Methods for detecting kidney disease

Abstract

To provide methods and apparatus for the determination, diagnosis, progression, and prognosis of kidney disease and mortality associated with kidney disease.SOLUTION: The disclosure includes methods for determining the renal function, in particular estimating the glomerular filtration rate (GFR), in an animal. The GFR can be useful in the diagnosis and treatment of kidney disease or kidney dysfunction. In various aspects, the disclosure is directed to the use of free symmetrical dimethylarginine (SDMA) and creatinine in blood samples from animals, particularly cats and dogs, to determine the glomerular filtration rate and kidney disease.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 61 / 874,011, filed on September 5, 2013.

[0002] This disclosure generally relates to the determination of kidney function. More particularly, this disclosure relates to methods for estimating glomerular filtration rate and diagnosing, predicting, and determining the progression of kidney disease.

Background Art

[0003] It is important to be able to measure kidney function quickly and accurately. For example, drug dosing must be appropriate for patients with renal insufficiency. Therefore, accurately assessing kidney function is necessary in clinical medicine. However, the diagnosis of renal insufficiency is hampered by the lack of a reliable marker for glomerular filtration rate (GFR) and / or available diagnostic tests. The most widely used method for measuring GFR is inulin clearance, but this test is cumbersome and expensive, which essentially reduces its usefulness in clinical practice. This also applies to radiolabeled isotope clearance tests. Therefore, in clinical practice, serum creatinine is usually used to evaluate kidney function. However, the use of serum creatinine has the drawback of inaccuracy because the data can vary relatively widely. [[ID=二十九]] [[ID=三三]]

Summary of the Invention

[0004] Therefore, the inventors recognize the need for a more accurate method for evaluating kidney function in the art.

[0005] ​​​​​​​​​​​In one embodiment, the present disclosure relates to a method for estimating glomerular filtration rate (GFR) in animals. The method relates to measuring the concentration of free SDMA in a blood sample from a subject. , measuring the concentration of creatinine in a blood sample from the subject, and creatinine The value obtained from the equation that includes the product of the concentration of and the concentration of free SDMA is used in animals. This includes comparing the glomerular filtration rate with one or more standard values ​​that correlate with it.

[0006] In various exemplary embodiments of the method described herein, the equation is creatinine It includes the reciprocal of the product of the concentration and the concentration of free SDMA. Also, the concentration of creatinine and / or Alternatively, the concentration of free SDMA can be weighted by calculation. The comparison step is This can be done using a microprocessor. The method also involves GF in the subject. By comparing R with GFR in one or more healthy subjects, renal function and kidney disease can be assessed. This also includes determining whether the patient has a kidney disorder or impaired renal function.

[0007] In yet another embodiment, the disclosure relates to renal disease or renal dysfunction in animals. The present invention relates to a diagnostic method. This method involves measuring the concentration of free SDMA in the serum of the subject. This involves measuring the concentration of creatinine in the serum of the subject, and the creatinine A first weighted value based on concentration, and a second weighted value based on the concentration of free SDMA. The product of the calculated value is compared to one or more standard values ​​that are correlated with kidney disease or renal dysfunction. This includes doing so.

[0008] In a particular exemplary embodiment, a first weighted value based on creatinine concentration The product of the first weighted value based on the creatinine concentration and the second weighted value based on the concentration of free SDMA is given by the formula PROD = (C RE) , , , , , , , ,

[0010] , , , , , × (SDMA) Q where PROD is the product, CRE is the concentration of creatinine, SDMA is the concentration of SDMA, P represents the weight given to CRE in the formula, and Q represents the weight given to SDMA in the formula. One or more standard values may be correlated with the reciprocal of the product.

[0009] A further aspect of the present disclosure relates to a method for calculating a value related to the diagnosis of kidney disease or kidney dysfunction in an animal subject. The method includes executing machine-readable instructions for calculating the product of a first weighted value based on the concentration of creatinine in a blood sample from the subject and a second weighted value based on the concentration of free SDMA in the blood sample from the subject. In a further aspect, the present disclosure relates to a method for determining whether an individual has a kidney disease. The method includes measuring the concentration [SDMA] of SDMA and the concentration [CRE] of creatinine in a serum sample from the individual, calculating the ratio [SDMA] / SDMA CUT , calculating the ratio [CRE] / CRE<00000​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ It is a value of F.

[0011] One method provided by this disclosure includes determining whether an individual has kidney disease. The method involves measuring the concentration of SDMA [SDMA] and creatinine in serum samples from individuals. Measuring the concentration of [CRE], [SDMA] / SDMA CUT Calculating the ratio, [CR E] / CRE CUT Calculate the ratio; the combined value is: C = [SDMA] / SDMA CUT +[CR E] / CRE CUT To calculate C is C CUT If it is larger than that, the individual has kidney disease. This includes the decision to use SDMA in the formula. CUT This is the cutoff value for SDMA, and C RE CUT This is the cutoff value for CRE, and C CUT This is the cutoff value for the combination.

[0012] Furthermore, this disclosure relates to a method for predicting premature death in animals, The method involves measuring the concentration of free SDMA in the serum of the subjects, and the serum of the subjects Measuring creatinine concentration, calculating the [SDMA] / [CRE] ratio, and This includes determining that if the ratio exceeds a cutoff value, the individual will die prematurely.

[0013] In one embodiment, the present disclosure relates to a method for determining mortality associated with kidney disease. The method involves measuring free SDMA in blood samples from patients, for example, dogs or cats. It is important to determine that if a patient has a blood SDMA concentration higher than the threshold, the patient may have renal disease. This includes determining that there is a high probability of death related to the disease. The method involves in a blood sample The process further includes the step of measuring creatinine and calculating the [SDMA] / [CRE] ratio. This may be the case, and here, the patient has a blood [SDMA] / [CRE] ratio higher than the threshold ratio. In such cases, the patient has a high probability of death related to kidney disease.

[0014] In another embodiment, the disclosure also relates to an apparatus for determining renal function in animals. The device uses SDMA analogs, or asymmetric dimethylarginine (ADMA), L - Intermingles one or more compounds selected from arginine and N-methylarginine. A first antibody conjugated to an SDMA-specific antibody that does not exhibit or substantially exhibits differential reactivity. The solid phase; and the creatinine detection reagent or antibody specific to creatinine conjugated thereto. It includes a second solid phase.

[0015] In a further embodiment, the present disclosure relates to a kit for determining renal function in animals. The kit contains one or more creatinine detection reagents, and one or more The SDMA detection reagent contains creatinine in one or more blood samples from animals. A set of one or more standard values ​​related to renal function, based on the product of the concentration and the concentration of SDMA. It may include "t".

[0016] Furthermore, this disclosure provides the reciprocal of the product of the creatinine concentration and the free SDMA concentration at runtime. A computing device having a storage device that includes software instructions for calculating The memory also stores the results of the calculation in one or more units representing the glomerular filtration rate in the animal subject. It may also include software instructions for comparing with standard values.

[0017] The attached drawings are included for further understanding of this disclosure and are incorporated herein. This specification includes an illustration of embodiments of the present disclosure, and together with a detailed description, the present invention This helps to explain the principle. The structural details of the present invention and how the present invention can be carried out. This does not attempt to provide more detail than is necessary for a basic understanding of the various methods. [Brief explanation of the drawing]

[0018] [Figure 1] This graph compares the results of ELISA for detecting SDMA with those of mass spectrometry. [Figure 2] This plot shows SDMA concentrations in healthy dogs and dogs with cancer, heart disease, or cardio-renal disease. The horizontal bars represent the cutoff value (determined as the mean SDMA concentration of the healthy dog ​​population + 2 standard deviations). [Figure 3] This plot shows SDMA concentrations in healthy cats and cats with kidney disease or cancer. The horizontal bars represent the cutoff value (determined as the mean SDMA concentration of the healthy cat population + 2 standard deviations). [Figure 4] This is a plot of absorbance at 280 nm against fraction number for the elution of SDMA cystamide protein conjugates (proteins are KLH(◆) or BSA(■)) from Sephadex G-25M gel filtration columns as described in the examples. [Figure 5] This is a plot of creatinine concentration versus GFR for the set of canine serum samples described in Example 6. [Figure 6] This is a plot of SDMA concentration versus GFR for the set of canine serum samples described in Example 6. [Figure 7] This is a plot of [creatinine]*[SDMA] versus GFR for the set of canine serum samples described in Example 6. [Figure 8]The following plots, using linear fit, show creatinine vs. GFR, 1 / SDMA vs. CGF, and 1 / [creatinine 0.37]*1 / [SDMA 0.43] vs. creatinine for the set of canine serum samples described in Example 6. [Figure 9] This is a plot of SDMA concentration versus GFR for the set of cat serum samples described in Example 7. [Figure 10] This is a plot of creatinine concentration versus GFR for the set of cat serum samples described in Example 7. [Figure 11] This is a plot of [creatinine]*[SDMA] versus GFR for the set of cat serum samples described in Example 7. [Figure 12] The following plots, using linear fit, show the values ​​of [creatinine] vs. GFR, 1 / SDMA vs. CGF, and 1 / [creatinine 1.2]*1 / [SDMA 0.39] vs. creatinine for the set of cat serum samples described in Example 7. [Figure 13] This graph shows improvements in sensitivity and specificity in methods for determining kidney disease. [Figure 14] This shows the correlation between SDMA (μg / dL) and creatinine (mg / dL) in dogs. [Figure 15] This shows serum creatinine and SDMA concentrations in a population of cats. [Figure 16] This shows serum creatinine and SDMA concentrations in cats over several years. [Figure 17] This shows serum creatinine and SDMA concentrations in cats over several years. [Figure 18] This shows serum creatinine and SDMA concentrations in cats over several years. [Figure 19] This Kaplan-Meier survival curve shows that cats with serum SDMA concentrations below 14 μg / dL survive approximately 1.6 times longer than cats with concentrations higher than 14 μg / dL. [Figure 20]This Kaplan-Meier survival curve shows that dogs with serum SDMA concentrations below 14 μg / dL survive approximately 2.6 times longer than dogs with concentrations higher than 14 μg / dL. [Modes for carrying out the invention]

[0019] In various aspects thereof, this disclosure relates to the determination of renal disease and renal disease-related mortality rates. Regarding diagnosis, progression, and prognosis. This disclosure relates to determining renal function in animals, in particular, This includes methods for estimating glomerular filtration rate (GFR). GFR is used in the diagnosis of kidney disease or renal dysfunction. It may be useful for diagnosis and treatment.

[0020] In various embodiments, this disclosure relates to animals for determining glomerular filtration rate and kidney disease. In particular, free symmetric dimethylarginine (SDM) in blood samples from cats and dogs. A) and the use of creatinine. In one embodiment, in a blood sample from an animal The product of creatinine concentration and free SDMA concentration is correlated with GFR and renal disease. It is possible. For example, the reciprocal of the product of the creatinine concentration and the free SDMA concentration (for example) However, 1 / [creatinine][SDMA]) is used, and surprisingly, the measurement of either one This method provides far greater accuracy in measuring glomerular filtration rate than the conventional method. This disclosure relates to the measurement of the concentration of free SDMA in blood samples from animal subjects; Measurement of creatinine concentration in blood samples; and creatinine concentration and free form The reciprocal of the product of the SDMA concentration is used as one or more standards for glomerular filtration rate in animals. This includes a method for determining the glomerular filtration rate of an animal by comparing it with a value. Other aspects of this disclosure This refers to the determination of kidney disease as described herein, using SDMA concentration alone or SDMA concentration. This includes the use of SDMA concentration in the ratio of degree to creatinine concentration.

[0021] SDMA is an endogenous nitric oxide synthase (NOS) inhibitor, specifically an asymmetric dimethylamine. It is a structural isomer of arginine (ADMA). Both ADMA and SDMA are L-arginine residues. SDMA is produced from nuclear methylation and released into the cytoplasm after protein degradation. By protein arginine methyltransferase 5 (PRMT5) and PRMT7 It is produced. Methylarginine, for example, SDMA, monomethylarginine, and AD Proteins containing MA are used in RNA processing, protein shuttle, and sil Involved in Gunar transmission (Bedford and Richard, Mol. Cell, 20 April 29, 2005, 18(3):263~72). Such methylated proteins Free SDMA produced from the solution is mainly excreted by renal excretion, whereas ADMA It is mostly metabolized. ADMA is a risk factor for coronary artery disease (CAD), for example, high Blood pressure, hypercholesterolemia, hyperhomocysteinemia, insulin resistance, age, and SDMA is strongly correlated with mean arterial pressure. SDMA is strongly correlated with parameters of renal function, such as glomerular filtration rate. It correlates with (GFR), inulin clearance, and creatinine clearance.

[0022] Therefore, one aspect of this disclosure relates the concentration of free SDMA in serum to the concentration of creatinine. This relates to a method for estimating glomerular filtration rate in animals by using both values. The reciprocal of the product of the values ​​(for example, 1 / [creatinine][SDMA]) is equal to creatinine or It correlates more accurately and linearly with GFR than SDMA concentration alone.

[0023] Many terms are defined below.

[0024] Ab is an antibody.

[0025] ADMA is asymmetric dimethylarginine. The structure of ADMA is:

[0026] [ka] That is the case.

[0027] BUN stands for blood urea nitrogen.

[0028] BSA is bovine serum albumin.

[0029] CMIA is a chemiluminescent magnetic immunoassay.

[0030] DCM is dichloromethane.

[0031] DIPEA is N,N-diisopropylethylamine.

[0032] DMF is dimethylformamide.

[0033] EIA is an enzyme immunoassay.

[0034] ELISA is an enzyme-linked immunosorbent assay.

[0035] EMI-MS is electrospray ionization mass spectrometry.

[0036] FPIA is a fluorescence-polarized immunoassay.

[0037] GFR stands for glomerular filtration rate.

[0038] HATU is (1H-7-azabenzotriazole-1-yl)-1,1,3,3-te It is tramethyluranium hexafluorophosphate methaminium.

[0039] KLH is keyhole limpet hemocyanin.

[0040] MEIA is a microparticle enzyme immunoassay.

