Method for determining copper concentration in biological samples

Abstract

The present disclosure relates to methods for measuring copper concentrations in biological samples. More particularly, the present disclosure relates to methods for measuring non-ceruloplasmin-bound copper and / or labile-bound copper concentrations in biological samples. Such methods are particularly useful in the management and treatment of metabolic-related diseases or disorders.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Application No. 62 / 899,498, filed September 12, 2019, U.S. Application No. 62 / 944,498, filed December 6, 2019, and U.S. Application No. 62 / 958,432, filed January 8, 2020, the disclosures of which are expressly incorporated herein by reference. [Background technology]

[0002] Field of Disclosure The present disclosure relates to methods for measuring copper concentration in a biological sample. More specifically, the present disclosure relates to methods for measuring free copper (e.g., non-ceruloplasmin bound copper concentration, labile bound copper concentration, or both) in a biological sample. Such methods are particularly useful in the management and treatment of metabolic-related diseases or disorders, including, but not limited to, Wilson's disease.

[0003] background Copper (Cu) is an essential element that plays a key role in the biochemistry of all living organisms, mainly involved in the transfer of electrons by specific cuproenzymes in important metabolic pathways. Copper reactivity also contributes to copper toxicity, so copper transport and cellular compartmentalization are specifically regulated. One such regulator is ceruloplasmin (CP), a copper-containing plasma ferroxidase that plays an essential role in mammalian iron homeostasis. This protein is a member of the multicopper oxidase family of enzymes that utilizes the electrochemistry of bound copper ions. It transports more than 90% of plasma copper, allowing the delivery of plasma copper to peripheral tissues and the excretion of copper into the bile. In general, normal adult serum ceruloplasmin levels are between 200 μg / mL and 400 μg / mL. Serum ceruloplasmin levels below the lower reference limit of 200 μg / mL are traditionally considered to be characteristic of Wilson's disease (WD).

[0004] Wilson's disease (also called hepatolenticular nucleus failure) is an inherited disorder of copper transport. Wilson's disease is caused by various genetic mutations in the Cu-loading enzyme ATP7B (human). ATP7B facilitates the movement of Cu into the CP and the excretion of Cu via the bile canaliculi. The resulting defect in the hepatic excretion pathway leads to copper accumulation in tissues such as the liver, kidney, central nervous system / brain, and cornea, and copper levels remain elevated without treatment. Specifically, copper accumulation exceeds the capacity of the CP, resulting in free non-ceruloplasmin-bound copper ("NCC"), which circulates in the blood and accumulates in tissues and organs. This NCC may be loosely bound to plasma proteins (e.g., albumin, transcuprein, and low molecular weight peptides or amino acids, etc.) to form complexes ("labile bound copper" or "LBC"). NCC and LBC contain "free copper" and may contribute to and suggest the copper toxicity observed in Wilson's disease.

[0005] Therefore, a tool for managing and treating patients with copper metabolism-related disorders, such as Wilson's disease, is the measurement of free copper levels. NCC is a biomarker and a recognized end point for evaluating copper control according to the American Association for the Study of Liver Diseases (AASLD) and the European Wilson's Disease Clinical Practice Guidelines. However, under these historically accepted treatment guidelines, NCC was not measured directly. Rather, only total blood copper and CP levels were measured directly, and then NCC was estimated from the following calculation:

number

[0006] The calculation assumes that six copper atoms are always bound to a single CP molecule, and the concentrations of NCC and ceruloplasmin are directly correlated. In reality, CPs can show considerable heterogeneity in the number of copper atoms associated per CP molecule. This formula for calculating NCC assumes six copper atoms bound per CP, but the copper / CP ratio varies depending on the pathology. In fact, six to eight copper atoms can be substantially bound to a CP, and in Wilson's disease, fewer than six copper atoms are usually associated per CP molecule.

[0007] As a result, this estimation method of determining NCC has proven problematic in the clinical setting and has been criticized for its inherent shortcomings. For example, using this method and formula can produce results with negative NCC that are physiologically and numerically impossible. However, despite the fact that negative values ​​of NCC are meaningless, negative values ​​were calculated and reported in 20-50% of patients evaluated with this method.

[0008] Therefore, there remains a need for an efficient and accurate method for determining the concentration of free copper in a biological sample.

[0009] In particular, there remains a need for methods to directly measure non-ceruloplasmin-bound copper (NCC), ceruloplasmin-bound copper (CP-Cu, also known as CPC), and labile bound copper (LBC) concentrations in patients with copper metabolism-related diseases and disorders, including but not limited to Wilson's disease. Summary of the Invention [Means for solving the problem]

[0010] The present disclosure provides an efficient and accurate method for measuring copper concentration in a biological sample. Accordingly, one aspect of the present disclosure provides a method for measuring labile bound copper in a biological sample. Such a method includes: removing ceruloplasmin from the biological sample to obtain a non-ceruloplasmin sample; contacting the non-ceruloplasmin sample with a chelating agent that binds to labile bound copper; Removing non-labile bound copper to obtain a labile bound copper sample; and Measuring copper concentration in labile copper samples Includes.

[0011] In at least one embodiment, a method for measuring labile bound copper in a biological sample comprises: contacting the biological sample with an immunocapture reagent that binds ceruloplasmin; removing the captured ceruloplasmin to obtain a non-ceruloplasmin sample; contacting the non-ceruloplasmin sample with a chelating agent that binds to labile bound copper; Removing non-labile bound copper to obtain a labile bound copper sample; and Measuring copper concentration in labile copper samples Includes.

[0012] A further aspect of the disclosure provides a kit for measuring copper concentration in a biological sample. In certain embodiments of this aspect, the kit comprises an immunocapture reagent and instructions for use. In certain other embodiments of this aspect, the kit comprises an immunocapture reagent, a chelating agent, and instructions for use.

[0013] The accompanying drawings are included to provide a further understanding of the disclosed methods and materials, and are incorporated in and constitute a part of this specification. The drawings illustrate one or more embodiments of the disclosure and, together with the description, serve to explain the principles and implementations of the disclosure. [Brief description of the drawings]

[0014] [Figure 1]Different measurements of copper in biological samples such as blood, serum, or plasma are shown. "Total Copper" is all copper in the biological sample. "Total Mo" is total molybdenum, which may generally serve as a surrogate for estimating the concentration of Mo-containing drugs (such as BC-TTM) since Mo-containing drugs are difficult to measure directly. "CP-Cu" (also known as "CPC") is the copper-ceruloplasmin complex. "ALB-Cu" (also known as "Albumin-Cu") is the copper-albumin complex. "PUF-Cu" (also known as "Pep-Cu") is plasma ultrafiltrate copper, which is generally a measurement of free copper in a sample extracted, e.g., by ultrafiltration, which contains low molecular weight peptides to which copper binds loosely. [Diagram 2] 1 illustrates the direct measurement of copper in a biological sample, in accordance with at least one embodiment of the present disclosure. [Figure 3-1] 1 shows the direct measurement of copper in a biological sample according to at least one embodiment of the present disclosure. "Pep-CU" (also known as "PUF-Cu") is free copper loosely bound to low molecular weight peptides or amino acids. "MAC" is Mo-albumin trimolecular bound copper. [Figure 3-2] 1 shows the direct measurement of copper in a biological sample according to at least one embodiment of the present disclosure. "UF" is ultrafiltration to remove Pep-Cu. "IP" is immunoprecipitation to remove Pep-Cu. [Figure 3-3] 1 illustrates the direct measurement of labile bound copper (including Pep-Cu and ALB-Cu) in biological samples in accordance with at least one embodiment of the present disclosure. [Figure 4] FIG. 1 shows NCC (non-ceruloplasmin bound copper) measured using the old NCC estimation method ("old") and the instantaneous method ("new") for baseline (untreated) plasma samples from Wilson's disease. [Diagram 5] FIG. 1 shows the ratio of CPC to CP in baseline plasma samples from Wilson's disease. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Before the disclosed methods and materials are described, it is to be understood that the aspects described herein are not limited to particular embodiments, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting, unless specifically defined herein.

[0016] definition Throughout this specification, unless the context indicates otherwise, the words "comprise" and "include" and variations thereof (e.g., "comprises," "comprising," "includes," "including") are understood to imply the inclusion of a stated component, function, element, or step or group of components, functions, elements, or steps, but not to exclude any other integer or step or group of integers or steps.

