[0168] Item 4. A composition comprising: a mouse anti-human IgG monoclonal antibody against CD163 conjugated with a fluorophore; ethylenediaminetetraacetic acid, at least one salt thereof, citric acid, at least one salt thereof, or a combination of any of the foregoing; A composition comprising:

[0169] Item 5. A composition comprising: a mouse anti-human IgG monoclonal antibody against CD163 conjugated with a fluorophore; ethylenediaminetetraacetic acid, at least one salt thereof, citric acid, at least one salt thereof, or a combination of any of the foregoing; Whole blood from a human patient; A composition comprising:

[0170] Item 6. A composition comprising: a mouse anti-human IgG monoclonal antibody against CD163 conjugated with a fluorophore; at least one anticoagulant; A composition comprising:

[0171] Item 7. The composition of Item 6, wherein the at least one anticoagulant is selected from ethylenediaminetetraacetic acid, at least one salt thereof, citric acid, at least one salt thereof, or any combination of the foregoing.

[0172] Item 8. The composition according to any one of Items 4 to 7, wherein the composition does not contain heparin.

[0173] Item 9. The composition according to any one of Items 4 to 8, wherein the composition does not contain allophycocyanin.

[0174] Item 10. The composition according to any one of Items 4 to 9, further comprising bovine serum albumin and sodium azide.

[0175] Item 11. The composition according to any one of Items 4 to 10, wherein the fluorophore is selected from fluorescein isothiocyanate, allophycocyanin, or a combination thereof.

[0176] Item 12. The composition of any one of items 4 to 10, wherein the fluorophore is selected from the fluorescent dye sold under the trademark Alexa Fluor® 488 (Molecular Probes, Inc., Eugene, Oregon), the fluorescent dye sold under the trademark Alexa Fluor® 647 (Molecular Probes, Inc., Eugene, Oregon); BUV395; BUV563; BUV615; BUV661; BUV737; BUV805; BV421; BV480; BV510; BV605; BV650; BV711; BV750; BV786; FITC; PE; PE-CF594; PerCP-Cy™ 5.5; R718, or a combination of two or more thereof.

[0177] Item 13. The composition of any one of Items 4 to 10, wherein the fluorophore is selected from the group consisting of fluorescein, rhodamine, lanthanide fluorophores, and derivatives thereof. Examples of fluorophores include, but are not limited to, fluorescein isothiocyanate (FITC) (e.g., 5-FITC), fluorescein amidite (FAM) (e.g., 5-FAM), eosin, carboxyfluorescein, erythrosine, Alexa Fluor® (e.g., Alexa 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 647, 660, 680, 700, or 750), carboxytetramethylrhodamine, tetramethylrhodamine, and sulforhodamine.

[0178] Item 14. The composition of any one of Items 4 to 10, wherein the fluorophore is selected from a fluorescent protein such as GFP, YFP, RFP, eGFP, mCherry, tdtomato, FITC, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 680, Alexa Fluor 750, Pacific Blue, coumarin, BODIPY FL, Pacific Green, Oregon Green, Cy 3, Cy 5, Pacific Orange, TRITC, Texas Red, R-phycoerythrin, allophycocyanin, or other fluorophores known in the art.

[0179] Item 15. The composition according to any one of Items 4 to 14, which does not contain a fluorescent dye sold under the trademark Alexa Fluor® 488 (Molecular Probes, Inc., Eugene, Oregon).

[0180] Item 16. The composition according to any one of Items 4 to 15, wherein the composition does not contain heparin.

[0181] Clause 17. The composition of any one of clauses 1 to 16, wherein the composition further comprises a second binding agent for one or more of scavenger receptors and their modulators conjugated to a second detectable moiety.

[0182] Clause 18. The composition of clause 17, wherein the second binding agent is selected from a single chain variable fragment, an antibody mimetic, an antibody fragment, an antibody, a monoclonal antibody, and combinations thereof.

[0183] Item 19. The composition of any one of Items 17 to 18, wherein the second detectable moiety is selected from a radioisotope, a stable isotope, a fluorophore, and combinations thereof.

[0184] Item 20. A method for diagnosing AD in a human patient, comprising: obtaining a blood sample from said human patient; Optionally, storing the blood sample on ice; optionally testing said blood sample within 3 hours of obtaining said blood sample; reacting the blood sample with a mouse anti-human IgG monoclonal antibody against CD163 conjugated with a fluorophore to obtain bound CD163; measuring the concentration of bound CD163 in the blood sample by fluorescence of the fluorophore; comparing said concentration to the range of concentrations of CD163 in healthy humans; observing a decrease in the concentration of CD163 in the blood sample; and thereby diagnosing AD in a human patient; 1. A method for diagnosing AD in a human patient, comprising:

[0185] Item 21. The method of Item 20, wherein measuring the concentration of bound CD163 comprises measuring the mean fluorescence intensity of bound CD163 on leukocytes.

[0186] Item 22. The method of any one of items 20 to 21, wherein comparing the concentrations comprises comparing the mean fluorescence intensity of bound CD163 for the human patient with the mean fluorescence intensity of bound CD163 for a pool of human participants with <25 CL as measured by amyloid PET imaging.

[0187] Clause 23. The method of any one of clauses 20 to 22, wherein observing a decrease in the concentration of CD163 in the blood sample comprises measuring a decrease in mean fluorescence intensity of at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, or at least about 19%, compared to the mean fluorescence intensity of bound CD163 for a pool of human participants with <25 CL measured by amyloid PET imaging.

[0188] Item 24. The method of any one of items 20 to 23, wherein the mouse anti-human IgG monoclonal antibody against CD163 conjugated to a fluorophore comprises a mouse anti-human CD163 antigen conjugated to a fluorescent dye sold under the trademark Alexa Fluor™ 647 (Molecular Probes, Inc., Eugene, Oregon).

[0189] Item 25. A method for determining the efficacy of a candidate AD treatment in a human patient, comprising: (A) obtaining a first blood sample from the human patient; Optionally, storing the first blood sample on ice; Optionally, testing the first blood sample within three hours of obtaining the first blood sample; reacting the first blood sample with a mouse anti-human IgG monoclonal antibody against CD163 conjugated to a fluorophore to obtain a first bound CD163; measuring a first concentration of a first bound CD163 in the first blood sample by fluorescence of the fluorophore; (B) administering a candidate AD treatment to the human patient; and (C) obtaining a second blood sample from the human patient; Optionally, storing the second blood sample on ice; Optionally, testing the second blood sample within three hours of obtaining the second blood sample; reacting the second blood sample with a mouse anti-human IgG monoclonal antibody against CD163 conjugated to a fluorophore to obtain a second bound CD163; measuring a second concentration of the second bound CD163 in the second blood sample by fluorescence of the fluorophore; (D) observing an increase in the first concentration of bound CD163 from the first concentration to the second concentration of bound CD163; (E) thereby determining the efficacy of the candidate AD treatment in a human patient.

[0190] Item 26. The method according to Item 25, wherein the candidate AD treatment is a phagocytosis promoter.

[0191] Item 27. The method according to Item 25, wherein the candidate AD treatment is a scavenger receptor agonist.

[0192] Item 28. The method according to any one of Items 25 to 27, wherein measuring the first concentration comprises measuring the first concentration on white blood cells, and measuring the second concentration comprises measuring the second concentration on white blood cells.

[0193] Item 29. A composition comprising a binding agent to CD91 conjugated to a detectable moiety.

[0194] Clause 30. The composition of clause 29, wherein the binding agent is selected from a single chain variable fragment, an antibody mimetic, an antibody fragment, an antibody, a monoclonal antibody, and combinations thereof.

[0195] Item 31. The composition of any one of Items 29 to 30, wherein the detectable moiety is selected from a radioisotope, a stable isotope, a fluorophore, and combinations thereof.