[0041] NOS is nitric oxide synthase.

[0042] PBS is phosphate-buffered saline.

[0043] RIA is a radioimmunoassay.

[0044] SDMA is symmetric dimethylarginine. The structure of SDMA is:

[0045] [ka] That is the case.

[0046] Free SDMA refers to SDMA that is not part of a polypeptide chain. Alternatively, multiple amino acid residues may be present in the polypeptide.

[0047] SLE is systemic lupus erythematosus.

[0048] TFA is trifluoroacetic acid.

[0049] The structure of arginine is

[0050] [ka] That is the case.

[0051] N-MMA is N-monomethylarginine, or simply N-methylarginine. The structure of N-monomethylarginine is

[0052] [ka] That is the case.

[0053] As used herein, the term "analog" generally refers to one or more individual atoms. This refers to a compound in which different atoms or different functional groups have been substituted. For example, the analogue may be a modified form of the analyte that can compete with the analyte for the receptor. The modification provides a means for the analyte to bind to another part, such as a label or solid carrier. The analyte analog can bind to the antibody in the same way as the analyte itself.

[0054] As used herein, the term "antibody" generally refers to a substance that reacts to exposure to an antigen, such as B phosphorus. This refers to a glycoprotein produced by pacyl cells that specifically binds to its antigen. The term "body" is used in its broadest sense, and in detail, it refers to a body that exhibits the desired biological activity. Noclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multiple specific antibodies This includes sex antibodies (e.g., bispecific antibodies) and antibody fragments.

[0055] When used herein, the terms "anti-SDMA," "anti-SDMA antibody moiety," or "anti-SDMA" are used. "DMA antibody fragments" and / or "anti-SDMA antibody variants," etc., are immunoglobulins. At least a portion of a phosphorus molecule, for example, one of the heavy chain or light chain constant regions, but not limited to any Complementarity Determination Region (CDR), Framework Region, or any part thereof Contains protein or peptide-containing molecules.

[0056] As used herein, the term "antibody fragment" refers to a portion of a full-length antibody, generally speaking. This refers to the antigen-binding or variable region. More specifically, for example, an antibody fragment is , Fab, Fab', F(ab')2, and Fv fragments; dia body (dia bodies; linear antibodies; single-chain antibody molecules; and multiple specific antibodies from antibody fragments Sex antibodies can be cited.

[0057] When used herein, the term "antigen" generally refers to an antibody specific to an antigen and appropriate conditions. This refers to substances that can react under certain conditions.

[0058] The term "analyte," as used herein, generally refers to the substance detected and / or measured. Refers to a substance or set of substances in a sample.

[0059] The term "animal," as used herein, generally refers to any animal, such as a human, and This refers to animals other than humans, such as cats, dogs, or horses.

[0060] The term "blood sample," as used herein, generally refers to, but is not limited to, blood samples. This refers to any blood-derived fluid sample, including whole blood, plasma, and serum, as described in this disclosure. To prepare the serum to be used, one or more serum samples are obtained from an animal subject. Serum samples can be obtained from animal subjects, for example, as blood samples, and then The serum can be separated and prepared. In certain embodiments, the serum is obtained from the blood. It can be measured without separation. As those skilled in the art will understand, a single sample obtained The liquid can be separated or used to measure both concentrations. Alternatively, multiple A number of samples can be obtained from animal subjects, and (at least) one sample is from Claire. The tinine concentration was measured, and (at least) one sample was found to have a free SDMA concentration. It is measured accordingly. In certain such cases, the sample is taken almost simultaneously (for example, It can be obtained from animals within 60 minutes, 30 minutes, or 10 minutes from each other.

[0061] As used herein, the term "cross-reactivity" generally refers to the individual antigen-binding sites of an antibody. The ability of a population of antibody molecules to react with two or more antigenic determinants, or to react with two or more antigens. It refers to the ability to react. Generally, cross-reactivity is when (i) a cross-reactive antigen is common with an immunoantigen. (ii) sharing an epitope, or (ii) a cross-reactive antigen is an epitope on an immunoantigen This occurs because it has structurally similar epitopes (multiple singularities).

[0062] The term "immunoassay," as used herein, generally refers to a method that produces a measurable reaction. This refers to a test that uses antibody-antigen complexes. The term "antibody-antigen complex" is sometimes used interchangeably with the term "immune complex." They can be used interchangeably. Generally, as an immunoassay, a non-competitive immunoassay is used. Examples include competitive immunoassays, homogeneous immunoassays, and heterogeneous immunoassays. In a competitive immunoassay, the unlabeled analyte in the test sample (or Antigens are measured in immunoassays by their ability to compete with labeled antigens. Unlabeled antigens interfere with the binding ability of labeled antigens because antibodies This is because the binding site is already blocked. In a "competitive immunoassay," the test The amount of antigen present in the sample is inversely related to the amount of signal produced by the label. Conversely, In non-competitive immunoassays, also known as "sandwich-type" immunoassays, The analyte binds between two highly specific antibody reagents to form a complex, and the amount of antigen It is directly proportional to the amount of signal associated with the complex. Separation of the bound antibody-antigen complex is necessary. The key immunoassay is generally called a "heterogeneous immunoassay," and involves antibody-antigen complex Immunoassays that do not require the separation of molecules are generally called "homogeneous immunoassays." The vendor will easily understand the various forms of immunoassays.

[0063] When used herein, the term “immune complex” generally refers to a complex that is accompanied by or associated with complement binding. This refers to a complex formed by the binding of an antigen and an antibody molecule. If one of them is labeled, the label is associated with the immune complex as a result of the binding of the antigen and antibody. Therefore, if an antibody is labeled, as a result of binding, the label is associated with the antigen. Similarly, if the antigen is labeled (for example, an analyte analog with a label), the anti As a result of the binding of the protozoan to the antibody, the label becomes associated with the antibody.

[0064] The term "label" as used herein means (for example, in covalent or non-covalent bonds) (On their own, or encapsulated) with the antibodies, SDMA analogs, or antigens of this disclosure, directly or A detectable compound, composition, or solid carrier that can be indirectly conjugated. It refers to the body. The label is detectable by itself (e.g., radioisotope label, chemiluminescent color). This may be an element, electrochemical label, metal chelate, latex particle, or fluorescent label. Furthermore, in the case of enzyme labeling, the chemical change of the detectable substrate compound or composition is affected. A medium may be used (for example, an enzyme, e.g., horseradish peroxidase, alkaline phosphine). (e.g., tase). The labels used in this disclosure are not limited to those, but include alkaline phosphatase. Glucose-6-phosphate dehydrogenase ("G6PDH"); horseradish Luoxidase (HRP); chemiluminescent substance, for example isolminol, fluorescent substances (fluorescers), such as fluoroceine and ro - These can be damine compounds; ribozymes; and dyes. The label may also be detectable in itself. Specific binding molecules that may be able to bind (e.g., biotin, avidin, streptavidin) Digoxigenin, maltose, oligohistidine, 2,4-dinitrobenzene, phenyl Arsenic acid (phenylarsenate), ssDNA, dsDNA, etc. may also be used. The label may be attached to another molecule or solid support, enabling the detection of the labeled molecule. The characteristics are selected. The use of the markers is by means of detection such as electromagnetic radiation or direct visualization. It can detect and, in some cases, provide a signal that can be measured.

[0065] As used herein, the term "monoclonal antibody" generally refers to a substantially homogeneous antibody. This refers to antibodies obtained from a group of bodies (i.e., antibodies that are identical to the individual antibodies that make up the group). Monoclonal antibodies are highly specific and target a single antigen site. Polyclonal antibody preparations typically contain different antibodies against different epitopes. In contrast, each monoclonal antibody targets a single epitope on the antigen. The modifier "monoclonal" simply refers to a characteristic of the antibody, and does not refer to an antibody produced by any specific method. This should not be interpreted as requiring the production of monoclonal antibodies. In detail, for example, monoclonal antibodies It may be produced by the hybridoma method or by the recombinant DNA method. Alternatively, they may be isolated from a phage antibody library using known techniques.

[0066] The term "polypeptide," as used herein, generally refers to molecules linked by peptide bonds. This term refers to molecules that have a specific amino acid sequence. This term is used for proteins, fusion proteins, and oligonucleotides. This includes peptides, cyclic peptides, and polypeptide derivatives. Antibodies and antibody derivatives are also included. As already stated in another paragraph, antibodies and antibody derivatives are poly They are treated as subclasses of peptides and polypeptide derivatives.

[0067] The term "solid carrier," as used herein, refers to the antibody or SDMA analog described herein. This refers to a non-aqueous matrix to which the support can adhere. Examples of solid supports include glass (for example, porous glass). Lath, synthetic and natural polymers, polysaccharides (e.g., agarose), polyacrylamide Polystyrene, polyvinyl alcohol and silicone, magnetic particles, latex particles, Chromatography strips, microtiter polystyrene plates, or conjugated antigens Any material that can be washed or separated from the antibody that is not bound to it. Examples include carriers that are partially or entirely formed of other materials. In certain embodiments, Depending on the application, the solid support may be the wells of the assay plate, or the purified water. It may also be a lam (for example, an affinity chromatography column).

[0068] A "receptor" is a specific spatial and polar structure of a molecule, such as an epitope or determinant region. This refers to any compound or composition that can recognize a specific position. Examples of receptors include antibodies and FabF. Examples include lag and other factors.

[0069] "Binding specificity" or "specific binding" refers to the substantial recognition of the first molecule by the second molecule. For example, polypeptides and polypeptide-specific polyclonal or monoclonal compounds Antibodies or antibody fragments (e.g., Fv, single-chain Fv, Fab', or F(a) b')2 refers to fragments). For example, "specificity" as used herein generally means Therefore, the ability of each antibody binding site to react with just one antigenic determinant, or the antibody molecule This refers to the ability of a population to react with a single antigen. Generally, in antigen-antibody reactions, High specificity exists. Antibodies have (i) the primary structure of the antigen, (ii) the isomer form of the antibody, (iii) It is possible to distinguish the differences in the secondary and tertiary structures of the antigen. Antibody-antigen reactions exhibiting specificity show low cross-reactivity.

[0070] "Substantial binding" or "effectively binding" refers to an assay under specific assay conditions. This refers to the degree of specific bonding or recognition between molecules in a mixture. In its broadest form, Qualitative bonding involves specific factors including the relative concentration of molecules, as well as incubation time and temperature. Under a set of assay conditions, the first molecule binds to the second molecule or the second molecule Lack of the ability to recognize, and the first molecule binding to the third molecule or recognizing the third molecule. This relates to the difference in ability, and this difference is relevant to performing meaningful assays that distinguish specific bindings. This is sufficient to make it possible. In another embodiment, in the sense of cross-reactivity, one The molecule has virtually no ability to bind to or recognize another molecule, and here Under specific assay conditions, the first molecule reacts with the second molecule, and then with the third molecule. In contrast, it exhibits a reactivity of less than 25%, less than 10%, less than 5%, or less than 1% of the observed reactivity. Specific binding can be detected by many well-known methods, such as immunohistochemical assays and enzymes. Binding immunosorbent assay (ELISA), radioimmunoassay (RIA), or Western blotting • This can be tested using a blot assay.

[0071] The term "salt," as used herein, refers to a compound formed between an acid and a basic functional group of a compound. It means a salt. Examples of salts include, but are not limited to, sulfates, citrates, and acetic acid. Salts, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acidic phosphoric acid Salt, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleic acid Salt, tannate, pantothenate, beetartrate, ascorbate, succinate, male phosphate, gentisinate, fumarate, gluconate, glucarate, saccharate, Formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, ben Zensulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'- Methylene-bis-(2-hydroxy-3-naphthoate)) is one example. The term "salt" is Furthermore, a compound having an acidic functional group, for example a carboxylic acid functional group, and an inorganic or organic base It also refers to the salts formed between them. Suitable bases are not limited to these, but alkali gold Genus, for example, sodium, potassium and lithium hydroxides; alkaline earth metals, for example For example, calcium and magnesium hydroxides; other metals, such as aluminum and Zinc hydroxide; ammonia; and organic amines, e.g., unsubstituted or hydroxysubstituted. Mono-, di-, or trialkylamines; dicyclohexylamines; tributylamines Pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine, mono -, bis-, or tris-(2-hydroxy-lower alkylamine), e.g., mono- , bis-, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert -Butylamine, or tris-(hydroxymethyl)methylamine, N,N-di-lower Alkyl-N-(hydroxy lower alkyl)-amines, for example, N,N-dimethyl-N- (2-hydroxyethyl)amine, or tris-(2-hydroxyethyl)amine; N -Methyl-D-glucamine; and amino acids, such as arginine and lysine. It is possible.

[0072] In certain methods described herein, the glomerular filtration rate of an animal subject is derived from the animal subject. The result of the equation considering the product of the creatinine concentration in the blood sample and free SDMA It is determined by comparing the following. For example, to determine GFR, creatinine The reciprocal of the product of the concentration and the concentration of free SDMA correlates with the glomerular filtration rate in animals. It can be compared with one or more standard values. This is described in more detail in Example 6 below. As explained above, there is a direct correlation between GFR and the reciprocal of the product of creatinine concentration and free SDMA concentration. A linear relationship exists. Therefore, a person skilled in the art can (for example, other animals of the same species or type) (Using) a linear relationship between GFR and 1 / ([creatinine][SDMA]) in animal subjects. We can set up an equation and use that equation to compare it with the reciprocal of the product of the measured concentrations. A standard value can be given for this. As those skilled in the art will understand, the comparison with the standard value is an equation. Only use 1 / ([creatinine][SDMA]) to calculate GFR. Includes. Or, creatinine and free SDMA relative to a known set of GFR values. We can determine a set of standard values ​​for the reciprocal of the product of concentrations, and use GFR for animals, creati By comparing the reciprocal of the product of the measured concentrations of nin and free SDMA with the standard value... This can be determined. In certain embodiments, the determination step is creatinine The reciprocal of the product of the concentrations of free SDMA and the equation, or the ratio of one or more standard values. This can be done using a microprocessor programmed to perform comparisons. The loprocessor typically operates based on input from the operator or detection device during runtime. A memory device containing software instructions that perform the function of calculating and comparing equations. It is a component of a computing device.

