Antibodies to symmetrically dimethylated arginine analytes and use thereof

Abstract

To provide tools that enable detection of symmetrically dimethylated arginine analytes.SOLUTION: The present invention provides an immunoassay method for symmetrically dimethylated arginine analytes. Specifically, the present invention provides a symmetric dimethylarginine derivative for signal-generating immunoassay systems. The compound of chemical formula 1 is represented by the following formula, where R1 is -Y-Z; Y is a linker; and Z is a labeled enzyme.SELECTED DRAWING: Figure 10

Description

[Technical field]

[0001] This application is a continuation of U.S. Provisional Application No. 62 / 828,769, filed April 3, 2019. § 119(e) to the date of filing, the disclosure of which is incorporated herein by reference.

[0002] The present invention relates to the field of immunoassays, in particular to immunoassays for the detection of symmetrically dimethylated arginine analytes. The present invention relates to antibodies that bind to symmetrically dimethylated arginine analytes that can be used in immunoassays. [Background technology]

[0003] Chronic kidney disease (CKD) is a common problem in dogs and cats. CKD cannot be cured. However, medical management is possible for patients with the disease. Focus on early detection of CKD, with the goal of initiating medical management and monitoring early in the course of the disease. Symmetric dimethylarginine ginine has recently been identified as a new renally excreted vasodilator useful for early detection of CKD in cats and dogs. It appeared as an ion marker.

[0004] Asymmetric dimethylarginine ne, ADMA; Fig. 2) and monomethylarginine ne, MMA or N-methylarginine; Figure 2), as well as symmetric dimethylarginine SDMA (Figure 1) is a post-translational agonist that regulates nearly all cellular proteins that contain arginine residues. It is derived from modification (methylation). After protein breakdown, the protein residues are excreted into the circulatory system. Kidney disease, cardiovascular disease, atherosclerosis Serum symmetric dimethylarginine is a marker for evaluating rheumatoid arthritis and other diseases. The potential uses of nin have been widely studied.

[0005] Symmetric dimethylarginine is eliminated from the body primarily through renal excretion. Unlike symmetric dimethylarginine, it is not affected by extrarenal factors, including lean body mass. Symmetric dimethylarginine is a cyclic amine that is partially bound to plasma proteins. Unlike dimethylarginine, it does not bind to proteins. Asymmetric dimethylarginine and the glomerulus The correlation between glomerular filtration rate (GFR) and glomerular filtration rate (GFR) is quite weak, but this is due to the fact that glomerular filtration is inhibited. This is likely due to the protein-bound fraction of asymmetric dimethylarginine.

[0006] Serum concentrations of symmetric dimethylarginine are also increased in human CKD patients. Dimethylarginine concentrations were found to be inversely proportional to GFR.

[0007] Circulatory metabolism of symmetric dimethylarginine and symmetric Nα-acetyl-dimethylarginine The product (Figure 1) was also identified, which also correlates with GRF. The acetylation of arginine occurs extracellularly to generate metabolic products.

[0008] Symmetric dimethylarginine is almost exclusively eliminated by the kidney and therefore has no effect on GFR. Therefore, symmetric dimethylarginine is already an ideal candidate for a marker in veterinary medicine. It is a new endogenous biomarker of kidney function that is widely used in clinical practice.

[0009] Enzyme-linked immunosorbent assay (ELISA) for quantification of symmetric dimethylarginine A) is commercially available. The method is used to quantify symmetric dimethylarginine. Addition of an acylation derivatization reagent to samples suspected of containing symmetric dimethylarginine The sample preparation step includes the steps of: quantitatively isolating symmetric dimethylarginines with an acylation reagent; The antibody used in the ELISA product is symmetric. It binds only to the Nα-acylated form of symmetric dimethylarginine and not to the free symmetric dimethylarginine. It does not bind to the

[0010] More recently, it has been reported that only free symmetric dimethylarginines are detected, whereas Nα-acylated symmetric Detection of free symmetric dimethylarginine using an antibody that does not cross-react with dimethylarginine A competitive immunoassay was developed to detect the free symmetric dimers of the antibody. Developed against a specific immunogen that produces antibodies specific only to methylarginine. It is a derivative related to the chemical modification of the acidic carboxyl group (-COOH) of dimethylarginine. Certain haptens are used. The antibodies react with free symmetric dimethylarginine (i.e., symmetric dimethylarginines that are not part of a polypeptide chain) It cross-links asymmetric dimethylarginine, L-arginine, and N-methylarginine. Shows no or virtually no reactivity. Summary of the Invention [Problem to be solved by the invention]

[0011] Symmetric dimethylarginine was measured using a liquid chromatography tandem mass spectrometer (LCMS). LC-MS / MS technology remains complex. This requires a high level of expertise on the part of the test developer and operator. S / MS assays are not fully automated and require significant sample preparation and time to results. This increases processing time compared to the automated assay for serum creatinine. do.

[0012] There is a continuing need to develop tools that allow for the detection of symmetrically dimethylated arginine analytes. can be. [Means for solving the problem]

[0013] The present disclosure provides an immunoassay method for a symmetrically dimethylated arginine analyte. In particular, The present disclosure relates to induction of symmetric dimethylarginines in signal generating immunoassay systems. The present disclosure also relates to the use of a method for producing an antibody for capturing the analyte. The term "symmetric dimethylarginine" as used herein relates to immunogenic uses of symmetric dimethylarginine. The "dimethylarginine analyte" is a nucleotide sequence that contains a common antibody-binding epitope for symmetric dimethylarginines. The epitope is an analyte that contains a symmetrically dimethylated arginine analyte. Analytes included in the assay include symmetric dimethylarginine (SDMA), symmetric Nα-acetyl-diamine Symmetric Nα-acylated dimethylarginines such as methylarginine and SDMA-Gl Included are SDMA-peptide derivatives such as y-Gly dipeptide.

[0014] In some embodiments, the present disclosure provides a method for the preparation of a dimethylarginine having a nitrogen atom of the α-amino group of a symmetric dimethylarginine. A symmetric dimethylarginine hapten alkylated at the base is provided (Figure 1). In an embodiment, the hapten produces an antibody specific for a symmetrically dimethylated arginine analyte. It is used to create

[0015] In some embodiments, the present disclosure provides a method for the preparation of a dimethylarginine having a nitrogen atom of the α-amino group of a symmetric dimethylarginine. A symmetric dimethylarginine derivative that is acylated on the aryl group is provided (Figure 1). In examples, the derivatives produce conjugates useful in the immunoassays described herein. It is used to.

[0016] In some embodiments, the present disclosure provides a method for the preparation of symmetric Nα-acetyl-dimethylarginine and other analogs. Polyclonal or monoclonal antibodies that specifically bind to symmetric dimethylarginine metabolites We provide antibody to the IgG antibody (Figure 1).

[0017] In some embodiments, the present disclosure provides free symmetric dimethylarginine and symmetric Nα- Polyclonal or monoclonal antibodies that specifically bind to acetylated dimethylarginine proteins Provide monoclonal antibodies (Figure 1).

[0018] In some embodiments, the antibody comprises asymmetric dimethylarginine, L-arginine, and It can specifically bind to one or more of the N-methylarginines (Figure 2).

[0019] In some embodiments, the present disclosure provides a method for the preparation of free symmetric dimethylarginine and SDMA-Gl Polyclonal antibodies that specifically bind to SDMA-peptide derivatives such as y-Gly dipeptide Alternatively, a monoclonal antibody is provided (Figure 3).

[0020] In some embodiments, the present disclosure provides a method for the preparation of symmetric dimethylarginines starting from symmetric dimethylarginines. Methods for the synthesis of aryl arginine haptens, immunogens and conjugates are provided. The synthesis is carried out by linking the α-amino nitrogen atom of a symmetric dimethylarginine with a linking group. This includes binding to a protein or label (e.g., a label enzyme) via a nucleic acid molecule.

[0021] An example of one embodiment of the present disclosure is a compound of formula 1: chemical formula 1 JPEG2025032213000002.jpg3733

[0022] During the ceremony: R 1 -YZ; and Y is a linking group, Z is hydrogen, OH, SH, S-acyl, O-alkyl, halogen, N H2, epoxy, maleimidyl, haloacetamide, carboxyl, activated carbo The antibody is selected from a xyl, an immunogenic carrier, a protein and a label.

[0023] Embodiments of the present disclosure include antibodies. In some examples, the antibodies of the present disclosure are and specifically binds to any compound of the present disclosure, including any compound of Formula 1 described herein. .

[0024] Nucleic acids encoding any of the antibodies of the present disclosure, expression vectors comprising the nucleic acids, and and a cell containing the expression vector.

[0025] Also provided is a method of producing an antibody of the disclosure, the method comprising: and culturing the cells of the present disclosure under conditions suitable for producing the antibody.

[0026] Aspects of the present disclosure further include compositions. The compositions of the present disclosure may include any of the antibodies, nucleic acids, , an expression vector and / or a cell.

[0027] Also provided is a method for determining the amount of at least one symmetrically dimethylated arginine analyte in a culture medium. In certain embodiments, the method comprises the step of: combining in a medium the arginine analyte and a sample suspected of containing an antibody of the present disclosure. The method includes detecting the presence or absence of a complex comprising a symmetrically dimethylated arginine analyte and an antibody. further comprising determining the absence or presence of a complex, wherein the presence of the complex indicates the presence of a symmetrically dimethylated complex in the sample. Indicates the presence of the arginine analyte.

[0028] Aspects of the present disclosure further include kits. According to some embodiments, the kits include a kit for detecting a small amount of The method is used to determine the amount of at least one symmetrically dimethylated arginine analyte. In some embodiments, the kit of the present disclosure comprises any of the antibodies of the present disclosure and at least one antibody in a sample. Guidelines for using antibodies to determine the amount of a symmetrically dimethylated arginine analyte The kit may further include any compound of formula 1 of the present disclosure. According to the present disclosure, the kit includes any compound of Formula 1 of the present disclosure, and at least To use the compound to determine the amount of one symmetrically dimethylated arginine analyte. The kit may further comprise any of the antibodies of the present disclosure. [Brief description of the drawings]

[0029] The present disclosure begins with the following detailed description taken in conjunction with the accompanying drawings, which form a part of this application. can be more fully understood.

[0030] Figure 1 shows the results for symmetric dimethylarginine and symmetric Nα-acetyl-dimethylarginine. The chemical structure is shown below.

[0031] FIG. 2 shows the chemical structure of asymmetric dimethylarginine, monomethylarginine, and arginine. The biological structure is shown.

[0032] FIG. 3 shows the chemical structure for the SDMA-Gly-Gly dipeptide.

[0033] FIG. 4 (left side) shows the α-amino nitrogen atom of a symmetric dimethylarginine according to an embodiment of the present invention. 4 shows SDMA-M (Nα-alkyl-SDMA) hapten modified with

[0034] FIG. 4 (right side) shows the α-amino nitrogen atom of a symmetric dimethylarginine according to an embodiment of the present invention. 4 shows the SDMA-SBAP (Nα-acyl-SDMA) hapten modified with

[0035] FIG. 5 shows the results of the present disclosure with respect to the SDMA-M (Nα-alkyl-SDMA) hapten. The reaction equation is shown below.

[0036] FIG. 6 shows an SDMA-SBAP (Nα-acyl-SDMA) hapten according to an embodiment of the present disclosure. The reaction equation for

[0037] FIG. 7 shows a reaction scheme for SDMA-M-SH-G6PDH according to an embodiment of the present disclosure.

[0038] FIG. 8 shows a reaction scheme for the SDMA-M-SH-KLH immunogen according to an embodiment of the present disclosure. .

[0039] FIG. 9 shows a reaction scheme for the SDMA-SBAP-SH-BSA immunogen according to an embodiment of the present disclosure. Shows.

[0040] FIG. 10 illustrates an antibody screening technique according to an embodiment of the present disclosure.

[0041] FIG. 11 shows rabbit polyclonal antibody #26494, monoclonal antibody #26494 according to an embodiment of the present disclosure. monoclonal antibody 1H2 / 1K4, monoclonal antibody 3H1 / 3K3, and monoclonal antibody 5 The graph shows the calibration curve for symmetric dimethylarginine using H1 / 5K1. vinegar.

[0042] FIG. 12 shows rabbit polyclonal antibody #21342, monoclonal antibody #21342 according to an embodiment of the present disclosure. monoclonal antibody 1H4 / 1K4, monoclonal antibody 8H1 / 8K3, and monoclonal antibody 1 Graph of calibration curve for symmetric dimethylarginine using 8H1 / 18K2 Shows.

[0043] FIG. 13 shows rabbit polyclonal antibody #26494, monoclonal antibody #26494 according to an embodiment of the present disclosure. monoclonal antibody 1H2 / 1K4, monoclonal antibody 3H1 / 3K3, monoclonal antibody 5H1 Calibration curve for symmetric Nα-acetyl-dimethylarginine using / 5K1 The graph shows:

[0044] FIG. 14 shows rabbit polyclonal antibody #21342, monoclonal antibody #21342 according to an embodiment of the present disclosure. monoclonal antibody 1H4 / 1K4, monoclonal antibody 8H1 / 8K3, and monoclonal antibody 1 Calibration of symmetric Nα-acetyl-dimethylarginine using 8H1 / 18K2 The graph shows the motion curve.

[0045] FIG. 15 shows rabbit polyclonal antibody #26494 (lower right) according to an embodiment of the present disclosure; Monoclonal antibody 1H2 / 1K4 (upper left), monoclonal antibody 3H1 / 3K3 ( (upper right) and symmetric dimethyl amines using monoclonal antibodies 5H1 / 5K1 (lower left). 1 is an experimental graph showing the recovery rate of small amounts of arginine added.

[0046] FIG. 16 shows rabbit polyclonal antibody #21342 (lower right) according to an embodiment of the present disclosure; Monoclonal antibody 1H4 / 1K4 (upper left), monoclonal antibody 8H1 / 8K3 ( Symmetrical dialysis using monoclonal antibody 18H1 / 18K2 (lower left) and monoclonal antibody 18H1 / 18K2 (upper right) 1 is an experimental graph showing the recovery rate of a small amount of added ethyl arginine.

[0047] FIG. 17 shows rabbit polyclonal antibody #26494 (lower right) according to an embodiment of the present disclosure; Monoclonal antibody 1H2 / 1K4 (upper left), monoclonal antibody 3H1 / 3K3 ( Symmetric Nα-acetylglucosamine esterase (upper right) and symmetric Nα-acetylglucosamine esterase (lower left) using monoclonal antibody 5H1 / 5K1 (lower left). 1 is an experimental graph showing the recovery rate of a small amount of ethyl-dimethylarginine added.

[0048] FIG. 18 shows rabbit polyclonal antibody #21342 (lower right) according to an embodiment of the present disclosure; Monoclonal antibody 1H4 / 1K4 (upper left), monoclonal antibody 8H1 / 8K3 ( (upper right) and symmetric Nα using monoclonal antibody 18H1 / 18K2 (lower left) 1 is an experimental graph of the recovery rate of small amounts of added β-acetyl-dimethylarginine.

[0049] FIG. 19 shows the monoclonal antibody 3H1 / 3K3 (left) and the monoclonal antibody 3H1 / 3K4 (right) according to the embodiment of the present disclosure. A small amount of SDMA-Gly-Gly dipeptide was detected using the local antibody 8H1 / 8K3 (right). 1 is an experimental graph of the amount added recovery rate.

[0050] FIG. 20 shows SDM using rabbit polyclonal antibody #27410 according to an embodiment of the present disclosure. A graph showing the experimental recovery rate of small amounts of symmetric Nα-acetyl-dimethylarginine. . DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] Before describing the present invention in more detail, it is to be understood that the invention is not limited to the particular embodiments described. It should be understood that the terms are self-explanatory and may be varied. It is to be understood that the terminology is for the purpose of describing particular embodiments only and is not intended to be limiting. and the scope of the present invention is limited only by the appended claims.