[0017] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0018] The term "antibody" as used herein refers to a protein capable of recognizing and specifically binding to at least one antigen. A normal or conventional mammalian antibody typically comprises a tetramer composed of two identical pairs of polypeptide chains, each pair consisting of one "light" chain (typically having a molecular weight of about 25 kDa) and one "heavy" chain (typically having a molecular weight of about 50-70 kDa). The terms "heavy chain" and "light chain" as used herein refer to any immunoglobulin polypeptide having sufficient variable domain sequence to confer specificity for a target antigen. The amino-terminal portion of each light and heavy chain typically contains a variable domain of about 100-110 or more amino acids that are typically involved in antigen recognition. When the antibody is derived from a non-human source, the variable domain may be subjected to further protein engineering to humanize the framework regions. The carboxyl-terminal portion of each chain typically defines a constant domain that is responsible for effector function. Thus, in a naturally occurring antibody, the full-length heavy chain immunoglobulin polypeptide comprises a variable domain (V H ) and three constant domains (C H1 , C H2 , and C H3 ), and C H1 and C. H2 wherein V comprises a hinge region between H The domain is located at the amino terminus of the polypeptide and is C H3 The domains are located at the carboxyl terminus, and full-length light chain immunoglobulin polypeptides contain a variable domain (V L ) and the constant domain (C L ), where V L The domain is located at the amino terminus of the polypeptide and is C LThe domain is at the carboxyl terminus. Antibodies as used herein may include, for example, polyclonal antibodies, monoclonal antibodies, chimerized or chimeric antibodies, humanized antibodies, primatized antibodies, deimmunized antibodies, and fully human antibodies. Antibodies can be made in or derived from any of a variety of species, for example, mammals such as humans, non-human primates (e.g., orangutans, baboons, chimpanzees), horses, cows, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. The antibodies may be purified or recombinant antibodies. The antibodies may also be engineered or antibody-like proteins (e.g., fusion proteins) that contain at least one immunoglobulin domain. The engineered or antibody-like proteins may also be bispecific or trispecific antibodies, or dimeric, trimeric, or multimeric antibodies, or diabodies, DVD-Ig, CODV-Ig, AFFIBODY® molecule antibody mimics, or nanobodies.

[0019] The term "antibody fragment" or "antigen-binding fragment" refers to an intact or full-length chain or a portion of an antibody, generally the target binding or variable region. Examples of antibody fragments include F ab , F ab’ , F (ab’)2 and F v As used herein, the term "functional fragment" is generally synonymous with "antibody fragment" and, with respect to antibodies, F v , F ab , F (ab’)2 It may refer to an antibody fragment such as.

[0020] As used herein, the term "patient" includes human and animal subjects.

[0021] As used herein, the term "therapeutic agent" or "therapeutic composition" refers to a compound or composition capable of eliciting a desired therapeutic effect when properly administered to a patient.

[0022] When used in reference to a therapeutic agent or composition, the term "effective amount" or "therapeutically effective amount" refers to an amount or dosage sufficient to produce a desired therapeutic result. More specifically, an effective amount is sufficient to inhibit one or more clinically defined pathological processes associated with the condition being treated over a period of time. The effective amount may vary depending on the specific therapeutic agent being used, and also depends on various factors and conditions related to the patient being treated and the severity of the disorder.

[0023] As used herein, the term "free copper" refers to free, non-ceruloplasmin-bound copper present in the patient's body, and includes NCC and LBC.

[0024] Wilson's Disease Symptoms and Treatment Patients with Wilson's disease may experience a combination of hepatic, neurological, psychiatric, and ocular symptoms. For example, hepatic symptoms are first seen in patients aged 9-13 years and may include both acute liver failure and chronic liver disease. Neurological signs and symptoms may include, for example, dysarthria, dystonia, tremor, and ataxia. Psychiatric symptoms may range, for example, from irritability to personality changes and depression. Corneal copper deposits, also known as Kayser-Fleischer rings, may also be present. Wilson's disease symptoms may also be present in other locations such as the patient's skin, joints, and kidneys. Additionally, a subpopulation of Wilson's disease patients is classified as "presymptomatic." Presymptomatic patients have the Wilson's disease gene mutation and associated biochemical abnormalities but are otherwise asymptomatic.

[0025] Without treatment, Wilson's disease is fatal, but with treatment, and if the patient is well compliant with treatment, life expectancy can be normal.

[0026] Treatment of Wilson's disease aims to remove copper accumulated in body tissues and then prevent copper from reaccumulating. D-penicillamine and trientine are two chelating agents that can be used to treat symptomatic Wilson's disease. D-penicillamine can be considered a first-line treatment, but some patients require switching to trientine after experiencing adverse events. Non-limiting examples of penicillamine include CUPRIMINE® (Valeant Pharmaceuticals, Inc.) and DEPEN® (Mylan Specialty LP). Trientine can also be used as a first-line treatment. Non-limiting examples of trientine include trientine hydrochloride (e.g., SYPRINE® (Valeant Pharmaceuticals, Inc.)) and trientine tetrahydrochloride (e.g., CUPRIOR® (gmp-orphan SA)). Once a patient's copper levels are reduced, the goal of treatment is to prevent copper reaccumulation and provide maintenance therapy. Zinc salts (non-limiting examples include GALZIN® (zinc acetate) (Teva Pharmaceuticals) and WILZIN® (zinc acetate dihydrate) (Recordati Rare Diseases)) can be used for maintenance treatment and can also be used as a first-line treatment for patients, including asymptomatic patients, to reduce copper absorption. In the past, ammonium tetrathiomolybdate has been investigated as a potential treatment option. Biscoline tetrathiomolybdate (BC-TTM), a copper-protein binder, can also be used to treat Wilson's disease. BC-TTM can rapidly form a highly specific copper-protein complex, detoxifying free copper in the liver and blood and promoting biliary excretion of copper. Finally, patients who present with acute liver failure, decompensated liver disease, or who do not respond to treatment may require liver transplantation.

[0027] Patients with Wilson's disease may be subject to dietary restrictions intended to limit the patient's copper consumption and copper accumulation. For example, liver should not be consumed during the decopperization period, as it can be very high in copper due to the high mineral content of the animal's diet. Shellfish have moderately high copper levels, and drinking water may also have high copper levels. If the patient's drinking water contains more than 0.1 mg of copper per liter, distilled or demineralized water should be used. The patient's diet may need to be adjusted to consume no more than 1 or 2 milligrams of copper daily. A copper-restricted diet may consist of foods with low copper content, excluding chocolate, nuts, shellfish, mushrooms, liver, molasses, broccoli, and copper-rich cereals and dietary supplements.

[0028] How to measure free copper In view of the present disclosure, the methods described herein can be configured by those skilled in the art to meet desired demands. In general, the disclosed methods provide improvements in the measurement of copper concentration in biological samples. For example, the disclosed methods provide efficient and accurate measurement of free copper in samples, eliminating some of the problems associated with currently used methods, such as biologically impossible negative values ​​of calculated NCC (which are based on erroneous assumptions from fully functional non-Wilson disease CP value characteristics). Certain embodiments of the disclosed methods provide accurate and reliable quantification of free copper, since the disclosed methods are direct measurements of free copper (i.e., not calculated estimates).

[0029] Thus, one aspect of the present disclosure provides a method for measuring the concentration of labilely bound copper in a biological sample, which comprises contacting the sample with an immunocapture reagent that binds ceruloplasmin, removing the immunocaptured ceruloplasmin to obtain a non-ceruloplasmin sample, contacting the non-ceruloplasmin sample with a chelator that binds labilely bound copper, removing the non-labilely bound copper to obtain a labilely bound copper sample, and measuring the copper concentration in the labilely bound copper sample.

[0030] In another embodiment of the present disclosure, a method for measuring NCC concentration in a biological sample is provided. Such a method is shown in FIG. 2. In this method, the sample is contacted with an immunocapture reagent that binds ceruloplasmin, and the captured ceruloplasmin is removed to obtain a non-ceruloplasmin sample. The NCC concentration is measured in the non-ceruloplasmin sample.

[0031] In general, any sample containing ceruloplasmin is a biological sample and can be used in the disclosed method. One of the characteristics of Wilson's disease is a serum ceruloplasmin concentration of less than 200 μg / mL. Thus, in some embodiments, the biological sample used in the disclosed method has a ceruloplasmin concentration of less than about 200 μg / mL. In some embodiments, the sample used in the disclosed method has a ceruloplasmin concentration in the range of about 200 μg / mL to about 400 μg / mL.

[0032] In certain embodiments, the sample is human plasma or human serum. In some embodiments, the sample is human plasma. In some embodiments, the sample is human serum. In some embodiments, the sample is mammalian plasma or mammalian serum.

[0033] As noted above, in certain embodiments of the methods of the present disclosure described herein, the sample is contacted with an immunocapture reagent that binds to ceruloplasmin. The captured ceruloplasmin is removed to obtain a non-ceruloplasmin sample. For example, in certain embodiments, the immunocapture reagent is a ceruloplasmin capture reagent.

[0034] In certain embodiments of the disclosed method described herein, the immunocapture reagent is an immunoprecipitation reagent. For example, in certain embodiments, an anti-ceruloplasmin immobilized solid support can be used as an immunoprecipitation reagent. In some embodiments, the immunoprecipitation reagent is a free anti-ceruloplasmin binding moiety configured to form a complex with ceruloplasmin and then immobilize on a solid support. Any suitable solid support known in the art can be used. For example, in certain embodiments, the solid support is at least one solid support selected from magnetic beads, agarose resin, chromatography plate, streptavidin plate, and titer plate. In at least one embodiment, the solid support is magnetic beads. In another embodiment, the solid support is selected from agarose resin, chromatography plate, streptavidin plate, and titer plate.

[0035] The solid support may be functionalized with one or more anti-ceruloplasmin reagents selected from monoclonal antibodies, polyclonal antibodies, chimerized or chimeric antibodies, humanized antibodies, primatized antibodies, deimmunized antibodies, fully human antibodies, bispecific antibodies, diabodies, antigen-binding fragments thereof, and peptides. In certain embodiments of the methods of the present disclosure described herein, the immunocapture reagent is at least one of a monoclonal or polyclonal goat anti-human ceruloplasmin antibody.