[0073] As those skilled in the art will understand, the reciprocal of the product of the creatinine concentration and the free SDMA concentration is, A comparison with one or more standard values ​​for the reciprocal of the product, which correlates with glomerular filtration rate, is possible. This includes numerical comparisons that are mathematically equivalent to comparisons like the one shown. For example, {constant × (1 / ([creatinine Comparisons using values ​​that represent [SDMA] and / or [constant × GFR] are also possible. The intention is to achieve, for example, that the comparison is based solely on the product ([creatinine][SDMA]). It is possible. In addition, a person skilled in the art can determine the fraction of the quotient (1 / ([creatinine][SDMA])) The inclusion of coefficients in the numerator and / or parentheses does not change the strength of the relationship with GFR. You will understand (for example, 2 / ([SDMA][Creatinine]), 1 / (2[SD MA [creatinine]) or 5 / (3 [SDMA] [creatinine])). Similarly, We also intend to examine the relationship between ([creatinine][SDMA]) and 1 / GFR.

[0074] In another embodiment, this disclosure is defined as follows: GFR ≈ 1 / (CRE × SDMA) Regarding the estimation of GFR using [this method].

[0075] Based on experimental results, this equation has a correlation coefficient (R-squared) of approximately 0.8347. Equation The following GFR ≈ (CRE) P × (SDMA) Q As derived, the exponents (P and Q) that maximize the correlation coefficient are P ≈ -1. 551102, and Q ≈ -0.2204409. For this set of exponents... The R-squared value of is 0.9116. As will be understood by those skilled in the art, P and Q are correlated by the correlation coefficient. This is a weighting coefficient that can be adjusted to maximize the effect.

[0076] Slightly changing the exponent does not seem to have a significant effect on R-squared. For example, If P = -1.5 and Q = -0.25, then R squared is 0.9114. Simply put, the ideal power conversion of creatinine and SDMA values ​​related to GFR is: GFR ≈ (CRE) -1.5 × (SDMA) -0.25 It takes this form.

[0077] In various embodiments, the weighting coefficients P and Q can be further adjusted. For example, P can vary from approximately -5 to less than approximately 0 (e.g., -0.01). In other words, P can vary from approximately -5 to any value between -5 and 0, but 0 It does not include. In certain non-limiting cases, P is approximately -4.0 to -0.1, and approximately -3.0 to It can vary from -0.5, approximately -2.0 to -1.0, and approximately -1.0 to 0. However, it does not include 0. Independently, Q can vary from -2.5 to approximately 0 (for example, -0.01). In other words, Q can range from approximately -2.5 to any value between -2.5 and 0. It can fluctuate, but does not include 0. In certain unrestricted cases, Q is approximately -2.0 to 0.1. Approximately -1.5 to -0.15, approximately -1.0 to -0.2, approximately -1.5 to -0.5, approximately -1 It can vary from 0.2 to -0.8, and from approximately -1.0 to 0, but does not include 0. .

[0078] In certain embodiments, glomerular filtration rate is used to determine renal function in animals. For example, glomerular filtration rate is used to diagnose kidney disease or kidney dysfunction in animals. It can be used for: kidney disease and kidney impairment (e.g., decreased renal function, renal failure, chronic kidney disease). Kidney disease, glomerulonephritis, diabetic nephropathy, interstitial nephritis, polycystic kidney disease, and hypertensive kidney disease ) tends to reduce overall renal function, including GFR, and using the methods described herein It can be used to make a diagnosis. For example, in animals that have or are suspected of having a disease. The glomerular filtration rate is the glomerular filtration rate in one or more, for example, in a population of healthy subjects. The quantity can be compared. In kidney disease and kidney impairment, the glomerular filtration rate of the subject is compared to that of a healthy person. It can be predicted when the glomerular filtration rate is less than (or more) certain values. In certain embodiments In this case, if the glomerular filtration rate is statistically significantly lower than the average value for a population of healthy animals of the same species, Combined (i.e., [creatinine]) P [SDMA] Q (Estimated using the correlation with), It can diagnose kidney disease or kidney dysfunction. In non-limited cases, the GFR of the target animal. If the difference from the mean GFR of a healthy population is greater than 2 standard deviations, then the mean GFR of a healthy population is considered to be greater than 2 standard deviations. It is statistically significantly lower than FR.

[0079] In one embodiment, this disclosure relates to GFR or to a condition that correlates with kidney disease or renal dysfunction. This concerns the accumulation of standard values ​​for the equation. As shown in Figure 7, the values ​​of the equation are GF This may relate to a standard curve correlated with R. In other embodiments, the value or standard curve is related to the kidney Related to disease or renal dysfunction. Normal values ​​are shown in tables or figures referenced by healthcare providers. The form of, or machine-readable, relating to the computing device described herein It can be expressed with commands.

[0080] In another embodiment, kidney disease or kidney impairment is diagnosed without an intermediate step in determining GFR. The diagnosis can be made using an equation that includes the product of the creatinine concentration and the SDMA concentration. Therefore, using the equation to obtain the value, we can determine if that value is related to the disease or impairment. It can be compared to a known standard value or set of standard values. In one embodiment, The calculations were performed at a reference laboratory, and the equations were derived from The value can be reported to a physician, veterinarian, or other animal healthcare provider. This refers to a set of one or more known values ​​that correlate with kidney disease or impaired renal function. It can be compared to that. In another embodiment, the reference laboratory may, for example, use a computer The device allows for comparison, and the final results can be reported to the doctor.

[0081] In another embodiment, the disclosure relates to samples taken from animals, for example, serum samples. Chronic kidney disease by combining SDMA and creatinine concentrations and related values. This relates to the diagnosis of kidney disease or kidney impairment, such as chronic kidney disease (CKD). The formula shows a sample that indicates kidney disease. Use the cutoff values ​​for SDMA and creatinine obtained from the threshold concentration. The threshold concentration is determined by taking a sample from an animal population and using the standard deviation of the population, as is well known in this field. The concentration of MA and creatinine can be determined by relating them to the disease state. In various embodiments, the cutoff value of the SDMA (SDMA CUT ) is approximately 10 to approximately It may be 20 μg / dL, and more specifically, approximately 10, 11, 12, 13, 14, 15 It may be 16, 17, 18, 19 or 20 μg / dL, and more specifically, about 1 A level of 4 μg / dL is also acceptable. The cutoff value for creatinine is approximately 1.3 to 2.5 mg. / dL, or approximately 1.7 to approximately 2.8 mg / dL, more specifically, approximately 1.7 , 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7 And it may be 2.8 mg / dL. Once the cutoff value is determined, SDMA The cutoff values ​​for SDMA and creatinine in patient samples were compared. The value (C) representing the combination of concentrations of SDMA is given by the following formula: C = [SDMA] / SDMA CUT +[C RE] / CRE CUT It can be obtained by C is C CUT If it is greater than that, the patient has kidney disease and Diagnosed.

[0082] C CUT This involves selecting a value that has the optimal combination of sensitivity and specificity for the assay. This is determined by the following. Figure 13 shows different C CUT The value relates to specificity and / or sensitivity. This indicates the extent of the impact. CUT This is the desired specificity for detecting kidney disease. And / or you can choose to give a sensitivity level. For example, as shown in Figure 13. Regarding the dataset, both the sensitivity and specificity of the detection are C CUT If = 1.6, it exceeds 90%. . Usually, C CUT Larger values ​​result in higher specificity but lower sensitivity. Conversely, C CUT Smaller values ​​usually result in lower specificity but higher sensitivity. It will result in spillage.

[0083] This disclosure or the means for determining GFR or for diagnosing renal disease or renal dysfunction This relates to a computing device that performs the above calculations. The computing device is The value is calculated at runtime from an equation that includes the product of the creatinine concentration and the free SDMA concentration. Includes storage for software instructions.

[0084] In another embodiment, the disclosure relates to premature or early death in patients or animals. This relates to a prediction method for predicting the normal cutoff value. In contrast, if the SDMA value rises to a much higher level than the CRE value, [SDMA If there is an abnormal disharmony between [CRE] and [Cat], the cat is at high risk of premature death. In one embodiment, the method involves determining that the [SDMA] / [CRE] ratio in serum is at a certain threshold T If it is greater than this, it gives a prediction of early death.

[0085] For example, [SDMA] is expressed in μg / dL (micrograms / deciliter), [ When CRE is expressed in mg / dL (milligrams / deciliter), T is approximately 4 Approximately 10 values ​​(i.e., approximately 4 μg / dL of SDMA: 1 mg / dL of creatinine) We may assume a SDMA concentration of 10 μg / dL and a creatinine concentration of 1 mg / dL. In the application method, the threshold T may be approximately 7 to 20, and more specifically, approximately 7, 8, 9 Even if you are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 Good. A person skilled in the art would know that the concentrations of CRE and / or SDMA are measured in units different from those shown above. When expressed in units, the threshold for [SDMA] / [CRE] is relevant to the purpose and prediction of the method. It is important to understand that it can be changed each time and proportionally without affecting usability. It will probably happen.

[0086] Furthermore, the risk of premature death increases with the value of the [SDMA] / [CRE] ratio. It can increase. For example, an individual with [SDMA] / [CRE]=40 will have [SDMA] / Individuals with [CRE]=12 may be at higher risk of premature death.

[0087] In addition, an abnormally sudden increase in [SDMA] is an indication of an increased risk of premature death. Similarly, abnormally high levels of [SDMA] are an indication of an increased risk of premature death. For example, Abnormally high levels of [SDMA] in cats are above approximately 25 μg / dL and approximately 30 μg / dL. The value can be greater than or approximately greater than 30 μg / dL.

[0088] In one embodiment, this disclosure relates to serum SDMA concentrations that predict mortality. For example, As shown in Figures 19 and 20, serum SDMA concentrations higher than 14 μg / dL are associated with feline stagnation. This indicates that it is related to the mortality rate of dogs. Therefore, this disclosure shows that the mortality rate is the most This relates to identifying an appropriate cutoff value for SDMA concentration, as also indicated. Therefore, the cutoff value is in the range of approximately 10-20 μg / dL, more specifically, approximately 12-18 μg / dL. It is in the range of g / dL, or approximately 14-16 μg / dL. Identification involves, for example, measuring the concentration of serum SDMA in each member of a group of dogs or cats. By repeating the measurements over several months or years until the death of each member of that group, It can be determined that all dogs or cats in that group are C He has been diagnosed with KD. Candidates with different SDMA concentration cutoff values ​​and thresholds are being considered for survival time. The ability to predict contractions is tested. Such tests include, for example, the Kaplan-Mayer test. This can be done by using survival curves.

[0089] The Kaplan-Meier survival curve can be used to represent the prediction of mortality. The Lahn-Meier curve is particularly useful when not all subjects are continuously studied. This is a general method for dealing with different survival periods (time until an event). Each subject has three variations. The number, that is, their continuous time, their at the end of the continuous time (event occurrence or termination). The condition is characterized by the group of studies they belong to (e.g., SDMA < 14 or ≥ 14). The event is typically a clinical outcome such as death or tumor disappearance. The duration of the study is determined by the event of interest. However, this is the period during which it may occur from the start. The study will determine the results for the remaining participants. Even if not, the event ends before all participants exhibit this phenomenon. Figures 19 and 20 show Cats and dogs with serum SDMA concentrations of less than 14 μg / dL Cats and dogs with the above concentrations lasted approximately 1.6 times and 2.6 times longer, respectively. This is a Kaplan-Meier survival curve that shows survival. In another embodiment, the present disclosure is Methods for determining the ratio of creatinine to SDMA in healthy and diseased animals, Furthermore, regarding the use of its ratio for determining kidney disease and kidney disease-related mortality. For example, in healthy animals, the concentration of SDMA (μg / dL) and the concentration of creatinine ( mg / dL is generally a ratio in the range of approximately 4:1 to 10:1 (μg / dL:mg / dL). However, in some patients with chronic kidney disease, SDMA levels correspond to creatinine levels. The value is considerably larger than expected, which may indicate disease progression. Therefore Disharmony in the SDMA:creatinine ratio may predict animal mortality. Figure 14 As shown, there is a strong correlation between SDMA and creatinine, and the normal ratio is less than 10. It is full (μg / dL: mg / dL). However, the SDMA concentration (μg / The ratio of dL to creatinine (mg / dL) indicates the progression of kidney disease and often leads to death. Garu.

[0090] Therefore, by using the ratio of serum SDMA concentration to creatinine concentration, the disease and / or mortality rates are predicted. Therefore, this disclosure is not related to kidney disease or kidney disease. This includes determining or predicting death from animals, particularly cats and dogs. To determine the concentrations of SDMA and creatinine in blood, for example, serum samples. It includes. Once the concentration is determined, compare the ratio of SDMA to creatinine with the cutoff ratio. This determines the presence, extent, or progression of kidney disease, and the likelihood of death as a result of kidney disease. It is possible. The cutoff ratio is approximately 5-15 (μg / dL of SDMA: mg / dL of Cryptomeria). Achinine), more specifically, about 7-13 or about 9-11, and even more specifically, about 10. Animals with an SDMA:creatinine ratio greater than 10 may be at risk of premature death. This is considered to be a high probability. Generally, the higher the ratio, the higher the likelihood of imminent death. Yes. For example, Figure 15 shows the SDMA:creatinine ratio for a group of cats. 2 cats The cats had ratios of approximately 19 and 34, respectively, and each died during the study period. Figure 16. Figures 17 and 18 show the period within approximately two years after their ratio was identified as being greater than approximately 10. The results of a long-term study of three cats that died are shown. One of these cats had a ratio of more than 20. It was identified as being large and died within about a month (Figure 18).