[0052] Where a range of numerical values ​​is provided, each intermediate value shall be included as a reference to the upper and lower limits of the range and any other clearly defined limits. between the indicated or intermediate values ​​and, unless the context requires otherwise, to one-tenth of the lower unit. It is understood that any smaller range may be included within the invention. The present invention is not limited to the above-mentioned ranges, and may be applicable to smaller ranges as well as specific ranges within the scope of the present invention. If the express range includes one or both of the limitations, the inclusion Any range excluding either or both of the limitations set forth herein is also encompassed by the invention.

[0053] Certain features of the invention that are, for clarity, described in the context of separate embodiments, may also be combined into a single embodiment. It is understood that the embodiments may be provided in combination in a single embodiment, to the contrary, for the sake of brevity. The various features of the invention described in the context may be used individually or in any suitable subcombination. All combinations of the embodiments of the present invention are provided by the present invention. are specifically included and each and every combination is separately and expressly disclosed. As disclosed herein, the combination can be, for example, a stable compound such as The definition includes compounds that can be prepared, isolated, characterized, and tested for biological activity. In addition, all subcombinations of the various embodiments and elements thereof (e.g., the variables The elements of the chemical groups listed in the examples illustrating the invention are also specifically encompassed by the present invention. Each of the subcomponents is disclosed herein as being individually and explicitly set forth in the claims. will be disclosed.

[0054] Unless otherwise defined, all technical and scientific terms used herein are understood to be within the meaning of the present invention. have the same meaning as commonly understood by a person of ordinary skill in the art to which they pertain. Any methods and materials similar or equivalent to those described herein may be used to practice the present invention. Alternatively, any of the methods and materials may be used for testing, the preferred methods and materials being described herein. All publications mentioned in disclose the methods and / or materials from which they are cited. and is hereby incorporated by reference for purposes of illustration.

[0055] As used in this specification and the appended claims, the singular forms "a," "an," and "the" refer to the same or similar elements unless expressly stated otherwise. , including multiple referents. The claims may be drafted to exclude any optional element. Thus, the statement may be used without regard to the recitation of claim elements or the use of "negative" limitations. as a precedent for the use of exclusive terms such as "only" and "solely" in connection with It is intended.

[0056] It will be appreciated that, for clarity, certain features of the invention which are described in the context of separate embodiments may also be combined into a single implementation. It is understood that the embodiments may be provided in combination in the examples. Conversely, for the sake of brevity, a single implementation may be provided. The various features of the invention which have been described in the context of the examples may be used individually or in any suitable subcombination. It may be offered in.

[0057] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing in this specification is intended to represent antecedent to this disclosure by virtue of prior invention. It should not be construed as an endorsement and the publication date provided may differ from the actual publication date. So you'll need to check that separately.

[0058] Before proceeding to a more detailed description of specific embodiments of the present disclosure, some terms will be defined.

[0059] (definition) Analyte Analyte refers to the compound or composition to be measured, the substance of interest. Analyte is a member of a specific binding pair (sbp). The component may be a monovalent or polyvalent ligand, typically an antigen or hapten, and may be one or more This may be a single compound or multiple compounds that share a common epitope or determinant site.

[0060] A sample suspected of containing the analyte Any sample reasonably suspected of containing the analyte may be analyzed by the methods of the present disclosure. The sample may include a human, animal or artificial sample. The sample does not inhibit the assay. The sample may be prepared in a suitable medium. Typically, the sample may include, but is not limited to, urine, whole blood, serum, plasma, brain spinal cord, or the like. It is an aqueous or natural fluid, such as cerebrospinal fluid or saliva. In some cases, the sample is serum.

[0061] Analyte quantity measurement Quantitative, semi-quantitative and qualitative methods and all other methods of determining an analyte shall be is regarded as a method of measuring the amount of, for example, Methods which detect only the presence or absence of an analyte are also considered to be within the scope of the present invention.

[0062] Synonyms for the phrase "measuring the amount of an analyte" contemplated within the scope of this disclosure are not limited. Detecting, measuring or determining the presence of an analyte; Detecting, measuring or determining the presence of an analyte detecting or determining the amount of the analyte; and detecting, measuring or determining the concentration of the analyte. This includes the step of:

[0063] Members of specific binding pairs A member of a specific binding pair (sbp member) is one of two different molecules. They bind specifically to surfaces or cavities and are complementary to the specific spatial and polar organization of other molecules. The members of a specific binding pair are a ligand and a receptor (anti-ligand). These are generally referred to as antigen-binding domains, sbp components and sbp partners. It is a component of an immunological pair such as an antibody.

[0064] Ligand Receptors refer to any organic compound that can occur naturally or be prepared. For example, In some contexts, the analyte is a ligand, and the present disclosure relates to the amount or concentration of the analyte of the ligand. A method for determining

[0065] Receptor A receptor is any compound or structure that can recognize a specific spatial and polar organization of molecules. Such organized regions of the molecule are called epitopes or determinant sites. Receptors include antibodies and enzymes.

[0066] Epitope An "epitope" is a molecule capable of inducing an immune response and binding to a specific antibody produced by that response. A hapten (symmetry) is a molecular region on the surface of an antigen that can bind to a target molecule. In conjunction with haptens such as dimethylarginine, non- Antibodies can be produced against the antigenic hapten molecule. Antibodies that recognize the "pitope" are produced.

[0067] linking group A linking group is a part of a structure that connects two or more substructures. A linking group provides a link between substructures. It has one or more uninterrupted chains of atoms that extend from one another. The atoms of the linking group themselves are not chemical bonds. The number of atoms in the linking group is determined by counting the atoms other than hydrogen.

[0068] Conjugate A conjugate may consist of two or more substructures joined together through a linking group to form a single structure. The linkage can be achieved by joining the subunits through a linking group. In the context of this disclosure, a conjugate is a molecule that binds to a hapten, an sbp member, or an analyte analog. Glucose-6-phosphate dehydrogenase (G6PDH) enzymes, e.g. G6PDH Mutant enzymes may be used in conjugates (see, e.g., U.S. Pat. No. 6,455,288, US Pat. No. and recombinant G6PDH described in US Pat. Nos. 6,090,567 and 6,033,890. In the context of this disclosure, G6PDH refers to an enzyme, such as a G6PDH enzyme, or a G6PDH label, etc. In some cases, the conjugates may be, but are not limited to, G6PDH, Alkaline phosphatase, β-galactosidase and horseradish radish) A label (e.g., a labeled protein) or hapten containing peroxidase, s The method includes attaching a chemical label, such as a fluorescent, luminescent or colorimetric label, to the bp component or to the analyte analogue. obtain.

[0069] join Conjugation is any process in which two subunits are linked together to form a complex. The conjugation process can consist of any number of steps, for example as described herein. Cut.

[0070] Hapten A hapten is capable of specifically binding to a corresponding antibody, but is generally not itself a suitable hapten for the preparation of antibodies. Antibodies that recognize haptens do not act as immunogens for the detection of haptens. It can be prepared for compounds constructed from butenes.

[0071] Symmetrically Dimethylated Arginine Analytes A "symmetrically dimethylated arginine analyte" is an antibody that binds to symmetrically dimethylarginine. -refers to an analyte having a binding epitope. Analytes include symmetric dimethylarginine (SDMA), symmetric Nα-acetyl-dimethylarginine (SDMA), Symmetric Nα-acylated dimethylarginines, such as arginine, and SDMA-Gly-G These include SDMA-peptide derivatives such as ly dipeptide.

[0072] derivative The term "derivative" refers to a chemical compound or compound made from a parent compound by one or more chemical reactions. refers to the molecule.

[0073] analogue The term "analog" refers to a compound that has a similar structure to another compound, but in certain components. refers to different compounds, which may differ in one or more atoms, functional groups, or substructures. , may be replaced by other atoms, groups or substructures.

[0074] Label A "label," "detector molecule," "reporter," or "detectable marker" is any molecule that can be detected. A label is any molecule that produces, or can be induced to produce, a signal that can be detected by an analyte. Receptors of other molecules, such as substances, immunogens, antibodies or receptors, or in particular haptens or antibodies The label can be conjugated directly or via a linking group to a molecule that can bind to the body. Non-limiting examples of labels include radioisotopes (e.g. 125 I ), enzymes (e.g., β-galactosidase, peroxidase), G6PDH (e.g., rice Recombinant recombinant proteins described in US Patent Nos. 6,455,288, 6,090,567, and 8903. G6PDH, mutant G6PDH, enzyme fragments, enzyme substrates, enzyme inhibitors, cofactors Enzymes, catalysts, fluorophores (e.g. rhodamine, fluorescein isothiocyanate or FI TC, or Dylight 649), dyes, chemiluminescent agents and luminescent agents (e.g., dioxane luciferin), or a sensitizer.

[0075] immunogen The term "immunogen" refers to a substance that can induce, produce or generate an immune response in an organism. .

[0076] immunogenic carrier As used herein, an "immunogenic carrier" refers to a general compound capable of binding a hapten at one or more positions. a specific protein that induces an immune reaction that allows the production of antibodies capable of specifically binding to said hapten; Examples of immunogenic carrier substances are proteins, glycoproteins, complex polyamino acids, etc. -Contains polysaccharides, particles, and nucleic acids that are recognized as foreign and induce an immunological response from the host. The polyamino-polysaccharides can be prepared by any of the conventional methods known for their preparation. It can be prepared from polysaccharides using

[0077] protein The terms "protein," "polypeptide," and "peptide" refer to amino acids of any length. The terms are used interchangeably in this application to refer to polymeric forms of polysaccharides. "Polypeptide", "peptide" and "protein" are genetically encoded or non-coded amino acids, chemically or biochemically modified or derived These may include derivatized amino acids, polypeptides with modified peptide backbones, and fusion proteins. do.

[0078] Signal Generation System A "signal producing system" refers to one or more components used in an assay for an analyte. and at least one component can have a detectable label (e.g., a mutant G The signal producing system detects the presence or absence of an analyte in a sample. The signal producing system produces a signal that is related to the amount of the signal. For purposes of this disclosure, typically G6P DH or labeled proteins (e.g., alkaline phosphatase, β-galactosidase or horseradish peroxidase) binds to Sbp components similar to the analyte. It is conjugated.

[0079] Other components of the signal producing system include substrates, enhancers, activators, and chemiluminescent compounds. The specific properties required for binding of ligands, cofactors, inhibitors, scavengers, metal ions, and signal generators These include heterozygous substances, coenzymes, substances that react with enzyme products, other enzymes, and catalysts.

[0080] The signal producing system may be detected by external means such as by visual inspection, measuring electromagnetic radiation, etc. In some cases, the signal producing system comprises a chromogenic substrate. and enzyme labels (e.g., mutant G6PDH enzymes), where the chromogenic substrate is UV- or UV-irradiated. or is enzymatically converted to a dye that absorbs light in the visible range.

[0081] Separated When used in relation to antibodies, "isolated" means that the antibody has been removed from any natural state and is therefore "human." It means that the original has been altered "by hand"; that is, if it occurs naturally, It can be altered or removed from the environment, or both. For example, a living animal may have been altered or removed from its environment. Naturally occurring antibodies that exist in nature in the wild are not "isolated" but are a natural co-occurring antibody. An identical antibody that has been separated from existing material is also referred to as "isolated" as that term is used herein. Antibodies may be generated in compositions, such as immunoassay reagents, that are not naturally occurring compositions. and among which remain isolated antibodies within the meaning of the term as used herein.

[0082] Cross-reactivity "Cross-reactivity" refers to the reaction of an antibody with an antigen not used to elicit the antibody. Cross-reactivity can be quantitatively determined by establishing a standard curve using known dilutions of the target analyte. It can be determined by immunoassay. The standard curve can then be used to measure the amount of the sample quantified under similar conditions. Calculate the apparent concentration of interfering substances present in various known amounts within the sample. Cross-reactivity is calculated based on the apparent concentration. This is the value obtained by dividing the degree by the actual concentration and multiplying the result by 100.

[0083] Calibration and Control Materials The term "calibration and control materials" refers to any standard that contains a known amount of analyte. A sample suspected of containing the analyte and the calibration material. The concentration of the analyte is determined by measuring the concentration of the analyte in unknown samples or in samples with known concentrations of the analyte. The results obtained for the sample containing the compound are compared with those obtained for the standard material and then calculated. This is typically done by constructing or generating a calibration curve.

[0084] sensitivity Limit of detection means the signal that can be distinguished from that obtained in the absence of analyte. The term is used to refer to the smallest amount of analyte that provides a

[0085] Spike-Recovery "Spike recovery" is the ratio of a known amount of analyte spiked (spiked) into a sample mixture to the Assays that measure the amount of analyte (recovery) in a sample mixture by comparison are shown. The concentration may be expressed as a concentration (ng / mL) or a percentage (%).

[0086] Substantial changes in enzyme activity When an enzyme is used as a label in an assay for an analyte, it is sufficient to detect the analyte. A significant change in enzyme activity. Typically, the activity of the enzyme is 10-100%, e.g., 20-99%, Or it decreases by 30-95%.

[0087] Inhibitory antibodies They bind to epitopes present on enzymes and inhibit the activity of the enzyme or enzyme-ligand complex. The antibody is capable of binding to the ligand and inhibiting the enzymatic activity of the enzyme-ligand conjugate. Distinguished from anti-ligand antibodies.

[0088] adjustment In an assay experiment, "modulation" involves the inclusion of an analyte that competes for the analyte-antibody binding sites. A hapten or analyte bound to a label, such as an enzyme and an analyte, in a sample suspected of having and regulates the amount of enzyme product formed (see FIG. 10).

[0089] Maximum Inhibition "Maximal inhibition" refers to the inhibition of the number of epitopes present on the enzyme when an excess of antibody is added to the assay. and an enzyme, or enzyme-ligand bond, when it binds to the signal obtained in the absence of analyte. It refers to an antibody that can inhibit the activity of the complex.

[0090] Auxiliary substances A variety of auxiliary substances are commonly used in the assays of the present disclosure. Not only is it present in the assay medium, but it also acts as a stabilizing agent for the assay medium and assay components. In many cases, in addition to the additives, albumin or surfactants, especially non-ionic surfactants, are also present. Additional protein-binding agents, such as hydrophobic surfactants, binding enhancers, e.g., polyalkylene glycols, etc. It can be seen.

[0091] (Compounds, conjugates and their synthesis) Homogeneous enzyme immunoassays demonstrate that enzyme activity can be strongly regulated by the binding of sbp partners. The present disclosure is useful for homogeneous immunoassays. The present invention provides enzyme-sbp component conjugates and antibodies for performing such assays.

[0092] In certain embodiments, the protein immunogen is synthesized to contain symmetrically dimethylated arginine moieties. The antibody is used to prepare an antibody specific for a compound, such as a precipitate, which is then reacted with a suspected sample containing the analyte. Labeling bonds used in methods for detecting symmetrically dimethylated arginine analytes in samples containing A combination of one or more of the symmetrically dimethylated aryl groups described above may be prepared and used in the method. Effective screening of samples for the presence of ginine analytes can be achieved.

[0093] The immunogen and label conjugate are linked to the α-amino nitrogen atom of symmetric dimethylarginine. The compound may include a derivative of symmetric dimethylarginine linked to a protein or label. In some cases, the conjugates are referred to herein as protein conjugates or label conjugates, respectively. This may happen.

[0094] Compounds of the present disclosure include compounds useful for producing antibodies according to the present disclosure. The compounds shown include conjugates useful in the immunoassays described herein. In the above, the compound includes a compound of the following formula 1: JPEG2025032213000003.jpg3733

[0095] During the ceremony: R 1 Ha-YZ; Y is a linking group; and Z is hydrogen, OH, SH, S-acyl, O-alkyl, halogen, NH2, epoxy, ma Laimidyl, haloacetamide, carboxyl, activated carboxyl, immunogenicity carrier The antibody is selected from an antibody, a protein, and a label.