[0036] A particular anti-ceruloplasmin reagent, such as an anti-ceruloplasmin antibody, may be selected by evaluating the efficiency of the reagent to bind CP. For example, an anti-ceruloplasmin reagent may be selected based on its efficiency of depleting CP from a biological sample. In certain embodiments, an anti-CP antibody that shows high efficiency of CP depletion by measuring ceruloplasmin in a biological sample after immunocapture may be used in the methods of the present disclosure. In at least one exemplary embodiment, an antibody having the MS parameters provided in Table 1 may be classified as having high efficiency of CP depletion (e.g., high CP binding). Thus, in certain embodiments, an anti-ceruloplasmin reagent (e.g., an anti-ceruloplasmin antibody) may deplete at least 90% of the CP, e.g., at least 92% or at least 94% of the CP, from the total CP in a biological sample. In certain embodiments, an anti-ceruloplasmin reagent may deplete at least 95% of the CP, e.g., at least 96% or at least 97% of the CP, from the total CP in a biological sample. In certain embodiments, the anti-ceruloplasmin reagent can deplete at least 98% of the CP, such as at least 98.5% or at least 99% of the CP, or even more than 99% of the CP, from the total CP in the biological sample.

[0037] [Table 1]

[0038] Generally, the measurement of copper concentration can be performed using inductively coupled plasma mass spectrometry (ICP-MS). Other analytical methods suitable for measuring copper concentration can be used, including, but not limited to, inductively coupled plasma optical emission spectrometry (ICP-OES) and Zeeman graphite furnace atomic absorption spectrometry (GFAAS).

[0039] In certain embodiments of the disclosed methods described herein, an internal standard (IS) is introduced into the labile bound copper sample or non-ceruloplasmin sample prior to measuring the copper concentration. In certain embodiments, the internal standard comprises at least one of copper, rhodium, and indium. In certain embodiments, the internal standard comprises at least one of copper and rhodium.

[0040] One of the therapeutic agents used to treat Wilson's disease is bischoline tetrathiomolybdate (BC-TTM), which acts by binding and removing serum copper with the tetrathiomolybdate anion. When tetrathiomolybdate binds to copper associated with proteins in tissues or blood, a tightly bound trimolecular complex with protein / copper (typically albumin / copper) is formed. The formation of this tetrathiomolybdate-copper-albumin trimolecular complex is characteristic of the mechanism of action of BC-TTM and distinguishes it from chelators that do not form protein complexes with copper. As a result, molybdenum (Mo) has been used as a surrogate measure to estimate BC-TTM exposure and adjust the effective therapeutic dose. To assess NCC in patients treated with BC-TTM, NCC was measured using a 10-μg / mL ELISA. corrected The concept was incorporated into the BC-TTM study, which uses plasma Mo as a measure of the tetrathiomolybdate-copper-albumin trimolecular complex. NCC corrected is calculated as follows:

number

[0041] The disclosed method further allows for direct quantification of NCC even in patients undergoing BC-TTM. For example, the disclosed method also allows for direct measurement of copper concentration in tetrathiomolybdate-copper-albumin trimolecular complex (MAC or Mo-Alb-Cu). Such an embodiment is shown diagrammatically in Figures 3A and 3B.

[0042] Thus, in certain embodiments, the method of the present disclosure described herein further comprises contacting the non-ceruloplasmin sample with a molybdenum capture reagent to obtain a molybdenum sample. The molybdenum capture reagent may be a chelating competitive reagent or a surfactant. In certain embodiments, the method further comprises measuring the molybdenum-bound copper concentration in the molybdenum sample. The copper concentration can be measured as described above for measuring copper in the non-ceruloplasmin sample. For example, the copper concentration of the molybdenum sample is measured using inductively coupled plasma mass spectrometry. The accurate non-ceruloplasmin-bound copper concentration can be obtained by subtracting the copper concentration of the molybdenum sample from the copper concentration of the non-ceruloplasmin sample. In some embodiments, the non-ceruloplasmin sample is subjected to ultrafiltration or contacted with an immunocapture reagent before contacting with the molybdenum capture reagent to remove plasma ultrafiltrate copper.

[0043] In the methods of the present disclosure described herein, ceruloplasmin is removed by an immunocapture reagent to obtain a non-ceruloplasmin sample and an immunocaptured ceruloplasmin sample. In certain embodiments, the immunocaptured ceruloplasmin sample can be further evaluated. For example, in certain embodiments, the methods of the present disclosure further include measuring the ceruloplasmin concentration of the immunocaptured ceruloplasmin sample. Typically, the ceruloplasmin concentration is measured using mass spectrometry. Other analytical methods suitable for measuring protein concentration in a sample can also be used. In certain embodiments, the mass spectrometry or other analytical method has an analyte (i.e., ceruloplasmin) detection limit of at least about 5 μg / mL. In certain embodiments, the measurement of the ceruloplasmin concentration is performed using liquid chromatography mass spectrometry (LC-MS). In other embodiments, the methods of the present disclosure further include measuring the copper concentration of the immunocaptured ceruloplasmin sample. The copper concentration can be measured as described above for measuring copper in a non-ceruloplasmin sample. For example, the copper concentration of the immunocaptured ceruloplasmin sample is measured using inductively coupled plasma mass spectrometry.

[0044] In certain embodiments, the disclosed method described herein further comprises contacting the non-ceruloplasmin sample with a chelating agent that binds to labilely bound copper present in the sample. In at least one embodiment, such a chelating agent does not bind to copper present in the MAC. The MAC can then be removed from the sample, leaving a sample containing labilely bound copper ("labilely bound copper sample").

[0045] Such an embodiment is shown diagrammatically in FIG. 3C. In FIG. 3C, the first row shows various bound forms of copper present in a biological sample, such as Pep-Cu, albumin-Cu, Mo-Alb-Cu trimolecular complex, and CP-Cu. The second row performs immunocapture of CP to separate the CP-Cu fraction from the NCC fraction (containing Pep-Cu, albumin-Cu, and Mo-Alb-Cu trimolecular complex). In at least one embodiment, the amount of CP-Cu and / or CP in the CP-Cu fraction can be measured. The NCC fraction shown in the third row of FIG. 3C can be subjected to chelation and filtration to separate the Mo-Alb-Cu trimolecular complex from the LBC fraction (containing Pep-Cu and albumin-Cu). In at least one embodiment, the amount of Mo and / or Cu in the Mo-Alb-Cu fraction can be measured. In at least one embodiment, the amount of Cu in the LBC fraction may be measured according to the methods described herein to obtain the amount of labile bound copper in the sample.

[0046] The chelating agent used in the methods described herein may be selected from any chelating agent that binds labilely bound copper, such as, by way of non-limiting example, trientine hydrochloride, trientine tetrahydrochloride, penicillamine, and ethylenediaminetetraacetic acid (also known as EDTA). In at least one embodiment, the chelating agent comprises EDTA.

[0047] After addition of the chelating agent, the resulting sample may be optionally mixed and / or incubated. The MAC may be removed from the non-ceruloplasmin sample by any suitable technique known to those of skill in the art, including, but not limited to, filtration. In at least one embodiment, the sample is centrifuged after removal of the MAC.

[0048] Biomarkers of copper metabolism The present disclosure provides a biomarker of copper metabolism. The free copper concentration in a biological sample indicates the concentration of free copper concentration circulating in the patient's blood and possibly accumulating in the patient's tissues and organs. Thus, the NCC and / or LBC measured by the methods described herein comprise a biomarker of the patient's copper metabolism. More specifically, the NCC and / or LBC measured by the methods described herein can be used to diagnose, identify, or monitor patients with copper metabolism-related disorders or diseases described herein. In at least one such embodiment, the copper metabolism-related disorder or disease is Wilson's disease.

[0049] In certain embodiments, the biomarkers disclosed herein may be measured using the methods for measuring NCC and / or LBC disclosed herein.

[0050] The biomarkers disclosed herein may be compared to a particular validated reference range for free copper concentration in a patient. In at least one embodiment, the biomarkers are compared to a particular set of validated reference ranges for free copper concentration in subpopulations of a particular patient population of interest, such as, for example, ethnicity, age, sex, comorbidities, and other factors.

[0051] Kits for measuring copper concentration Another aspect of the present disclosure provides a kit for measuring copper concentration in a biological sample. More specifically, the present disclosure provides a kit for measuring free copper concentration (e.g., NCC and / or LBC) in a biological sample.

[0052] In certain embodiments of this aspect, a kit for measuring NCC in a biological sample comprises an immunocapture reagent as described herein and instructions for use. In at least one embodiment, the instructions for use include a specific validated reference range for the free copper concentration. In at least one further embodiment, the instructions for use include a specific validated reference range for the free copper concentration in a subpopulation of a particular population of subjects, such as, for example, ethnicity, age, sex, comorbidities, and other factors.

[0053] In certain other embodiments of this aspect, a kit for measuring LBCs in a biological sample comprises an immunocapture reagent as described herein, a chelator as described herein, and instructions for use as described herein.

[0054] In certain other embodiments of this aspect, a kit for measuring LBC in a biological sample comprises an immunocapture reagent as described herein, and instructions for use as described herein, In certain such other embodiments, for example, the instructions include instructions for using a chelator as described herein to obtain a labile bound copper sample.