[0091] Once diagnosed with kidney disease or kidney dysfunction, this method is for the treatment of kidney disease or kidney dysfunction. This may include applying the treatment to the animal. Examples of treatments include dialysis and kidney transplantation. Antibiotic therapy (for example, if renal dysfunction is due to an underlying infection), therapeutic diet Treatment of potential systemic inflammatory diseases, infections, or neoplastic diseases (e.g., renal dysfunction, If it is due to protein-losing nephropathy; fomepazole or administration of ethanol (for example, in cases of ethylene glycol poisoning); administration of ACE inhibitors In addition, a moderately protein-restricted diet and / or omega-3 fatty acid supplementation (e.g., for proteinuria) In cases of hyperphosphatemia; administration of phosphate binders and / or a phosphate-restricted diet; I Treatment with V-infusion, subcutaneous fluid therapy, low-protein diet, and / or H2 receptor antagonist Stollen (for example, in cases of azotemia); amlodipine, atenolol, and / or AC E inhibitors (e.g., in the case of systemic hypertension); bicarbonates and / or citrates (e.g.) Regarding acidosis; vitamin D analogues, e.g., calcitriol or 1,2 Administration of 5-dihydroxyvitamin-D) and a phosphate binder (preferably not Ca-based). , and / or phosphorus-restricted diet (e.g., in the case of renal secondary hyperparathyroidism); and / or H2 receptor antagonists and / or recombinant human erythropoietin Administration (possible together with iron supplementation) (for example, in cases of anemia) is one example.

[0092] In certain embodiments, the concentration of free SDMA is such that, by reference, they are all The U.S. Provisional Patent Application No. 61 / 0, filed on 7 August 2008, is incorporated herein by reference. 86,870, U.S. Patent Application No. 12 / 512,479, filed on July 30, 2009. , and U.S. Patent Application Publication No. 2010 / 003527, published on February 11, 2010. The method is determined using immunological methods, apparatus, and kits described in section 4. This includes a control, calibration material, or standard material containing one or more SDMA analogs. This can be done. In particular, the method can be used with microplates and lateral plates, but is not limited to this. This can be achieved by using immunoassay techniques well known to those skilled in the art, including the use of a low-power apparatus. It is possible. Animal subjects from which samples can be obtained for detecting SDMA include: This includes both humans and non-human animals (e.g., companion animals, livestock, etc.). SDMA Determining disease status related to the presence or quantity of [the substance] is done in both human and non-human animal subjects. It can be done regarding this.

[0093] Solid-phase assays are a commonly used binding assay technique. The presence of the analyte is conjugated. This is indicated by the binding of the analyte to the jugate and / or immobilized complementary binding member. There are several assay devices and procedures that are used. In one particular embodiment, The regulated binding member (e.g., anti-SDMA antibody) is attached to the solid phase during the assay, for example. Reaction wells, dipsticks, test strips, flow-through pads, paper, fiber matrix It bonds to or becomes bonded to custard or other suitable solid materials in the sample. The binding reaction between free SDMA and immobilized antibody is performed by adding a certain amount of SDMA analog to the sample. This is determined by the labeling of SDMA analogs, and the SDMA analog is conjugated to the label. This includes contacting a mixture of the sample and an SDMA analog with a solid phase, and then the mixture and The solid phase is incubated to bind the immobilized antibody, SDMA, and SDMA analog. After incubation, unbound reactants are removed from the solid phase. The amount of label bound to the antibody by the body is measured. It is inversely proportional to the amount of free SDMA in the sample.

[0094] Immobilization of one or more antibodies against SDMA onto an apparatus or solid carrier is performed by the antibody This is done so that the sample, diluent, and / or washing procedure do not wash them away. Alternatively, multiple antibodies can be adsorbed by physical adsorption (i.e., without the use of a chemical linker), It can be attached to the surface by chemical bonding (i.e., using a chemical linker). Yes, it is possible. Chemical bonding can lead to stronger adhesion of antibodies to the surface, and surface bonding components It can give the offspring a clear orientation and form.

[0095] In another embodiment, the SDMA antibody produced in a specific species binds to a solid carrier. It binds to a solid support through interaction with a species antibody. In one particular embodiment, it is an anti-SD agent. MA antibodies are produced in rabbits, and the carrier is the anti-SDMA antibody produced in rabbits. It recognizes and binds to the anti-rabbit antibody. In this embodiment, the antibody is obtained from that species. It may also be in the form of antiserum. The anti-SDMA antibody is used before adding the sample to the solid phase. It can be attached to a solid phase containing the species antibody, or the anti-SDMA antibody can be added to the solid phase. Before doing so, it can also be mixed with the sample. In either case, the anti-SDMA antibody is It becomes bound to the solid phase by binding to the anti-species antibody on the solid phase.

[0096] In another embodiment, one or more labeled antibodies are attached to a solid support in the mixture. Before that, it can be mixed with the test sample. In this case, the SDMA analog is , the sample, diluent and / or adhere to the solid carrier so as not to be washed away by the washing procedure It can be done. The labeled antibody in the sample binds to the SDMA in the sample and Therefore, it cannot be used to bind to the SDMA analog on the solid support. After attaching the mixture to the solid support and appropriately incubating, the mixture is washed from the solid support. Antibodies that were not bound to SDMA in the sample will bind to the SDMA analog on the solid support. The presence or amount of SDMA in the sample is inversely proportional to the amount of antibody bound to the SDMA analog. The signal associated with the label on the antibody can be measured by an appropriate method. After attaching the mixture to the solid support and appropriately incubating, the mixture is washed from the solid support. Antibodies that were not bound to SDMA in the sample will bind to the SDMA analog on the solid support. The presence or amount of SDMA in the sample is inversely proportional to the amount of antibody bound to the SDMA analog. The signal associated with the label on the antibody can be measured by an appropriate method.

[0097] Figure 1 shows a comparison between ELISA for detecting SDMA in pooled canine sera spiked with SDMA and SDMA detection using mass spectrometry. As shown, the SDMA concentration values obtained using the ELISA described herein strongly correlate with the SDMA concentration values obtained using MS. As shown, the SDMA concentration values obtained using the ELISA described herein strongly correlate with the SDMA concentration values obtained using MS. The SDMA concentration values obtained using the ELISA described herein strongly correlate with the SDMA concentration values obtained using MS.

[0098] The detection of the antibody-antigen complex can be achieved by various techniques well known to those skilled in the art, such as nephelometry, enzyme labeling, radioactive labeling, luminescence, or fluorescence. Immunoassay methods are known to those skilled in the art and include, but are not limited to, radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence polarization immunoassay (FPIA), microparticle enzyme immunoassay (MEIA), enzyme multiplied immunoassay technique (EMIT) assay, immunoturbidimetric or agglutination assay, colloidal gold-based immunoassay including lateral flow devices, and chemiluminescent magnetic immunoassay (CMIA). In RIA, the antibody or antigen is labeled with radioactivity and used in a competitive or non-competitive format. In EIA, the antibody or antigen is an enzyme that converts a substrate into a product with a measurable signal, such as a color change. The detection of the antibody-antigen complex can be achieved by various techniques well known to those skilled in the art, such as nephelometry, enzyme labeling, radioactive labeling, luminescence, or fluorescence. Immunoassay methods are known to those skilled in the art and include, but are not limited to, radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence polarization immunoassay (FPIA), microparticle enzyme immunoassay (MEIA), enzyme multiplied immunoassay technique (EMIT) assay, immunoturbidimetric or agglutination assay, colloidal gold-based immunoassay including lateral flow devices, and chemiluminescent magnetic immunoassay (CMIA). Immunoassay methods are known to those skilled in the art and include, but are not limited to, radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence polarization immunoassay (FPIA), microparticle enzyme immunoassay (MEIA), enzyme multiplied immunoassay technique (EMIT) assay, immunoturbidimetric or agglutination assay, colloidal gold-based immunoassay including lateral flow devices, and chemiluminescent magnetic immunoassay (CMIA). In RIA, the antibody or antigen is labeled with radioactivity and used in a competitive or non-competitive format. In EIA, the antibody or antigen is an enzyme that converts a substrate into a product with a measurable signal, such as a color change. Immunoassay methods are known to those skilled in the art and include, but are not limited to, radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence polarization immunoassay (FPIA), microparticle enzyme immunoassay (MEIA), enzyme multiplied immunoassay technique (EMIT) assay, immunoturbidimetric or agglutination assay, colloidal gold-based immunoassay including lateral flow devices, and chemiluminescent magnetic immunoassay (CMIA). In RIA, the antibody or antigen is labeled with radioactivity and used in a competitive or non-competitive format. In EIA, the antibody or antigen is an enzyme that converts a substrate into a product with a measurable signal, such as a color change. In RIA, the antibody or antigen is labeled with radioactivity and used in a competitive or non-competitive format. In EIA, the antibody or antigen is an enzyme that converts a substrate into a product with a measurable signal, such as a color change. In RIA, the antibody or antigen is labeled with radioactivity and used in a competitive or non-competitive format. In EIA, the antibody or antigen is an enzyme that converts a substrate into a product with a measurable signal, such as a color change. In EIA, the antibody or antigen is an enzyme that converts a substrate into a product with a measurable signal, such as a color change. ​​Labeled. In FPIA, the antigen is labeled with a fluorescent label and is labeled from the sample. It competes with antigens that are not present. The amount of analyte being measured is inversely proportional to the amount of signal being measured. In MEIA, solid-phase microparticles are coated with antibodies against the target antigen and capture the analyte. It is used for this purpose. The antibody for detection is labeled with an enzyme, as in the EIA method. The concentration of the analyte being measured is proportional to the amount of signal being measured. Then, when the chemiluminescent label is conjugated to an antibody or antigen and combined with its substrate... It generates light. CMIA can be set to conflicting or non-conflicting formats, respectively. This yields results that are inversely or directly proportional to the amount of analyte present.

[0099] The use of test strips immersed in reagents in specific binding assays is also well known. In the method, the test sample is attached to a portion of the test paper, and the test paper material is moved, or it is absorbed by it. Therefore, the analyte to be detected or measured is absorbed by the test sample itself. Possibly using an eluent that may be a body or added separately, in the material or the It passes along the material. The analyte is fixed in place by the complementary bonding members of the analyte. It moves to the capture or detection area on the test paper. The degree to which the analyte binds at the detection area is determined by the test. Using conjugates that can be incorporated into the test paper or attached separately. It can be determined. In one embodiment, antibodies specific to SDMA are fixed at a distance. It is immobilized on a body carrier. After adding the sample, detection of the SDMA-antibody complex on the solid carrier is performed. This may be done by any means known in the art. For example, by reference the whole thing is revealed. U.S. Patent No. 5,726,010, incorporated in the details, is an example of a lateral flow apparatus, S NAP® Immunoassay Equipment (IDEXX Laboratories) is listed. They are doing it.

[0100] Other detection techniques involve immersing magnetic particles or microbeads, for example, in superparamagnetic iron oxide. Polymer beads are used. These beads, for example, bind to specific bonding partners of the analyte. The beads attach to the target analyte in the sample being tested, and are then typically magnetic. Therefore, it is isolated or separated from the solution. Once isolated, it can be directly, optically, or Alternatively, other tests may be conducted using a camera, including observing specific images or labels. It is possible.

[0101] In further embodiments, SDMA analogs, particularly thiol-containing and hydroxyl-containing SDMA analogs containing amino acids and carboxylates are used to convert SDMA into other molecules (carboxylates). (Target to be denjugated), for example, can be linked to an activating protein, such as SDMA. It forms a conjugate. The SDMA analogs described herein form a conjugate with SDMA. Targets to be targeted, such as proteins, polypeptides, detectable labels, and solid supports. It can be linked to and results in an SDMA conjugate. The SD described herein Use MA conjugates to create antibodies for use in SDMA-specific immunoassays. It is possible. The antibody is arginine, ADMA and / or monomethylarginine They have little to no cross-reactivity with SDMA. SDMA analogs are SDMA It can also be conjugated with labels used in immunoassays specific to that substance.

[0102] The SDMA analogs may have, for example, the following structure

[0103] [Chemical formula] (where x and y are integers in the range from 1 to 5) and may have.

[0104] According to one embodiment, the SDMA analog has the following general formula

[0105] [Chemical formula] [where R1 may be a thiol (or protected thiol), hydroxyl (or protected hydroxyl), amino (or protected amino) group, or carboxylate (including carboxylic acid) or protected carboxylate group] and has. [[ID=Y29]]

[0106] Suitable thiol, hydroxyl, amino and carboxylate protecting groups, for example, those described in T.W. Greene et al., Protective Groups in Organic Synthesis, 3rd Edition (1999) are known to those skilled in the art. )]]

[0107]

[0108] [Chemical formula] or a salt thereof. The compound of formula (3) provides an available thiol that can react with a conjugate target containing a suitable "thiol reaction site", i.e., a site that reacts with a thiol group. For example, maleimide, alkyl halide and aryl halide, etc.

[0109] ​Furthermore, α-haloacyls can react with thiols to form thioethers, for example. This is a typical thiol reaction site. Similarly, pyridyl disulfide reacts with thiols, Mixed disulfides can be formed.

[0109] In another embodiment, R1 is X-R2, where X is -S-, -O-, -N- , or -COO-, where R2 is thiol, hydroxyl, amino, or carboxy It is a label containing a silate-reactive group.

[0110] In one embodiment, R1 is X-R2, where X is -S-, -O-, -N-, Alternatively, it is -COO-, where R2 is a thiol, hydroxyl, amino, or carboxyl. This protein has functional groups that include rate-reactive groups.

[0111] In one embodiment, SDMA is conjugated with a carrier protein and "haptic Ten-carriers form immunogens, which trigger an immune response to epitopes containing SDMA. It can be used to stimulate. Examples of immunoprotozoa include, but are not limited to, those listed above. However, BSA, KLH, and ovalbumin are examples. Haptens are immunogenic proteins. Protocols for conjugating with the material are publicly known in this field (e.g., Anti Bodies: A Laboratory Manual, E. Harlow and D. Lane, ed., Cold Spring Harbor Laboratory (Cold (See Spring Harbor, NY, 1988, pp. 78-87.)

[0112] In one embodiment, the SDMA analog is a maleimide-activated protein, for example, maleimide Imide-activated keyhole limpet protein (KLH) or maleimide-activated bovine blood It is conjugated with clear albumin (BSA).