[0096] In some embodiments, Z is a protein. For example, the protein may be an immunogenic carrier. The immunogenic carrier may be conjugated to a symmetric dimethylarginine hapten, This allows for the production of antibodies capable of specifically binding to the hapten. The antibodies may be selected from hemocyanins, globulins, albumins, and polysaccharides. In some cases, the immunogenic carrier is bovine serum albumin (BSA). The carrier was Keyhole Limpet Hemocyanin. In certain embodiments, the immunogenic carrier is adapted to include one or more functional groups. The functional group on the modified immunogenic carrier may be modified to a linking group in the compound of formula 1. is a reactive functional group that facilitates attachment of an immunogenic carrier to the

[0097] In some embodiments, the symmetric dimethylarginine hapten is The nitrogen atom of the α-amino group of the nin is alkylated (Figure 1). In the present study, the linking group was attached to the nitrogen atom of the α-amino group of symmetric dimethylarginine (i.e., R 1 -Alkyl or substituted alkyl groups attached to a nitrogen atom. In an embodiment, the hapten generates an antibody specific for a symmetrically dimethylated arginine analyte. It is used to

[0098] In certain embodiments, Z is a label. The label generates or is capable of generating a detectable signal. is a molecule that can be induced to produce. For example, the label can be alkaline phosphatase. As selected from β-galactosidase and horseradish peroxidase In some embodiments, the label is an enzyme such as glucose-6-phosphate dehydrogenase. In some cases, G6PDH is an enzyme that is a protease inhibitor (G6PDH) compared to the wild-type form. For example, a mutant G6PDH containing one or more amino acid residue substitutions. DH contains cysteine ​​substitutions, e.g., in each subunit of the G6PDH enzyme. In some cases, the linking group may be attached to the G6PDH enzyme at a cysteine ​​residue. In some embodiments, the label may be modified to include one or more functional groups. The functional group on the label is a reactive group that facilitates attachment of the label to the linking group in the compound of Formula 1. It is a functional base.

[0099] In some embodiments, the symmetric dimethylarginine derivative is The nitrogen atom of the α-amino group of the amine is acylated (Figure 1). , the linking group was attached to the nitrogen atom of the α-amino group of symmetric dimethylarginine (i.e. R 1 - nitrogen atom) acyl or substituted acyl groups. In such cases, the derivatives may be used to generate conjugates useful in the immunoassays described herein. I can.

[0100] In certain embodiments, Z is a protein. The protein may be, for example, two or more, three or more More than, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, etc. It may contain any number of amino acid residues, such as dipeptides, tripeptides, etc. In some embodiments, the protein may be modified to include one or more functional groups. The functional groups on the protein are reactive groups that facilitate attachment of the protein to the linking group in the compound of formula 1. In some cases, the protein is acylated. In some cases, the protein is The protein is alkylated.

[0101] The linking group may contain about 1 to 25 atoms (excluding hydrogen atoms) or may contain a chain of 2 to 15 atoms (excluding hydrogen atoms), each independently selected from carbon, oxygen, sulfur, nitrogen, halogen, and phosphorus. In some embodiments, the linking group contains 1 to 15 carbon atoms and / or 0 to 6 heteroatoms. Examples of linking groups include, but are not limited to, -(CH2) n C(O)-, -C(O)(CH2) n -, -C(O)(CH2) n -NHC(O)-, -C(O)(CH2) n NHC(O)(CH2) n -, -(CH2) n SCH2C(O)-, -(CH2) n C(O)NH(CH2) n -, -(CH2) n NHC(O)-, -(CH2) n NHC(O)(CH2) n -, NH(CH2) n C(O)-, -(CH2) n - and -(CH2) n (Heterocyclyl)S(CH2) n In certain embodiments, the linking group is -C(O)-, where n is an integer from 1 to 10, including acid salts thereof. n NHC(O)(CH2) n -, for example, -C(O)(CH2CH2)NHC(O)(CH2)-. In certain embodiments, the linking group is -(CH2) n (Heterocyclyl)S(CH2) n C(O)-, for example -(CHCHCHCHCH)(2,5-dioxopyrrolidin-1-yl)S(CH)C(O)-.

[0102] The number of heteroatoms in the linking group is from 0 to 6, for example from about 1 to 5, or from 2 to 5, or The number of heteroatoms may range from 1 to 5. The linking agent may be aliphatic or aromatic. When present, oxygen is bonded to carbon, sulfur, nitrogen, or phosphorus as iodine or oxy. nitrogen may be present in any of the nitro, nitroso or aryl groups bonded to carbon, oxygen, sulfur or phosphorus; or amino; sulfur can be analogous to oxygen. Phosphorus can be analogous to phosphonates and phosphatides. The linking group may be attached to carbon, sulfur, oxygen or nitrogen, such as a phosphate mono- or diester. Common functional groups that form covalent bonds between molecules to be conjugated with are alkylamines, Amidine, thioamide, ether, urea, thiourea, guanidine, azo, thioether esters, amides and thiols, carboxylates, sulfonates and phosphates It is an ester.

[0103] In certain embodiments, the linking group has a non-oxocarbonyl group, including nitrogen and sulfur analogs. In this case, the phosphate group, the amino group, the alkylating agent such as halo or tosyl alkyl, Oxy(hydroxyl or sulfur analogue, mercapto)oxocarbonyl (e.g., aldehyde hydrides or ketones, or activated olefins, such as vinyl sulfones, or α-, β- Unsaturated esters, these functional groups include amine groups, carboxyl groups, activated olefins, alkyl groups The amine and the carboxylic acid, or their nitrogen derivatives, can be linked to a carboxylic acid. When a conductor or a phosphate is linked, amides, amidines, and phosphoramides are formed. When a mercaptan and an activated olefin are linked, a thioether is formed. When a mercaptan and an alkylating agent are combined, a thioether can be formed. When an aldehyde and an amine are combined under reducing conditions, an alkylamine can be formed. When a carboxylate or phosphate is linked to an alcohol, an ester can be formed. Such linking groups are described, for example, in Cautrecasas, J. Biol. Chem (1970) 2 45:3059.

[0104] To develop an assay for symmetric dimethylarginine, The chemical structure of the analyte is used. For example, asymmetric dimethylarginine is It has two methyl groups added to one of the terminal nitrogen atoms of the aryl group (see Figure 2). Methylarginine has a single methyl group on one of the terminal nitrogen atoms (Figure 2). Dimethylarginine has two methyl groups, one of which is attached to the terminal nitrogen of the guanidine group. Specific symmetric methylation of the chemical structure modulates the immunogen and This allows them to be maintained in order to produce antibodies.

[0105] The present disclosure relates to a method for producing a symmetric diamine by modifying the nitrogen atom of the α-amino group of a symmetric dimethylarginine. The present invention provides a method for designing methylarginine haptens and immunogens. and SDMA-SBAP are shown in Figure 4. The amine nitrogen of symmetric dimethylarginine The arrangement of the linking groups in is symmetrical since the analytes share a symmetrical dimethylated guanidine group. To provide an antibody that may specifically react with a dimethylarginine analyte. The present disclosure provides symmetric dimethylarginines useful in the various types of immunoassays described herein. arginine derivatives (i.e., symmetrically dimethylated arginine analytes) and immunogens (e.g., (See Figure 10 for an example.)

[0106] Compounds useful for generating antibodies and conjugates according to the present disclosure include those compounds having the general structure described below. The compounds of formula 1 can be prepared by conventional methods. The following reaction schemes are merely illustrative of the methods and are not intended to limit the disclosure. do not have.

[0107] a) Hapten Attachment of a maleimide functional group to the nitrogen atom of the α-amino group of symmetric dimethylarginines. This was achieved through the use of 4-maleimido-1-butanal (4), shown in Scheme 1. The preparation of this compound is described in Example 1. For the alkylation reaction of 4-maleimide with AcOH and NaBH3CN, The resulting mixture can be added to a solution of mid-1-butanal (4) and symmetric dimethylarginine. The resulting mixture can be stirred at room temperature for 3 hours. The reaction is quenched with water and purified by reverse phase chromatography. The hapten SDMA-M can be obtained by synthesis (Figure 4).

[0108] The bond of the linking group to the nitrogen atom of the α-amino group of symmetric dimethylarginine is shown by the reaction scheme Through the use of N-tert-(butoxycarbonyl)-β-alanine (6) shown in 2 This may be achieved by the synthesis of N-tert-(butoxycarbonyl)-β-alanine (6) and diamine The acylation of ethyl arginine is also described in Example 2. The deprotection described in Example 2 is Then, it was reacted with N-succinimidyl bromoacetate (9) in DMF and DIPEA to give The solvent can be removed under vacuum to give compound (8). The crude product can be purified by ethanol. After dissolving in CN and water, the product SDMA-SBA was purified by reverse phase chromatography. The product (SDMA-SBAP) can be synthesized by the reaction of thiol with 1,2-dihydro-1,2-trimethylsilyl-2,2-tetrahydrofuran (1,2-dihydro-2,2-tetramethylsilyl) with 1,2-dihydro-1,2-trimethylsilyl-2,2-tetra ... It can be linked to a containing protein.

[0109] Removal of the t-Boc protecting group from the linking group can yield compound (8). are described in detail in patents and articles in the technical literature. of Peptide Synthesis” (M.Bodanszky,Sprin ger Verlag,Berlin,Heidelberg,New York,To See, for example, t-butoxycarbonyl, t-butyl ether ... t-Boc, fluorenylmethyloxycarbonyl (Fmoc), acetamide Acm, triphenylmethyl, benzyloxycarbonyl, biphenyl Phenylisopropyloxycarbonyl, 1-amyloxycarbonyl, isobornyloxycarbonyl dicarbonyl, alpha-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-Nitrophenylsulfenyl, 2-cyano-1,1-dimethylethoxycarbonyl, bromobenzyloxy, carbamyl, formyl, etc. The particular protecting group selected is It may vary depending on the nature of the reaction being carried out and the reaction conditions such as temperature, pH, etc.

[0110] b) Immunogen Maleimide-functionalized haptens (e.g., SDMA-M) can be conjugated to proteins. N-Succinimidyl S-acetylthioacetate (SATA) was used to gate the Activation of protein lysine residues by acylation of the epsilon-nitrogen followed by hydroxylation Subsequent hydrolysis of the S-acetyl group with an amine generates the nucleophilic sulfhydryl group. Conjugation of sulfhydryl-activated proteins with maleimide-derivatized haptens has been shown to be This is done through the Michael addition reaction. A suitable protein (immunogenic carrier) is including, but not limited to, keyhole limpet hemocyanin, bovine thyroglobulin and ovalbumin. I can't.

[0111] The compound SDMA-M provides a maleimide functional group for thiol modification of thiol-containing proteins. The nitrogen atom of the α-amino group of symmetric dimethylarginine is linked to a symmetric diamine. The synthesis of the methylarginine immunogen SDMA-M-SH-KLH was performed as shown in FIG. Starting with the synthesis of SDMA-M, the preparation of which is disclosed in Example 1. The reaction of acetyl-S-acetylthioacetate with amines from keyhole limpet hemocyanin (KLH) was , which can generate protected sulfhydryls for subsequent reaction with SDMA-M. It can be deprotected with hydroxylamine. Sodium phosphate (0.1M, pH=8. 0) The reaction of thiol-modified KLH with SDMA-M in buffer was as shown in Figure 8. , the desired immunogen SDMA-M-SH-KLH can be produced. KLH can be purified by chromatography on a buffered Sephadex G-25 column. The concentration of the immunogen SDMA-M-SH-KLH is not limited, but may be purified using Pierce Pharmaceutical's e (trademark) Rapid Gold BCA Protein Assay Kit and other proteins The immunogen SDMA-M-SH-KLH can be used to measure the level of antibody production. The antibody can be used to immunize rabbits.

[0112] c) Enzyme conjugate The hapten SDMA-SBAP, which contains a bromoacetamide functional group, reacts with tandem thiol groups. SDMA-SBA can be used to react with cysteine-containing proteins, including G6PDH. The conjugation of P is shown in FIG.

[0113] The hapten SDMA-M can be used to prepare an immunogen. The immunogen SDMA-M-SH-KLH was used to prepare a G6PDH conjugate. In a specific embodiment, the antibodies produced can be used in an enzyme-based assay format. may show good regulation with symmetrically dimethylated arginine analytes. In this study, the immunogen SDMA-M-SH-KLH was used to successfully produce antibodies, which The antibody has potential use in the enzyme-based symmetric dimethylarginine immunoassay described below. It may provide a display having a variety of uses.

[0114] Antibodies and their preparation Embodiments of the present disclosure include antibodies that specifically bind to symmetrically dimethylated arginine analytes. Optionally, the antibody is selected from asymmetric dimethylarginine, L-arginine and N-arginine. In some embodiments, the present disclosure provides a method for specifically binding to one or more of the following amino acids: The antibody may be any of the compounds of this disclosure, including any of the compounds of Formula 1 described elsewhere herein. It specifically binds to any compound.

[0115] The term "antibody" (used interchangeably with "immunoglobulin") refers to an antibody or any Isotype (e.g., IgG (e.g., IgG1, IgG2, I Antibodies to immune groups such as IgG1, IgG2, IgG3, IgG4, IgE, IgD, IgA, IgM, etc. Polyclonal (e.g. rabbit polyclonal) and monoclonal antibodies that are globulins Preparation of whole antibodies (e.g., antibodies comprised of tetramers) is then performed by dissociating heavy and light chain polypeptides. single chain antibodies (e.g., scFv); including but not limited to, single chain F Specificity for compounds including v(scFv), Fab, (Fab´)2, (SCFV´)2 Fragments of antibodies that maintain heterologous binding (e.g., whole or single chain antibody fragments); and dimers; chimeric antibodies; monoclonal antibodies, human antibodies; and antibody and non-antibody proteins. In some embodiments, the antibody is an IgA antibody, and the IgA antibody is an IgA antibody. selected from gG, Fv, single chain antibody, scFv, Fab, F(ab´)2 or Fab´ Antibodies are often linked to members of specific binding pairs, such as biotin (biotin-avidin specific binding). The nucleotide sequence may be further conjugated to another moiety, such as a pair member.

[0116] Immunoglobulin polypeptides include kappa and lambda light chains and alpha, gamma (IgG 1, IgG2, IgG3, IgG4), delta, epsilon and mu heavy chains, or other types Full length immunoglobulin "light chains" (usually about 25 kDa or about 214 amino acids) The NFα-terminal amino acid is a variable region of about 110 amino acids and a COOH-terminal cDNA. The full length immunoglobulin "heavy chain" (approximately 150 kDa or approximately The variable region (approximately 116 amino acids) and the heavy chain (approximately 446 amino acids) are similar to each other. It includes one of the constant regions, for example gamma (approximately 330 amino acids).

[0117] The immunoglobulin light or heavy chain variable region is also called the "complementarity determining region" or "CDR". Each nucleotide sequence is composed of three hypervariable regions, called "framework" regions (FR), interrupted by three hypervariable regions. The extent of the framework regions and CDRs are defined (see "Sequences of Proteins of Immunological Interest,” E.Ra bat et al, US Department of Health and Hu man Services, (1991 and LefranC et al.IMGT, Country ImMunoGeneTics information system (registered trademark) .Nucl.Acids Res.,2005,33,D593-D597)). IMG IM, including how the T system was formulated and how it compares to other systems For more information on the GT system, see imgt.cines.fr / textes / IM GTScientificChart / Numbering / IMGTnumberin The different light chains are available on the World Wide Web at gsTable.html. The sequences of the framework regions of the heavy chains are relatively conserved within a species. The framework region of an antibody, which is the framework region of the chains that connects them, defines the positions of the CDRs. CDRs play a key role in determining and aligning the target site of an antigen. All CDRs and frameworks provided by this disclosure are provided unless otherwise indicated. Unless otherwise specified, the term is defined by IMGT.

[0118] Thus, an "antibody" may refer to, for example, an immunoglobulin gene or A protein having one or more polypeptides that can be genetically encoded by a fragment of The recognized immunoglobulin genes include kappa, lambda, alpha, and gamma. , delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable The light chains are classified as kappa or lambda. The heavy chains are classified as gamma, mu, They are classified as either alpha, delta or epsilon, which in turn are immunoglobulin classes. This defines IgG, IgM, IgA, IgD, and IgE, respectively.