[0196] Item 32. Use of a binding agent against CD163 for the diagnosis of AD in a human patient in need thereof.

[0197] Clause 33. The use of clause 32, wherein the binding agent is selected from a single chain variable fragment, an antibody mimetic, an antibody fragment, an antibody, a monoclonal antibody, and combinations thereof.

[0198] Item 34. The use according to any one of items 32 to 33, wherein the binding agent is conjugated to a detectable moiety.

[0199] Clause 35. The use of clause 34, wherein the detectable moiety is selected from a radioisotope, a stable isotope, a fluorophore, and combinations thereof.

[0200] Item 36. A binding agent against CD163 for the diagnosis of AD in a human patient in need thereof.

[0201] Clause 37. The binding agent to CD163 according to clause 36, wherein said binding agent is selected from a single chain variable fragment, an antibody mimetic, an antibody fragment, an antibody, a monoclonal antibody, and combinations thereof.

[0202] Clause 38. The binding agent to CD163 according to any one of clauses 36 to 37, wherein the binding agent is conjugated to a detectable moiety.

[0203] Clause 39. The binding agent to CD163 according to Clause 38, wherein the detectable moiety is selected from a radioisotope, a stable isotope, a fluorophore, and combinations thereof.

[0204] Item 40. A method for diagnosing AD in a human patient, comprising: obtaining a blood sample from said human patient; Optionally, storing the blood sample on ice; optionally testing said blood sample within 3 hours of obtaining said blood sample; reacting the blood sample with a mouse anti-human IgG monoclonal antibody against CD91 conjugated to a fluorophore to obtain bound CD91; measuring the concentration of bound CD91 in the blood sample by fluorescence of the fluorophore; comparing said concentration to the range of concentrations of CD91 in healthy humans; observing an increase in the concentration of CD91 in the blood sample; and thereby diagnosing AD in a human patient; 1. A method for diagnosing AD in a human patient, comprising:

[0205] Item 41. The method of Item 40, wherein measuring the concentration of bound CD91 comprises measuring the mean fluorescence intensity of bound CD91 on leukocytes.

[0206] Clause 42. The method of any one of clauses 40 to 41, wherein comparing the concentrations comprises comparing the mean fluorescence intensity of bound CD91 for the human patient with the mean fluorescence intensity of bound CD91 for a pool of human participants with <25 CL as measured by amyloid PET imaging.

[0207] Paragraph 43. The method of any one of paragraphs 40 to 42, wherein observing an increase in the concentration of CD91 in the blood sample comprises measuring an increase in mean fluorescence intensity of at least about 44%, at least about 50%, at least about 55%, or at least about 59%, compared to the mean fluorescence intensity of bound CD91 for a pool of human participants with <25 CL as measured by amyloid PET imaging.

[0208] Item 44. A method for determining the efficacy of a candidate AD treatment in a human patient, comprising: (A) obtaining a first blood sample from the human patient; Optionally, storing the first blood sample on ice; Optionally, testing the first blood sample within three hours of obtaining the first blood sample; The first blood sample is reacting with a mouse anti-human IgG monoclonal antibody against CD91 conjugated with a fluorophore to obtain a first bound CD91; measuring a first concentration of the first bound CD91 in the first blood sample by fluorescence of the fluorophore; (B) administering a candidate AD treatment to the human patient; and (C) obtaining a second blood sample from the human patient; Optionally, storing the second blood sample on ice; Optionally, testing the second blood sample within three hours of obtaining the second blood sample; reacting the second blood sample with a mouse anti-human IgG monoclonal antibody against CD91 conjugated to a fluorophore to obtain second bound CD91; measuring a second concentration of the second bound CD91 in the second blood sample by fluorescence of the fluorophore; (D) observing a decrease in the first concentration of bound CD91 from the first concentration to the second concentration of bound CD91; (E) thereby determining the efficacy of the candidate AD treatment in a human patient.

[0209] Item 45. The method of Item 44, wherein the candidate AD treatment is a phagocytosis promoter.

[0210] Item 46. The method according to Item 44, wherein the candidate AD treatment is a scavenger receptor agonist.

[0211] Item 47. The method according to any one of Items 44 to 46, wherein measuring the first concentration comprises measuring the first concentration on white blood cells, and measuring the second concentration comprises measuring the second concentration on white blood cells.

[0212] Clause 48. Use of a binding agent against CD91 for the diagnosis of AD in a human patient in need thereof.

[0213] Clause 49. The use of clause 48, wherein the binding agent is selected from a single chain variable fragment, an antibody mimetic, an antibody fragment, an antibody, a monoclonal antibody, and combinations thereof.

[0214] Clause 50. The use of any one of clauses 48 to 49, wherein the binding agent is conjugated to a detectable moiety.

[0215] Clause 51. The use of clause 50, wherein the detectable moiety is selected from a radioisotope, a stable isotope, a fluorophore, and combinations thereof.

[0216] Item 52. A binding agent against CD91 for the diagnosis of AD in a human patient in need thereof.

[0217] Clause 53. The binding agent to CD91 of clause 52, wherein said binding agent is selected from a single chain variable fragment, an antibody mimetic, an antibody fragment, an antibody, a monoclonal antibody, and combinations thereof.

[0218] Clause 54. The binding agent to CD91 according to any one of clauses 51 to 53, wherein said binding agent is conjugated to a detectable moiety.

[0219] Clause 55. The binding agent to CD91 according to Clause 54, wherein the detectable moiety is selected from a radioisotope, a stable isotope, a fluorophore, and combinations thereof.

[0220] Item 56. A composition comprising a binding agent for MerTK conjugated to a detectable moiety.

[0221] Clause 57. The composition of clause 56, wherein the binding agent is selected from a single chain variable fragment, an antibody mimetic, an antibody fragment, an antibody, a monoclonal antibody, and combinations thereof.

[0222] Item 58. The composition of any one of Items 56 to 57, wherein the detectable moiety is selected from a radioisotope, a stable isotope, a fluorophore, and combinations thereof.

[0223] Item 59. A method for diagnosing AD in a human patient, comprising: obtaining a blood sample from said human patient; Optionally, storing the blood sample on ice; optionally testing said blood sample within 3 hours of obtaining said blood sample; reacting the blood sample with a mouse anti-human IgG monoclonal antibody against MerTK conjugated to a fluorophore to obtain bound MerTK; determining the concentration of bound MerTK in the blood sample by fluorescence of the fluorophore; comparing said concentration to the range of MerTK concentrations in healthy humans; observing a decrease in the concentration of MerTK in the blood sample; thereby diagnosing AD in a human patient; 1. A method for diagnosing AD in a human patient, comprising:

[0224] Clause 60. The method of clause 59, wherein measuring the concentration of bound MerTK comprises measuring the mean fluorescence intensity of bound MerTK on white blood cells.

[0225] Clause 61. The method of any one of clauses 59 to 60, wherein comparing the concentrations comprises comparing the mean fluorescence intensity of bound MerTK for the human patient with the mean fluorescence intensity of bound MerTK for a pool of human participants with <25 CL as measured by amyloid PET imaging.

[0226] Clause 62. The method of any one of clauses 59 to 61, wherein observing a decrease in the concentration of MerTK in the blood sample comprises measuring a decrease in mean fluorescence intensity of at least about 12% compared to the mean fluorescence intensity of bound MerTK for a pool of human participants with <25 CL as measured by amyloid PET imaging.