[0113] In one embodiment, the compound of formula (3) is a maleimide-activated protein, for example, ma Reimide-activated keyhole limpet protein (KLH) or maleimide-activated bovine It is conjugated with serum albumin (BSA).

[0114] Therefore, in certain embodiments, the compound of formula (3) and maleimide-activated protein The conjugate with quality is given by the following formula:

[0115] [ka] (In the formula, m is an integer.) It has.

[0116] Typically, m is greater than 5. However, the value of m can be varied. For example, m is a Murray, commercially available from Sigma-Aldrich in St. Louis, MO. In mido-activated BSA, there are approximately 15 maleimide groups per protein; m is Si In maleimide-activated KLH, which is commercially available from gma-Aldrich, the protein It has approximately 80 maleimide groups per unit; m is Thermo of Rockford, IL Scientific Pierce Protein Research Produ In maleimide-activated BSA commercially available from cts, approximately 15% per protein It is a maleimide group in the range of approximately 25; m is Thermo Scientific P Commercially available from ierce Protein Research Products In maleimide-activated KLH, there are more than approximately 400 maleimide groups per protein. Ri;m is commercially available from AGS Scientific in San Diego, CA. In maleimide-activated KLH, the range of ma is approximately 150 to 300 per protein. It is a reimide group. Generally, m is the number of available amine groups present in the immunoprotoprotein. The number of available amines is limited by the number of immunoprotoproteins, polyamines, and condiments. It can be increased by terminating.

[0117] In one embodiment, the protein is BSA, and m is greater than approximately 5. In this embodiment, the protein is BSA, and m is greater than approximately 10. The protein is BSA, and m is greater than approximately 25. In one embodiment, tan The protein is BSA, and m is greater than approximately 50. In one embodiment, the protein is , BSA, and m is greater than approximately 75. In one embodiment, the protein is BSA And m is in the range of about 5 to about 80. In one embodiment, the protein is BS A is greater than approximately 75. In one embodiment, the protein is BSA. m is in the range of about 10 to about 80. In one embodiment, the protein is in BSA Yes, m is greater than approximately 75. In one embodiment, the protein is BSA, and m It is in the range of approximately 20 to approximately 80. In one embodiment, the protein is BSA. , m is greater than approximately 75. In one embodiment, the protein is BSA, and m is, It falls within the range of approximately 30 to 80.

[0118] In one embodiment, the protein is KLH, and m is greater than about 5. In this state, the protein is KLH, and m is greater than approximately 50. The protein is KLH, and m is greater than approximately 100. In one embodiment, The protein is KLH, and m is greater than approximately 200. In one embodiment, the protein The quality is KLH, and m is greater than approximately 300. In one embodiment, the protein is It is KLH, and m is greater than about 400. In one embodiment, the protein is KLH And m is greater than about 500. In one embodiment, the protein is KLH. , m is greater than approximately 600. In one embodiment, the protein is KLH, and m is , greater than approximately 700. In one embodiment, the protein is KLH, and m is approximately 8 Greater than 00. In one embodiment, the protein is KLH, and m is about 5 to about It is in the range of 800. In one embodiment, the protein is KLH, and m is about 5. It is in the range of approximately 600. In one embodiment, the protein is KLH, and m is approximately It is in the range of 5 to about 400. In one embodiment, the protein is KLH, and m is , in the range of approximately 5 to approximately 200. In one embodiment, the protein is KLH, m is in the range of about 5 to about 100. In one embodiment, the protein is KLH Furthermore, m is in the range of approximately 100 to approximately 200. In one embodiment, the protein is K LH is in the range of approximately 100 to approximately 300. In one embodiment, protein The quality is KLH, and m is in the range of approximately 100 to approximately 400. In various embodiments, The protein is KLH, and m is approximately 100 to approximately 500, approximately 100 to approximately 600, approximately In the range of 100 to approximately 700, approximately 100 to approximately 800, or approximately 100 to approximately 1,000. be.

[0119] The conjugate of the compound of formula (3) and maleimide-activated protein is well known to those skilled in the art. It can be characterized using the following method (for example, Sigma-Aldrich Technical Bulletin for Maleimide Activat Refer to the ed BSA, KLH Conjugation Kit (catalog number MBK1). I want to be illuminated.

[0120] In an alternative embodiment, the SDMA analog is thiol, hydroxyl, amino or It is linked to a detectable label via a carboxylate group. The label is itself detectable Capabilities (e.g., radioisotope labeling, chemiluminescent dyes, electrochemical labeling, metal chelates, It may be latex particles, or fluorescently labeled, or in the case of enzyme labeling, detectable It may catalyze the chemical change of a substrate compound or composition (for example, an enzyme, for example) (e.g., horseradish peroxidase, alkaline phosphatase). The label itself is a test Specific binding molecules that may be able to bind (e.g., biotin, avidin, streptavidin) N, digoxigenin, maltose, oligohistidine, 2,4-dinitrobenzene, fe It may also be naric acid, ssDNA, dsDNA, etc. SDMA is well known to those skilled in the art. The method can be used to concatenate it with a detectable label. For example, an SDMA analog. This is derived from freeze-dried horseradish powder containing more than 200 units per 1 mg of protein. Maleimide-activated peroxidase (St. Louis, MO's Sigma-Aldri The product is commercially available from ch (catalog number P1709), and you should follow the instructions in the product manual. It can be connected to ( ).

[0121] An analogue of equation (3) is obtained by the following illustrative synthesis scheme (1) to form SDMA (Gibbs Prepared from (commercially available from EMD Chemicals Inc. in town, NJ) It may also be used.

[0122] [ka]

[0123] The primary and secondary amino groups of SDMA are converted into di-tert-butyl dicarbonate. It is protected by reacting with (Boc2O). The resulting tert-butoxyca Rubonyl (BOC)-protected SDMA ((Boc3)-SDMA, 1) is then applied to the resin. To cause a bond to form. For example, (Boc3)-SDMA(1) is linked to dimethylformamide (DMF). The 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl Chiluranium hexafluorophosphate methamaminonium (HATU) and N,N-di In the presence of sopropylethylamine (DIPEA), (Boc3)-SDMA(1) is saturated. Stain-4-methoxytrityl resin (EMD Chem, Gibbstown, NJ) By bringing it into contact with (commercially available from icals Inc.), it connects with the resin. It is possible to bond (Boc3)-SDMA cystamid to the resin. e) (2) is obtained. B on (Boc3)-SDMA cystamide (2) bound to the resin. The OC protecting group is removed, and the resulting resin-bound SDMA cystamide is, for example, dichloro Using trifluoroacetic acid in lomethane, it is cleaved from the resin and SDMA cystamide (3 This resulted in a reaction with hydrochloric acid, which converted to hydrochloride salt (4).

[0124] Analogues of the above formulas A through D can be prepared using the same method as described in Scheme 1. It is possible.

[0125] Next, the maleimide-activated protein is reacted with SDMA-cystamide (3), and the following steps are taken: The SDMA cystamide protein conjugate described in Scheme II is obtained. :

[0126] [ka] (In the formula, n is an integer in the range of 1 to 3, and m is an integer as defined above.)

[0127] The resulting conjugates are not limited to those, but can be used in column chromatography, for example. For example, Sephadex as a solid carrier (e.g., Sephadex G-25M) Gel filtration column chromatography using commercially available products from Sigma-Aldrich It can be purified using methods known to those skilled in the art, including fees.

[0128] The conjugate of analogues A through D is performed using a method similar to that described in Scheme 2. It can be manufactured by [this method].

[0129] Conjugation of analogs of formulas A to D with maleimide-activated KLH or maleimide-activated BSA Using jugate as an immunogen, an antibody that substantially binds to SDMA (i.e., anti-SDMA) is used. DMA antibody) containing ADMA, L-arginine, and / or N-methylarginine This can yield antibodies that show no or substantially no cross-reactivity with . Formula (3) The conjugate of an analog of this with maleimide-activated KLH or maleimide-activated BSA An antibody that, when used as an immunogen, substantially binds to SDMA (i.e., an anti-SDMA antibody). Such antibodies can result from ADMA, L-arginine, and / or It shows no or substantially no cross-reactivity with N-methylarginine.

[0130] Anti-SDMA antibodies useful in the methods, apparatus, and kits disclosed herein include ADMA and arginine. , and / or have little or no cross-reactivity to monomethylarginine It is characterized by binding to SDMA with high affinity without any need for assistance. Therefore, in this specification This description concerns isolated, genetically modified, synthetic, and / or in vivo-generated substances. This invention relates to a developed anti-SDMA antibody, as well as a method for producing and using such an antibody, and for diagnosis. This includes compositions, methods, and apparatus for use and therapy. The anti-SDMA antibodies described herein are For example, renal function, for example, decreased renal function, renal failure, glomerular filtration rate (GFR), inulin As a marker of clearance and creatinine clearance, as well as for renal impairment / disease Diseases such as chronic kidney disease, glomerulonephritis, diabetic nephropathy, interstitial nephritis, polycystic kidney disease, and It is also useful as a diagnostic marker for hypertensive kidney disease.

[0131] In one embodiment, the resulting antibody is a free SDMA (i.e., polypeptide). It can detect SDMA (which is not part of the chain), ADMA, L-arginine, and / or shows no or substantially no cross-reactivity with N-methylarginine. As shown herein, the antibodies described herein are based on equal concentrations of antigens, ADMA, L-A It shows less than 1% cross-reactivity with arginine and / or N-methylarginine. As is generally understood, the effect of cross-reactivity is due to the immune antigens (SDM) in the test sample. A) Cross-reactive antigens (e.g., ADMA, L-arginine, and / or It depends on the relative abundance of N-methylarginine. For example, high cross-reactivity of about 50%. It is acceptable if the concentration of the immune antigen is 100 times higher than the concentration of the cross-reactive antigen. Conversely, a low cross-reactivity of about 1% is when the concentration of the cross-reactive antigen is lower than the concentration of the immune antigen. If the cross-reactivity is 100 times, it could be a problem. Therefore, the effect of cross-reactivity will be analyzed. This must be considered in the context of the relative abundances of any cross-reactive antigens and immunoantigens in the sample. In various embodiments of this disclosure, cross-reactivity is SDMA or SDMA-like. It does not affect the substantial binding between the body and anti-SDMA antibodies.

[0132] The method for producing antibodies involves using one or more SDMA conjugates as immunogens. This method may include using it to stimulate an immune response. Using Tocol, administer one or more SDMA conjugates to animals. For example, as described in Example 3 below, appropriate antibodies can be obtained from animal body fluids (multiple fluids are possible). This includes separation. Alternatively, using SDMA conjugates in phage display. By using this method, phages exhibiting appropriate antibodies on their surfaces can be selected, and then a small number of However, the isolation of nucleic acid sequences encoding various domain regions of appropriate antibodies continues. Display methods are well known to those skilled in the art (for example, Antibody Phage Dis play;Methods in Molecular Biology, Vol,17 8, O'Brien, Philippa M.; Aitken, Robert (Eds. (See 2002). Monoclonal antibodies against SDMA are generally used in this field. It can be prepared by known methods.

[0133] The SDMA analogs described herein are used in receptor binding assays, for example, in immunoassays on SDMA. To provide a detectable conjugate for use in the assay, it can be linked to a label. Yes, it is possible. Similarly, anti-SDMA antibodies can also be used in receptor binding assays, for example, in SDMA immunoassays. It can be linked to a label to provide a detectable anti-SDMA antibody for use in the sieve. The SDMA analog and anti-SDMA antibody are labeled using methods well known to those skilled in the art. It can be linked. For example, Immunochemical Protocols; Methods in Molecular Biology, Vol.295, RB (Urns, ed., 2005). Detectable SDMA conjugate or detectable anti-SD MA antibodies emit signals related to the presence or amount of SDMA in the test sample. It can be used in various homogeneous, sandwich, competitive, or non-competitive assay formats. It is possible.

[0134] In certain embodiments, the immunoassay method is a competitive immunoassay for the detection of anti-SDMA antibodies. This is an epidemic assay. A competitive immunoassay can be performed using the following exemplary method. Samples containing potential anti-SDMA antibodies from bodily fluids are transmitted via a solid carrier and conjugate. A conjugated SDMA analog, and an anti-SDMA antibody conjugated with a detectable label. Bring them into contact. The target anti-SDMA antibody present in the sample is conjugated with the solid carrier. For binding with SDMA analogs, anti-SDMA was conjugated with a detectable label. It competes with the antibody. The amount of label bound to the solid support is the same as that of the unbound antibody and solid support. This can be determined after separation. In an alternative embodiment, a competitive immunoassay is This is carried out by the following exemplary method: extracting potentially anti-SDMA antibodies from animal body fluids. The sample consists of an SDMA analog linked to a detectable label, followed by a solid carrier and conjugate. The sample is brought into contact with the gated antibody. The anti-SDMA antibody in the sample is linked to a detectable label. It competes with anti-SDMA antibodies on a solid carrier for binding to the SDMA conjugate. In either case, the signal obtained indicates the amount of the desired SDMA antibody present in the sample and It is the opposite relationship.

[0135] Of course, other methods for measuring free SDMA may be used in the methods described herein. It is possible. SDMA itself can predict disease (see Figures 2 and 3). sea ​​bream).

[0136] The concentration of creatinine in serum can be measured by various methods, as is known to those skilled in the art. It can be determined. For example, Catalyst Dx (trademark) Chemistry A Nalyzer or VetTest® Chemistry Analyzer Dry slides suitable for creatinine testing, for example, IDEXX Laboratories It can be used in conjunction with commercially available products from ories. Other analytical instruments and RIDE, for example, is available from Ortho Clinical Diagnostics. The VITROS® 950 analyzer and VITROS® CREA slides are also available. Can be used: Enzymatic wet assay It can also be used. For example, a person skilled in the art can use an Integra 800 analyzer to perform enzyme humidification. A chemical method can be used. One specific assay involves detection at 552 nm, and Creatininase / creatinase / monkey with absorbance blanking at 659 nm Based on the cosine oxidase system. Those skilled in the art will know of a colorimetric method, for example, one based on picric acid. For example, the Jaffe assay can also be used. Other methods known to those skilled in the art, for example , each incorporated herein by reference U.S. Patent Publication No. 2005 / 026657 The items described in U.S. Patent No. 4 and U.S. Patent No. 4,818,703 also concern creatinine concentration. It can be used to measure creatinine concentration. In certain embodiments, creatinine concentration The measurement is performed using isotope dilution mass spectrometry.