[0119] A typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair consisting of one "light chain" (approximately 25 The N-terminus of each chain is the N-terminal domain of the antigen recognition Define a variable region of about 100 to 110 or more amino acids primarily responsible for The variable light chain (V L ) and variable heavy chain (V H The terms "antibody" and "antibody" refer to these light and heavy chains, respectively. Point.

[0120] Antibodies are not only complete globulins, but also genetically coded and diverse peptides. It contains a number of well-characterized fragments that can be generated by digestion with ribosomal enzymes. Thus, for example, pepsin cleaves antibodies at the disulfide bonds in the hinge region to produce F(ab) ´2. A dimer of Fab, a light chain connected to VH-CHI by a disulfide bond. F(ab)´2 is reduced under mild conditions to break the disulfide bond in the hinge region. The (Fab')2 dimer may be converted to a Fab' monomer by breaking the Fab The monomer is essentially a Fab with part of the hinge region (as in other antibody fragments). For a more detailed explanation, see Fundamental Immunology, WEP (See, e.g., Aul, ed., Raven Press, NY (1993)). Although the fragment is defined in terms of digestion of the complete antibody, a person of ordinary skill in the art would understand The Fab' fragment can be synthesized de novo using chemical or recombinant DNA methodologies. It is understood that the term antibody as used herein is therefore not limited. However, Fab´2, IgG, IgM, IgA, scFv, dAb, nanobody, unibody They may be synthesized de novo using recombinant DNA methodologies, including duplexes and by modification of whole antibodies. In certain embodiments, the antibodies of the present disclosure include antibody fragments produced by Ig Fv, single chain antibody, scFv, Fab, F(ab')2, and Fab' .

[0121] When referring to antibodies, the terms "specifically bind," "specific," "immunoreactive," and "immunoreactive" are used. The terms "immunoreactivity" and "antigen-binding specificity" refer to the ability of a given antigen to be bound to a given antigen in the presence of a heterogeneous population of antigens. To determine the presence of a highly preferential antigen binding to the antigen or its fragments, Thus, under the specified immunoassay conditions, a specific antibody will react with a specific Under these conditions, the antibody binds to a given antigen and does not bind appreciably to other antigens present in the sample. Specific binding to an antigen may require an antibody that is selected for its specificity for a particular antigen. For example, antibodies can specifically bind to compounds and not to other molecules present in a sample. It does not show comparable binding.

[0122] In some embodiments, the antibodies of the present disclosure have an affinity or K a (i.e., 1 / M units When a compound binds or associates with a molecule with a specific binding interaction (the equilibrium binding constant of the specific binding interaction), For example, 10 5 M -1 The above is "specifically binds." In a specific embodiment, the antibody , about 10 6 M -1 , 10 7 M -1 , 10 8 M-1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 , 10 12 M -1 , or 10 13 M -1 Above K a Combined with "High affinity" binding is defined as binding that is at least 10 7 M -1 More than, at least 10 8 M -1 Below Top, at least 10 9 M -1 More than, at least 10 10 M -1 More than, at least 10 11 M -1 More than, at least 10 12 M -1 More than, at least 10 13 M -1 More than K a One Alternatively, affinity is expressed in M ​​units (e.g., 10 -5 M or 10- 13 M or less) of a specific binding interaction with In some embodiments, specific binding occurs when the antibody is at least about 10 -5 M or less, about 10 -6 M and below, about 10 -7 M or less, 10 -8 M or less, or about 10 -9 M, 10 -10 M, 10 -11 M , or 10 -12 K below M D This means that the compound binds to the antibody. The binding affinity of the antibody can be determined by conventional techniques, such as competitive ELISA (enzyme-linked immunosorbent assay). ), equilibrium dialysis, surface plasmon resonance (SPR) technology (e.g., BIAcore2000 instrument) By using a radioimmunoassay or other similar method (using a device or general procedures as described by the manufacturer), This can be easily determined.

[0123] Whether a first antibody "competes" with a second antibody for binding to a compound is well known in the art. Competitive antibodies can be readily determined using known competitive binding assays, for example, antibody competition assays. For example, the first antibody sample may be bound to a solid support. A sample of a second antibody that is suspected of competing with the first antibody is then added. When labeled and unlabeled antibodies bind to separate, distinct sites on the compound, In this case, the labeled antibody will be bound at the same level regardless of the presence of a suspected competing antibody. However, if the interaction sites are identical or overlapping, unlabeled antibodies will compete to bind to the compound. If there is too much unlabeled antibody present, the amount of labeled antibody bound will be low. There is almost no case.

[0124] For purposes of this disclosure, a competing antibody is one that inhibits binding of the antibody to the compound by at least about 50%, but not by at least about 60%. 0% or more, about 70% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or at least about 99% reduction. As is well known in the art and described, for example, in Harlow and Lane, Antib odies,A Laboratory Manual,Cold Spring Ha rbor Laboratory Press,Cold Spring Harbor ,New York,1988,567-569,1988,ISBN0-87969- The assay can be made quantitative using purified antibodies. A standard curve can be established by titrating one antibody itself, i.e., the same antibody is used as a standard. It is used as both a recognition and a competitor. The capacity of the labeled competing antibody may be titrated. The results are plotted and the desired binding inhibition is determined. The concentrations required to achieve the degree of harm may be compared.

[0125] According to some embodiments, the antibodies of the present disclosure have the following binding profiles for binding to a compound of Formula 1: Competes with antibodies containing: V containing the amino acid sequence GFSLSSY (SEQ ID NO:2) H CDR1 V containing the amino acid sequence DIKTGDR (SEQ ID NO:3) H CDR2, and V containing the amino acid sequence ARVYVSGNDHYDL (SEQ ID NO: 4) H CDR3 The variable heavy chain (V H ) polypeptides; and V containing the amino acid sequence QSISNY (SEQ ID NO:6) L CDR1, V containing the amino acid sequence RAS (SEQ ID NO:7) L CDR2, and V containing the amino acid sequence QLGYTYSNVENA (SEQ ID NO:8) L CDR3 The variable light chain (V L ) Polypeptides.

[0126] In particular embodiments, the antibody is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6. , SEQ ID NO:7, and SEQ ID NO:8.

[0127] According to some embodiments, the antibody has a sequence similar to that of the amino acid sequence set out in SEQ ID NO:1 but not identical to that of the amino acid sequence set out in SEQ ID NO:1. ≥ 75%, ≥ 80%, ≥ 85%, ≥ 90%, or ≥ 95% identity A variable heavy chain (VH) comprising an amino acid sequence having H ) polypeptide; and the polypeptide set forth in SEQ ID NO:5 70% or more, 75% or more, 80% or more, 85% or more, 90% or more of the amino acid sequence or a variable light chain (V) having an amino acid sequence with 95% or more identity L ) a polypeptide include.

[0128] According to some embodiments, the antibodies of the present disclosure have the following binding profiles for binding to a compound of Formula 1: Competes with antibodies containing: V containing the amino acid sequence GFSLSSY (SEQ ID NO:2) H CDR1 V containing the amino acid sequence DIKTGDR (SEQ ID NO:3) H CDR2, and V containing the amino acid sequence ARVYVSGNDHYDL (SEQ ID NO: 4) H CDR3 The variable heavy chain (V H ) polypeptides; and V containing the amino acid sequence QSISNY (SEQ ID NO:6) L CDR1, V containing the amino acid sequence RAS (SEQ ID NO:7) L CDR2, and V containing the amino acid sequence QLGYTYTNVENA (SEQ ID NO: 10) L CDR3 The variable light chain (V L ) Polypeptides.

[0129] In particular embodiments, the antibody is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6. , SEQ ID NO:7, and SEQ ID NO:10.

[0130] According to some embodiments, the antibody has a sequence similar to that of the amino acid sequence set out in SEQ ID NO:1 but not identical to that of the amino acid sequence set out in SEQ ID NO:1. ≥ 75%, ≥ 80%, ≥ 85%, ≥ 90%, or ≥ 95% identity A variable heavy chain (VH) comprising an amino acid sequence having H ) polypeptide; and the polypeptide set forth in SEQ ID NO:9 70% or more, 75% or more, 80% or more, 85% or more, 90% or more of the amino acid sequence or a variable light chain (V) having an amino acid sequence with 95% or more identity L ) Polypeptide Includes.

[0131] According to some embodiments, the antibodies of the present disclosure have the following binding profiles for binding to a compound of Formula 1: Competes with antibodies containing: V containing the amino acid sequence GFSFSSTK (SEQ ID NO: 12) H CDR1 V containing the amino acid sequence CIGTDT (SEQ ID NO: 13) H CDR2, and V containing the amino acid sequence ARSSSSTGYYNL (SEQ ID NO: 14) H CDR3 The variable heavy chain (V H ) polypeptides; and V containing the amino acid sequence QSIRSY (SEQ ID NO: 16) L CDR1, V containing the amino acid sequence YAS (SEQ ID NO: 17) L CDR2, and V containing the amino acid sequence HDYYTFTDND (SEQ ID NO: 18) L CDR3 The variable light chain (V L ) Polypeptides.

[0132] In particular embodiments, the antibody is selected from the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO: It contains the six CDRs set out in SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18.

[0133] According to some embodiments, the antibody has: % or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more identity A variable heavy chain (V H ) polypeptide; and the polypeptide set forth in SEQ ID NO:15 70% or more, 75% or more, 80% or more, 85% or more, 90% or more of the amino acid sequence or a variable light chain (V L ) Polypeptide Includes do.

[0134] According to some embodiments, the antibodies of the present disclosure have the following binding profiles for binding to a compound of Formula 1: Competes with antibodies containing: V containing the amino acid sequence GFSFSSTK (SEQ ID NO: 12) H CDR1 V containing the amino acid sequence CIGVGSRGS (SEQ ID NO: 20) H CDR2, and V containing the amino acid sequence ARSSTTGYYIL (SEQ ID NO:21) H CDR3 The variable heavy chain (V H ) polypeptides; and V containing the amino acid sequence ESIYSY (SEQ ID NO:23) L CDR1, V containing the amino acid sequence KAS (SEQ ID NO:24) L CDR2, and V containing the amino acid sequence QNYYTFTEND (SEQ ID NO: 25) L CDR3 The variable light chain (V L ) Polypeptides.

[0135] In particular embodiments, the antibody is selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: It contains the six CDRs set out in SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25.

[0136] According to some embodiments, the antibody has: % or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more identity A variable heavy chain (V H ) polypeptide; and the polypeptide set forth in SEQ ID NO:22 70% or more, 75% or more, 80% or more, 85% or more, 90% or more of the amino acid sequence or a variable light chain (V L ) Polypeptide Includes do.

[0137] According to some embodiments, the antibodies of the present disclosure have the following binding profiles for binding to a compound of Formula 1: Competes with antibodies containing: V containing the amino acid sequence GFSFWR (SEQ ID NO:27) H CDR1 V containing the amino acid sequence CIDGGNTNR (SEQ ID NO:28) H CDR2, and V containing the amino acid sequence ARVRLGNNDYIDL (SEQ ID NO:29) H CDR3 The variable heavy chain (V H ) polypeptides; and V containing the amino acid sequence QSISNY (SEQ ID NO:6) L CDR1, V containing the amino acid sequence RAS (SEQ ID NO:7) L CDR2, and V containing the amino acid sequence QQGYNWDLDGA (SEQ ID NO:31) L CDR3 The variable light chain (V L ) Polypeptides.

[0138] In particular embodiments, the antibody is selected from the group consisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, It contains the six CDRs set out in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:31.

[0139] According to some embodiments, the antibody has: % or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more identity A variable heavy chain (V H ) polypeptide; and the polypeptide set forth in SEQ ID NO:30 70% or more, 75% or more, 80% or more, 85% or more, 90% or more of the amino acid sequence or a variable light chain (V L ) Polypeptide Includes do.

[0140] Variable heavy chain (V H ) polypeptide, variable light chain (V L ) polypeptide, and amino acid sequence of CDR The acid sequences are provided in Table 1 below.

[0141] Table 1-V H , V L and CDR amino acid sequences JPEG2025032213000004.jpg149121

[0142] JPEG2025032213000005.jpg172121

[0143] JPEG2025032213000006.jpg56121

[0144] In certain examples, the antibodies of the present disclosure are capable of binding to metabolic products of symmetric dimethylarginine. Metabolites of interest include, but are not limited to, symmetric Nα-acetyl-diamine In some embodiments, the antibodies of the present disclosure include , and their reactivity to SDMA in more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, and 3%. >0%, >35%, >40%, >45%, or >50% Ac-SDMA responsiveness .

[0145] Embodiments of the present disclosure further include nucleic acids. In certain embodiments, the nucleic acids of the present disclosure are V that does not contain any of the CDRs of the antibodies listed in Table 1 H and / or V L and the variable heavy chain (V H ) polypeptide, variable light chain (V L ) Polype The nucleotide sequences encoding the exemplary antibodies of the present disclosure encode a polypeptide, a nucleotide sequence, or both. Examples of nucleic acids having the sequence are provided in Table 2 below.

[0146] Table 2 - Nucleotide sequences JPEG2025032213000007.jpg150121

[0147] JPEG2025032213000008.jpg151121

[0148] Also provided is an expression vector comprising any of the nucleic acids of the present disclosure. , the V of the antibody of the present disclosure in a host cell H and / or V L The present invention provides a method for the development of a novel V of the disclosed antibody H and / or V L Expression of natural or synthetic nucleic acids encoding the Type V H and / or V L The nucleic acid encoding the promoter (constitutive or inducible) This is accomplished by incorporating the construct into an expression vector in operable linkage to a gene that is functionally functional. The vector is suitable for replication and integration in prokaryotes, eukaryotes or both. The zoning vector is V H and / or V L A transcription factor useful for regulating the expression of nucleic acids encoding It contains transcription and translation terminators, initiation sequences, and promoters. At least one independent terminator sequence, both in eukaryotes and in prokaryotes Sequences permitting replication of the vector, i.e. shuttle vectors, and in prokaryotic and eukaryotic systems. The general expression cassette contains a selection marker for both the See, e.g., et al. (1989). In order to obtain high levels of expression of cloned nucleic acids, Generally, a strong promoter to direct transcription, a ribosome binding site for translation initiation, and a transcription factor It is common to construct an expression plasmid that contains transcription / translation terminators.

[0149] Thus, embodiments of the present disclosure further include cells, e.g., recombinant host cells. In embodiments, a cell is provided that includes any of the nucleic acids and / or expression vectors disclosed herein. According to some embodiments, the variable heavy chain (V H ) encoding a polypeptide and a first nucleic acid encoding the variable light chain (V L a second nucleic acid encoding a polypeptide; In certain embodiments, a cell is provided comprising a first expression vector comprising a first nucleic acid, and A cell is provided that comprises a second expression vector that comprises the second nucleic acid. The cells of the present disclosure can be any of the known in the art. Methods such as electroporation, lipofection, microinjection, Introducing one or more of the nucleic acids and / or expression vectors of the present disclosure into a host cell via insertion or the like. It can be produced in

[0150] Also provided is a method of making an antibody of the present disclosure. In certain embodiments, the method comprises: The cells of the disclosure (e.g., recombinant host cells) are cultured under conditions suitable for the cells to express the antibody. To culture the cells so that the antibodies are expressed, Suitable conditions for the incubation may vary. The conditions may include an appropriate temperature (e.g., 32°C-42°C, such as 37°C). ) and pH (e.g. pH 7.0-7.7, such as pH 7.4) with the appropriate percentage of CO2, e.g. In an environment having 3% to 10%, such as 5%, an appropriate medium (e.g., cell culture plate or its well), a suitable container (e.g., DMEM, RPMI, MEM, IMDM, D This may include culturing the cells in a cell culture medium such as MEM / F-12.