[0227] Item 63. A method for determining the efficacy of a candidate AD treatment in a human patient, comprising: (A) obtaining a first blood sample from the human patient; Optionally, storing the first blood sample on ice; Optionally, testing the first blood sample within three hours of obtaining the first blood sample; The first blood sample is reacting with a mouse anti-human IgG monoclonal antibody against MerTK conjugated with a fluorophore to obtain a first bound MerTK; measuring a first concentration of the first bound MerTK in the first blood sample by fluorescence of the fluorophore; (B) administering a candidate AD treatment to the human patient; and (C) obtaining a second blood sample from the human patient; Optionally, storing the second blood sample on ice; Optionally, testing the second blood sample within three hours of obtaining the second blood sample; reacting the second blood sample with a mouse anti-human IgG monoclonal antibody against MerTK conjugated to a fluorophore to obtain a second bound MerTK; measuring a second concentration of the second bound MerTK in the second blood sample by fluorescence of the fluorophore; (D) observing an increase in the first concentration of the first bound MerTK to the second concentration of the second bound MerTK; (E) thereby determining the efficacy of the candidate AD treatment in a human patient.

[0228] Item 64. The method of Item 63, wherein the candidate AD treatment is a phagocytosis promoter.

[0229] Item 65. The method of Item 63, wherein the candidate AD treatment is a scavenger receptor agonist.

[0230] Item 66. The method of any one of items 63 to 65, wherein measuring the first concentration comprises measuring the first concentration on white blood cells, and measuring the second concentration comprises measuring the second concentration on white blood cells.

[0231] Item 67. Use of a binding agent to MerTK for the diagnosis of AD in a human patient in need thereof.

[0232] Clause 68. The use of clause 67, wherein the binding agent is selected from a single chain variable fragment, an antibody mimetic, an antibody fragment, an antibody, a monoclonal antibody, and combinations thereof.

[0233] Clause 69. The use of any one of clauses 67 to 68, wherein the binding agent is conjugated to a detectable moiety.

[0234] Clause 70. The use of Clause 69, wherein the detectable moiety is selected from a radioisotope, a stable isotope, a fluorophore, and combinations thereof.

[0235] Item 71. A binding agent against MerTK for the diagnosis of AD in a human patient in need thereof.

[0236] Clause 72. The binding agent to MerTK of Clause 71, wherein said binding agent is selected from a single chain variable fragment, an antibody mimetic, an antibody fragment, an antibody, a monoclonal antibody, and combinations thereof.

[0237] Clause 73. A binding agent to MerTK according to any one of clauses 84 to 85, wherein the binding agent is conjugated to a detectable moiety.

[0238] Clause 74. The binding agent to MerTK of Clause 73, wherein the detectable moiety is selected from a radioisotope, a stable isotope, a fluorophore, and combinations thereof.

[0239] Item 75. A composition comprising: a first binding agent for CD59 conjugated to a first detectable moiety; a second binding agent to CD163 conjugated to a second detectable moiety; A composition comprising:

[0240] Clause 76. The composition of Clause 75, wherein the first binding agent and the second binding agent are independently selected from a single chain variable fragment, an antibody mimetic, an antibody fragment, an antibody, a monoclonal antibody, and combinations thereof.

[0241] Clause 77. The composition of any one of clauses 75-76, wherein the first detectable moiety, the second detectable moiety, and the third detectable moiety are independently selected from a radioisotope, a stable isotope, a fluorophore, and combinations thereof.

[0242] Item 78. A composition comprising: a monoclonal antibody against CD59 conjugated to a first fluorophore; a monoclonal antibody against CD163 conjugated to a second fluorophore; A composition comprising:

[0243] Item 79. A composition comprising: a mouse anti-human IgG monoclonal antibody against CD59 conjugated to a first fluorophore; a mouse anti-human IgG monoclonal antibody against CD163 conjugated to a second fluorophore; A composition comprising:

[0244] Item 80. The composition of any one of items 78 to 79, further comprising at least one anticoagulant.

[0245] Clause 81. The composition of any one of clauses 78 to 80, wherein the at least one anticoagulant comprises ethylenediaminetetraacetic acid, at least one salt thereof, citric acid, at least one salt thereof, or any combination of the foregoing.

[0246] Item 82. The composition according to any one of Items 78 to 81, wherein the composition does not contain heparin.

[0247] Item 83. The composition according to any one of Items 78 to 82, wherein the composition does not contain allophycocyanin.

[0248] Item 84. The composition of any one of Items 78 to 83, wherein the composition does not contain a fluorescent dye sold under the trademark Alexa Fluor® 647 (Molecular Probes, Inc., Eugene, Oregon).

[0249] Item 85. The composition of any one of Items 78 to 84, further comprising bovine serum albumin and sodium azide.

[0250] Item 86. The composition of any one of Items 78 to 85, wherein the composition does not contain a fluorescent dye sold under the trademark Alexa Fluor® 488 (Molecular Probes, Inc., Eugene, Oregon).

[0251] Item 87. The composition of any one of Items 78 to 86, wherein the first fluorophore is R-phycoerythrin.

[0252] Item 88. The composition of any one of items 78 to 87, wherein the second fluorophore is a fluorescent dye sold under the trademark Alexa Fluor® 647 (Molecular Probes, Inc., Eugene, Oregon).

[0253] Item 89. The composition of any one of Items 78 to 88, further comprising whole blood from a human patient.

[0254] Item 90. A kit comprising: a first binding agent for CD59 conjugated to a first detectable moiety; a second binding agent to CD163 conjugated to a second detectable moiety; Includes a kit.

[0255] Clause 91. The kit of Clause 90, wherein the first binding agent and the second binding agent are independently selected from a single chain variable fragment, an antibody mimetic, an antibody fragment, an antibody, a monoclonal antibody, and combinations thereof.

[0256] Clause 92. The kit of any one of clauses 90-91, wherein the first detectable moiety and the second detectable moiety are independently selected from a radioisotope, a stable isotope, a fluorophore, and combinations thereof.

[0257] Item 93. A kit comprising: a monoclonal antibody against CD59 conjugated to a first fluorophore; a monoclonal antibody against CD163 conjugated to a second fluorophore; Includes a kit.

[0258] Item 94. A kit comprising: a mouse anti-human IgG monoclonal antibody against CD59 conjugated to a first fluorophore; a mouse anti-human IgG monoclonal antibody against CD163 conjugated to a second fluorophore; Includes a kit.

[0259] Item 95. A kit according to any one of Items 93 to 94, wherein the composition does not contain a fluorescent dye sold under the trademark Alexa Fluor® 488 (Molecular Probes, Inc., Eugene, Oregon).

[0260] Item 96. The kit according to any one of Items 93 to 95, wherein the first fluorophore is R-phycoerythrin.

[0261] Item 97. The kit of any one of Items 93 to 96, wherein the second fluorophore is a fluorescent dye sold under the trademark Alexa Fluor® 647 (Molecular Probes, Inc., Eugene, Oregon).

[0262] Paragraph 98. A method for measuring the relative expression of CD59 and CD163 in a human patient, comprising: obtaining a sample of whole blood from said human patient; The sample a first binding agent to CD59 conjugated to a first detectable moiety, and with a second binding agent for CD163 conjugated to a second detectable moiety; forming a binding CD59 and binding CD163; measuring the concentration of the first detectable moiety and the second detectable moiety; Thereby, the relative expression of CD59 and CD163 is determined; 1. A method for measuring the relative expression of CD59 and CD163 in a human patient, comprising:

[0263] Item 99. The method of Item 98, wherein measuring the concentration includes measuring the concentration on white blood cells.