[0137] Several methods are known for determining GFR. For example, GFR can be determined by reference. The entirety of Perrone et al., Am.J. Kidney D is incorporated herein by reference. isease, vol.16, pp. 224-225 (1990) and Levey et al., JA It is described in m.Soc.Nephrol., vol.4, pp. 1159-1171 (1993). As stated, 125 It can be determined as the renal clearance of I-iotalamate. Urine collection-based methods also contain other exogenous substances, for example, 51 Cr-EDTA, 99 Tc-DTPA Use including measuring the renal clearance of iohexol or inulin. This can be done. The GFR value obtained by any of these methods is the method described herein. To obtain a calibration curve or standard values ​​to be used, a curve is created for samples collected at approximately the same time. The reciprocal of the product of the reatinine concentration and the free SDMA concentration can be used to correlate the results.

[0138] The following are for illustrative purposes only and do not represent the present invention as described in the broader terms above. This is not intended to limit the scope. All references cited in this disclosure are references to the following: This is incorporated herein by reference. [Examples]

[0139] Example 1: Synthesis of SDMA cystamide (3) and SDMA cystamide hydrochloride (4) SDMA cystamide (3) was prepared according to the synthesis route described in Scheme 1.

[0140] (BOC)3-SDMA(1): 4.36 g (20 mmol) of di-tert-butyl To a 20 mL solution of dioxane (Boc2O) of dicarbonate, add 10 mL of 5.0 N 550 mg (2.0 mmol) of N,N-dimethylarginine dihydride dissolved in NaOH Lochloride (SDMA) (EMD Chemicals, Gibbstown, NJ) The commercially available product from nc. was added dropwise over 30 minutes while stirring at room temperature. The reaction mixture was stirred overnight. Then, 30 mL of dichloromethane was added to the reaction mixture. DCM) and 30 mL of water were added, and the pH was adjusted to 6.5 with acetic acid (AcOH). The CM layer was separated, washed with brine, and dried with anhydrous Na2SO4. Then, DCN was subjected to reduced pressure. The residue was removed below to obtain a solid. The obtained solid was washed twice with 10 mL of hexane. Then, The solid was dried in a vacuum to obtain 800 mg of pale yellow solid. The next reaction requires further purification. It was not required. The solid was characterized by mass spectrometry. ESI-MS: 525.7 M+Na) + , 503.6 (M+1) + , 403.5(M-Boc+1) + , 303.5( M-2Boc+1) + .

[0141] (Boc)3-SDMA-cystamine-resin(2):15 mL of dimethylformamide (DMF) contains 600 mg (1.2 mmol) of (Boc)3-SDMA(1) and 627 mg (1.6 mmol) of 2-(1H-7-azabenzotriazol-1-yl)-1, 1,3,3-Tetramethyluranium hexafluorophosphate methaminium (HATU ) Mixture of 420 μL (2.4 mmol) N,N-diisopropylethylamine ( DIPEA was added. The resulting mixture was then stirred for 20 minutes under a dry N2 atmosphere. Separately, cystamine 4-methoxytrityl resin (1.0g) (Gibbstown) (Commercially available from EMD Chemicals Inc. in NJ) expands D The mixture was washed using MF. Then, the expanded resin was added to the reaction mixture, and the reaction mixture was prepared. The mixture was then gently shaken for 3 hours under an N2 atmosphere. The resin was then collected by filtration and 5 mL of D2 was added. The samples were washed sequentially with MF, 5 mL of methanol, and 5 mL of DCM.

[0142] SDMA-Cystamide (3): Modified resin with 15mg of 90% trifluoroacetic acid (TFA). L was added, the resulting mixture was gently shaken for 2 hours, and then filtered. The resin was then mixed with 3 mL of TFA. Washed twice with / DCM(1:1(v / v)). Combined the filtrate and washed-off materials. Then, it was added to 200 mL of cold ether to obtain a precipitate. The obtained precipitate was centrifuged. The sample was collected and dried under reduced pressure to obtain 300 mg of SDMA-cystamide (3). SDMA-cystamide (3) was characterized by mass spectrometry. EIS-MS:262. 4(M+1)+, 132.0(M+2)+.

[0143] SDMA-cystamide hydrochloride (4): SDMA-cystamide 3 (300 mg), 1. The mixture was reconstituted with 5 mL of 0N HCl, and the resulting mixture was freeze-dried to obtain a pale yellow solid as a foamy substance. I got it.

[0144] Other SDMA analogs were prepared using the same general procedure described above.

[0145] Example 2: Conjugation of SDMA cystamide (3) and maleimide-activated protein Yon A. For conjugating SDMA cystamide (3) with maleimide-activated KLH General procedure 1. Maleimide-activated KLH (Sigma-Aldrich, St. Louis, MO) Slowly open the commercially available vial (catalog number K0383) and release the vacuum. Removed. Return the contents of the vial with 2.1 mL of water, and add 5 mg / mL of maleimide-activated KLH. 20 mM solution containing 30 mM NaCl, 2 mM EDTA, and 80 mM sucrose A solution was obtained in sodium phosphate buffer (pH 6.6). 3. Conjugation buffer (Sigma-Aldrich, St. Louis, MO) By rehydrating the commercially available product (catalog number C3957) with 10 mL of water, 20 mM sodium phosphate with 100 mM EDTA and 80 mM sucrose A conjugation buffer solution (pH 6.6) was prepared. 4. Approximately 0.8 mg of hapten (i.e., SDMA cystamide (3)) in 0.5 mL of ko It was dissolved in the denaturation buffer. To determine the binding efficiency (hapten-total), 50 The obtained peptide solution was maintained in μl. The maintained hapten solution was stored at 2–8°C. . 5. Immediately transfer the hapten solution from Step 4 to the reaction vial equipped with a stirring bar, and then to Step 2. The mixture was mixed with maleimide-activated KLH solution. The resulting mixture was heated under a gentle stream of nitrogen for about 1... The air was removed while stirring for 2 minutes. 6. Close the reaction vial and continue stirring at room temperature for 2 hours or overnight at 2-8°C. 7. Step 6 (Hapten-Free) 100 μl of the conjugation reaction product, measuring the binding efficiency. It was maintained for the decision.

[0146] B: For conjugating SDMA cystamide (3) with maleimide-activated BSA General procedure 1. Maleimide-activated BSA (Sigma-Aldrich, St. Louis, MO) Slowly open the commercially available vial (catalog number B7542) and release the vacuum. Removed. Return the contents of the vial with 2.1 mL of water, and add 5 mg / mL of maleimide-activated BSA. 20 mM solution containing 30 mM NaCl, 2 mM EDTA, and 80 mM sucrose A solution was obtained in sodium phosphate buffer (pH 6.6). 3.5 mg of hapten (i.e., SDMA cystamide (3)) in 0.5 mL of conjugate It was dissolved in the saturation buffer (prepared as described in step A3). Binding efficiency (H For the determination of the butene (whole), 50 μl of the obtained peptide solution was maintained. The hapten solution was stored at 2-8°C. 4. Immediately transfer the hapten solution from Step 3 to the reaction vial equipped with a stirring bar in Step 2. The mixture was mixed with maleimide-activated BSA solution. The resulting mixture was heated under a gentle stream of nitrogen for about 1... The air was removed while stirring for 2 minutes. 5. Close the reaction vial and continue stirring at room temperature for 2 hours or overnight at 2-8°C. 6. Step 5 (Hapten-Free): 100 μl of the conjugation reaction product, measuring the binding efficiency. It was maintained for the decision.

[0147] Isolation of C:KLH or BSA conjugates 1. Container of phosphate-buffered saline (PBS) (Sigma-Aldrich, St. Lo The contents of the product sold commercially by UIS and MO (catalog number P3813) are in a 1-liter container. It dissolved in water. 2. Sephadex G-25M gel filtration column (Sigma-Aldrich, St Supporting Louis, which is commercially available from MO (catalog number B4783), in a beaker. Ta. 3. Remove the top cap of the column and cut open the bottom end of the column to allow excess liquid to flow out. We made sure not to deplete the column. The column was equilibrated with 4.30 mL of PBS. 5. The reaction mixture from Example 2A or 2B was attached to the column. 6. Using a total volume of approximately 10 mL, elute the column with PBS and extract approximately 0.5–1.0 mL of image. The fractions were collected. The presence of protein in each fraction was determined by measuring the absorbance of each fraction at 280 nm. This was done to monitor the situation. 7. The fractions containing protein were combined. Figure 4 shows the absorbance versus fraction number for protein K. This graph shows exemplary dissolution profiles for LH (◆) and BSA (■). be. 8. The protein-containing fraction was divided into smaller portions and stored frozen at -20°C.

[0148] D. Assay for determining binding efficiency 1. Cysteine ​​standard assay - To estimate the binding efficiency between analogs and cysteine ​​peptides. A standard curve was created using known concentrations of cysteine. The assay was performed at pH 8.0 It reacts with a sulfhydryl group to produce a chromophore that has maximum absorbance at 412 nm, 5, Based on the reaction of 5'-dithiobis(2-nitrobenzoic acid) (DTNB or Elman's reagent) There it was. The subsequent steps are as follows: a. DTNB buffer is provided by DTNB buffering agents (Sigma-Aldrich, St. Louis). s, the contents of the vial (catalog number D4179) sold by MO, 10 mL It was prepared by dissolving it in water. b. Next, DTNB reagent (Sigma-Aldrich, St. Louis, MO) A commercially available product (catalog number D8130) is used, starting from step a, with 5 ml of DTNB buffer. It was dissolved in L. c. Immediately before use, add 32 mg of L-cysteine ​​hydrochloride monohydrate (S) to the cysteine ​​solution. IGMA-Aldrich, St. Louis, and MO are commercially available (catalog number) It was prepared by dissolving C7880)) in 1 mL of water. The resulting L-cysteine Dilute the hydrochloride solution sequentially with water to obtain diluted stocks in the range of 0.4 to 0.04 mg / mL. The solution was obtained. The diluted stock solution was used immediately. d. 50 μL of diluted stock solution was added to a labeled test tube. The test tubes I had were used as blanks. e. Next, add 0.1 mL of water and 0.75 mL of DTNB buffer (pH 8.0) to each test tube. ), and immediately add 0.1 mL of DTNB reagent solution (1 mg / mL) to a volume of 1 m³. The final cysteine ​​standard assay solution for L was obtained. f. The contents of each test tube were mixed. g. The absorbance of each cysteine ​​standard assay solution was determined at 412 nm. If the value exceeded a certain limit, the sample was diluted and the assay was repeated. The absorbance at h.412nm was plotted against the cysteine ​​concentration (mg / mL), and the standard was used. A curve was obtained. The straight portion of the standard curve corresponds to the cysteine ​​concentration range of 2 to 20 μg / ml. This was used to determine hapten-total and hapten-free.

[0149] 2. Hapten assay - Note: If the sample is the highest in the cysteine ​​standard assay... If the cysteine ​​level was higher than the standard, the sample was diluted and the assay was repeated. . a. 50 μl of the following solution was added to a properly labeled test tube: (i) DTNB buffer (blank) (ii) Diluted peptide sample (hapten - total, KLH conjugation, performed) (Starting from step A4 of Example 2) (iii) Hapten-KLH (Hapten-free, KLH conjugation, Example 2) (Starting from Step A7) (iv) Diluted peptide sample (hapten - total, BSA conjugation, Example 2) (Starting from step B3) (v) Hapten-BSA (Hapten-free, BSA conjugation, step of Example 2) (From B6) b. Add 0.1 mL of water and 0.75 mL of DTN to each labeled test tube from step (a). B buffer (pH 8.0), and immediately 0.1 ml of DTNB reagent solution (1 mg / mL). Adding [the substance] yielded a final hapten assay solution in volume 1 mL. c. The contents of each test tube were mixed. d. The absorbance of the solution in each labeled test tube was determined at 412 nm. If the absorbance exceeds 1.4 If this occurred, the sample was diluted and the assay was repeated. e. Next, the hapten-total concentration is calculated using the standard curve obtained above in the 1h term. The absorbance was determined from the measured values. The absorbance measured in test tube (ii) and test tube (iv) The hapten totals for KLH and BSA were determined using luminosity, respectively. Using the absorbances measured for test tube (iii) and test tube (v), respectively, Hapten-free fusion for KLH and BSA was determined. Then, the undiluted solution was analyzed. The peptide concentration and binding efficiency of the solution were calculated as described in the following calculations.

[0150] 3.Calculation To estimate peptide concentration and binding efficiency, the above-mentioned (cysteine ​​standard assay) A standard curve was created using known cysteine ​​concentrations. In this calculation, cysteine One mole is equal to one mole of sulfhydryl-containing hapten.

[0151] The following formula was used. % binding efficiency = {(hapten (conjugated) / hapten (total)} × 100 = [{Hapten {Phten (total) - Hapten (free)} / Hapten (total)} × 100 Hapten (total) = Peptide (total) μmole / ml Hapten (free) = Peptide (free) μmole / ml Hapten (conjugated) = Hapten (total) - Hapten (free) (Sigma-Aldrich Technical Bulletin for Ma leimide Activated BSA,KLH Conjugation Ki See also t (catalog number MBK1). Same as described in Examples 2A to 2D. Using the same general procedure, conjugate KLH and BSA with other SDMA analogs. The efficiency of the process can be measured.