[0055] In certain embodiments, the kits disclosed herein may be used to identify or diagnose patients with copper metabolism-related disorders or diseases, hi other certain embodiments, the kits disclosed herein may be used to monitor free copper in a patient over time. EXAMPLES

[0056] Certain aspects of the present disclosure are further illustrated by the following examples, which should not be construed as limiting the disclosure either in scope or spirit to the particular methods and materials described therein.

[0057] Example 1: Direct Measurement of Non-Ceruloplasmin Copper (NCC) Bioanalytical Assay Non-ceruloplasmin copper concentrations in human serum / plasma were determined using inductively coupled plasma mass spectrometry (ICP-MS) with a continuous dynode detector.

[0058] 114 plasma samples were obtained from a Phase 3 clinical trial of Wilson's disease patients prior to treatment with BC-TTM (pre-dose samples). Plasma samples were also obtained from 120 healthy volunteers (60 under 18 years of age ("pediatric") and 60 over 19 years of age ("adult").

[0059] Tosyl-activated magnetic beads are commercially available (ThermoFisher, formerly Dynabeads) and are coated with goat anti-human ceruloplasmin polyclonal antibody from Bethyl Laboratories, Inc. (Montgomery, TX). Twenty microliters of human plasma sample was diluted with 200 μL of coated magnetic beads suspended in PBST solution (phosphate-buffered saline (PBS) + 0.01% Tween®-20). Dilution was performed in a round-bottom plate for 90 minutes at room temperature with shaking at 1000 rpm. After incubation, the magnetic beads were separated from the supernatant by a magnetic stand for 5 minutes at room temperature, and then 200 μL of the resulting CP-free supernatant was transferred to a 15 mL metal-free plastic centrifuge tube.

[0060] The Cu concentration in the supernatant was measured directly as described below. The CP / magnetic bead fraction was briefly washed, and CP was quantitatively eluted according to standard procedures to provide a solution of CP and CP-Cu (immunocaptured CP sample).

[0061] 100 μL of standard samples containing copper (Cu-STD) and ceruloplasmin (CP-STD), quality control samples containing copper (Cu-QC) and ceruloplasmin (CP-QC), and blank samples were each added to a 15 mL metal-free plastic centrifuge tube. To each tube containing a plasma sample, blank, STD, or QC, 20 μL of internal standard containing copper (CU-IS) and ceruloplasmin (CP-IS) and 900 μL of 0.1% nitric acid were added, and the tubes were vortexed for about 5 minutes. The tubes were then centrifuged at 12,000 rpm for 5 minutes at room temperature, and the supernatant was transferred to a metal-free plastic tube suitable for ICP-MS analysis or LS-MS analysis on an Agilent 7800 / 7900 or Agilent 8900 autosampler.

[0062] For copper, data acquisition was performed using Mass Hunter software (Mass Hunter 4.2 Workstation software version C.01.02). For ceruloplasmin, data acquisition was performed using Analyst Software version 1.6.2 or equivalent (Applied Biosystems-MDS SCIEX) and chromatograms were integrated using Analyst. Results for plasma samples are reported in the appropriate bioanalytical method specified concentration units.

[0063] Analysis of CP-free supernatants gave non-ceruloplasmin-bound copper concentrations (NCC). Analysis of immunocaptured CP samples gave CP-Cu and CP concentrations.

[0064] In 114 pre-dose samples from a Phase 3 clinical trial, all pre-dose samples yielded detectable NCC levels, none had negative NCC values, and none had NCC values ​​below the limit of quantification (BQL). The lowest NCC detected was 0.2 μM, and the assay sensitivity was determined to be 0.08 μM. Previous analyses reported negative NCC results in 40% of samples.

[0065] For plasma samples from 120 healthy volunteers (60 under 18 years of age ("children") and 60 over 19 years of age ("adults")), the lowest NCC detected was 0.2 μM, with an overall range from 0.08 μM to 15.7 μM from baseline clinical trial samples (i.e., samples from untreated Wilson's disease patients), corresponding to 5 ng / mL to 1000 ng / mL.

[0066] To verify the accuracy and precision of the method, the NCC method was performed on different days, by different analysts, using multiple QC samples (n=18) for each concentration level. The method demonstrated both precision and accuracy at all concentrations evaluated.

[0067] Example 2: Direct Measurement of Non-Ceruloplasmin Copper (NCC) Bioanalytical Assay Clinical trial blood samples from (untreated) Wilson's disease patients were obtained by sterile venipuncture (or intravenous administration) and stored at 4°C or on ice until plasma was obtained by centrifugation at 2,000g for 30 minutes at 4°C. Plasma samples were stored at -70°C until analysis was performed. Samples were analyzed according to the process of Example 1. The data is shown in Figure 4 and is paired with previously reported NCC obtained by the previous estimation method. According to the old NCC estimation method, approximately 40% of subjects were determined to have negative baseline NCC levels, which is biologically impossible and complicates the evaluation of Wilson's disease treatment. In comparison, the method according to Example 1 showed a higher sensitivity to eliminate negative values ​​of NCC and quantify the reduction of free Cu.

[0068] Baseline (untreated) Wilson's disease plasma samples (n=42) were also assessed for the CP-Cu to CP ratio to determine the molar ratio of copper to ceruloplasmin protein (Figure 5). The mean CP-Cu / CP was 4.29 with a standard deviation of 0.92. This contrasts with the historically assumed value of 6 Cu per ceruloplasmin. (See Arredondo M. et al., Ceruloplasmin, an indicator of copper status, Biol Trace Elem Res, 2008 Summer;123(1-3):261-9.)

[0069] Example 3: Labile Bound Copper (LBC) Bioassay In this two-step bioassay, plasma samples were first subjected to bead immunocapture of CP, then to chelation, followed by filtration to remove the Mo-Alb-Cu trimolecular complex. This method provides (for the first time) a direct measurement of free Cu species LBC.

[0070] Preparation of immunocapture reagents Step 1: Preparation of 20 mg / mL bovine serum albumin (BSA) (trace copper) Approximately 20 mg / mL BSA in water is prepared by dissolving approximately 0.4 g of BSA in approximately 20 mL of purified H in a metal-free tube. 2 BSA was prepared by dissolving in 0. Approximately 4000 μL of 20 mg / mL BSA solution was added to the 30K filters (4 mL filter volume) and centrifuged at approximately 4000 x g for approximately 25 minutes. The filtrate was discarded. Approximately 3600 μL of 100 mM EDTA solution was added to each filter and the resulting solution was vortexed for approximately 30 seconds and then spun at approximately 4000 x g for approximately 25 minutes. The filtrate was discarded and the steps of adding EDTA, vortexing, and then spinning were repeated four more times.

[0071] The filtrate was then discarded, approximately 3600 μL of purified water was added to the filter, and the resulting solution was vortexed for approximately 30 seconds and then centrifuged at approximately 4000×g for approximately 15 minutes. The steps of discarding the filtrate, adding purified water, vortexing, and then centrifugation were repeated four more times. The resulting solution was stored at approximately −80° C.

[0072] Step 2: Prepare blocking buffer: 1X PBS containing 0.01% Tween®-20 Approximately 0.1 mL of Tween®-20 was added to approximately 1000 mL of 1×PBS buffer. The resulting solution was mixed well and stored at approximately room temperature.

[0073] Step 3: Prepare coupling buffer Coupling Buffer A: 0.1 M borate buffer, pH 9.5. Dissolve approximately 100 mL of 0.5 M borate buffer pH 9.5 in approximately 400 mL of H 2 The solution was mixed well and stored at approximately 4°C.

[0074] Coupling Buffer C: 3 M Ammonium Sulfate in Coupling Buffer A. Approximately 39.6 g of ammonium sulfate (MW 132.14) was dissolved in approximately 70-80 mL of Coupling Buffer A. The pH was adjusted to 9.5 using 10 M sodium hydroxide solution. An amount of Coupling Buffer A was added such that the final volume was approximately 100 mL. The solution was mixed well and stored at approximately 4 °C.

[0075] Coupling Buffer D (5 mg / mL trace copper BSA in 50 mM Tris-HCl, pH 8.5). Approximately 5 mL of 20 mg / mL (trace copper) BSA and 1 mL of 1 M Tris-HCl, pH 8.5 was added to 14 mL of water. The solution was mixed well and stored at approximately 4° C.

[0076] Step 4: Preparation of goat anti-human ceruloplasmin coated 50 mg beads Approximately 30 mg / mL M-280 tosyl-activated magnetic beads (Dynabeads®, Invitrogen) were mixed well. Approximately 1670 μL (equivalent to approximately 50 mg) of beads were transferred to a 2 mL Eppendorf tube. The tube was placed on a magnetic stand and the beads were allowed to settle for approximately 30 seconds. The supernatant was removed. The beads were washed with 1.5 mL of coupling buffer A with a magnetic stand. The bead suspension was vortexed to completely suspend the beads. Approximately 500 μL of coupling buffer A and approximately 600 μL of antibody (0.6 mg 1 mg / mL goat anti-human ceruloplasmin antibody (Bethyl)) were added to the beads. Approximately 500 μL of coupling buffer C was then added to the antibody-bead mixture. The mixture was vortexed well. The mixture was incubated overnight (approximately 18 hours) at 37° C. with rotation. The tube was placed on a magnetic stand and the supernatant was removed. Approximately 1 mL of coupling buffer D was added and the solution was incubated at approximately 37° C. for approximately 1 hour with rotation. The tube was placed on a magnetic stand and the supernatant was removed. Using the magnetic stand, the beads were washed three times with approximately 1.5 mL of blocking buffer. The beads were resuspended in approximately 1.25 mL of blocking buffer to a final bead concentration of approximately 40 mg / mL.