[0264] Paragraph 100. A method for measuring the relative expression of CD59 and CD163 in a human patient, comprising: obtaining a sample of whole blood from said human patient; The sample a monoclonal antibody against CD59 conjugated to a first fluorophore, and contacting with a monoclonal antibody against CD163 conjugated to a second fluorophore; forming a binding CD59 and binding CD163; measuring the fluorescence of the first fluorophore and the second fluorophore; Thereby, the relative expression of CD59 and CD163 is determined; 1. A method for measuring the relative expression of CD59 and CD163 in a human patient, comprising:

[0265] Paragraph 101. A method for measuring the relative expression of CD59 and CD163 in a human patient, comprising: obtaining a sample of whole blood from said human patient; The sample a mouse anti-human IgG monoclonal antibody against CD59 conjugated to a first fluorophore, and contacting with a mouse anti-human IgG monoclonal antibody against CD163 conjugated to a second fluorophore; forming a binding CD59 and binding CD163; measuring the fluorescence of the first fluorophore and the second fluorophore; Thereby, the relative expression of CD59 and CD163 is determined; 1. A method for measuring the relative expression of CD59 and CD163 in a human patient, comprising:

[0266] Paragraph 102. The method of any one of paragraphs 100 to 101, wherein the relative expression of CD59 and CD163 is the relative expression of CD59 and CD163 on leukocytes.

[0267] Clause 103. A method for diagnosing AD in a human patient, comprising obtaining a blood sample from the human patient; Optionally, storing the blood sample on ice; optionally testing said blood sample within 3 hours of obtaining said blood sample; The blood sample reacting with a mouse anti-human IgG monoclonal antibody against CD59 conjugated with a first fluorophore to obtain bound CD59; reacting with a mouse anti-human IgG monoclonal antibody against CD163 conjugated with a second fluorophore to obtain bound CD163; measuring the concentration of bound CD59 and the concentration of bound CD163 in the blood sample by fluorescence of the first fluorophore and the second fluorophore; observing an increase in the concentration of bound CD59 compared to the range of CD59 concentrations in healthy humans; observing a decrease in the concentration of bound CD163 compared to a range of concentrations of CD163 in healthy humans; thereby diagnosing AD in human patients; and 1. A method for diagnosing AD in a human patient, comprising:

[0268] Item 104. The method of Item 103, wherein measuring the concentration includes measuring the concentration on white blood cells.

[0269] Item 105. The method of any one of Items 103 to 104, wherein measuring the concentration of bound CD59 comprises measuring the mean fluorescence intensity of bound CD59.

[0270] Item 106. The method of any one of Items 103 to 105, wherein measuring the concentration of bound CD163 comprises measuring the mean fluorescence intensity of bound CD163.

[0271] Clause 107. The method of any one of clauses 103 to 106, wherein observing the increase in the concentration of bound CD59 comprises comparing the mean fluorescence intensity of bound CD59 for the human patient with the mean fluorescence intensity of bound CD59 for a pool of human participants with <25 CL as measured by amyloid PET imaging.

[0272] Clause 108. The method of clause 107, wherein observing the increase in the concentration of bound CD59 comprises measuring an increase in mean fluorescence intensity of at least about 27%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 78%, compared to the mean fluorescence intensity of bound CD59 for a pool of human patients with <15 CL as measured by amyloid PET imaging.

[0273] Clause 109. The method of any one of clauses 103 to 108, wherein observing the decrease in the concentration of bound CD163 comprises comparing the mean fluorescence intensity of bound CD163 for the human patient with the mean fluorescence intensity of bound CD163 for a pool of human participants with <25 CL as measured by amyloid PET imaging.

[0274] Clause 110. The method of clause 109, wherein observing the decrease in the concentration of bound CD163 comprises measuring a decrease in mean fluorescence intensity of at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, or at least about 19%, compared to the mean fluorescence intensity of bound CD163 for a pool of human patients with <15 CL measured by amyloid PET imaging.

[0275] Paragraph 111. A method for determining the efficacy of a candidate AD treatment in a human patient, comprising: (A) obtaining a first blood sample from the human patient; Optionally, storing the first blood sample on ice; Optionally, testing the first blood sample within three hours of obtaining the first blood sample; The first blood sample is reacting with a mouse anti-human IgG monoclonal antibody against CD59 conjugated with a first fluorophore to obtain a first bound CD59; reacting with a mouse anti-human IgG monoclonal antibody against CD163 conjugated with a second fluorophore to obtain the first bound CD163; measuring a first concentration of the first bound CD59 and a first concentration of the first bound CD163 in the first blood sample by fluorescence of the first fluorophore and the second fluorophore; (B) administering a candidate AD treatment to the human patient; and (C) obtaining a second blood sample from the human patient; Optionally, storing the second blood sample on ice; Optionally, testing the second blood sample within three hours of obtaining the second blood sample; The second blood sample is reacting with a mouse anti-human IgG monoclonal antibody against CD59 conjugated with a first fluorophore to obtain a second bound CD59; reacting with a mouse anti-human IgG monoclonal antibody against CD163 conjugated with a second fluorophore to obtain a second bound CD163; measuring a second concentration of the second bound CD59 and a second concentration of the second bound CD163 in the second blood sample by fluorescence of the first fluorophore and the second fluorophore; (D) observing a decrease in the human patient from the first concentration of the first binding CD59 to the second concentration of the second binding CD59; observing an increase in first bound CD163 from the first concentration to the second concentration of second bound CD163; (E) thereby determining the efficacy of the candidate AD treatment in a human patient.

[0276] Clause 112. The method of clause 111, wherein the candidate AD treatment is a phagocytosis promoter.

[0277] Clause 113. The method of clause 111, wherein the candidate AD treatment is a scavenger receptor agonist.

[0278] Item 114. The method of any one of items 111 to 113, wherein measuring the first concentration comprises measuring the first concentration on white blood cells, and measuring the second concentration comprises measuring the second concentration on white blood cells.

[0279] Item 115. Use of a binding agent against CD59 and a binding agent against CD163 for the diagnosis of AD in a human patient in need thereof.

[0280] Clause 116. The use of clause 115, wherein the binding agent is selected from a single chain variable fragment, an antibody mimetic, an antibody fragment, an antibody, a monoclonal antibody, and combinations thereof.

[0281] Item 117. The binding agent for CD59 is conjugated to a first detectable moiety; the binding agent to CD163 is conjugated to a second detectable moiety; Item 117. Use according to any one of items 115 to 116.

[0282] Clause 118. The use of Clause 117, wherein the first detectable moiety and the second detectable moiety are independently selected from a radioisotope, a stable isotope, a fluorophore, and combinations thereof.

[0283] Paragraph 119. A method for diagnosing AD in a human patient, comprising: obtaining a blood sample from said human patient; Optionally, storing the blood sample on ice; optionally testing said blood sample within 3 hours of obtaining said blood sample; The blood sample reacting the scavenger receptor with a mouse anti-human IgG monoclonal antibody against the scavenger receptor conjugated with a fluorophore to obtain a bound scavenger receptor; measuring the concentration of the bound scavenger receptor in the blood sample by fluorescence of the fluorophore; observing a decrease in the concentration of the bound scavenger receptor compared to a range of scavenger receptor concentrations in a healthy human; thereby diagnosing AD in said human patient; and 1. A method for diagnosing AD in a human patient, comprising:

[0284] Paragraph 120. A method for diagnosing AD in a human patient, comprising: obtaining a blood sample from said human patient; Optionally, storing the blood sample on ice; optionally testing said blood sample within 3 hours of obtaining said blood sample; The blood sample reacting with a plurality of antibody-fluorophore conjugates, each of the plurality of antibody-fluorophore conjugates comprising a mouse anti-human IgG monoclonal antibody against a scavenger receptor conjugated to a fluorophore; thereby forming a bound scavenger receptor; measuring the concentration of the bound scavenger receptor in the blood sample by fluorescence of the fluorophore; observing a decrease in the concentration of at least one bound scavenger receptor relative to a range of concentrations of a corresponding scavenger receptor in a healthy human; thereby diagnosing AD in said human patient; and 1. A method for diagnosing AD in a human patient, comprising:

[0285] Paragraph 121. The method of any one of paragraphs 119 to 120, wherein the scavenger receptor is selected from CD163, MerTK, CD18, and combinations thereof.