[0151] In addition, a polyclonal antibody that specifically binds to any of the novel immunogens represented by Chemical Formula 1 is also disclosed. A method for preparing a clonal antibody is provided. The antibody-containing immune serum is prepared by subjecting the clonal antibody to a method for preparing a clonal antibody. This involves immunization of animals such as rabbits and sheep with the appropriate immunogen, followed by an appropriate waiting period as suggested. Antisera are obtained by well-established techniques for obtaining antisera from the blood of animals immunized via immunotherapy. The paper is based on the findings of Parker, Radioimmunoassay of Biologica lly Active Compounds,Prentice-Hall(Engle Wood Cliffs, NJ, US, 1976), Butler, J. Imm. unol.Meth.7:1 24(1975);Broughton and Str. ong,Clin.Chem.22:726 732(1976);and Playf air, et al., Br. Med. Bull. 30:24 31 (1974) Immunization procedures are well established in the art and are described by David Wild (Nature Press, 1999). The Immunotherapy and Immunology Review, edited by the Immunology Publishing Group, 2000 Assay Handbook, 2nd Edition, and many other references cited therein. The degree of antibody purification required will depend on the desired application program. For many purposes, no purification is necessary.

[0152] The collected serum was analyzed using either a symmetric dimethylarginine protein conjugate or an ELISA format. or other symmetric dimethylarginine conjugates in a homogeneous enzyme immunoassay format. It is possible to test for the presence of antibodies that specifically bind to the dimethylated arginine analyte. The technique can be used generally for symmetrically dimethylated arginine analytes as described herein. The present invention can be applied to generate polyclonal antibodies that are specific to the target gene and evaluate their usefulness. Certain antibodies are useful for the detection or determination (including selective quantification) of symmetric dimethylarginines. The present invention is useful as a reagent for immunoassays.

[0153] A monoclonal antibody, particularly a monoclonal antibody that specifically binds to the immunogen of formula 1, is provided. The monoclonal antibodies for which the following procedure is used are described in the literature by Kohler et al. nd Milstein, Nature 265:495 497, 1975 A review of monoclonal antibody technology is provided in Lymphocyte Immunology, 1999. Hybridomas, ed. Melchers, et al. Springer-V erlag(New York1978),Nature266:495(1977), ScienCe 208:692(1980) and Methods of Enzymo logy 73(part B):3 46 (1981). After injecting the injection into an animal such as a heron or mouse and waiting a sufficient amount of time, the animal is sacrificed and the spleen cells are extracted. Alternatively, spleen cells from non-immunized animals can be sensitized to the immunogen in vitro. Spleen cell chromosomes that code for the sequences for immunoglobulins are typically purified using non-ionic washes. fusing spleen cells with myeloma cell lines in the presence of an agent such as polyethylene glycol; The cells produced, including the fused hybridomas, can be compressed with The cells are grown in a selective medium such as HAT-medium, and the surviving immortalized cells are the limiting The cells are grown in the medium using dilution conditions. and the supernatants are screened for monoclonal antibodies with the desired specificity. The hybridoma cells are injected into the peritoneal cavity of a mammalian host that will receive the cells, and the ascites fluid is collected. There are various techniques to improve the yield of monoclonal antibodies, such as isolating ascites fluid from If there are insufficient amounts of monoclonal antibody in the host, the antibody may be harvested from the host's blood. Alternatively, cells producing the desired antibodies can be cultured in a hollow fiber cell culture device or a spinner flask device. Both are well known in the art. There are a variety of conventional methods for the isolation and purification of monoclonal antibodies from materials (see above). (See Kohler and Milstein).

[0154] Using the following procedure, recombinant monoclonal antibodies, particularly those specifically binding to the immunogen of formula 1, were prepared. Monoclonal antibodies that combine with the B-cell antigens can be prepared. Peripheral blood mononuclear cells (PBMCs) were isolated from rabbit whole blood and cultured on the same day. Culture and plate single B cells in 40 x 96 well plates. The plates were incubated in B cell culture medium for 7 days at 37°C / 5% CO2, after which the top The supernatant was screened by indirect ELISA against the SDMA-SBAP-BSA antigen. The original positive wells were determined. The antigen-positive wells were stored in RNA lysis buffer at -80°C. The mRNA was purified using the Dynabeads mRNA DIRECT purification kit (Am B cell wells selected by SDMA-SB cDNA was synthesized and PCR was performed twice. The antibody variable region cDNA for cloning was prepared using the above procedure. The variable region cDNA was cloned into a mammalian expression vector containing rabbit heavy and light chain constant regions, respectively. The expression constructs were co-transfected into HEK293 cells to express SDMA-SBA Cell culture supernatants were analyzed by indirect ELISA against the P-BSA antigen. The antibodies were purified from the supernatant by conventional approaches for antibody purification. The antibodies can be purified by chromatography, e.g., DEAE chromatography, ABx chromatography, etc. Antibodies may be purified by known techniques such as filtration. The assay can be performed using, for example, a homogeneous enzyme immunoassay format as shown in FIG. and screening may be performed taking into account properties such as binding inhibition, curve magnitude, and cross-reactivity. .

[0155] Selected positive rabbit monoclonal antibodies were subjected to DNA sequencing. The rabbit IgG heavy chain sequence is approximately 1200 bp and can be reliably sequenced at the 5´ end. The rabbit kappa light chain is approximately 700 bp and has a 5´ Reliable full-length variable sequences can be obtained from sequencing of all heavy chains. and kappa chain variable region sequences were translated. H and V L The produced amphibian The amino acid sequences are provided in Table 1 above.

[0156] composition The present disclosure also provides compositions. According to some embodiments, the compositions of the present disclosure include This includes any compound, antibody, nucleic acid, expression vector and / or cell.

[0157] In certain embodiments, the compositions of the present disclosure comprise any compound of the present disclosure present in a liquid medium. The liquid medium includes water, a buffer solution, or other aqueous medium. It is a liquid medium containing salts (e.g., NaCl, MgCl2, KCl, MgSO4), buffers, etc. (Tris buffer, N-(2-hydroxyethyl)piperazine-N´-(2-ethoxyethyl) 2-(N-morpholino)ethanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES) , 2-(N-morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-morpholino)ethanesulfonic acid sodium salt (MES), phorino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl- 3-aminopropanesulfonic acid (TAPS), etc.), solubilizers, detergents (e.g., Tween -20 or other non-ionic detergents), nuclease inhibitors, protease inhibitors, glycerol One or more additives, such as a chelating agent, may be present in the composition.

[0158] In certain embodiments, the compositions of the present disclosure may be used in any of the homogeneous enzyme immunoassays described herein. The present invention can be used as a reagent for performing any of the immunoassays of the present disclosure, including the immunoassays of the present disclosure. A composition comprising any compound of Formula 1, any antibody of the present disclosure, or both, is useful in the immunoassay. In some implementations, the reagents may be used to perform the assay, e.g., for stability, convenience, etc. To give just one example, the compounds of the present disclosure may be provided in a lyophilized form for multiplication. Compositions comprising any of the compounds of Formula 1, any of the antibodies of the present disclosure, or both, can be used in No. 5,413,732, the disclosure of which is incorporated herein by reference for any purpose. The entirety of which is incorporated herein by reference. In summary, the spherical bodies are used, for example, to homogenize reagents. can be prepared by forming a uniform drop of the solution (e.g., 2 to 4 mL). 50 μL); Distribute uniform measurement droplets into unstirred cryogenic liquid (e.g., liquid nitrogen) to obtain droplets collecting the frozen particles from the cryogenic liquid; and and lyophilizing to form a plurality of lyophilized reagent spheres.

[0159] Immunoassays Aspects of the present disclosure include any compound of Formula 1 of the present disclosure and / or any antibody of the present disclosure. In certain embodiments, the compounds and / or antibodies are administered in culture medium, For example, a medium containing a biological sample of interest is assayed for at least one symmetrically dimethylated arginine. For example, in some embodiments, the A method for determining the amount of at least one symmetrically dimethylated arginine analyte in vivo is provided. The method further comprises the step of: The method includes binding a sample to any of the antibodies of the present disclosure. determining the presence or absence of a complex comprising the analyte and the antibody, The presence of the analyte indicates the presence of a symmetrically dimethylated arginine analyte in the sample. In the present disclosure, the medium further comprises any compound of Formula 1. For example, the medium may comprise a symmetric dimer. A counter molecule having a methylarginine moiety and a detectable label (e.g., an enzyme such as G6PDH). It may further comprise a symmetric dimethylarginine conjugate.

[0160] A sample suspected of containing at least one symmetrically dimethylated arginine analyte may be In particular embodiments, the sample is whole blood, serum, plasma, urine, sputum, semen, This includes saliva, ocular fluid, cerebral fluid, spinal fluid, amniotic fluid, tissue culture medium, and the like, and dilutions thereof.

[0161] The present disclosure provides a method for determining the presence of a symmetrically dimethylated arginine analyte in a sample suspected of containing the analyte. Immunoassay methods for assessing the presence or absence of Immunoassays can be in a variety of formats. Immunoassays are also known as homogeneous immunoassays. The assays disclosed herein include both sandwich and competitive assays. Immunoassays include sandwich and competitive assays, as well as certain assays involving immunoprecipitation. Other types of assays may be implemented. In a specific embodiment, an immunoassay is a homogeneous immunoassay, in which the assay reagents and the sample form a homogeneous assay mixture. The components are mixed together to obtain a final product.

[0162] In certain embodiments, the immunoassay detects symmetrically dimethylated amino acids in a biological fluid sample. A homogeneous enzyme immunoassay system for use in assaying arginine analytes. Thus, the immunoassay involves the binding of symmetrically dimethylated arginine analytes to antibody binding sites in a sample. Between Nα-acylated-SDMA and / or Nα-alkylated-SDMA labeled against In some embodiments, the label is a protein, such as an enzyme. For example, In one non-limiting example, the label is an enzyme whose activity can be measured spectrophotometrically. The assay shown binds the enzyme glucose-6-phosphate dehydrogenase (GlcNAc) to antibody binding sites. G6PDH) labeled with Nα-acylated-SDMA and / or Nα-alkylated-S In certain embodiments, the enzyme activity is decreased when bound to the antibody, and the sample is The concentration of the symmetrically dimethylated arginine analyte can be measured in terms of enzyme activity within the In some cases, the active enzyme is nicotinamide adenine dinucleotide (NAD + )of This converts it to NADH, resulting in a change in absorbance that can be measured by a spectrophotometer. Endogenous serum G6PDH is a cofactor for NAD + The bacteria used in the assay (Leuc onostoc mesenteroides enzymes, so immunoassays Does not hinder.

[0163] Generally, to detect the presence (or absence) of a symmetrically dimethylated arginine analyte in a sample, The disclosed immunoassay for detecting arginine residues comprises adding to a reaction mixture (i) a symmetrically dimethylated arginine moiety. (ii) a sample suspected of containing a symmetrically dimethylated arginine analyte specific A complex is formed between the specifically binding antibody and a symmetrically dimethylated arginine analyte that may be present in the sample. The method can also be performed by adding an antibody that forms a complex. The presence (or absence) of the complex is determined by detecting the presence or absence of a symmetric dimethyl ether in the sample. In addition, the presence (or absence) of the complex formed may indicate The amount of arginine present in the sample is evaluated to determine the concentration of the symmetrically dimethylated arginine analyte present in the sample. (e.g., the concentration of symmetrically dimethylated arginine analytes from the subject from whom the sample was obtained) (to provide an assessment of serum or tissue concentration). The presence and / or amount of complexes can be determined by direct (e.g., by detecting bound antibodies in a complex) or indirectly (e.g., symmetric dimethyl When the arginine enzyme conjugate is not bound to an antibody, the symmetric dimethylarginine enzyme conjugate The activity of the enzyme was assessed by combining the symmetrically dimethylated arginine analyte with the arginine analyte. A detectable amount of arginine from the sample that indicates that the arginine is bound by the symmetrically dimethylated arginine analyte. (See, for example, FIG. 10.)

[0164] Generally, the immunoassays of the present disclosure include assays using a medium (e.g., an assay medium or assay reaction medium). mixture) under conditions that permit the formation of a stable complex between the analyte and the antibody in the sample, It involves combining a symmetrically dimethylated arginine analyte antibody with the sample.

[0165] The assay can be performed in solution or on a solid (insoluble) support (e.g., polystyrene, nitro Cellulose or beads) may be used, and any conventional method (e.g., Current Protocols in Immunology,Coligan et al., ed.; John Wiley & Sons, New York, 1992.) The methods include ELISA (enzyme-linked immunosorbent assay), IRMA (immunoradiometric assay), and In certain embodiments, the assay includes a ELISA (Radioimmunoassay) and a RIA (Radioimmunoassay). is performed in solution, e.g., the assay is not bound to a solid support or without binding assay reagents. It is held.

[0166] When the assay is performed in solution, the test sample (and optionally the control sample) contains the analyte and and an affinity reagent complex for a period of time sufficient to form an asymmetrically dimethylated arginine analyte. As mentioned above, symmetrically dimethylated arginines may be incubated with the antibody. The analyte antibody may contain a detectable label, such as a radionuclide, a fluorescent substance, or an enzyme. The sample is then treated to separate the counter ions from the excess, unreacted symmetrically dimethylated arginine analyte antibody. Isolate the symmetric dimethylated arginine analyte-antibody complex (e.g., with a secondary antibody (e.g., anti- -immunoglobulin antiserum) and centrifugation to precipitate the secondary complexes, or Sepharose® or immobilized on a solid substrate such as a plastic well (by binding to an affinity surface such as a secondary unlabeled antibody). Symmetrically dimethylated arginine analytes Detection of the symmetrically dimethylated arginine analyte antibody bound to the arginine analyte can be accomplished in a variety of ways. If necessary, a substrate for the detectable label may be added to the sample.

[0167] When the assay uses a solid support, the support may comprise a symmetric dimethyl group bonded to the support surface. The antibody (or conjugate) can be a carboxylated arginine analyte. is a method for determining the amount of symmetrically dimethylated arginine analyte from a sample (or a sample obtained by a competitive binding assay, etc.) The antibody (which may be unbound and present in the reaction mixture) is bound to the solid support through specific binding to the antibody. The insoluble support facilitates stable, water-resistant attachment to the antibody or suitable target molecule. It is capable of binding soluble dimethylarginine conjugates and is capable of being separated from soluble materials. or any method compatible with the total detection of symmetrically dimethylated arginine analytes in a sample. The composition is:

[0168] The surface of the support may be solid or porous and may have any suitable shape. Dimethylarginine analyte antibody, or symmetrically a dimethylarginine conjugate, is bound to the antibody. Examples of suitable insoluble supports include beads, e.g., magnetic beads, membranes and microtiter plates. These include glass, plastics (e.g., polystyrene), polysaccharides, nylons, The substrate may be composed of polyvinyl chloride or nitrocellulose.

[0169] The assay reagents consist solely of the symmetrically dimethylated arginine analyte antibodies disclosed herein. The assay reagents may include a second antibody which may be selectively detectably labeled. After that, the support is treated with a blocking agent to remove the support in areas not occupied by the assay reagents. Suitable blocking agents are non-interfering proteins such as bovine serum albumin, casein, gelatin, etc. Or, use detergents such as Tween, NP40, or TX100 at non-interfering concentrations. The blocking treatment can reduce non-specific binding.

[0170] The assays of the present disclosure include both qualitative and quantitative assays. Common quantitative methods include: The analyte is mixed with a predetermined amount of reagent antibody to obtain a known antibody within the expected range for the sample being tested. The relationship determined using a conventional sample containing a known amount of analyte is used to determine the amount of analyte in the original sample. Qualitative assays involve correlating the amount of analyte with the amount of complex formed. The sample is considered to be included or excluded if it is higher or lower than the threshold level set by the sample. Unless otherwise specified, in this disclosure, " The act of "measuring" or "determining" includes both qualitative and quantitative determinations.