[0077] Plasma sample preparation Human plasma (lithium heparin) samples were obtained from BioIVT. Human plasma samples essentially contained endogenous LBCs and were therefore pre-screened to determine LBC content. Diluted plasma samples were then prepared using conventional methods.

[0078] [Table 2]

[0079] [Table 3]

[0080] [Table 4]

[0081] Immunocapture of CP Each plasma sample to be analyzed was thawed to approximately room temperature, if necessary, and then gently vortexed well. Approximately 20 μL of each sample was placed into a well on a 96-well protein low binding plate. Approximately 200 μL of M-280 tosyl-activated beads (Dynabeads) pre-coated with anti-human ceruloplasmin antibody (Bethyl Laboratories) in blocking buffer prepared as described above was added to each well. The plate was sealed and centrifuged at approximately 500 rpm for approximately 1 minute. The plate was incubated on a plate shaker at a speed of approximately 1000 rpm at approximately 25° C. for approximately 1.5 hours. The beads were then removed from each well by KingFisher Flex. The remaining solution on each well of the 96-well plate contained the NCC (CP-free) supernatant.

[0082] Chelation-filtration Approximately 200 μL of NCC supernatant from each sample was transferred to a clean metal-free tube, and then approximately 60 μL of chelate spiking solution (43.3 mM EDTA and 433.3 μM L-histidine) was added to each sample. The samples were gently mixed well and incubated at approximately 37° C. for approximately 1 hour. Optionally, the tubes could be centrifuged. Each incubated sample was transferred to a washed 30K MWCO centrifugal filter (regenerated cellulose membrane) (Millipore, AmiconUltra) and centrifuged at approximately 14,000×g for approximately 35 minutes at approximately 25° C. Approximately 200 μL of the filtrate was transferred to a new clean metal-free plastic tube and approximately 600 μL of H were added to each sample. 2 0.1%HNO in O 3 was added to a metal-free plastic tube. To each of the above metal-free tubes, about 10 μL of 100 ng / mL rhodium internal standard ("rhodium IS spike") was added. Each tube was then centrifuged at approximately 3500 rpm for about 1 minute and vortexed to mix well.

[0083] Preparation of calibration standards and diluted QC samples in diluent Diluent for mass spectrometry (MS) (0.1% HNO) as described in Tables 5 and 6 below 3 Calibration standards and QC samples in were prepared using conventional methods in the art.

[0084] [Table 5]

[0085] Cu standard stock solution: Cu standard stock (1000 μg / mL) Cu standard stock 1 (10 μg / mL) Cu standard stock 2 (1 μg / mL) Cu standard stock 3 (100ng / mL)

[0086] Mo standard stock solution Mo standard stock (1001 μg / mL) Mo standard stock 1 (100 μg / mL) Mo standard stock 2 (5 μg / mL) Mo standard stock 3 (1000ng / mL) Mo standard stock 4 (60ng / mL)

[0087] [Table 6]

[0088] Calibration standard samples were diluted by adding approximately 100 μL of each calibration standard to approximately 1265 μL of diluent (0.1% HNO 3 ) were mixed with diluent (0.1% HNO 3 Approximately 100 μL of each QC sample in 0.1% HNO 2 was added to approximately 1265 μL of diluent and mixed well. Approximately 200 μL of each of the following samples was placed in a metal-free plastic tube and vortexed well. A double blank sample (200 μL 0.1% HNO 2 3 ), blank sample (200 μL 0.1% HNO 3 ), diluent (0.1% HNO3 ) of each calibration standard sample and each QC sample diluted. 2 0.1% HNO in O 3 Approximately 600 μL (610 μL for the double blank) was added to each metal-free plastic tube. Approximately 10 μL of Rhodium IS spike was added to each metal-free tube except the double blank. Each tube was centrifuged at approximately 3500 rpm for approximately 1 minute. After centrifugation, each metal-free tube was vortexed to mix well.

[0089] Sample Analysis and Results Samples were analyzed by ICP-MS analysis using an Agilent 8900 equipped with Auto Tune operated under the conditions and parameters summarized in Tables 7 and 8. A centralized MicroMist nebulizer was used and the spray chamber temperature was maintained at approximately 2° C. The analysis was performed in He mode.

[0090] [Table 7]

[0091] [Table 8]

[0092] The LBC bioassay procedure was repeated six times for each type of sample (but only twice for the calibration standard samples). The measured LBC and Mo concentration values ​​for the samples are shown in Table 9 below.

[0093] [Table 9-1]

[0094] [Table 9-2]

[0095] [Table 9-3]

[0096] Example 4: Validation of NCC and LBC bioanalytical assays Validation of the NCC bioanalytical assay for measuring non-ceruloplasmin copper (NCC) and ceruloplasmin copper (CPC) in lithium heparinized human plasma by ICP-MS was performed. Validation of the LBC bioanalytical assay for measuring labile bound copper (LBC) in lithium heparinized human plasma by ICP-MS was also performed.

[0097] Sample preparation Since copper is an endogenous component of human blood, with total concentrations ranging from approximately 800 to approximately 1200 ng / mL in normal human plasma (approximately 20 to 100 ng / mL for non-ceruloplasmin copper), calibration standards in human plasma could not be prepared without significant interference from endogenous levels of copper. Therefore, calibration standards were prepared using 0.1% nitric acid in water ("diluent"). For the same reason, QC samples were also prepared in diluent. However, to ensure the accuracy and precision of the assay method, including the immunocapture process, matrix QC samples were prepared by diluting plasma (of established copper concentrations) with phosphate-buffered saline (PBS) or by spiking additional copper into prescreened lithium-heparinized human plasma, taking into account the average endogenous concentration levels, to achieve samples with the same Cu levels as the LLOQ QC, low QC, mid QC, and high QC samples.

[0098] Copper can only be transported to ceruloplasmin in living subjects with enzymatic assistance. Therefore, preparation of validation samples of CPC would be difficult. Only evaluation of matrix effects was performed on CPC samples.

[0099] Biological matrices: Blank lithium heparinized human plasma, 100% hemolyzed blank lithium heparinized human plasma, and blank lithium human whole blood were obtained from BIOIVT.

[0100] Blank human plasma was prepared and used during method validation. Pooled plasma was pre-screened to determine endogenous concentration levels of non-ceruloplasmin copper. The average endogenous concentration in the pooled pre-screened plasma was considered when preparing the matrix QC samples.

[0101] For hemolysis evaluation, 2% hemolyzed plasma was prepared from 100% hemolyzed lithium heparin human plasma diluted 50-fold with pooled non-hemolyzed plasma. The average endogenous concentration in pre-screened 2% hemolyzed plasma was considered when preparing medium and high QC samples for hemolysis evaluation. Plasma (pooled and individual lots) was stored at -20°C ± 8°C. Whole blood lots were stored at 4°C ± 4°C.

[0102] Concentrations of Calibration Standards: Calibration standards were prepared in diluent at the copper concentration levels listed in Table 10. In addition, the calibration standards included a "double blank" (no internal standard) sample in diluent and a "blank" (with internal standard) sample in diluent.

[0103] [Table 10]

[0104] Concentrations of QC samples: QC samples were prepared at the copper concentration levels listed in Table 11. LLOQ QC, Low QC, Mid1 QC, Mid2 QC, and High QC samples were prepared in diluent (collectively "Diluent QC Samples"). Matrix LLOQ QC, Matrix Low QC, Matrix Mid QC, and Matrix High QC samples (collectively "Matrix QC Samples") were prepared in plasma diluted with phosphate buffered saline (PBS) buffer or by spiking copper into pre-screened lithium heparin human plasma taking into account the average endogenous concentration levels.

[0105] Additional in-matrix QC and matrix-high QC samples (300 ng / mL and 750 ng / mL) were prepared in pre-screened 2% hemolyzed plasma for evaluation of hemolytic effect. 2% hemolyzed plasma was prepared from 100% hemolyzed lithium heparin human plasma (blank human plasma diluted 50-fold with pooled non-hemolyzed plasma). The average endogenous concentration in pre-screened 2% hemolyzed plasma was considered when preparing the additional in-matrix QC and matrix-high QC samples.

[0106] [Table 11]

[0107] Ceruloplasmin immunocapture: Goat anti-human ceruloplasmin antibodies were immobilized on Dynabeads® using the method described in step 4 of Example 3, except that in the final resuspension step, BSA buffer (0.5 mg / mL BSA and 0.01% Tween®-20 in PBS) was used instead of blocking buffer. Each matrix QC sample was subjected to CP immunocapture according to the “CP immunocapture” method described in Example 3, except that in the LBC assay, after about 1.5 hours of incubation, the sample plate was further centrifuged at about 500 rpm for about 1 minute, and then the beads were removed using the KingFisher Flex System. The NCC supernatant of each matrix QC sample was retained and subjected to NCC quantification in the NCC assay as described below.