[0286] Paragraph 122. The method of any one of paragraphs 119 to 121, wherein the scavenger receptor is expressed on a leukocyte.

[0287] Paragraph 123. A method for determining the efficacy of a candidate AD treatment in a human patient, comprising: (A) obtaining a first blood sample from a human patient; Optionally, storing the first blood sample on ice; optionally, testing the first blood sample within three hours of obtaining the first blood sample; A first blood sample is reacting with a first aliquot of a plurality of antibody-fluorophore conjugates, each of the plurality of antibody-fluorophore conjugates comprising a mouse anti-human IgG monoclonal antibody against a scavenger receptor conjugated to a fluorophore; thereby forming a first bound scavenger receptor; measuring a first concentration of a first bound scavenger receptor in the first blood sample by fluorescence of the fluorophore; (B) administering a candidate AD treatment to the human patient; and (C) obtaining a second blood sample from the human patient; Optionally, storing the second blood sample on ice; optionally, testing the second blood sample within three hours of obtaining the second blood sample; A second blood sample is reacting with a second aliquot of the plurality of antibody-fluorophore conjugates; thereby forming a second bound scavenger receptor; measuring a second concentration of a second bound scavenger receptor in a second blood sample by fluorescence of the fluorophore; (D) observing an increase in the first concentration of the first bound scavenger receptor to a second concentration of the second bound scavenger receptor in the human patient; (E) thereby determining the efficacy of the candidate AD treatment in a human patient.

[0288] Item 124. The method of Item 123, wherein the candidate AD treatment is a phagocytosis promoter.

[0289] Item 125. The method of Item 123, wherein the candidate AD treatment is a scavenger receptor agonist.

[0290] Item 126. The method of any one of items 123 to 125, wherein measuring the first concentration comprises measuring the first concentration on white blood cells, and measuring the second concentration comprises measuring the second concentration on white blood cells.

[0291] Paragraph 127. The method of any one of paragraphs 123 to 126, wherein the scavenger receptor is selected from CD163, MerTK, CD18, and combinations thereof. Item 128. A composition comprising: A composition comprising a first binding agent to CD18 conjugated to a first detectable moiety.

[0292] Clause 129. The composition of Clause 128, wherein the first binding agent is selected from a single chain variable fragment, an antibody mimetic, an antibody fragment, an antibody, a monoclonal antibody, and combinations thereof.

[0293] Paragraph 130. The composition of any one of paragraphs 128-129, wherein the first detectable moiety is selected from a radioisotope, a stable isotope, a fluorophore, and combinations thereof.

[0294] Item 131. A composition comprising: A composition comprising a monoclonal antibody against CD18 conjugated to a first fluorophore.

[0295] Item 132. A composition comprising: A composition comprising a mouse anti-human IgG monoclonal antibody against CD18 conjugated to a first fluorophore.

[0296] Item 133. The composition of any one of Items 131 to 132, further comprising whole blood from a human patient.

[0297] Item 134. A kit comprising: A kit comprising a first binding agent for CD18 conjugated to a first detectable moiety.

[0298] Clause 135. The kit of Clause 134, wherein the first binding agent is selected from a single chain variable fragment, an antibody mimetic, an antibody fragment, an antibody, a monoclonal antibody, and combinations thereof.

[0299] Clause 136. The kit of any one of clauses 134-135, wherein the first detectable moiety is selected from a radioisotope, a stable isotope, a fluorophore, and combinations thereof.

[0300] Item 137. A kit comprising: A kit comprising a monoclonal antibody against CD18 conjugated to a first fluorophore.

[0301] Item 138. A kit comprising: A kit comprising a mouse anti-human IgG monoclonal antibody against CD18 conjugated to a first fluorophore.

[0302] Paragraph 139. A method for measuring the relative expression of CD18 in a human patient, comprising: obtaining a sample of whole blood from said human patient; The sample contacting the cells with a monoclonal antibody against CD18 conjugated to a first fluorophore; forming a bound CD18; measuring the fluorescence of the first fluorophore; and thereby determining said relative expression of CD18; 1. A method for measuring the relative expression of CD18 in a human patient, comprising:

[0303] Paragraph 140. A method for measuring the relative expression of CD18 in a human patient, comprising: obtaining a sample of whole blood from said human patient; The sample contacting the cells with a mouse anti-human IgG monoclonal antibody against CD18 conjugated to a first fluorophore; forming a bound CD18; measuring the fluorescence of the first fluorophore; and thereby determining said relative expression of CD18; 1. A method for measuring the relative expression of CD18 in a human patient, comprising:

[0304] Paragraph 141. The method of any one of paragraphs 139 to 140, wherein said relative expression of CD18 is the relative expression of CD18 on leukocytes.

[0305] Paragraph 142. A method for diagnosing AD in a human patient, comprising: obtaining a blood sample from said human patient; Optionally, storing the blood sample on ice; optionally testing said blood sample within 3 hours of obtaining said blood sample; The blood sample reacting with a mouse anti-human IgG monoclonal antibody against CD18 conjugated with a first fluorophore to obtain bound CD18; determining the concentration of the bound CD18 in the blood sample by fluorescence of the first fluorophore; observing a decrease in the concentration of the bound CD18 compared to the range of concentrations of CD18 in healthy humans; thereby diagnosing AD in said human patient; and 1. A method for diagnosing AD in a human patient, comprising:

[0306] Clause 143. The method of clause 142, wherein measuring the concentration comprises measuring the concentration on white blood cells.

[0307] Item 144. Use of a binding agent to CD18 for the diagnosis of AD in a human patient in need thereof.

[0308] Clause 145. The use of clause 144, wherein the binding agent is selected from a single chain variable fragment, an antibody mimetic, an antibody fragment, an antibody, a monoclonal antibody, and combinations thereof.

[0309] Section 146. 146. The use of any one of paragraphs 144 to 145, wherein the binding agent to CD18 is conjugated to a first detectable moiety.

[0310] Clause 147. The use of Clause 146, wherein the first detectable moiety is selected from a radioisotope, a stable isotope, a fluorophore, and combinations thereof.

[0311] Paragraph 148. A method for determining the efficacy of a candidate AD treatment in a human patient, comprising: (A) obtaining a first blood sample from the human patient; Optionally, storing the first blood sample on ice; Optionally, testing the first blood sample within three hours of obtaining the first blood sample; The first blood sample is reacting with a mouse anti-human IgG monoclonal antibody against CD18 conjugated with a first fluorophore to obtain a first bound CD18; measuring a first concentration of the first bound CD18 in the first blood sample by fluorescence of the first fluorophore; (B) administering a candidate AD treatment to the human patient; and (C) obtaining a second blood sample from the human patient; Optionally, storing the second blood sample on ice; Optionally, testing the second blood sample within three hours of obtaining the second blood sample; reacting the second blood sample with a mouse anti-human IgG monoclonal antibody against CD18 conjugated to a first fluorophore to obtain second bound CD18; measuring a second concentration of the second bound CD18 in the second blood sample by fluorescence of the first fluorophore; (D) observing an increase in the first concentration of bound CD18 from the first concentration to the second concentration of bound CD18; (E) thereby determining the efficacy of the candidate AD treatment in a human patient.

[0312] Clause 149. The method of clause 148, wherein the candidate AD treatment is a phagocytosis promoter.

[0313] Clause 150. The method of clause 148, wherein the candidate AD treatment is a scavenger receptor agonist.

[0314] Item 151. The method of any one of items 148 to 150, wherein measuring the first concentration comprises measuring the first concentration on white blood cells, and measuring the second concentration comprises measuring the second concentration on white blood cells.