[0171] Immunoassays used alone or in combination in the assays described herein include The reagents include, but are not limited to, symmetrically dimethylated arginine analyte antibodies, symmetrically dimethylated arginine Arginine conjugates and symmetrically dimethylated arginine analytes (e.g., for competitive binding assays) The immunoassay reagents may be provided in a buffered aqueous solution. Contains additional ingredients such as surface active additives, organic solvents, defoamers, buffers, surfactants and antimicrobial agents. The surface active additives may include a surface active agent that maintains hydrophobic or poorly soluble compounds in solution and prevents the formation of hydrophobic or poorly soluble compounds in solution. Examples of components that can be introduced to stabilize the glycerol include β-lactoglobulin (BLG) and β-lactoglobulin (BLG). ) or polyethylene glycol (PEG); Tween-20, Plura facA38, TritonX-100, Pluronic25R2, rabbit serum albumin Antifoaming agents and surfactants such as RSA, bovine serum albumin (BSA) and carbohydrates Examples of organic solvents may include methanol and other alcohols. Exemplary buffers include HEPES, borate, and ethyl acetate. , phosphates, carbonates, Tris, barbital, etc. Antimicrobial agents are also used as immunoassay reagents. Extends the shelf life of

[0172] Symmetric dimethylarginine conjugates and / or symmetric dimethylarginine conjugates for use as reagents in assays The dimethylated arginine analyte antibody can be insolubilized by binding to a solid support. This may involve particulates or particles that can be kept in suspension but are separated by physical means such as centrifugation or microfiltration. The wall of the medium contains a reagent for high molecular weight carriers that can be removed by chemical means. Thus, the attachment is through one or more covalent bonds. The attachment does not have to be covalent, but at least long enough to withstand separation techniques (including washing) that may be part of the assay procedure. In some cases, a solid support may be required. The subject compounds may be symmetrically dimethylarginine conjugates and / or symmetrically dimethylated arginine analytes. The antibody may be functionalized to contain reactive groups that facilitate attachment to a solid support. Non-limiting examples of reactive groups that can be used include -COOH, -NH2, -C(O)H, -SH, etc. In some embodiments, the symmetric dimethylarginine conjugates and / or the symmetric dimethylarginine conjugates The lysed arginine analyte antibody can be conjugated to a protein carrier and The protein carrier can be conjugated to a solid support, thus forming a symmetric dimer. Indirectly attaching methylarginine conjugates and / or symmetrically dimethylated arginines to a solid support Specific particulate materials include agarose, polystyrene, and cellulose. These include, but are not limited to, polyacrylamide, latex particles, magnetic particles and fixed red blood cells. Non-limiting examples of commercially available matrices include Sepharose (registered trademark) (Pharmacia), Poros resin (Roche Molecular lar Biochemicals), Actigel Superflow (trademark) ) resin (Sterogene Bioseparations Inc.) and Dyna These include, but are not limited to, beads (trademark) (Dynal Inc.). In certain embodiments, the selection of the solid support is based on factors such as stability, capacity, and binding capacity of the coupled antibody. Accessibility, flow rate (or capacity to disperse the resin in the reaction mixture), and ease of separation The number of possible outcomes may depend on one or more of the following:

[0173] As previously mentioned, there are many immunoassays for the detection of symmetrically dimethylated arginine analytes. Generally, immunoassays involve the addition of a medium (e.g., a reaction mixture or assay Within a mixture) a sample (i.e., suspected of containing a symmetrically dimethylated arginine analyte) sample) together with one or more immunoassay reagents (e.g., at least one asymmetric dimethyl The term "reaction mixture" or "assay mixture" refers to a process in which a reaction mixture is mixed with a sample (i.e., a soluble arginine analyte and an antibody). "Substance" generally refers to a substance suspected of containing a symmetrically dimethylated arginine analyte as exemplified in this disclosure. The term refers to a combination of a sample having symmetric dimethyl ethers and one or more immunoassay reagents. The present invention facilitates detection of the presence or absence of a substituted arginine analyte, the detection being qualitative or quantitative. The immunoassay reagent(s) may be in solution or attached to a support (e.g., a matrix such as beads). The reaction mixture is generally an aqueous solution, although it may be immobilized. The mixture may contain other components that can be separated, such as buffers, reagents, etc.

[0174] Immunoassays are generally classified into one of a variety of methods. For example, immunoassays include: They can be classified according to the detection method used, e.g., enzyme immunoassay, radioimmunoassay, etc. These include immunoassays, fluorescence polarization immunoassays, chemiluminescence immunoassays, and turbidity assays. Another classification method is based on the assay procedure used, i.e. competitive assay format, sandwich assay, or ELISA. Depending on the assay format and on the precipitation or agglutination principles. For these assays, washing steps are included in the procedure (so-called heterogeneous assays) or the reaction and detection An additional distinction is made whether the extraction is carried out without a washing step (so-called homogeneous assay). Particular assays are described in detail below.

[0175] Immunoassays may be described as heterogeneous or homogeneous. "One-immunoassay" is a method in which the complex is not separated from the unreacted reactants, but instead the presence of the complex is detected. is gained or lost as a result of incorporation of at least one of the reactants into the complex A homogeneous assay is an analytical method that uses different reagents to detect Fluorescence and fluorescence quenching pairs; enzyme and enzyme inhibitor pairs of other reagents; chromophores and chromophores of other reagents and latex agglutination assays.

[0176] The specific homogeneous assay is a quantitative homogeneous enzyme assay in which symmetric methylarginines are bound to the active enzyme. In some embodiments, the symmetric to symmetric methylarginine conjugates are used as immunoassays. The binding of the analyte antibody to the dimethylated arginine can be used to determine the enzyme activity of the conjugate in a qualitative or quantitative manner. The bonds are arranged to affect the symmetric dimethylated arginine analyte. When premixed with the antibody, the antibody forms a complex with the symmetrically dimethylated arginine analyte, Thus, the activity of the enzyme in the conjugate can be inhibited by the The amount of symmetrically dimethylated arginine analyte present in the sample may be related to the amount of symmetrically dimethylated arginine analyte present in the sample.

[0177] G6PDH is a particular enzyme useful in the assay. DH is a peptide that has one or more lysine residues deleted or substituted, or one or more cysteine ​​residues introduced. It is a naturally occurring mutant of G6PDH that has been isolated from Leuconostoc mese. nteroides G6PDH is a NAD + or NADPH + By using D-G A dimeric enzyme catalyzing the oxidation of leucono-delta-lactone-6-phosphate. NAD + The property of using the enzyme is + Human G6 that effectively uses only It is distinguished from PDH, and for example, in the presence of human G6PDH, as in human-derived samples, L.meta senteroides-specific G6PDH activity can be measured. The two specific bacterial genera to choose from are Leuconostoc and Zymomona. s. Among these, L. mesenteroides, L. citreum, L. lactis, L. dextranicum and Z. mobilis are of interest, Specific examples include L. mesenteroides, L. citreum and L. lactis. Another example of a homogeneous assay system is a cloned enzyme-donor immunoassay. It is a request.

[0178] Symmetric dimethylarginine derivatives bearing thiol-reactive groups were prepared as previously described. and glucose-6-phosphate dehydrogenase (G6PDH) mutant enzymes. The mutant enzymes can be reacted to form the respective enzyme conjugates (see, for example, FIG. 7). By the procedures described in U.S. Pat. Nos. 6,090,567 and 6,033,890. available from the same publication, the disclosures of which are incorporated herein by reference.

[0179] In some embodiments, the immunoassay comprises a symmetric dimethylarginine moiety and a detectable The method further comprises adding to the sample a symmetric dimethylarginine conjugate having a suitable label. The presence or absence of the symmetrically dimethylated arginine analyte in the sample is detected by detecting the detectable label. The detectable label may comprise an enzyme, and detection may involve assaying the activity of the enzyme. In a particular embodiment, the enzyme is a dehydrogenase such as G6PDH. It is.

[0180] Luminescent oxygen channeling assay (LOCI) is a method in which a biotinylated antibody against an analyte is streptavidin. Leptavidin binds to the coated donor beads, and a second antibody against the analyte is attached to the donor beads. LOCI is a chemiluminescent homogeneous immunoassay directly conjugated to receptor beads. In the presence of the assay material, the two beads are brought into close proximity. Excitation of the beads generates singlet oxygen molecules that trigger a series of chemical reactions in the LOCI receptor beads. to produce a detectable light emission peak at 615 nm (Ullman, E. F. et al. (1996)Clin.Chem.42,1518-1526).

[0181] Symmetrically dimethylated arginine analyte antibody and analyte in separation-based or "heterogeneous" assays For complex detection, the complex formed must be physically separated from the unreacted analyte, the unreacted antibody, or both. This involves the process of separating the

[0182] In a heterogeneous immunoassay, the complex of the antibody and the symmetrically dimethylated arginine analyte is The proteins are first formed in the fluid and then captured by a solid-phase reagent or separated by methods such as gel filtration or precipitation. Alternatively, one of the reagents may be isolated based on modified physical or chemical properties, such as The reagents are allowed to bind to the solid phase before contacting it with other reagents, and the solid phase is then washed free of unreacted reagents. Separation-based assays typically involve the detection or quantification of complexes. Suitable labels include the use of labeled derivatives or labeled antibodies to facilitate labeling. 12 5 Radioisotopes such as I, enzymes such as peroxidase and β-galactosidase, and The separation step involves the separation of unreacted labeling reagents. This involves removing the labeling reagent that is present in a complex form. The amount of labeling in the complex is either measured directly or , can be estimated by the amount left unreacted.

[0183] The assays of the present disclosure include both sandwich and competitive assays. Typically, the analyte to be measured is a first antibody or other antibody that is ultimately used to separate the complex. Between one reagent and another, such as a second antibody, used as a marker for the separated complex Competitive assays involve the formation of a complex in which the analyte being measured binds to another molecule, such as an antibody. The system includes a derivative of the analyte that competes with the reagent to be bound. An example of a competitive assay using is described in US Pat. No. 3,817,837.

[0184] The compounds and methods of the presently disclosed embodiments also allow for the detection of chromatographically specific chromatographically specific peptides using lateral flow chromatography ( This includes the use of these materials in later flow chromatography techniques. Lateral flow chromatography has been shown to be effective for symmetrically dimethylated arginine analytes. a membrane strip containing a detection device, such as a non-isotopic signal generating moiety; A patient sample may be applied to the membrane strip. The sample interacts with the detection device to produce a result. The results may include the absence of symmetric dimethylated arginine analytes in the sample, the absence of symmetric dimethylated arginine analytes in the sample, The presence of symmetrically dimethylated arginine analytes and / or symmetrically dimethylated arginine in the sample. A variety of things can be represented, including the concentration of an analyte.

[0185] Certain embodiments provide for the generation of symmetrical sequences within a sample through the use of lateral flow chromatography. A method for qualitatively determining the presence or absence of a dimethylated arginine analyte is provided. In this embodiment, the basic design of the qualitative lateral flow device is as follows: 1) Test The sample pad is where the sample is applied. The sample pad is treated with chemicals such as buffers or salts. The sample is then processed to prepare a sample for reaction with the conjugate, test and control reagents when redissolved. 2) Conjugate release pads are generally antibody colloids. The material is a polyester or glass fiber material that has been treated with a conjugation reagent, such as a reactive gold conjugate. The general process for treating conjugate pads is to impregnate them and then dry them before use. During use, liquid sample added to the test redissolves the conjugate and flows through the membrane. 3) The membrane substrate is generally made of nitrocellulose or a type of membrane with an immobilized antibody capture component. 4) The wicking pad is made of a material similar to that of plasma and whole blood. The impregnation process is generally used to prepare samples and separate the cells. The absorbent pad is used to treat the pad with a reagent to promote separation. 6) The upper layer and membrane The rain system is laminated to a plastic support with an adhesive material that acts as a structural member. do.

[0186] Certain embodiments provide for the generation of symmetric dimers within a sample through the use of lateral flow chromatography. A method for qualitatively determining the presence of a methylated arginine analyte is provided. The membrane strips have antibodies specific for symmetrically dimethylated arginine analytes. The sample pad is a conjugate release pad containing colloidal gold particles. The antibody may be conjugated to a non-isotopic signal generating moiety such as lateral flow chromatography. Other detection moieties that are useful in laboratory environments include dyes, colored latex particles, and fluorescently labeled latex. These include photocatalytic particles, nonisotopic signal generating moieties, etc. The trip further comprises a capture line, where a symmetrically dimethylated arginine analyte antigen or In contrast, dimethylarginine conjugates are immobilized on the strip. The immobilization is optionally achieved through covalent attachment to the membrane strip via a linking group. In another embodiment, immobilization is via non-covalent attachment to the membrane strip. In another embodiment, the symmetrically dimethylated arginine is immobilized on the capture line. The analyte is bound to a reactive partner, such as an immunogenic carrier, such as BSA.

[0187] When a sample from a patient is applied to the sample pad, it binds to the antibody on the conjugate release pad. The solution can be mixed together to form a solution that can be quenched by capillary action across the membrane. chromatographically migrating. A symmetrically dimethylated arginine analyte is present in the sample. In this case, a symmetrically dimethylated arginine analyte antibody complex may be formed, which is When the solution reaches the capture line, it is transported across the membrane by the action of symmetric dimethylation. Symmetrical conformation that prevents the arginine analyte antibody complex from moving toward the restricted binding site of the antibody The symmetric dimethylated arginine analyte may compete with the dimethylated arginine analyte. If a high degree of cleavage is present in the sample, it may fill the limited antibody binding sites. In certain cases, this may Inhibits formation of colored antibody-non-mobile symmetrically dimethylated arginine analyte complexes at the capture line Therefore, the absence of color in the capture line indicates that there is no symmetric dimethylation in the sample. Indicates the presence of arginine assay material.

[0188] If the sample does not contain the symmetrically dimethylated arginine analyte, the solution is Upon reaching the capture line, the colored antibody-non-migrating symmetrically dimethylated arginine analyte complex In some cases, the formation of this complex at the capture line indicates that the sample This is evidence of the absence of symmetrically dimethylated arginine analytes.

[0189] Certain embodiments provide for the generation of symmetrical sequences within a sample through the use of lateral flow chromatography. The present invention provides a method for quantitatively determining the amount of a dimethylated arginine analyte. Patent No. 4,391,904; No. 4,435,504; No. 4,959,324; No. 5 ,264,180; 5,340,539; and 5,416,000. No. 6,399,623, the disclosure of which is incorporated herein by reference. In some embodiments, the antibody can be fixed for the entire length of the membrane strip. If the lip is made of paper, the antibodies can be covalently attached to the membrane strip. If the brain strip is made of nitrocellulose, the antibodies may be, for example, hydrophobic and hydrophobic. The membrane strip may be non-covalently bound through electrical interactions. The trip is a conjugate containing a symmetrically dimethylated arginine analyte bound to a detector moiety. In a specific embodiment, the detector portion may include a horseradish and enzymes such as human redox reaction product (HRP).

[0190] In a particular embodiment, a sample from a patient is symmetrically mixed with a conjugate release pad. A membrane strip capable of binding methylated arginine analyte / detector molecules to form a solution. This solution is chromatographically transported by capillary action across the membrane. If a symmetrically dimethylated arginine analyte is present in the sample, the sample may be moved to the symmetrically dimethylated arginine analyte. Both the methylated arginine analyte and the symmetrically dimethylated arginine analyte / detector molecule are antibodies. The symmetrically dimethylated arginine analyte competes for a limited number of binding sites. If sufficient concentrations are present in the sample, they may fill the limited antibody binding sites. , the symmetrically dimethylated arginine analyte / detector molecule continues on the membrane strip. The membrane strip is then loaded with symmetrically dimethylated arginine analytes / detectors. The shorter the migration distance of the detector molecule, the lower the concentration of symmetrically dimethylated arginine analyte in the sample. and vice versa. Symmetrically dimethylated arginine analyte / detector molecules When an enzyme is included, the migration length of the symmetrically dimethylated arginine analyte / detector molecule is proportional to the enzyme group. The enzyme reaction product can be detected by applying the enzyme to the membrane strip. , may be utilized to determine the concentration of a symmetrically dimethylated arginine analyte in a sample. In a particular embodiment, the chromogenic substrate for the enzyme, such as modified N,N-dimethylaniline, Immobilized on a membrane brain strip, 3-methyl-2-benzothiazolinone hydrazone was It is passively applied to the membrane, alleviating the need for separate reagents for visualization of the color reaction.