[0108] In the NCC assay, the CP and CPC immobilized on the coated beads were eluted for CP and CPC quantification. To perform the elution, the removed beads were washed twice with about 300 μL of PBS containing 0.01% Tween®-20 ("blocking buffer"), followed by about 300 μL of water. The CP on the beads was eluted with about 200 μL of 30 mM HCl for about 10 min.

[0109] Approximately 10 μL of GAYPLSIEPIG[(13 C 5 , 15 A N)Val]R internal standard spike (2 μg / mL GAYP peptide in water) was added to each sample except for the double blank sample, which had about 10 μL of water added instead. The resulting samples were centrifuged at about 500 rpm for about 1 minute and vortexed at a low setting for about 1 minute. About 50 μL of each sample was transferred to a new well of a new plate for further evaluation, and the remaining solution was stored at about -70 °C for CPC quantification as described below. A dilution factor of about 1.29 could be considered for the calculation of CPC.

[0110] For the LBC assay, each NCC supernatant retained from the immunocapture step was subjected to chelation and filtration according to the “chelation-filtration” method described in Example 3.

[0111] Quantification of NCCs, CPCs, and LBCs: Quantification of NCCs, CPCs, and LBCs was performed by ICP-MS method using rhodium as the internal standard (or "IS"). In preparation for quantification, the calibration standards and diluent QC samples were diluted with diluent. Approximately 100 μL of each calibration standard was added to diluent and mixed well to create the diluted calibration standard samples (dilution can be scaled up or down by a dilution factor of approximately 13.5). The same procedure was repeated with each diluent QC sample to create the diluted diluent QC samples.

[0112] Each sample (double blank, blank, diluted calibration standard, diluted diluent QC sample, NCC supernatant sample from the immunocapture step, and eluted CPC solution from the immunocapture step (except for 155 μL used for eluted CPC solution)) was pipetted into a metal-free plastic tube and vortexed thoroughly.

[0113] Diluent was added to each of the metal-free plastic tubes. Approximately 10 μL of 100 ng / mL rhodium internal standard ("rhodium IS spike") was added to each tube, except for the double blank sample. Each tube was centrifuged at approximately 3500 rpm for approximately 1 minute.

[0114] For quantification of NCC and CPC, autotune and tune check of the Agilent 7700x ICP-MS was performed using tuning solution (Agilent, 28-1GSX2). For quantification of LBC, autotune and tune check of the Agilent 8900x ICP-MS was performed using tuning solution (Agilent, 30-182GSX2). A centralized MicroMist nebulizer was used and the temperature of the spray chamber was maintained at approximately 2°C. Analysis was performed in He mode.

[0115] Data Evaluation The following validation parameters were evaluated: linearity, sensitivity, intra- and inter-run accuracy and precision, stability, matrix effect (selectivity), hemolytic effect, batch volume, injection carryover, and chelate robustness.

[0116] To perform the evaluation, counts per second (CPS) were determined by MassHunter® Data Acquisition / Processing Software 4.2. Analyte concentrations were obtained from calibration curves constructed by plotting CPS against concentration using Watson LIMS (version 7.3).

[0117] Concentrations were calculated using linear regression according to the following formula: y=ax+b During the ceremony: y = CPS ratio of analyte / internal standard a = slope of the corresponding standard curve x = concentration of analyte (ng / mL) b = the intercept of the corresponding standard curve 1 / x 2 is used as a weighting factor.

[0118] The following formulas were used to calculate accuracy and precision: Accuracy:

number

number

[0119] The linearity of the method was evaluated in the linear range of 5.00–1000 ng / mL of copper. Linear regression (weighting factor 1 / x 2 ) was used to generate a best fit to the concentration-detector response relationship of copper in the diluent. All calibration curves were fitted with a coefficient of determination (R 2 ) ≥ 0.98 and met the predefined acceptance criteria.

[0120] Sensitivity: Validation was performed with a target LLOQ of 5.00ng / mL for copper in diluent (LLOQ QC) and a measured background concentration of 5.00ng / mL+ for copper in human plasma (matrix LLOQ QC). To assess sensitivity, six QC samples (for each of the diluent LLOQ QC sample and matrix LLOQ QC sample) prepared at the concentration level of LLOQ were analyzed as part of the individual accuracy and precision within the run and concentrations were calculated with a calibration curve. In addition, to calculate the nominal concentration of the matrix LLOQ QC sample, the diluent matrix used to prepare the matrix LLOQ QC sample was also analyzed in the same batch run (n=6) and the average endogenous concentration was determined. Results showed that the method met the predefined acceptance criteria for sensitivity in diluent (accuracy within ±20.0%, %CV ≤ 20.0%) and for sensitivity in human plasma (accuracy within ±25.0%, %CV ≤ 25.0%). Thus, the method was sensitive enough to measure the LLOQ concentrations of the analytes in either diluent or human plasma.

[0121] The intra- and inter-run accuracy and precision were verified by analyzing QC samples at different concentration levels in different runs on different days. Six replicate sample preparations were analyzed at each concentration level. The intra- and inter-run accuracy and precision of the method were verified at the following QC concentration levels: -Five QC concentration levels prepared in diluent: 5.00ng / mL (LLOQ QC), 15.0ng / mL (Low QC), 250ng / mL (Mid QC), 150ng / mL (Mid 1 QC), 300ng / mL (Mid 2 QC), and 750ng / mL (High QC). -Four QC concentration levels prepared in screened human plasma: 5.00ng / mL+mean measured background concentration (matrix LLOQ QC), 15.0ng / mL+mean measured background concentration (matrix low QC), 250-300ng / mL (matrix medium QC), and 750ng / mL (matrix high QC).

[0122] The results showed that the intra- and inter-run accuracy and precision of the method met the predefined acceptance criteria in diluent (% bias within ±15.0% (within ±20.0% for LLOQ) and %CV ≤15.0% (20.0% for LLOQ)) and in human plasma (% bias within ±20.0% (within ±25.0% for LLOQ) and %CV ≤20.0% (25.0% for LLOQ)).

[0123] Matrix effect (selectivity) is defined as the suppression or enhancement of analyte ionization due to the presence of matrix components in a biological sample. Matrix effects were evaluated for CPC, NCC, and LBC by extracting single replicates of blank human plasma and spiking each lot with mid QC concentration levels (250 ng / mL) and / or high QC concentration levels (750 ng / mL for copper) in addition to endogenous concentration levels after extraction (measured during the same batch run). Matrix coefficients were calculated according to the following formula:

number

[0124] Hemolytic effect: To evaluate the effect of hemolysis on the quantification results, in-matrix and matrix-rich QC samples were prepared in pre-screened 2% hemolyzed plasma. Three replicates of in-matrix and matrix-rich QC samples in hemolyzed plasma were extracted and analyzed. Results met the predefined acceptance criteria: mean concentration within ±20.0% of the nominal concentration and %CV ≤ 20.0%.

[0125] Bench-top and short-term stability: To assess bench-top and short-term stability, QC samples at multiple concentration levels were first stored at -70°C ± 10°C for approximately 5-11 days. Three aliquots of each level of frozen QC samples were then thawed on the bench-top at ambient room temperature, and three separate aliquots of each level of frozen QC samples were thawed in a refrigerator at 4°C ± 4°C for 17.5-19 hours. Each aliquot was assayed against calibration standards. Non-ceruloplasmin copper and labile bound copper were considered stable if the mean of the concentrations obtained at each level was within ± 20.0% of the nominal concentration and the %CV was 20.0% or less.

[0126] Results showed that the NCC QC sample was stable for 19 hours at ambient room temperature and at 4° C.±4° C. Results further showed that the LBC QC sample was stable for 17.5 hours at ambient room temperature and at about 4° C.

[0127] QC freeze / thaw stability: Stability through four freeze / thaw cycles was evaluated at two QC levels (in-matrix QC and matrix-high QC prepared in prescreened human plasma) at each concentration level. QC samples were stored at -70°C ± 10°C and subjected to four freeze / thaw (ambient temperature) cycles and concentrations were measured against calibration standards. Samples from the first freeze cycle were kept in the freezer for at least 24 hours before thawing for 60 minutes at ambient room temperature. Subsequent freezing periods were for a minimum of 12 hours duration before thawing under the same conditions as the first cycle. QC samples were considered stable if the average of the concentrations obtained at each level was within ± 20.0% of the nominal concentration and the %CV was 20.0% or less. Results from cycle 4 showed that the QC samples were stable for at least four freeze / thaw cycles.

[0128] Stability of whole blood: The stability of NCC was evaluated at endogenous levels in human whole blood (lithium heparin as anticoagulant) at three time points (0, 60, and 120 min) in an ice-water bath (wet ice) and at ambient room temperature. Blood samples were stored in an ice-water bath and at ambient room temperature for 0, 60, and 120 min, and then centrifuged at about 3500 rpm, about 4° C., for about 15 min to separate plasma. Samples (n=3 separate tubes per storage condition / time point) were then assayed according to the NCC bioanalytical assay method described in this Example 4. The mean CPS ratios of whole blood samples stored in an ice-water bath and at ambient room temperature for up to 120 min were within the acceptance criteria of ±20.0%. Therefore, it can be concluded that NCC in human whole blood was stable for 120 min in an ice-water bath and at ambient room temperature.