[0315] Item 152. A composition comprising: one or more first binding agents for one or more of CD33, CD35, CD36, CD59, CD91, P2X7 and RAGE conjugated to one or more first detectable moieties; one or more second binding agents for one or more of CD18, CD163 and MerTK conjugated to one or more second detectable moieties; A composition comprising:

[0316] Item 153. A composition comprising: one or more monoclonal antibodies against one or more of CD33, CD35, CD36, CD59, CD91, P2X7 and RAGE conjugated to one or more first fluorophores; one or more monoclonal antibodies against one or more of CD18, CD163 and MerTK conjugated to one or more second fluorophores; A composition comprising:

[0317] Item 154. The composition of any one of items 152 to 153, further comprising at least one anticoagulant.

[0318] Item 155. The composition of any one of Items 152 to 154, further comprising whole blood from a human patient.

[0319] Item 156. A kit comprising: one or more first binding agents for one or more of CD33, CD35, CD36, CD59, CD91, P2X7 and RAGE conjugated to one or more first detectable moieties; one or more second binding agents for one or more of CD18, CD163 and MerTK conjugated to one or more second detectable moieties; Includes a kit.

[0320] Item 157. A kit comprising: one or more monoclonal antibodies against one or more of CD33, CD35, CD36, CD59, CD91, P2X7 and RAGE conjugated to one or more first fluorophores; one or more monoclonal antibodies against one or more of CD18, CD163 and MerTK conjugated to one or more second fluorophores; Includes a kit.

[0321] Item 158. A composition comprising: one or more monoclonal antibodies against one or more scavenger receptors and their regulators CD163, CD91, P2X7, MerTKCD59, CD18 conjugated to different fluorophores; the fluorophores have different but compatible emission and excitation spectra in multicolor flow cytometry analysis; composition.

[0322] Item 159. A composition comprising: A composition comprising one or more first binding agents for one or more scavenger receptors and their regulators CD163, CD91, P2X7, MerTKCD59, CD18 conjugated to different first detectable moieties.

[0323] Item 160. A kit comprising: 1. A kit comprising one or more monoclonal antibodies against one or more of CD18 and CD59 conjugated to one or more first fluorophores.

[0324] Item 161. A kit comprising: A kit comprising one or more first binding agents for one or more of CD18 and CD59 conjugated to one or more first detectable moieties.

[0325] Item 162. A composition comprising: 1. A composition comprising one or more monoclonal antibodies against one or more of CD91, CD163, P2X7 and MerTK conjugated to one or more first fluorophores.

[0326] Item 163. A composition comprising: A composition comprising one or more first binding agents for one or more of CD91, CD163, P2X7 and MerTK conjugated to one or more first detectable moieties.

[0327] Item 164. A kit comprising: 1. A kit comprising one or more monoclonal antibodies against one or more of CD91, CD163, P2X7 and MerTK conjugated to one or more first fluorophores.

[0328] Item 165. A kit comprising: 1. A kit comprising one or more first binding agents for one or more of CD91, CD163, P2X7 and MerTK conjugated to one or more first detectable moieties.

[0329] Item 166. A composition comprising: one or more monoclonal antibodies against one or more of CD91, CD163, P2X7 and MerTK conjugated to one or more first fluorophores; one or more monoclonal antibodies against one or more of CD18 and CD59 conjugated to one or more second fluorophores; and A composition comprising:

[0330] Item 167. A composition comprising: one or more first binding agents for one or more of CD91, CD163, P2X7 and MerTK conjugated to one or more first detectable moieties; one or more second binding agents for one or more of CD18 and CD59 conjugated to one or more second detectable moieties; A composition comprising:

[0331] Item 168. A kit comprising: one or more monoclonal antibodies against one or more of CD91, CD163, P2X7 and MerTK conjugated to one or more first fluorophores; one or more monoclonal antibodies against one or more of CD18 and CD59 conjugated to one or more second fluorophores; and Includes a kit.

[0332] Item 169. A kit comprising: one or more first binding agents for one or more of CD91, CD163, P2X7 and MerTK conjugated to one or more first detectable moieties; one or more second binding agents for one or more of CD18 and CD59 conjugated to one or more second detectable moieties; Includes a kit.

[0333] Item 170. A kit comprising: A kit comprising a first binding agent for one or more of CD163, CD91, and MerTK conjugated to a first detectable moiety.

[0334] Clause 171. The kit of Clause 170, wherein the first binding agent is selected from a single chain variable fragment, an antibody mimetic, an antibody fragment, an antibody, a monoclonal antibody, and combinations thereof.

[0335] Clause 172. The kit of any one of clauses 170-171, wherein the first detectable moiety is selected from a radioisotope, a stable isotope, a fluorophore, and combinations thereof.

[0336] Item 173. A composition comprising: a first binding agent for CD59 conjugated to a first detectable moiety; a second binding agent to CD163 conjugated to a second detectable moiety; A composition comprising:

[0337] Clause 174. The composition of Clause 173, wherein the first binding agent and the second binding agent are independently selected from a single chain variable fragment, an antibody mimetic, an antibody fragment, an antibody, a monoclonal antibody, and combinations thereof.

[0338] Clause 175. The composition of any one of clauses 173-174, wherein the first detectable moiety and the second detectable moiety are selected from a radioisotope, a stable isotope, a fluorophore, and combinations thereof.

[0339] Item 176. A composition comprising: a monoclonal antibody against CD59 conjugated to a first fluorophore; a monoclonal antibody against CD163 conjugated to a second fluorophore; A composition comprising:

[0340] Item 177. A composition comprising: a mouse anti-human IgG monoclonal antibody against CD59 conjugated to a first fluorophore; a mouse anti-human IgG monoclonal antibody against CD163 conjugated to a second fluorophore; A composition comprising:

[0341] Item 178. The composition of any one of items 176 to 177, further comprising at least one anticoagulant.

[0342] Clause 179. The composition of any one of clauses 176 to 178, wherein the at least one anticoagulant is selected from ethylenediaminetetraacetic acid, at least one salt thereof, citric acid, at least one salt thereof, or any combination of the foregoing.

[0343] Item 180. The composition of any one of Items 176 to 179, further comprising bovine serum albumin and sodium azide.

[0344] Item 181. The composition of any one of Items 176 to 180, further comprising phosphate buffered saline, gelatin, and sodium azide.

[0345] Item 182. The composition of any one of items 176 to 181, wherein the composition does not contain a fluorescent dye sold under the trademark Alexa Fluor® 488 (Molecular Probes, Inc., Eugene, Oregon).

[0346] Item 183. The composition of any one of Items 176 to 182, wherein the first fluorophore is R-phycoerythrin.

[0347] Item 184. The composition of any one of items 176 to 183, wherein the second fluorophore is a fluorescent dye sold under the trademark Alexa Fluor® 647 (Molecular Probes, Inc., Eugene, Oregon).

[0348] Item 185. The composition of any one of Items 176 to 184, further comprising whole blood from a human patient.

[0349] Item 186. A kit comprising: a first binding agent for CD59 conjugated to a first detectable moiety; a second binding agent to CD163 conjugated to a second detectable moiety; Includes a kit.

[0350] Clause 187. The kit of Clause 186, wherein the first binding agent and the second binding agent are independently selected from a single chain variable fragment, an antibody mimetic, an antibody fragment, an antibody, a monoclonal antibody, and combinations thereof.

[0351] Clause 188. The kit of any one of clauses 186-187, wherein the first detectable moiety and the second detectable moiety are independently selected from a radioisotope, a stable isotope, a fluorophore, and combinations thereof.

[0352] Item 189. A kit comprising: a monoclonal antibody against CD59 conjugated to a first fluorophore; a monoclonal antibody against CD163 conjugated to a second fluorophore; Includes a kit.