[0152] Example 3: Method for producing anti-SDMA antibodies The immunization protocol for producing anti-SDMA antibodies was carried out according to the following protocol. 6 Six California breed rabbits were immunized with SDMA conjugate. Three of them were immunized with SDMA conjugated with BSA (Rabbits #155, #156) , and 157), the remaining three rabbits were immune with SDMA conjugated with KLH. Epidemic-treated (rabbits #152, #153, ​​and #154) (prepared as described in Example 2) ( ) For primary immunization, each rabbit was given 1 ml of complete Freund's adjuvant mixed with... Also, 0.5 mg of SDMA conjugate in 1 ml of phosphate-buffered saline (PBS) The drug was injected. 20-30 intradermal injections were administered to the shaved back of each rabbit. In the hind legs, 0. A booster immunization was performed with 25 mg of immunogen. Booster immunization injections were given monthly after the initial injection. 7-10 days after immunization, 5 ml of test blood was collected from each rabbit. Product blood collection (p (Roduction bleeds) 40 ml, after the third booster immunization injection, antiserum titer When the ratio was greater than approximately 1:2000, samples were taken from each rabbit. Antiserum titers were used in the assay. The dilution of the antiserum that has the steepest slope on the calibration curve is the one shown.

[0153] Example 4: Characterization of anti-SDMA antibodies In Example 3, to evaluate the specificity of the antibody obtained by the procedure described above, SDMA, Reactivity to ADMA, L-arginine, and / or N-methylarginine, The results were measured using a combined ELISA assay (Table 1).

[0154] ADMA-2HCl, SDMA-2HCl, N-methylarginine acetate (Sigma (catalog number M7033), or L-arginine (Sigma, catalog number A50) 06) was dissolved in PBS to prepare 1 mg / ml stock solutions. Standard solutions of 100 μg / ml, 10 μg / ml, and 1 μg / ml were prepared from the stock solution. It was prepared in PBS.

[0155] 50 μl of SDMA-HRP conjugate (described in Example 5 below), 50 μl ADMA, SDMA, N-methylarginine or L-arginine (as described above, 1 (concentration of ~100 μg / ml), and 50 μl of rabbit anti-SDMA antibody in serum (potency 1: 3000) sheep anti-rabbit IgG (Portland, ME Beacon Ana Pre-coated with (commercially available from lytical Systems Inc.) It was continuously added to each well of a 96-well polystyrene microwell plate. After incubation at room temperature for 30 minutes, the wells were placed in PBST (phosphate-buffered saline). It was washed four times with 0.05% Tween.

[0156] Next, 100 μl of 3,3',5,5'-tetramethylbenzidine (Madi (commercially available from Promega Corporation in WI) After incubation at room temperature for 30 minutes, add 100 μl of reaction stop solution (1N HCl). ) was added and the absorbance was measured using BioTek's ELX808 (Winooski, VT) plate. Measurements were taken at 450 nm using a TREEDer. The data was processed using Softmax software. Quantification using (Molecular Devices, Sunnyvale, CA) did.

[0157] ADMA in concentrations of 0 μg / mL, 1 μg / mL, 10 μg / mL, and 100 μg / mL. Determine the absorbance values ​​obtained for SDMA, N-methylarginine, or L-arginine. The data was then plotted. The concentration of SDMA at which the absorbance value decreased by 50% (SDMA at 0 μg / mL) With respect to the maximum absorbance obtained (i.e., IC50), the absorbance value of A is reduced by 50%. Divide by the respective concentrations (IC50) of DMA, N-methylarginine, or L-arginine. The obtained value was multiplied by 100 to obtain the "% cross-reactivity" value. <50% absorbance reduction When observed at concentrations of 100 μg / mL or less, it was found that the cross-reactivity was <1%. (See Table 1.)

[0158] As shown in Table 1, all six rabbits' anti-SDMA serum samples contained ADMA and N-methylal. It showed <1% cross-reactivity to ginine or L-arginine.

[0159] [Table 1]

[0160] In the same experiment as described in Examples 1 to 4, but using ADMA instead of SDMA. But antibodies were produced. However, in order to produce antibodies, ADMA-protein conjugate Using this method, it is not specific to free ADMA and is useful for assays that measure ADMA. Antibodies that were not present were produced.

[0161] In another experiment using only polyclonal antibodies from rabbit number 154, the antibodies The specificity was determined more rigorously using the method described above. (See Table 2 for this data.) The specificity of the antibody from rabbit number 154 is much higher than that shown in Table 1 above. To indicate that.

[0162] [Table 2]

[0163] Example 5: Competitive immunoassay for detecting SDMA levels in vivo Serum samples are obtained from animals that have undergone routine physical examinations and routine chemical reaction tests. Provided by an animal hospital / veterinary research institute.

[0164] SDMA-HRP conjugates were prepared according to the following procedure. 1. Maleimide-activated horseradish per mg of protein >200 units Dase freeze-dried powder (Sigma-Aldrich St. Louis, commercially available from MO) (Product number P1709) is mixed with 0.15M NaCl and 0.1M sodium phosphate. Rehydrate in um (pH 7.0) to a concentration of 2-5 mg / mL. The buffer solution was sterilized before use. The water used to prepare the buffer solution was degassed and purged with argon, and was slightly heavy. It did not contain any metals or other oxidizing agents. To protect the coupling reaction from light, amber was used. The procedure was performed using an amber-colored vial. 2. Dissolve the SDMA analog (3) in the same buffer used in step 1, and immerse for 2-5 minutes. Solutions with concentrations of mg / mL were prepared. Generally, 1 to 2 mg per mole of sulfhydryl compound. A molar amount of peroxidase compound was used. The molecular weight of the peroxidase was approximately 40,000. It was 0. 3. Combine the solution from Step 1 with the solution from Step 2, and set the resulting solution to room temperature. Gently stirred for 3 hours. Then, 1M 2-mercaptoethanol (Sigma-Al Drich St. Louis is available commercially from MO (catalog number M6250). The solution was to add 2-mercaptoethanol so that the final concentration would be 0.0015M. Therefore, the unreacted maleimide groups were blocked, and the resulting solution was stirred for approximately 15 minutes. 4. Next, 0.3M N-ethylmaleimide (Sigma-Aldrich St.L Ouis, commercially available from MO (catalog number D8654), N-ethyl maleate By adding the mid to the solution from step 3 so that the final concentration of mid is 0.003 M, This blocked the unreacted sulfhydryl groups. 5. Next, the obtained SDMA-HRP conjugate solution was subjected to chromatography (S Example of conjugating DMA analog (3) with maleimide-activated KLH and BSA By using the same procedure described above, or by following the manufacturer's instructions for use. Dialysis to PBS (Spectra / Por3, MWCO 3500, Spectrum Replaced by PBS (Labs, Rancho Dominguez, CA). The resulting solution was then freeze-dried.

[0165] Lin, FT et al., Biochemistry, 18(4), 690 (1979); Kitagawa, T et al., Chem. Pharm. Bull., 29(4), 1131( 1981); Duncan, RJS et al., Anal. Biochem., 132, 6 8 (1983); and Palmer, JL et al., J. Biol. Chem., 238. (7) See also 2393 (1963).

[0166] 50 μl of SDMA-HRP conjugate, 50 μl of serum sample (or calibration) Quality, SDMA2HCl, commercially available from Calbiochem in San Diego, CA. (and) and 50 μl (potency 1:3000) of rabbit anti-SDMA antibody in serum were used. Dianti-rabbit IgG (Portland, ME Beacon Analytical S A 96-well polystainless steel (commercially available from ystems Inc.) pre-coated The solution was continuously added to individual wells of a ethylene microwell plate. The mixture was then allowed to stand at room temperature for 30 minutes. After incubation, the wells were filled with PBST (phosphate-buffered saline, 0.05% tweed). It was washed four times with (en).

[0167] Next, 100 μl of 3,3',5,5'-tetramethylbenzidine (Madi (commercially available from Promega Corporation in WI) After incubation at room temperature for 30 minutes, add 100 μl of reaction stop solution (1N HCl). ) was added and the absorbance was measured using BioTek's ELX808 (Winooski, VT) plate. Measurements were taken at 450 nm using a TREEDer. The data was processed using Softmax software. Quantification using (Molecular Devices, Sunnyvale, CA) The calibration curve was created using a series of SDMA standards (e.g., 0, 0.05 μg / mL, 0.15 μg / mL). This is produced by processing with g / mL, 0.45 μg / mL, and 1.35 μg / mL. Success. The unknown sample was quantified using a calibration curve. The results are summarized in Table 3.

[0168] [Table 3]

[0169] In Table 3, the condition "renal disease" means that the sample taken from the animal was outside the normal reference range. This indicates that the patient has exceeded the limits of creatinine and blood urea nitrogen (BUN), and is in a "healthy" state. "The sample taken from the animal had normal (reference range) creatinine levels and blood This indicates the blood urea nitrogen (BUN) level. In dogs, the upper limit of the normal reference range is BU The N level was 27 mg / dL, and the creatinine level was 1.8 mg / dL. In this case, the upper limit of the normal reference range is 34 mg / dL for BUN, and creatinine The level of n was 2.3 mg / dL.

[0170] The results in Table 3 show that SDMA levels were elevated in dogs and cats with renal impairment. This indicates that SDMA is a marker for diagnosing kidney disease in animals. It can be used as -.

[0171] Example 6: Analysis of glomerular filtration rate in dogs based on creatinine and free SDMA concentrations. Serum samples are used to identify heterozygous individuals with X-linked hereditary neuropathy (XLHN). The sample was taken from female dogs (n=20) in the REA. XLHN is a mutation in the COL4A5 gene. This is caused by a difference in the mosaic development of type IV collagen peptides in female dogs. It causes the development of glomerular proteinuria at present and between 3 and 6 months of age (Nabity et al.). J Vet Intern Med 2007;21:425~430). Creatinine The concentration of SDMA was also measured in each sample.

[0172] As described above, the creatinine concentration of the sample was measured using IDEXX's dry slide technology. It was measured using [a specific method / tool].

[0173] The free SDMA concentration in the sample was determined as follows. The mobile phase of LCMS was (A) 10 mL of propionic acid and 250 μL of trifluoroacetic acid in 1 L of water; and ( B) 10 mL of propionic acid and 250 μL of trifluorocarbon in 1 L of acetonitrile It was acetic acid. 2.5 ng / mL of deuterated asymmetric dimethylarginine (dA) in water. An internal standard called DMA was prepared. The STD (standard) curve was used for 20 μg / mL SDMA. The solution is mixed in, then diluted to varying concentrations from 1.56 μg / dL to 100 μg / dL. The STD curve was created using treated dog serum by obtaining 9 points of the resulting 9-point curve. To measure, place the 100 μL sample (i.e., serum sample or standard solution) into the machine. Transferred to microtubes. 10 μL of internal standard solution and 200 μL of mobile phase B were added to each tube. It was added to the tube. The tube was vortexed and mixed, then left for 30 minutes, and then 2 The mixture was centrifuged at 13000g for 20 minutes at 5°C. The supernatant was then mixed with 2 mL of amber-colored HPCL. The samples were transferred to an AL and analyzed by LC-MS. LC-MS was then analyzed by HPLC and AB. Sciex API-4000 (Scan type MRM, positive polarity, turbospray) Performed in CAN mode (operated by Q1 resolution=unit and Q3 resolution=unit). The column used was a 150 × 4.6 PVA SIL column, and the flow rate was 1 mL / min. Yes, and the gradient was a flat 90:10 (B:A). The chromatogram was obtained at ambient temperature. It lasted for 9 minutes.

[0174] The actual GFR of animals was measured using the iohexole clearance method. I received an injection of oxol.

[0175] Blood samples were collected at various time intervals, and iohexol in the serum was analyzed by HPLC. It was measured.

[0176] Three data points were collected for each dog: creatinine concentration (mg / dL) versus GFR. Figure 5 shows a four-parameter logistic (4PL) plot of (ml / min / kg). R of these data 2 The value is 0.94, and the concentration range is 0.5 to 3.0 mg / dl. The standard error for this range is 0.12, which is about 5% of the total range.

[0177] Figure 6 shows the results for SDMA concentration (μg / dl) versus GFR (ml / min / kg). The 4APL fit to the MA-GFR relationship is R 0.95. 2 Give a value and SDMA The standard error for the range of 5 to 40 μg / dL is 1.7. This error is approximately the same as the error across the entire range. It is 5%.

[0178] Figure 7 shows the combination of creatinine and SDMA values ​​using a simple multiplication of those values. The combined results show that the relationship between creatinine alone or SDMA alone and GFR is not shown. It shows improvement in the relationship. [Creatinine]* The application of the 4PL model to the relationship between [SDMA] and GFR. This is R 0.98 2 Give a value, [creatinine] * [SDMA] 0-90 μg / dL The standard error for the range is 2.8. This error is due to the GFR and for these dogs. This represents approximately 3% of the total range of relationships.

[0179] Figure 8 shows the 1 / [creatinine] ratio using a straight line fit. P* 1 / [SDMA] Q portion The analysis is shown. Using linear regression, P was 0.37 and Q was 0.43. R of the combination 2 0.83 for 1 / [creatinine] only, and 0.83 for 1 / [SDMA] only. The value was 0.87, compared to 0.85.

[0180] Example 7: Analysis of glomerular filtration rate in cats based on creatinine and free SDMA concentrations Using 10 cats, each with 1 to 4 data points, we measured SDMA values ​​and creatine levels. Whether the combination of Nin values ​​correlates better with GFR than the individual marker values ​​alone. The following were evaluated. SDMA, creatinine, and GFR were measured as described above.

[0181] Figure 9 shows the results for SDMA concentration (μg / dl) versus GFR (ml / min / kg). The application of 4PL to the MA-GFR relationship is R = 0.73 2 Given a value, the standard error is, The value for SDMA in the 15 μg / dL range is 2.3. This error represents approximately 15% of the entire range. be.

[0182] Figure 10 shows the results for creatinine concentration (mg / dl) versus GFR (ml / min / kg). The 4PL fit to the relationship between creatinine and GFR is 0.82.2 Give a value The standard error is 0.15 for the 1.5 mg / dL range of SDMA. This error is, This represents approximately 10% of the entire range.