[0129] Injection carryover testing is used to evaluate the degree of carryover of the analyte of interest from one sample to the next in each analytical run. During each validation run, replicate double blank samples (prepared in diluent) were injected along with a high standard from a set of calibrators. For results to be acceptable, the analyte CPS of the injected carryover sample must be 25% or less of the average analyte CPS of the LLOQ (standard 1 sample) per NCC and 20% or less of the average analyte CPS of the LLOQ per LBC. For the same carryover sample, the IS CPS must be 10% or less of the average IS CPS per NCC and 5% or less of the average IS CPS per LBCC from the batch calibration standards and QC samples to be acceptable. All evaluations met the predefined acceptance criteria.

[0130] Batch size robustness: A validation run was used to mimic the batch size of the sample analysis run. A total of 150-199 samples, including both standard curve and bracketing QC samples, were run in the batch. Matrix low, medium, and high QC samples were prepared in pre-screened human plasma and used to assess the maximum batch size.

[0131] To calculate the nominal concentrations of the matrix-low QC samples (measured per injection cycle for n = 6 matrix-low QC samples during this batch run), the diluted matrix used to prepare the matrix-low QC samples was also analyzed (n = 6) and the average endogenous concentration was determined.

[0132] Standards and QC samples met the general batch acceptance criteria for the sample analytical run (overall accuracy must be within 20% of the nominal value for QCs prepared in plasma or diluted plasma; CV for QCs prepared in plasma or diluted plasma must be ≤ 20%; at least 50% of the QC samples must be within 20% of the nominal concentration at each concentration level of QCs prepared in plasma or diluted plasma, and no more than 1 / 3 of all QC samples must be out of specification).

[0133] Selectivity was assessed by spiking Matrix Mid QC level concentrations (250ng / mL) into six separate lots of human plasma with a singlet above the LBC endogenous level. Six lots of unspiked plasma samples were analyzed in the same batch run with a singlet. The formula to calculate the LBC recovery of spiked Matrix QC is:

number

[0134] The Chelation Robustness Test examined whether longer incubation times during the chelation step would affect the stability of the Mo-Alb-Cu trimolecular complex ("TPC"), resulting in the release of copper in the LBC fraction. Six replicates of in-matrix and matrix-high QC samples spiked with TPC were evaluated. During sample preparation, the tested samples were kept in the chelation solution for 2 hours (currently, a 1-hour incubation time is used in the LBC method) prior to the next step. Extracted samples were measured against freshly prepared calibration standards.

[0135] Six replicates of in-matrix and matrix-high QC samples (no TPC spike) were analyzed in the same batch run to evaluate the effect of a longer chelation step (at least 2 hours, but with the same concentration of EDTA) on copper recovery. The results show that an incubation time of at least 2 hours for the chelation step does not affect the stability of the TPC.

[0136] Results from method validation showed adequate intra- and inter-run accuracy and precision, sensitivity, linearity, bench-top stability, short-term stability (4°C ± 4°C), matrix effect, maximum batch size assessment, freeze / thaw stability, carryover assessment, whole blood stability, and analyte interference at the IS. Direct NCC and CPC quantification was determined to be suitable for the measurement of non-ceruloplasmin copper and ceruloplasmin copper in lithium heparin human plasma. The LBC bioassay method was also determined to be suitable for the measurement of labile bound copper in lithium heparin human plasma.

[0137] Example 5: Direct measurement of non-ceruloplasmin copper (NCC) in plasma samples from 24 healthy volunteers Plasma samples were obtained from a Phase 1 clinical trial of 24 healthy adult volunteers. Samples were assayed according to the NCC bioassay process outlined in Example 4. The results of the NCC concentrations of the 24 healthy adult volunteers are shown in Table 12 below.

[0138] [Table 12]

[0139] Example 6: Complete verification of healthy Cu plasma levels Unaffected, healthy individuals are subjected to blood sampling and testing according to one or more of the previous examples. The resulting Cu levels are assessed and subdivided according to ethnicity, age, sex, comorbidities, and other factors. Reference levels are determined using the standard deviation of each subpopulation. A minimum of 120 individuals per subgroup are assessed.

[0140] Example 7: Wilson's disease diagnostic kit The phlebotomist provides sterile copper-free blood collection centrifuge tubes (i.e., standard vacutainers) and venipuncture to obtain a blood sample from the individual. The sample is spun down to separate the plasma, and the cell-free plasma is provided to a testing laboratory that supplies a Cu measurement kit. Kit components include standard concentrations of anti-ceruloplasmin antibody-coated magnetic beads, EDTA standard concentration solutions, and instructions for use. Specific valid reference ranges are provided for specific subgroups of interest.

[0141] Example 8: Treatment of Wilson's Disease A patient presenting with a condition believed to be related to copper metabolism has a blood sample taken and analyzed according to one or more of the previous examples. A patient with Wilson's disease is treated with at least one therapeutic agent selected from at least one of BC-TTM, trientine hydrochloride, trientine tetrahydrochloride, zinc (or a salt thereof), and / or penicillamine, and during treatment, blood tests are performed over time (weekly, biweekly, monthly, etc.) according to one or more of the above examples, and the dosage is adjusted to maintain therapeutic levels of the therapeutic agent and satisfactory Cu / NCC / LBC levels.

[0142] Various aspects of the present disclosure are further illustrated by the non-limiting embodiments set forth in the following claims, in each case the features of the claims can be combined in a manner that is not inconsistent with the specification and is not logically contradictory.

[0143] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in this respect will be suggested to those skilled in the art and are to be incorporated within the spirit and scope of this application and the appended claims. All publications, patents, and patent applications cited herein are incorporated by reference for all purposes. The present invention provides, for example, the following items. (Item 1) 1. A method for measuring a labile bound copper concentration in a biological sample, comprising: contacting the biological sample with an immunocapture reagent that binds to ceruloplasmin to form immunocaptured ceruloplasmin; removing said immunocaptured ceruloplasmin to obtain a non-ceruloplasmin sample; contacting the non-ceruloplasmin sample with a chelating agent that binds labilely bound copper; removing the non-labilely bound copper to obtain a labilely bound copper sample; and Measuring the copper concentration in said labile bound copper sample. A method comprising: (Item 2) 2. The method of claim 1, wherein the sample comprises human plasma or human serum. (Item 3) 4. The method of claim 2 or 3, wherein the immunocapture reagent comprises an immunoprecipitation reagent. (Item 4) 4. The method according to any one of items 1 to 3, wherein the immunocapture reagent comprises a ceruloplasmin capture reagent. (Item 5) 5. The method of claim 4, wherein the ceruloplasmin capture reagent comprises at least one reagent selected from a monoclonal antibody, a polyclonal antibody, a bispecific antibody, a diabody, a peptide, and an antigen-binding fragment thereof. (Item 6) 5. The method of claim 4, wherein the ceruloplasmin capture reagent comprises at least one monoclonal or polyclonal goat anti-human ceruloplasmin antibody. (Item 7) 5. The method of claim 4, wherein the immunocapture reagent comprises an anti-ceruloplasmin immobilized solid support. (Item 8) 5. The method of claim 4, wherein the immunoprecipitation reagent comprises a free anti-ceruloplasmin reagent configured to form a complex with ceruloplasmin and then immobilize on a solid support. (Item 9) 9. The method according to claim 7 or 8, wherein the solid support comprises magnetic beads. (Item 10) 9. The method of claim 7 or 8, wherein the solid support comprises at least one solid support comprising at least one of an agarose resin, a chromatography plate, a streptavidin plate, and a titer plate. (Item 11) 11. The method according to any one of items 1 to 10, wherein the measurement of the copper concentration in the labile bound copper sample comprises the use of inductively coupled plasma mass spectrometry (ICP-MS). (Item 12) 12. The method according to any one of items 1 to 11, further comprising introducing an internal standard into the labile bound copper sample prior to measuring the copper concentration. (Item 13) Item 13. The method of item 12, wherein the internal standard comprises at least one of copper and rhodium. (Item 14) 14. The method according to any one of items 1 to 13, wherein the recovering of the immunocaptured ceruloplasmin further comprises obtaining an immunocaptured ceruloplasmin sample. (Item 15) 15. The method of claim 14, further comprising measuring the concentration of ceruloplasmin in the immunocaptured ceruloplasmin sample. (Item 16) Item 16. The method of item 15, wherein measuring the ceruloplasmin concentration comprises using mass spectrometry. (Item 17) 17. The method of claim 16, wherein the mass spectrometry has an analyte detection limit of at least about 5 μg / mL. (Item 18) 18. The method of claim 16 or 17, wherein measuring the ceruloplasmin concentration comprises using liquid chromatography mass spectrometry (LC-MS). (Item 19) 19. The method according to any one of items 15 to 18, wherein the concentration of ceruloplasmin in the biological sample is less than about 200 μg / mL. (Item 20) 20. The method of any one of items 15 to 19, further comprising measuring the copper concentration in the immunocaptured ceruloplasmin sample. (Item 21) 21. The method of claim 20, wherein measuring the copper concentration in the immunocaptured ceruloplasmin sample comprises using inductively coupled plasma mass spectrometry. (Item 22) 22. The method according to any one of items 1 to 21, wherein the chelating agent comprises at least one of penicillamine, trientine hydrochloride, trientine tetrahydrochloride, and EDTA. (Item 23) 23. The method of claim 22, wherein the chelating agent comprises EDTA. (Item 24) 24. The method according to any one of items 1 to 23, wherein the removal of non-labile bound copper further comprises obtaining a non-labile bound copper sample. (Item 25) 25. The method of any one of claims 24, further comprising measuring the copper concentration in the non-labile bound copper sample. (Item 26) 25. The method of any one of claims 24, wherein the non-labile bond copper sample comprises molybdenum. (Item 27) 27. The method of claim 26, further comprising measuring the molybdenum concentration in the non-labile bound copper sample. (Item 28) A kit for measuring copper concentration in a biological sample, comprising an immunocapture reagent and instructions for use. (Item 29) 29. The kit of claim 28, wherein the immunocapture reagent comprises an immunoprecipitation reagent. (Item 30) 30. The kit according to any one of items 28 to 29, wherein the immune capture reagent comprises a ceruloplasmin capture reagent. (Item 31) 31. The kit of claim 30, wherein the ceruloplasmin capture reagent comprises at least one reagent selected from a monoclonal antibody, a polyclonal antibody, a bispecific antibody, a diabody, a peptide, and an antigen-binding fragment thereof. (Item 32) 31. The kit of claim 30, wherein the ceruloplasmin capture reagent comprises at least one monoclonal or polyclonal goat anti-human ceruloplasmin antibody. (Item 33) 31. The kit of claim 30, wherein the immunocapture reagent comprises an anti-ceruloplasmin immobilized solid support. (Item 34) 31. The kit of claim 30, wherein the immunoprecipitation reagent comprises a free anti-ceruloplasmin reagent configured to form a complex with ceruloplasmin and then immobilize it on a solid support. (Item 35) 35. The kit according to item 33 or 34, wherein the solid support comprises magnetic beads. (Item 36) 35. The kit according to item 33 or 34, wherein the solid support comprises at least one solid support comprising at least one of an agarose resin, a chromatography plate, a streptavidin plate, and a titer plate. (Item 37) A kit for determining copper concentration in a biological sample, comprising an immunocapture reagent, a chelating agent, and instructions for use. (Item 38) 38. The kit of claim 37, wherein the immunocapture reagent comprises an immunoprecipitation reagent. (Item 39) 39. The kit according to any one of items 37 to 38, wherein the immunocapture reagent comprises a ceruloplasmin capture reagent. (Item 40) 40. The kit of claim 39, wherein the ceruloplasmin capture reagent comprises at least one reagent selected from a monoclonal antibody, a polyclonal antibody, a bispecific antibody, a diabody, a peptide, and an antigen-binding fragment thereof. (Item 41) 40. The kit of claim 39, wherein the ceruloplasmin capture reagent comprises at least one monoclonal or polyclonal goat anti-human ceruloplasmin antibody. (Item 42) 40. The kit of claim 39, wherein the immunocapture reagent comprises an anti-ceruloplasmin immobilized solid support. (Item 43) 40. The kit of claim 39, wherein the immunoprecipitation reagent comprises a free anti-ceruloplasmin reagent configured to form a complex with ceruloplasmin and then immobilize it on a solid support. (Item 44) 44. The kit according to item 42 or 43, wherein the solid support comprises magnetic beads. (Item 45) 44. The kit according to item 42 or 43, wherein the solid support comprises at least one solid support comprising at least one of an agarose resin, a chromatography plate, a streptavidin plate, and a titer plate. (Item 46) 46. ​​The kit according to any one of items 37 to 45, wherein the chelating agent comprises at least one of penicillamine, trientine hydrochloride, trientine tetrahydrochloride, and EDTA. (Item 47) 46. ​​The kit according to any one of items 37 to 45, wherein the chelating agent comprises EDTA.