[0353] Item 190. A kit comprising: a mouse anti-human IgG monoclonal antibody against CD59 conjugated to a first fluorophore; a mouse anti-human IgG monoclonal antibody against CD163 conjugated to a second fluorophore; Includes a kit.

[0354] Item 191. The kit of any one of Items 189 to 190, wherein the kit does not include a fluorescent dye sold under the trademark Alexa Fluor® 488 (Molecular Probes, Inc., Eugene, Oregon).

[0355] Item 192. The kit of any one of Items 189 to 191, wherein the first fluorophore is R-phycoerythrin.

[0356] Clause 193. The kit of any one of clauses 189 to 192, wherein the second fluorophore is a fluorescent dye sold under the trademark Alexa Fluor® 647 (Molecular Probes, Inc., Eugene, Oregon).

[0357] Item 194. Use of a binding agent against CD59 and a binding agent against CD163 for the diagnosis of AD in a human patient in need thereof.

[0358] Clause 195. The use of Clause 194, wherein the binding agent is selected from a single chain variable fragment, an antibody mimetic, an antibody fragment, an antibody, a monoclonal antibody, and combinations thereof.

[0359] Item 196. The binding agent for CD59 is conjugated to a first detectable moiety; the binding agent to CD163 is conjugated to a second detectable moiety; Item 194-195. Use according to any one of items 194-195.

[0360] Clause 197. The use of Clause 196, wherein the first detectable moiety and the second detectable moiety are independently selected from a radioisotope, a stable isotope, a fluorophore, and combinations thereof.

[0361] Item 198. A composition comprising: a first binding agent to CD91 conjugated to a first detectable moiety; a second binding agent for CD59 conjugated to a second detectable moiety; a third binding agent to CD163 conjugated to a third detectable moiety; A composition comprising:

[0362] Clause 199. The composition of Clause 198, wherein the first binding agent, the second binding agent, and the third binding agent are independently selected from a single chain variable fragment, an antibody mimetic, an antibody fragment, an antibody, a monoclonal antibody, and combinations thereof.

[0363] Clause 200. The composition of any one of clauses 198-199, wherein the first detectable moiety, the second detectable moiety, and the third detectable moiety are independently selected from a radioisotope, a stable isotope, a fluorophore, and combinations thereof.

[0364] Item 201. A composition comprising: a monoclonal antibody against CD91 conjugated to a first fluorophore; a monoclonal antibody against CD59 conjugated to a second fluorophore; a monoclonal antibody against CD163 conjugated to a third fluorophore; and A composition comprising:

[0365] Item 202. A composition comprising: a mouse anti-human IgG monoclonal antibody against CD91 conjugated to a first fluorophore; a mouse anti-human IgG monoclonal antibody against CD59 conjugated to a second fluorophore; a mouse anti-human IgG monoclonal antibody against CD163 conjugated to a third fluorophore; A composition comprising:

[0366] Item 203. The composition according to any one of items 201 to 202, further comprising at least one anticoagulant.

[0367] Clause 204. The composition of any one of clauses 201 to 203, wherein the at least one anticoagulant is selected from ethylenediaminetetraacetic acid, at least one salt thereof, citric acid, at least one salt thereof, or any combination of the foregoing.

[0368] Item 205. The composition of any one of Items 201 to 204, further comprising bovine serum albumin and sodium azide.

[0369] Item 206. The composition according to any one of Items 201 to 205, further comprising phosphate buffered saline, gelatin, and sodium azide.

[0370] Item 207. The composition of any one of items 201 to 206, wherein the first fluorophore is fluorescein isothiocyanate.

[0371] Item 208. The composition of any one of items 201 to 207, wherein the composition does not contain a fluorescent dye sold under the trademark Alexa Fluor® 488 (Molecular Probes, Inc., Eugene, Oregon).

[0372] Item 209. The composition of any one of items 201 to 208, wherein the second fluorophore is R-phycoerythrin.

[0373] Item 210. The composition of any one of items 201 to 209, wherein the third fluorophore is a fluorescent dye sold under the trademark Alexa Fluor® 647 (Molecular Probes, Inc., Eugene, Oregon).

[0374] Item 211. The composition of any one of Items 201 to 210, further comprising whole blood from a human patient.

[0375] Item 212. A kit comprising: a first binding agent to CD91 conjugated to a first detectable moiety; a second binding agent for CD59 conjugated to a second detectable moiety; a third binding agent to CD163 conjugated to a third detectable moiety; Includes a kit.

[0376] Clause 213. The kit of Clause 212, wherein the first binding agent, the second binding agent, and the third binding agent are independently selected from a single chain variable fragment, an antibody mimetic, an antibody fragment, an antibody, a monoclonal antibody, and combinations thereof.

[0377] Clause 214. The kit of any one of clauses 212-213, wherein the first detectable moiety, the second detectable moiety, and the third detectable moiety are independently selected from a radioisotope, a stable isotope, a fluorophore, and combinations thereof.

[0378] Item 215. A kit comprising: a monoclonal antibody against CD91 conjugated to a first fluorophore; a monoclonal antibody against CD59 conjugated to a second fluorophore; a monoclonal antibody against CD163 conjugated to a third fluorophore; and Includes a kit.

[0379] Item 216. A kit comprising: a mouse anti-human IgG monoclonal antibody against CD91 conjugated to a first fluorophore; a mouse anti-human IgG monoclonal antibody against CD59 conjugated to a second fluorophore; a mouse anti-human IgG monoclonal antibody against CD163 conjugated to a third fluorophore; Includes a kit.

[0380] Item 217. The kit according to any one of Items 215 to 216, wherein the first fluorophore is fluorescein isothiocyanate.

[0381] Item 218. The kit of any one of Items 215 to 217, wherein the kit does not include a fluorescent dye sold under the trademark Alexa Fluor® 488 (Molecular Probes, Inc., Eugene, Oregon).

[0382] Item 219. The kit of any one of Items 215 to 218, wherein the second fluorophore is R-phycoerythrin.

[0383] Clause 220. The kit of any one of clauses 215 to 219, wherein the third fluorophore is a fluorescent dye sold under the trademark Alexa Fluor® 647 (Molecular Probes, Inc., Eugene, Oregon).

[0384] Item 221. Use of a binding agent against CD91, a binding agent against CD59 and a binding agent against CD163 for the diagnosis of AD in a human patient in need thereof.

[0385] Clause 222. The use of Clause 221, wherein the binding agent is selected from a single chain variable fragment, an antibody mimetic, an antibody fragment, an antibody, a monoclonal antibody, and combinations thereof.

[0386] Item 223. The binding agent for CD91 is conjugated to a first detectable moiety; the binding agent to CD59 is conjugated to a second detectable moiety; wherein said binding agent to CD163 is conjugated to a third detectable moiety. Item 221-222. Use according to any one of items 221-222.

[0387] Clause 224. The use of Clause 223, wherein the first detectable moiety, the second detectable moiety, and the third detectable moiety are independently selected from a radioisotope, a stable isotope, a fluorophore, and combinations thereof.

[0388] Item 225. A composition comprising: a first binding agent to CD91 conjugated to a first detectable moiety; a second binding agent for CD59 conjugated to a second detectable moiety, and a third binding agent for CD163 conjugated to a third detectable moiety; A composition comprising:

[0389] Clause 226. The composition of Clause 225, wherein the first binding agent, the second binding agent, and the third binding agent are independently selected from a single chain variable fragment, an antibody mimetic, an antibody fragment, an antibody, a monoclonal antibody, and combinations thereof.

[0390] Clause 227. The composition of any one of clauses 225-226, wherein the first detectable moiety, the second detectable moiety, and the third detectable moiety are independently selected from a radioisotope, a stable isotope, a fluorophore, and combinations thereof.