[0191] Fluorescence polarization assay The FPIA technique is based on competitive binding. Japanese Patent Nos. 4,593,089, 4,492,762, 4,668,640 and No. 4,751,190, the disclosures of which are incorporated herein by reference. It is used.

[0192] The FPIA technique can be used to identify the presence of symmetrically dimethylated arginine analytes. and can be used in an assay to quantify the amount of symmetrically dimethylated arginine analyte in a sample. In part, the rotational properties of molecules in solution are such that the degree of polarization is directly proportional to the size of the molecule. Therefore, the larger the molecule, the more polarization there can be. Fluorescent or other luminescent labeled symmetrically dimethylated amines that are small and rapidly rotating in solution. When linearly polarized light is used to excite the arginine analyte, the emitted light can be highly depolarized. Photolabeled symmetrically dimethylated arginine analytes interact with or bind to antibodies When the antibody is too large, the rotation slows down and the emitted light can become highly polarized. This is to significantly and measurably increase the amount of unlabeled symmetrically dimethylated amines in the samples. Increasing the amount of arginine analyte increases the fluorescence with the symmetrically dimethylated arginine analyte antibody. The amount of light emitted within the sample upon reduced binding of the labeled symmetrically dimethylated arginine analyte is The relationship between the concentration and polarity of the unlabeled symmetrically dimethylated arginine analyte in the sample may be reduced. The quantitative relationship was determined by calibration bias with known concentrations of symmetrically dimethylated arginine analytes. The FPIA can therefore measure and set the light value for symmetrically dimethylated alginates in the sample. The present invention can be used to identify the presence and concentration of an analyte.

[0193] This is called immunoturbidimetric assay. The homogeneous particulate immunoassay technology is based on the aggregation of particles and compounds in solution. Or the compounds may aggregate, increasing particle size and increasing the turbidity of the solution. Thus, the symmetrically dimethylated arginine analyte antibody is capable of detecting symmetrically dimethylated arginine in the sample. It can be used in conjunction with microparticles to assess the presence and optionally the amount of an analyte. The immunoassay may be performed using blood, blood hemolysate, serum, plasma, tissue and / or It is useful to perform homogeneous microparticle immunoassays on other samples. The procedure was performed using a dimethylated arginine analyte and loaded onto microparticles, and a symmetrically dimethylated arginine was used. The antibody to the arginine analyte can be loaded onto microparticles. Microparticle immunoassays or immunoturbidimetric assays are used to measure the agglutination of substances within a sample. Immunoturbidimetric assay techniques are described, for example, in U.S. Pat. Nos. 7,209, 6,514,770, and 6,248,597. , the disclosures of which are incorporated herein by reference. The assay includes photoattenuation, nephelometry, and These include rometric or turbidimetric measurement methods.

[0194] The CEDIA method (ThermoFisher) is a method for determining the β-D from E. coli to bind to an antibody capable of binding to a methylated arginine analyte. -galactoside galactohydrolase or β-galactosidase ("β-gal"), etc. Symmetric dimethylarginine containing an inactive engineered enzyme donor ("ED") fragment The method is based on the competition between an arginine conjugate and a symmetrically dimethylated arginine analyte in a biological sample. If a symmetric dimethylated arginine analyte is present in the sample, it will bind to the antibody and form an ED-derivative bond. The combined ED portion is left free and the reaction mixture is added to the β-D-galactoside galactohydrolase. or can restore the enzymatic activity of β-gal and bind to the enzyme acceptor ("EA") fragment. Active enzymes, including ED and EA, undergo quantifiable reactions when exposed to appropriate substrates. An example of a substrate is galactose, which can be cleaved by the active enzyme into CPR. and chlorophenol red-β-D-galactopyranoside ("CPRG"), The CPR is measured by absorbance at a wavelength of about 570 nm. If the analyte is not present in the sample, the antibody binds to the ED-derivative conjugate and produces the ED fragment. This may inhibit the binding of the EA fragment to the enzyme and inhibit the recovery of enzyme activity. The resulting absorbance change is proportional to the amount of symmetrically dimethylated arginine analyte in the sample.

[0195] Competitive assays using chemiluminescent microparticle immunoassay ("CMIA") technology are It may also be used to assess whether a methylated arginine analyte is present in a sample. The presence and / or amount of an analyte can be determined in a sample using the CMIA technique. The assay comprises detecting symmetrically dimethylated arginine moieties capable of binding to a symmetrically dimethylated arginine analyte. This includes the use of precipitating antibodies, which are suitable for separation by filtration, precipitation and / or other means. The antibody may be attached to a particle, such as a particle or a magnetic particle. Alternatively, the antibody may be attached to a suitable chemiluminescent moiety. A tracer that may contain symmetric dimethylarginine or a derivative is used to detect symmetric dimethylarginine on the particles. The free dimethylated arginine in the patient sample was determined for a limited amount of dimethylated arginine analyte antibody. The sample, tracer and antibody particles interact to form a complex that can be easily washed with water. After the unbound tracer is removed in the step, the tracer bound to the antibody particles is The amount can be measured by chemiluminescence, where chemiluminescence is expressed in relative light units (RULEs). The amount of chemiluminescence is inversely proportional to the amount of free assay in the patient sample, the concentration of which is known. The analyte values ​​are determined by constructing a standard curve.

[0196] According to some embodiments, biological fluids (e.g., whole blood, serum, plasma, urine, sputum, semen, saliva, fluid, crystalline fluid, cerebral fluid, spinal fluid, amniotic fluid, tissue culture medium, etc., and dilutions thereof) A homogeneous enzyme immunoassay for assaying SDMA is provided. The SDMA in the fluid binds to the antibody binding site via an enzyme (e.g., glucose-6-phosphatase). Nα-acylated-SDMA or Nα-labeled with G6PDH Based on the competition between alkylation and SDMA. Enzyme activity is reduced when bound to antibody. In biological fluids, the concentration of SDMA can be measured by enzyme activity. For example, active G6PDH is nicotinamide adenine dinucleotide (NAD + ) converted to NADH and spectroscopically This results in an absorbance change that can be measured using a photometer. + It only interacts with bacterial enzymes. To avoid interference from endogenous serum G6PDH, the bacteria Leuconostoc mesenteroides enzyme is used in the assay.

[0197] In certain embodiments, the method includes any of the homogeneous enzyme immunoassays described herein. In the immunoassays of the present disclosure, one or more of the reagents are provided in lyophilized form. By way of example only, any of the compounds of formula 1 of the present disclosure, any of the antibodies of the present disclosure, or or a composition comprising both, the disclosure of which is incorporated herein by reference in its entirety for all purposes. As described in U.S. Pat. No. 5,413,732, incorporated herein by reference, lyophilized reagents may be used. The reagent may be provided in the form of a sphere (or "bead"). Briefly, the sphere may be, for example, A uniform droplet measurement (e.g., 2 to 50 mL) of the solution can be made. 1 μL); Distribute uniform measurement droplets into an unstirred cryogenic liquid (e.g., liquid nitrogen) to freeze the droplets. collecting the frozen droplets from the cryogenic liquid; and freeze-drying the frozen droplets. and drying to form a plurality of lyophilized reagent spheres.

[0198] For example, in some embodiments, including those in which one or more reagents are provided in lyophilized form, According to the present disclosure, a centrifugal analyzer including a microfluidic rotor (or "disk") is used to perform the immunoassay of the present disclosure. For example, the immunoassays of the present disclosure can be used to assay plasma or serum by subjecting the plasma or serum to one or more Distributing reagents (e.g., as described above) and plasma or serum into a plurality of individual test wells. The chambers and passages required to separate whole blood into plasma are precisely measured. Radially outward from the metering chamber for delivering a quantity of blood and / or diluent to the separation chamber. The separation chamber contains a radially outwardly directed cell trap. Upon spinning, the cellular components of the whole blood are sequestered in a cell trap. The separated plasma is then The separation and dispensing steps described above are typically performed using a rotating This occurs as a result of the centrifugal force generated by the rotor. The lyophilized reagent spheres described above are particularly suitable for quantifying plasma or diluted plasma. In addition, urine, sputum, semen, saliva, crystalline fluid, cerebral fluid, spinal fluid, amniotic fluid, tissue culture medium, etc. Useful for a variety of other biological fluids, food and industrial chemicals, etc. Details regarding the centrifugal analyzer, including the rotor, are described, for example, in U.S. Pat. No. 5,061, Nos. 381, 5,173,193; 5,122,284 and 5,186,844 No. 6,393,363, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0199] In some embodiments, the immunoassay is pre-mixed between the first and second chambers in the rotor. To deliver a measured volume of liquid (e.g., a biological sample such as whole blood, serum, or plasma) A centrifugal analyzer containing a microfluidic rotor containing siphons for The siphon is located radially inward of the radially innermost point of the fluid in the first chamber. The rotor may include an elbow through which fluid does not flow as the rotor rotates. Once the rotor has stopped, the capillary tube draws in the fluid immediately around the elbow, forming a siphon. When the rotor restarts, the centrifugal force is transferred to the flow in the metering chamber. Drain the remaining fluid in the metering chamber until the fluid level is at the same radial distance as the siphon outlet. The siphon is placed in such a way that the inlet of the siphon on the first chamber is The siphon may be designed to be radially outward of the siphon outlet on the two chambers. The location of the inlet and outlet of the siphon provides certain advantages. For example, the inlet of the siphon is The final position of the meniscus of the fluid in the first chamber after being transferred to the second chamber is always The meniscus is therefore minimized, resulting in different flow rates. Measurement inaccuracies associated with different shapes of meniscus in different bodies are minimized. As is evident, the fluid column in the siphon is stable but easily breaks when the rotor is perturbed. Therefore, all siphons are antistable. If the train of fluid is interrupted, Centrifugal force causes the fluid contained in the siphon to flow to the radially outermost point. For the front siphon, this point is the siphon outlet. The size of the cavity may be increased by up to 100 mm, which may transfer a volume of fluid to the receiving chamber. In a siphon, the radially outermost point of the siphon is the siphon inlet. When the fluid line breaks, the fluid flows back into the first chamber, so the amount that was not measured is This avoids the problem of fluid transfer in any of the methods / immunoassays of the present invention. The present invention relates to a centrifuge rotor including a siphon for transferring a pre-measured volume of liquid. Additional details regarding the analyzer are described in U.S. Pat. No. 7,998,411. , the entire contents of which are incorporated herein by reference for all purposes.

[0200] The symmetric dimethylarginine derivatives, conjugates, antibodies, immunogens and / or Alternatively, other conjugates may be used, including enzyme or fluorescent and / or homogeneous immunoassays. These include, but are not limited to, rapid lateral flow assays, and antibody arrays and as yet undeveloped formats. It is also suitable for many other heterogeneous immunoassays using a variety of detection systems.

[0201] Diverse immunodiagnostics utilizing symmetric dimethylarginine derivatives, conjugates, antibodies and immunogens Although the assays are described herein, the assays can also be modified. Various variations of steps or acts for carrying out the immunoassay are contemplated by the embodiments described herein. Further information relating to assay formats can be found in David Wild(The Immunoassay Handbook, 4th Editorial n Published Date:31st January2013,Elsevi This is explained in the paper "Er ScienCe."

[0202] kit

[0013] Embodiments of the present disclosure further include kits. In some embodiments, the kits include The method is used to determine the amount of at least one symmetrically dimethylated arginine analyte. The kit may include any compound of Formula 1 of the present disclosure, any antibody of the present disclosure, or both. do.

[0203] In certain embodiments, the amount of one or more symmetrically dimethylated arginine analytes in a sample is determined. and a kit for determining whether or not a patient is a patient having a pulmonary function using the antibody, the kit comprising any of the antibodies disclosed herein. To provide guidance for determining the amount of one or more symmetrically dimethylated arginine analytes in a sample. According to some embodiments, the antibodies are capable of binding to symmetric dimethylarginine metabolites (non-limiting examples include and specific for symmetric Nα-acetyl-dimethylarginine (Ac-SDMA) The kit further provides guidance for determining the amount of symmetric dimethylarginine metabolites. In certain embodiments, the kits containing the antibodies of the present disclosure may include any of the compounds of Formula 1 of the present disclosure. In some embodiments, the compound further includes a compound in which Z is a label, e.g., an enzyme, e.g. Glucose-6-phosphate dehydrogenase (G6PDH).

[0204] Also provided is a kit for determining the amount of one or more symmetrically dimethylated arginine analytes in a sample. The kit includes any compound of Formula 1 of the present disclosure, and is adapted to detect one or more Guidelines for using the compounds to determine the amount of a symmetrically dimethylated arginine analyte are provided. In certain embodiments, the compound is provided wherein Z is a label, e.g., an enzyme, e.g. For example, glucose-6-phosphate dehydrogenase (G6PDH). According to some embodiments, the antibody may further comprise any of the following antibodies: A non-limiting example of a metabolite of arginine is symmetric Nα-acetyl-dimethylarginine (Ac-S The kit specifically binds to dimethylarginine (DMA), and determines the amount of symmetric dimethylarginine metabolites. The method may further include guidelines for determining the

[0205] According to some embodiments, the kits of the present disclosure include, for example, symmetric dimethylarginine and Analogues of symmetric Nα-acetyl-dimethylarginine, etc., are symmetrically dimethylated in the sample. The kit is useful for properly performing an assay for the measurement of an arginine analyte. ) symmetric dimethylarginine and compounds of formula 1 described herein (e.g., symmetric (b) an antibody generated to specifically bind to the test compound (a soluble dimethylarginine conjugate); and The method may include determining the amount of symmetrically dimethylated arginine analyte in the sample. In an embodiment, the kit also includes a conjugate of the compound of Formula 1, wherein the conjugate is a target Optionally, the kit also includes a detection label, such as a G6PDH identification. The kit also includes auxiliary reagents for determining the analyte. The antibody of the kit is represented by formula 1 described in the present application. It is an antibody raised against the compound.

[0206] To improve the versatility of immunoassays, the kit reagents are designed with ratios of reagents that are optimized for the method and assay. The same or different compositions may be prepared in liquid or lyophilized form to provide substantial optimization of the effect of the lyophilization. In a specific embodiment, the kit may be provided in a packaged combination in a container for use in the present invention. The disclosure includes the disclosed antibodies, compounds of the present disclosure, or both, and is hereby incorporated by reference in its entirety for all purposes. The freeze-dried method described in U.S. Pat. No. 5,413,732, which is incorporated herein by reference, The reagents provided in the kit are each in a separate container. Alternatively, multiple reagents may be combined in one or more containers depending, for example, on the cross-reactivity and stability of the reagents. In some embodiments, the compounds of formula 1 described herein (e.g., , symmetric dimethylarginine conjugates) are present in lyophilized form. In the method, the antibody is present in a lyophilized form. For example, the compound of formula 1 is In the lyophilized composition (which may further comprise one or more excipients, buffers, stabilizers, etc.), The antibody is then mixed with a second lyophilized composition (which may further comprise an enzyme substrate and one or more excipients, buffers, etc.). The first lyophilized composition and the second lyophilized composition may be present in the presence of a buffer, a stabilizer, etc. The items may be provided in a single kit, for example in a package or container for single use.

[0207] The kit may contain other separately packaged reagents, such as auxiliary enzyme substrates, to perform the assay. The relative amounts of the various reagents in the kit may vary depending on the method / immunoassay (e.g. For example, the reaction occurring during homogeneous enzyme immunoassays can be substantially optimized and the assay can be further The concentrations of reagents may be varied over wide ranges to provide for substantially optimizing the sensitivity of the assay. In some circumstances, one or more of the reagents in the kit may be used to perform a method or assay according to the invention. Lyophilized solutions containing excipients are generally used to provide a reagent having a suitable concentration for The kit may be provided in a dry powder form. The kit may further include instructions for the method of the present invention as described above. obtain.