[0183] Figure 11 combines creatinine and SDMA values ​​using a simple multiplication of those values. The combined results show the relationship between GFR and creatinine alone or SDMA alone. It indicates an improvement in the relationship. [Creatinine] * The 4PL application of the relationship between [SDMA] and GFR is The value of R is 0.89. 2 Give a value, [creatinine] * [SDMA] 40 μg / dL range The standard error for the range is 3.9. This error is approximately 10% of the total range.

[0184] Figure 12 shows the 1 / [creatinine] ratio using a straight line fit. P* 1 / [SDMA] Q of The analysis is shown below. Using linear regression, P was 1.2 and Q was 0.95. R of the combination 2 This is 0.44 for 1 / [creatinine] only, and 0.44 for 1 / [SDMA] only. The value was 0.95, compared to 0.65.

[0185] Example 8: Combinations of CRE and SDMA cutoff values ​​in the diagnosis of kidney disease Improvement of sensitivity and / or specificity The kidney disease status of 113 cats was assessed by the International Renal Inte Provided by the rest Society (IRIS), for dogs and cats Algorithm for Staging of Chronic Kidney The diagnosis and treatment were determined and categorized according to the disease (CKD). For each cat, at various times... One to six serum samples collected at a single point were used for creatinine [CRE] and / or SDM. A was analyzed. 194 samples were compared with 61 normal (i.e., non-CKD) animals. The samples came from cats. 182 samples were taken from 55 cats with chronic kidney disease (CKD). That was the case.

[0186] In this embodiment, the cutoff values ​​for SDMA and CRE are determined and used CKD was diagnosed. The cutoff value is when an individual is diagnosed with kidney disease based on this specific test. This indicates the threshold concentration of the serum. CUT This is the cutoff value for SDMA. SDMA and SDMA CUT This is measured in μg / dL (micrograms / deciliter). For example, SDMA. CUT It may be approximately 14 μg / dL, or approximately 10-20 μg / dL. g / dL is also acceptable.

[0187] CRE CUT This is the cutoff value for CRE. CRE and CRE CUT is in mg / dL It is measured. For example, CRE CUT The range is approximately 2.0 mg / dL to 2.4 mg / dL. It is also acceptable to have a concentration of approximately 1.7-2.8 mg / dL.

[0188] For SDMA only, cutoff value (SDMA CUT ) was set to 14 μg / dL Using this value, there is a 10.3% false positive rate for normal cats, and CKD Regarding the test, there was a 26.9% false negative rate (see Table 4).

[0189] [Table 4]

[0190] For creatinine only, the cutoff value (CRE CUT Set the concentration to 2.4 mg / dL. This value indicates that for normal cats, there is a 0.0% false positive rate, and for CKD... 43.4% of cats exhibited false negatives (see Table 5).

[0191] [Table 5]

[0192] C CUT This is the cutoff value for the combination value C. The creatinine and SDMA values ​​are lowered. They were combined according to the formula. Combination value C = [SDMA] / SDMA CUT +[CRE] / CRE CUT

[0193] C CUT It does not have a unit of measurement. For example, C CUT It was 1.5, 1.7, or 2.0 It may be 1.3 to 2.5.

[0194] C CUT When set to 1.5, there is a 12.4% false positive rate for normal cats. Regarding cats with CKD, there was a 1.6% false negative rate (see Table 6). CUT When set to 1.7, there is a 3.5% false positive rate for normal cats, and CKD Regarding cats, there was a 14.3% false negative rate (see Table 7). C CUT 2. When set to 0, there is a 3.5% false positive rate for normal cats, and for cats with CKD... Furthermore, 33.5% of the results were false negatives (see Table 8).

[0195] [Table 6]

[0196] [Table 7]

[0197] [Table 8]

[0198] C CUT To determine the appropriate value of the combination, the estimated sensitivity and specificity of the combination value are used. CUT This was plotted against (see Figure 13). C is C CUT If it is greater than (>) Therefore, the individual is diagnosed with kidney disease. Based on each diagnostic cutoff value, S Combining DMA and CRE values ​​improves the detection of kidney disease in animals. This results in high sensitivity and / or specificity.

[0199] Example 9: Determination of the creatinine-to-SDMA ratio in healthy or diseased animals In healthy animals, the concentrations of SDMA (μg / dL) and creatinine (mg / dL) The ratio is generally in the range of approximately 4:1 to 10:1 (μg / dL:mg / dL). In patients with chronic kidney disease, this ratio exceeds 10:1, which may indicate disease progression. It is possible.

[0200] In this study, the long-term trends of SDMA and creatinine in dogs with CKD The following was observed: 24 dogs with CKD were selected according to the following criteria, namely age (9.4-1 8.3 years old); persistent azotemia (>3 months); GFR; physical examination; serum creatinine, Based on urine tests, this was included in the study.

[0201] We provide all dogs with quality care, including optimal nutrition, veterinary health management, and daily exercise. It was maintained with [this method]. After being diagnosed with CKD, the dog was given dog food PRESCRIPTION DIET(registered trademark) k / d(registered trademark) (Hill's Pet Nutrition) They fed it (Topeka, Kansas, Inc.).

[0202] Samples were collected regularly (2-3 times a year) from these dogs. The samples were frozen and stored. Creatinine was stored using the COBAS® analyzer by enzymatic colorimetric method. This was measured. SDMA was measured by precipitating the serum sample with acetonitrile, and W Aters XBridge C18 (5μm 4.6 * 30) Using the column Except for the above, measurements were taken by LCMS. Mobile phase A was prepared in 0.1% formic acid in water. It consists of 0.5 mM perfluoroheptanoic acid, and mobile phase B is 0.1 mM perfluoroheptanoic acid in acetonitrile. The solution was % formic acid, the gradient was from 100% B to 100% A, and the execution time was 4 minutes. Figure 14 shows the correlation between DMA (μg / dL) and creatinine (mg / dL).

[0203] Example 10: The difference between SDMA levels and creatinine levels in some cats with CKD Harmony Disadvantages in the SDMA:creatinine ratio may predict mortality in animals. Yes, for example, in cats with CKD, the observed SDMA levels correspond to the creatinine levels. The concentration was higher than the predicted concentration based on the values. As shown in Figure 14, the interval between SDMA and creatinine A strong correlation exists, and the normal ratio is less than 10 (μg / dL:mg / dL). In the study, the ratio was determined using 26 cats with CKD. These 26 cats were... The diagnosis of CKD was based on physical examination, serum creatinine, and urinalysis. (See Figure 15) As mentioned, it is not documented whether two of the 26 cats were euthanized or died from illness. However, the patient had an SDMA:creatinine ratio greater than 10 and died during the follow-up period.

[0204] Example 11: SDMA:Creatinine ratio in predicting mortality in cats with CKD In this study, long-term trends in SDMA and creatinine in cats with CKD The following was observed: 18 cats with CKD were selected based on the following criteria, namely, at least 3 months Persistent hyperazotemia; or GFR >30% lower than the median GFR of a normal cat. Patients were included in the study based on non-azotemia or calcium oxalate kidney stones.

[0205] We provide all cats with optimal nutrition, veterinary health care, daily exercise, and a suitable environment. and maintained with quality care including regular opportunities for behavioral enrichment. Diagnosed with CKD. Afterwards, PRESCRIPTION DIET(registered trademark) c / d(registered trademark) Food (Hill's Pet Nutrition, Inc., Topeka, Kan. I fed them sas.

[0206] Blood and urine samples were collected from these cats at various points in time, frozen, and stored. Nin was measured using an enzymatic colorimetric method with a COBAS® analyzer. DMA was measured by LCMS as described above.

[0207] In each of the 18 cats, the SDMA concentration reached 14 μg / dL for the first time. When it exceeds that, the 12 cats have an SDMA:creatinine ratio greater than 10:1 Six of the cats had an SDMA:creatinine ratio of 10:1 or less. Therefore, from the day the SDMA concentration first reaches or exceeds 14 μg / dL, death The period leading up to the day of death was observed for all cats except for two. These two cats were considered to have reached the end of the study. They were still alive at the time, and therefore the last day of the study was used as a substitute for the date of death of these two cats. It became so.

[0208] Twelve cats with an SDMA:creatinine ratio greater than 10:1 had a median lifespan. The value was 13.9 months (mean 18.7; range = 1.8~47.4). Less than 10:1 Six cats with the specified SDMA:creatinine ratio had a median survival time of 18.7 months (average). The mean was 18.9 (range = 8.7~28.7). Therefore, S is greater than 10:1. Cats with a DMA:creatinine ratio had an SDMA:creatinine ratio of 10:1. The mortality rate is higher than that of cats. Figures 16, 17, and 18 show the SDMA:creatinine ratio. Of the more than 10 cats from this study, three (Cat #13, Cat #8, and Cat #14) The following shows the time course of the SDMA:creatinine ratio over several years. From the day the blood glucose level first reached at least 14 μg / dL, cat #13 died at 27.2 months. Cat #8 died at 29.4 months old, and cat #14 died at 12.3 months old. Autopsy At the time of the final measurement, the above ratio of the three cats ranged from approximately 17 to 34.

[0209] Example 12: Prediction of mortality using SDMA and creatinine Figures 19 and 20 show cats using an SDMA cutoff value of 14 μg / dL. (From the study described in Example 11) and dogs (From the study described in Example 9) The Plan-Meier survival curve is shown. Figure 19 shows serum SDM at least 14 μg / dL. Cats with a concentration of A have a shortened lifespan and a high probability of death. 14μ Cats with serum SDMA levels below g / dL should not have serum SDMA levels of 14 μg / dL or higher. The cats lived about 1.6 times longer than those that had creatinine. In this study, creatinine was found to be the cause of death in cats. The mortality rate could not be predicted (reference cutoff value 2.1 mg / dL).

[0210] Figure 20 shows the capsular cysts of dogs with serum SDMA concentrations higher or lower than 14 μg / dL. The Lann-Meier survival curve is shown. In this study, the subjects had SDMA < 14 μg / dL. Dogs lived 2.6 times longer than dogs with SDMA ≥ 14 μg / dL. The drug could not predict mortality (reference cutoff value 1.5 mg / dL).

[0211] The embodiments described above are illustrative only, and all possible embodiments and applications of the present invention are shown. This does not mean that it is an exhaustive list of possible forms of modification. Without departing from the scope and spirit of the invention, various modifications of the methods and systems of the present invention described above. Or modified versions will be obvious to those skilled in the art. The present invention will be described in connection with specific embodiments. However, the claimed invention should not be excessively limited to such specific embodiments. It should be understood that, in fact, various modifications of the above embodiments for carrying out the present invention are This is obvious to those skilled in the art.

[0212] The present invention is not limited to the specific methods, protocols, and reagents described herein. This should be understood because they can change as those skilled in the art would recognize. The terms used herein are for the purpose of describing specific embodiments only, and this It should also be understood that this is not intended to limit the scope of the invention. When used in writing and in the attached claims, the singular form "one (a, an)" " and "the" imply multiple references unless otherwise explicitly indicated by the context. Please also note this. Therefore, for example, to "a linker" The references are to one or more linkers and their equivalents known to those skilled in the art.

[0213] Unless otherwise defined, all technical and scientific terms used herein refer to the present invention. This has the same meaning as that commonly understood by those skilled in the art in the relevant technical field. Embodiments and various features, as well as their advantageous details, refer to non-limiting embodiments. This is more fully explained and / or illustrated in the attached drawings and further detailed in the following description. The features shown in the drawings are not necessarily to scale, and even if not explicitly stated in this specification, As the vendor recognizes, features of one embodiment can be used in other embodiments. This should be noted.

[0214] Any numerical value described herein is such that its lowest and highest values ​​are separated by at least two units. This includes all values ​​that increase by one unit from the lowest value to the highest value. The concentration of a certain component, or the value of a process variable, such as magnitude, angle, pressure, time, etc. For example, if it says 1-90, more specifically 20-80, and even more specifically 30-70 Values ​​such as 15-85, 22-68, 43-51, and 30-32 are explicitly listed herein. This is intended to mean that. For values ​​less than 1, 1 unit is appropriately 0.0001, 0 It is thought to be 0.001, 0.01, or 0.1. These are simply what is specifically intended. This is just one example, and it represents all possible values ​​between the smallest and largest values ​​listed. The combinations should be considered to be similarly specified in this application.

[0215] This document describes specific methods, apparatus, and materials, but does not describe any similar or equivalent methods, apparatus, or materials described herein. The methods and materials described above can be used in the implementation or testing of the present invention. The disclosure of all references and publications used is as if each of them were a reference to... They are incorporated individually, and by reference, they are all clearly incorporated into this specification. It can be done.

Claims

1. A device for determining renal function in animals, comprising: a first solid phase to which an antibody specific to SDMA is conjugated, which does not or substantially have cross-reactivity with SDMA analogs or one or more compounds selected from the group consisting of asymmetric dimethylarginine (ADMA), L-arginine, and N-methylarginine; and a second solid phase to which a creatinine detection reagent or an antibody specific to creatinine is conjugated.

2. The creatinine detection reagent contains picric acid or its salt, or a substance specific to creatinine. The apparatus according to claim 1, wherein the antibody is an antibody.

3. The apparatus according to claim 1 or 2, wherein the apparatus is a microplate and a lateral flow assay.

4. The apparatus according to any one of claims 1 to 3, wherein the solid phase is selected from a reaction well, a dipstick, a test strip, a flow-through pad, paper, and a fiber matrix.

5. The apparatus according to any one of claims 1 to 4, wherein the antibody specific to SDMA has less than 1% cross-reactivity with one or more compounds selected from the group consisting of asymmetric dimethylarginine (ADMA), L-arginine, and N-methylarginine.

6. Use of the apparatus according to any one of claims 1 to 5 in an immunoassay.

7. The use according to claim 6, wherein the immunoassay is selected from radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence-polarized immunoassay (FPIA), microparticle enzyme immunoassay (MEIA), enzyme amplification immunoassay technology (EMIT) assay, immunoturbidity or agglutination assay, colloidal gold-based immunoassay including a lateral flow apparatus, and chemiluminescence magnetic immunoassay (CMIA).

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