[0391] Item 228. The first binding agent to CD91 is a monoclonal antibody to CD91, and the first detectable moiety is a first fluorophore; the second binding agent to CD59 is a monoclonal antibody to CD59, and the second detectable moiety is a second fluorophore; the third binding agent to CD163 is a monoclonal antibody to CD163, and the third detectable moiety is a third fluorophore. Item 226. The composition according to item 225.

[0392] Item 229. The monoclonal antibody against CD91 is a mouse anti-human IgG monoclonal antibody against CD91; the monoclonal antibody against CD59 is a mouse anti-human IgG monoclonal antibody against CD59; the monoclonal antibody against CD163 is a mouse anti-human IgG monoclonal antibody against CD163; Item 229. The composition according to item 228.

[0393] Item 230. The composition of any one of Items 228 to 229, wherein the first fluorophore is fluorescein isothiocyanate.

[0394] Item 231. The composition of any one of Items 228 to 230, wherein the second fluorophore is R-phycoerythrin.

[0395] Item 232. The composition of any one of items 228 to 231, wherein the third fluorophore is a fluorescent dye sold under the trademark Alexa Fluor® 647 (Molecular Probes, Inc., Eugene, Oregon).

[0396] Item 233. The composition of any one of Items 225 to 232, further comprising whole blood from a human patient.

[0397] Item 234. The composition of any one of items 225 to 233, further comprising at least one anticoagulant.

[0398] Item 235. A method for diagnosing AD in a human patient, comprising obtaining a blood sample from the human patient; Optionally, storing the blood sample on ice; optionally testing said blood sample within 3 hours of obtaining said blood sample; The blood sample reacting with a mouse anti-human IgG monoclonal antibody against CD91 conjugated with a first fluorophore to obtain bound CD91; reacting with a mouse anti-human IgG monoclonal antibody against CD59 conjugated with a second fluorophore to obtain bound CD59; reacting with a mouse anti-human IgG monoclonal antibody against CD163 conjugated with a third fluorophore to obtain bound CD163; measuring the concentration of bound CD91, the concentration of bound CD59, and the concentration of bound CD163 in the blood sample by fluorescence of the first fluorophore, the second fluorophore, and the third fluorophore; observing an increase in the concentration of bound CD91 compared to the range of CD91 concentrations in healthy humans; observing an increase in the concentration of bound CD59 compared to the range of CD59 concentrations in healthy humans; observing a decrease in the concentration of bound CD163 compared to a range of concentrations of CD163 in healthy humans; thereby diagnosing AD in human patients; and 1. A method for diagnosing AD in a human patient, comprising:

[0399] As mentioned above, although detailed embodiments of the present invention are disclosed herein, it should be understood that the disclosed embodiments are merely exemplary of the invention, which may be embodied in various forms. It will be understood that many modifications and other variations are within the intended scope of the invention as claimed below. Moreover, the foregoing description of various embodiments does not necessarily imply exclusion. For example, "some" embodiments may include all or part of "other" and "further" embodiments within the scope of the present invention. Also, "a" does not mean "only one," but "a" can mean "one and more." In certain embodiments, for example, the following are provided: (Item 1) A kit comprising: a first binding agent to CD91 conjugated to a first detectable moiety; a second binding agent for CD59 conjugated to a second detectable moiety; a third binding agent to CD163 conjugated to a third detectable moiety; Includes a kit. (Item 2) 2. The kit of claim 1, wherein the first binding agent, the second binding agent, and the third binding agent are independently selected from a single-chain variable fragment, an antibody mimetic, an antibody fragment, an antibody, a monoclonal antibody, and combinations thereof. (Item 3) 3. The kit of any one of items 1 and 2, wherein the first detectable moiety, the second detectable moiety, and the third detectable moiety are independently selected from a radioisotope, a stable isotope, a fluorophore, and combinations thereof. (Item 4) the first binding agent to CD91 is a monoclonal antibody to CD91, and the first detectable moiety is a first fluorophore; the second binding agent to CD59 is a monoclonal antibody to CD59, and the second detectable moiety is a second fluorophore; The third binding agent to CD163 is a monoclonal antibody to CD163. and the third detectable moiety is a third fluorophore. Item 1. The kit according to item 1. (Item 5) the monoclonal antibody against CD91 is a mouse anti-human IgG monoclonal antibody against CD91; the monoclonal antibody against CD59 is a mouse anti-human IgG monoclonal antibody against CD59; the monoclonal antibody against CD163 is a mouse anti-human IgG monoclonal antibody against CD163; Item 4. The kit according to item 4. (Item 6) 6. The kit of any one of items 4 and 5, wherein the first fluorophore is fluorescein isothiocyanate. (Item 7) 6. The kit of any one of items 4 and 5, wherein the second fluorophore is R-phycoerythrin. (Item 8) 6. The kit of any one of items 4 and 5, wherein the third fluorophore is a fluorescent dye sold under the trademark Alexa Fluor® 647 (Molecular Probes, Inc., Eugene, Oregon). (Item 9) 1. A composition comprising: a first binding agent to CD91 conjugated to a first detectable moiety; a second binding agent for CD59 conjugated to a second detectable moiety; a third binding agent to CD163 conjugated to a third detectable moiety; A composition comprising: (Item 10) 10. The composition of claim 9, wherein the first binding agent, the second binding agent, and the third binding agent are independently selected from a single chain variable fragment, an antibody mimetic, an antibody fragment, an antibody, a monoclonal antibody, and combinations thereof. (Item 11) 11. The composition of any one of items 9 and 10, wherein the first detectable moiety, the second detectable moiety, and the third detectable moiety are independently selected from a radioisotope, a stable isotope, a fluorophore, and combinations thereof. (Item 12) the first binding agent to CD91 is a monoclonal antibody to CD91, and the first detectable moiety is a first fluorophore; the second binding agent to CD59 is a monoclonal antibody to CD59, and the second detectable moiety is a second fluorophore; the third binding agent to CD163 is a monoclonal antibody to CD163, and the third detectable moiety is a third fluorophore. Item 9. The composition according to item 9. (Item 13) the monoclonal antibody against CD91 is a mouse anti-human IgG monoclonal antibody against CD91; the monoclonal antibody against CD59 is a mouse anti-human IgG monoclonal antibody against CD59; the monoclonal antibody against CD163 is a mouse anti-human IgG monoclonal antibody against CD163; Item 13. The composition according to item 12. (Item 14) 14. The composition of any one of items 12 and 13, wherein the first fluorophore is fluorescein isothiocyanate. (Item 15) 14. The composition of any one of items 12 and 13, wherein the second fluorophore is R-phycoerythrin. (Item 16) 14. The composition of any one of items 12 and 13, wherein the third fluorophore is a fluorescent dye sold under the trademark Alexa Fluor® 647 (Molecular Probes, Inc., Eugene, Oregon). (Item 17) Use of a binding agent against CD91, a binding agent against CD59 and a binding agent against CD163 for the diagnosis of AD in a human patient in need thereof. (Item 18) 18. The use according to item 17, wherein the binding agent is selected from a single chain variable fragment, an antibody mimetic, an antibody fragment, an antibody, a monoclonal antibody, and combinations thereof. (Item 19) said binding agent to CD91 is conjugated to a first detectable moiety; the binding agent to CD59 is conjugated to a second detectable moiety; wherein said binding agent to CD163 is conjugated to a third detectable moiety. 19. The use according to any one of claims 17 and 18. (Item 20) 20. The use of item 19, wherein the first detectable moiety, the second detectable moiety, and the third detectable moiety are independently selected from a radioisotope, a stable isotope, a fluorophore, and combinations thereof.

Claims

[Claim 1] The invention described in the specification.

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