Claims

1. 1. A method for measuring a labile bound copper concentration in a biological sample, comprising: contacting the biological sample with an immunocapture reagent that binds to ceruloplasmin to form immunocaptured ceruloplasmin; removing said immunocaptured ceruloplasmin to obtain a non-ceruloplasmin sample; contacting the non-ceruloplasmin sample with a chelating agent that binds labilely bound copper; removing the non-labilely bound copper to obtain a labilely bound copper sample; and Measuring the copper concentration in said labile bound copper sample. A method comprising:

2. The method of claim 1 , wherein the sample comprises human plasma or human serum.

3. The method of claim 1 or claim 2, wherein the immunocapture reagent comprises an immunoprecipitation reagent.

4. The method of any one of claims 1 to 3, wherein the immunocapture reagent comprises a ceruloplasmin capture reagent.

5. 5. The method of claim 4, wherein the ceruloplasmin capture reagent comprises at least one reagent selected from a monoclonal antibody, a polyclonal antibody, a bispecific antibody, a diabody, a peptide, and an antigen-binding fragment thereof.

6. The method of claim 4 , wherein the ceruloplasmin capture reagent comprises at least one of a monoclonal or polyclonal goat anti-human ceruloplasmin antibody.

7. The method of claim 4 , wherein the immunocapture reagent comprises an anti-ceruloplasmin immobilized solid support.

8. 5. The method of claim 4, wherein the immunoprecipitation reagent comprises a free anti-ceruloplasmin reagent configured to form a complex with ceruloplasmin and then immobilize it on a solid support.

9. 9. The method of claim 7 or claim 8, wherein the solid support comprises magnetic beads.

10. 9. The method of claim 7 or claim 8, wherein the solid support comprises at least one solid support comprising at least one of an agarose resin, a chromatography plate, a streptavidin plate, and a titer plate.

11. A method according to any one of claims 1 to 10, wherein the measurement of the copper concentration in the labile bound copper sample comprises the use of inductively coupled plasma mass spectrometry (ICP-MS).

12. 12. The method of any one of claims 1 to 11, further comprising introducing an internal standard into the labile bound copper sample prior to measuring the copper concentration.

13. 13. The method of claim 12, wherein the internal standard comprises at least one of copper and rhodium.

14. The method of any one of claims 1 to 13, wherein harvesting the immunocaptured ceruloplasmin further comprises obtaining a sample of immunocaptured ceruloplasmin.

15. 15. The method of claim 14, further comprising measuring the concentration of ceruloplasmin in the immunocaptured ceruloplasmin sample.

16. 16. The method of claim 15, wherein measuring the ceruloplasmin concentration comprises the use of mass spectrometry.

17. 17. The method of claim 16, wherein the mass spectrometry has an analyte detection limit of at least 5 μg / mL.

18. 18. The method of claim 16 or 17, wherein measuring the ceruloplasmin concentration comprises the use of liquid chromatography mass spectrometry (LC-MS).

19. The method of any one of claims 15 to 18, wherein the concentration of ceruloplasmin in the biological sample is less than 200 μg / mL.

20. The method of any one of claims 15 to 19, further comprising measuring the copper concentration in the immunocaptured ceruloplasmin sample.

21. 21. The method of claim 20, wherein measuring the copper concentration in the immunocaptured ceruloplasmin sample comprises the use of inductively coupled plasma mass spectrometry.

22. 22. The method of any one of claims 1 to 21, wherein the chelating agent comprises at least one of penicillamine, trientine hydrochloride, trientine tetrahydrochloride, and EDTA.

23. 23. The method of claim 22, wherein the chelating agent comprises EDTA.

24. 24. The method of any one of claims 1 to 23, wherein the removing of non-labile bound copper further comprises obtaining a non-labile bound copper sample.

25. 25. The method of claim 24, further comprising measuring the copper concentration in said non-labile bound copper sample.

26. 25. The method of claim 24, wherein the non-labile bond copper sample comprises molybdenum.

27. 27. The method of claim 26, further comprising measuring the molybdenum concentration in the non-labile bound copper sample.

28. A kit for carrying out the method of claim 1, comprising: the immunocapture reagent binds to ceruloplasmin for immunocapture; said chelating agent binding to labilely bound copper; and User manual Including the kit.

29. The kit of claim 28 , wherein the immunocapture reagent comprises an immunoprecipitation reagent.

30. 30. The kit of claim 28 or claim 29, wherein the immunocapture reagent comprises a ceruloplasmin capture reagent.

31. 31. The kit of claim 30, wherein the ceruloplasmin capture reagent comprises at least one reagent selected from a monoclonal antibody, a polyclonal antibody, a bispecific antibody, a diabody, a peptide, and an antigen-binding fragment thereof.

32. 31. The kit of claim 30, wherein the ceruloplasmin capture reagent comprises at least one monoclonal or polyclonal goat anti-human ceruloplasmin antibody.

33. The kit of claim 30 , wherein the immunocapture reagent comprises an anti-ceruloplasmin immobilized solid support.

34. 31. The kit of claim 30, wherein the immunoprecipitation reagent comprises a free anti-ceruloplasmin reagent configured to form a complex with ceruloplasmin and then immobilize on a solid support.

35. 35. The kit of claim 33 or claim 34, wherein the solid support comprises magnetic beads.

36. 35. The kit of claim 33 or claim 34, wherein the solid support comprises at least one solid support comprising at least one of an agarose resin, a chromatography plate, a streptavidin plate, and a titer plate.

37. The kit of any one of claims 28 to 36, wherein the chelating agent comprises at least one of penicillamine, trientine hydrochloride, trientine tetrahydrochloride, and EDTA.

38. The kit of any one of claims 28 to 36, wherein the chelating agent comprises EDTA.

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