[0208] The description of certain exemplary embodiments of the kit uses the term "and / or," which means This means that the kit may or may not include each item mentioned. The terminology is used for brevity. Generally, immunoassay kits are used to identify the immunogen of the analyte. one or more antibodies against, e.g., symmetric dimethylarginine, and the analyte, e.g., symmetric di One or more enzyme conjugates corresponding to the enzyme conjugates of the methylarginine derivatives (e.g., label-conjugated Includes the body.

[0209] In certain embodiments, the analyte symmetric dimethylarginine and / or symmetric dimethyl A kit for assaying for a metabolic product of arginine is provided. In combination, the antibody may include: (i) an antibody raised against a compound of formula 1; and ii) Conjugates of derivatives of analytes. Yet another embodiment of the present disclosure is a packaged combination. As an example, symmetric dimethylarginine and / or symmetric dimethylarginine A kit for assaying a metabolite of: (i) an antibody raised against a derivative of the analyte is provided. and (ii) a conjugate of a hapten of the analyte, wherein the hapten is a compound of formula 1. ).

[0210] The compounds, methods and kits disclosed herein are useful for detecting symmetric dimethylarginines by immunoassay. In certain embodiments, the immunoassay is used for early detection and monitoring. The present invention provides an automated test system that can be easily adapted to conventional laboratory equipment in a short time. Thus, an antibody against a symmetrically dimethylated arginine analyte is used to provide the immunoassay. The derivatives and immunogens react with symmetric dimethylarginines through the corresponding antibodies. It was designed to confer specific reactivity against

[0211] The instructions included in the kit may be recorded on a suitable recording medium. For example, the instructions may be on paper or The instructions may be printed on a substrate such as plastic. In the kit, the labeling of the kit container or a component thereof may be present (i.e., on the packaging). In another embodiment, the guidelines include The software can be stored on a suitable computer readable storage medium, e.g. a portable flash drive, It exists as an electronic data file stored on a DVD, CD-ROM, floppy disk, etc. In other embodiments, the actual instructions are not present in the kit, but are obtained from a remote source. The example embodiment provides a means for viewing and listening to the guideline, for example, via the Internet. The kit includes a web address where the instructions and / or Similarly, the means for obtaining the pointers are recorded on a suitable substrate.

[0212] Working Example The following examples are provided to those of ordinary skill in the art to provide a complete disclosure and description of how to make and use the present invention. The present invention is presented for the sole purpose of providing the present invention to those having skill in the art. It is not intended to be limiting in scope. The experiments described below are either all or the only experiments performed. In conjunction with the numbers used (e.g. amounts, temperatures, etc.) Efforts have been made to ensure accuracy, but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is degrees Celsius, pressure is The force is at or near atmospheric pressure. "Average" means the arithmetic mean. Standard abbreviations, e.g. b p, base pair; kb, kilobase; pl, picoliter; s or sec, seconds, minutes; h or hr, time; aa, amino acid; kb, kilobase; bp, base pair; nt, nucleotide ;im, intramuscular;ip, intraperitoneal;sc, subcutaneous; etc.

[0213] The following abbreviations are used in connection with the compounds, conjugates and immunogens: DCM is dichloromethane; DMF is N,N-dimethylformamide; EDTA is ethylenediaminetetraacetate acid; KLH is keyhole limpet hemocyanin; SATA is N-succinimidyl S-acetylglucosamine acetate; TFA is trifluoroacetate; EDCI is 1-ethyl-3(3-dimethyl Aminopropyl)carbodiimide hydrochloride; NHS is N-hydroxysuccinimide mide; DTT is dithioerythritol; G6PDH is glucose-6-phosphate dehydrogenase ; EtOAc is ethyl acetate; BSA is bovine serum albumin; DMO is Dess-Ma rtin periodinane; MeCn is acetonitrile; EA is ethyl acetate; tB oc is tert-butyloxycarbonyl protecting group; TLC is thin layer chromatography; M eOH is methanol; AcOH is acetic acid; PBST is phosphoric acid containing Tween-20. TMB is 3,3´,5,5´-tetramethylbenzidine; PBMC is peripheral blood Blood mononuclear cells.

[0214] General synthesis procedure Provides general known chemical synthesis schemes and conditions useful for synthesizing the disclosed compounds A variety of general references are available (e.g., Smith and Marc h,March´s AdvanCed Organic Chemistry:Rea ctions,Mechanisms,and Structure,Fifth Ed ition,Wiley-InterscienCe,2001; or Vogel,A Textbook of Practical Organic Chemistry ,InClude Qualitative Organic Analysis,Fo Urth Edition, New York: Longman, 1978).

[0215] The compounds described herein may be purified by chromatography, e.g., HPLC, preparative thin layer chromatography, Chromatography, Flash Column Chromatography and Ion Exchange Chromatography The ionic liquid can be purified by any purification protocol known in the art, including ionic liquid. Any suitable stationary phase may be used, including normal phase and reverse phase, as well as resins. In the embodiments of the present invention, the disclosed compounds are chromatographed on silica gel and / or alumina. For example, Introduction to Modern Law iquid Chromatography,2nd Edition,ed.LR Snyder and J.J. Kirkland, John Wiley and Sons , 1979; and Thin Layer Chromatography, ed. E.S. See tahl, Springer-Verlag, New York, 1969.

[0216] During the preparation of the target compound, it is necessary to protect sensitive or reactive groups on any relevant molecule. This may be necessary and / or desirable. ive Groups in Organic Chemistry”, Plenum Press, London and New York1973, T.W. Greene, and PGM Wuts, "Protective Groups in Organic "Synthesis", third edition, Wiley, New York 1999, “The Peptides”;Volume 3(Editors:EG Ross and J. Meienhofer), Academic Press, London. n and New York 1981, in "Methoden der Orga" nischen Chemie”, Houben-Weyl, 4th edition, Vol. 15 / 1, Georg Thieme Verlag,Stuttgard1974,H.-DJ akubke and H. Jescheit,” Aminosauren, Pepti de,Proteine”,Verlag Chemie,Weinheim,Deer field Beach, and Basel 1982, and / or in Joche n Lehmann, "Chemie der Kohlenhydrate:Mono saccharide and derivate",Georg Thieme Ve The conventional procedures for protecting groups disclosed in previous working papers such as Rlag and Stuttgart 1974 The protecting groups can be removed at a suitable subsequent step using methods known in the art. It may be removed by.

[0217] The target compounds are prepared from commercially available starting materials and / or by conventional synthetic methods. The compounds disclosed herein can be synthesized through a variety of different synthetic routes using the starting materials described above. Various examples of synthetic routes that can be used to synthesize the compounds shown are shown in the following reaction schemes: It has been done.

[0218] Example 1 Preparation of SDMA-M hapten 1st stage JPEG2025032213000009.jpg3170

[0219] Of the 12 mL of saturated NaHCO3 (1), (534 mg, 6.0 mmol, 1.0 equiv.) (2) (930 g, 6.0 mmol, 1.0 equiv.) was added to a solution of (2) (930 g, 6.0 mmol, 1.0 equiv.) while maintaining the temperature at 0°C. ) was added in small portions. Upon completion of the addition, the resulting solution was stirred at 0° C. for 1.5 h. LC-M S indicated that all starting material was converted to the target material. The mixture was warmed to room temperature and extracted with EtOAc. The crude product was purified by silica gel column chromatography. (3) (0 The compound was obtained as a clear oil (0.91 g, 91% yield). The structure was: 1 Confirmed by HNMR.

[0220] 2nd stage JPEG2025032213000010.jpg2952

[0221] Dess -Martin oxidation reagent (4.5 g, 10.6 mmol, 2.0 equiv.) and NaHCO3 The mixture was stirred at 25°C for 3 h. TLC showed that most of the starting material was transferred to the target product. The reaction mixture was filtered and the resulting filtrate was concentrated in vacuo. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 0330-04 (0.35 g, 40% yield) was purified as a clear oil. The product (4) was extremely air sensitive and was used immediately in the next step. The structure was confirmed by LC-MS.

[0222] 3 stages TIFF2025032213000011.tif2475

[0223] Of MeOH, (4) (668 mg, 4 mmol, 2.0 equiv.) and symmetric dimethyl aryl A solution of sodium arginine (SDMA) (404 mg, 2 mmol, 1.0 equiv.) was diluted with AcOH (20 0 μL) and NaBH3CN (150 mg, 2.4 mmol, 1.2 equiv.) were added. The resulting mixture was stirred at room temperature for 3 h. LC-MS showed that most of the starting material was the target product. The reaction was quenched with water and immediately run on a Biotage reverse phase column. SDMA-M (326 mg, TFA salt) was purified by chromatography as a clear oil. The compound was obtained in a yield of 35%. The structure is: 1 Confirmed by HNMR. 1 HNMR (400MH z, D20): δ6.81(s, 2H), 3.97(M, 1H), 3.51(m, 2H) , 3.21(m, 2H), 3.08(m, 2H), 2.78(s, 6H), 1.98(m , 2H), 1.64(m, 6H). (See Figure 5).

[0224] Example 2 Preparation of SDMA-SBAP haptens 1st stage TIFF2025032213000012.tif1670

[0225] In DMF (18 mL), (5) (1.4 g, 7.2 mmol, 1.0 equiv.) and N- A solution of hydroxysuccinimide (NHS) (1 g, 8.7 mmol, 1.2 equiv.) was added to E DCI (2.78 g, 14.5 mmol, 2.0 equiv.) was added. The resulting mixture The mixture was stirred at room temperature for 12 hours. LC-MS showed that all starting materials were transformed into the target products. The solution was poured into H2O (100 mL) and extracted with EA (100 mL). The organic phase was washed with H2O and saturated saline. The organic solvent was removed under reduced pressure and dried in vacuum. This gave the desired product (6) (2.0 g, 95.2% yield). The structure is: 1 HNMR Confirmed.

[0226] 2nd stage TIFF2025032213000013.tif2580

[0227] Of the DMF (5 mL), symmetric dimethylarginine (SDMA) (400 mg, 2.0 (6) (630 mg, 2.2 mmol, 1.1 equiv.) was added to a solution of 1.2 mmol of 1.2 sucrose (1.0 equiv.) The reaction mixture was stirred at 60° C. for 12 h. LC-MS showed that all the starting material was Transformation to the target material was demonstrated. The solvent was removed under vacuum. The crude product was purified by M After dissolving in eCN and water (7.5 mL, 1 / 1, v / v), the mixture was subjected to Biotage reverse phase chromatography. The pure product (7) (650 mg, TFA The salt (66.7% yield) is used as a clear oil. The structure is: 1 Confirmed by HNMR .

[0228] 3 stages TIFF2025032213000014.tif2568

[0229] of (7) (650 mg, 1.33 mmol, 1.0 equiv) in MeCN (10 mL). To the solution was added TFA (4 mL). The reaction mixture was stirred at room temperature (RT) for 30 min. LC -MS showed that all the starting material was transformed into the target product (8). The solvent was removed under reduced pressure and dried in vacuo to give the desired product in quantitative yield (as the TFA salt). The structure was confirmed by LC-MS.

[0230] 4th stage TIFF2025032213000015.tif2579

[0231] (8) (258 mg, 0.87 mmol, 1.0 equiv.), N-succinimidyl bromo Acetate (9) (205 mg, 0.87 mmol, 1.0 equiv.) and DIPEA (0. 3 mL, 1.74 mmol, 2.0 equiv) in DMF (2 mL) at 30 °C for 12 h. LC-MS showed that all starting material was transformed into the target product. The solvent was removed under vacuum. The crude product was dissolved in MeCN and water (3.5 mL, 1 / 1, v / v). v) and purified by Biotage reverse phase chromatography (conventional method) to obtain a pure The pure product SDMA-SBAP (175 mg, TFA salt, 39.6% yield) was obtained as a clear aqueous solution. The structure was: 1 This was confirmed by HNMR. 1 HNMR (400MHz, D20)δ4.36-4.32(m, 1H), 3.83(s, 2H), 3.45(t, J =6.4, 2H), 3.18-3.15(m, 2H), 2.76(s, 6H), 2.49 (t, J=6.4, 2H), 1.87-1.74(m, 1H), 1.73-1.58(m , 3H). The reaction scheme is shown in Figure 6.

[0232] Example 3 Preparation of SDMA-M-SH-KLH immunogen Hapten SDMA-M is a compound that is a derivative of KLH in which a thiol group has been chemically introduced. N-Succinimidyl-S-acetylthioacetate was protected React with the primary amine of sulfhydryl-added KLH. Protect with hydroxylamine Deprotection of the thiolated sulfhydryls yielded the desired thiolated SH-KLH (Figure 8). The conjugation of the hapten SDMA-M and SH-KLH produces the immunogen SDMA-M-SH-KLH. (See FIG. 8).

[0233] a) Preparation of SH-KLH Lyophilized KLH (20 mg) was reconstituted in deionized water and diluted in 1M carbonate-bicarbonate buffer. The pH was adjusted to 8.6 using buffer. A solution of KL was prepared (4.67 mg dissolved in 92 μL of DMF at a concentration of 220 mM). Slowly add N-succinimidyl S-acetylthioacetate to the H solution over 4 hours. The reaction mixture was stirred at room temperature while adding the acetone, and then incubated in a cold room (4°C) for an additional 16 hours. Stirred.

[0234] Deacylation to generate sulfhydryls for crosslinking was performed using 200 μL of deacylated 100% PBS. Cetylation solution (12.5 mM, 0.7M hyaluronan in NaH2P04-Na2HPO4 buffer solution) The contents were mixed and the reaction was allowed to stand at room temperature. The reaction was incubated for 2 hours to produce the product SH-KLH (see Figure 8). Finally, EDTA was added to a concentration of 1 mM.

[0235] b) Preparation of SDMA-M-SH-KLH Immunogen Dithiothreitol solution was added to the SH-KLH solution at a total concentration of 1 mM to form disulfide bonds. The pH was adjusted to 7.2 using 1M carbonate-bicarbonate buffer. The -KLH solution was added with mAb dissolved in DMF (0.2 mL, 9.6 mg dissolved in DMF). 146 μl of the imide derivative hapten SDMA-M was slowly added over a period of 4 to 5 hours. The reaction was continued overnight at 4°C. The mixture was cooled to 2-8°C with 4 L of NaH2P04-N a10,000 MWCO Slide in 2HPO4 buffer 12.5 mM, pH 7.0 - Purified by dialysis using A-Lyzer® dialysis cassettes (Pierce) This procedure yielded the immunogen SDMA-M-SH-KLH (see Figure 8).

[0236] Example 4 Preparation of SDMA-SBAP-SH-G6PDH conjugate The hapten SDMA-SBAP, prepared as described in Example 2, was fused to the mutant G6 PDH (U.S. Patent Nos. 6,455,288, 6,090,567, 6,033,8 90) or by chemical reactions as described in Example 3. It was designed for the introduction of thiol-groups into G6PDH as described previously.

[0237] SDMA-SBAP hapten (7 mg, 0.018 mmol) was dissolved in DMF (0.21 mL The solution was stirred at room temperature for 30 minutes. was used as follows:

[0238] Dithiothreitol solution was added to the mutant G6PDH at a concentration of 2 mM to break down disulfide bonds. The cysteine ​​thiol group linked to the sulfhydryl group in the reaction was reduced. The solution (0.9 mg, 1.5 mL) was adjusted to pH 7.2 with 1 M carbonate-bicarbonate buffer and ca. Mix with 340-fold molar excess (0.07 mL) of SDMA-SBAP hapten. The mixture was allowed to gently stir for 16 hours at 4° C. Excess SDMA-SBAP hapten was Sepha in 12.5 mM NaH2PO4-Na2HP04 buffer (pH 7.0) The reaction mixture was passed through a dex G-50 column to separate the enzyme-hapten conjugate. The column fractions containing the enzyme-hapten conjugate were pooled by measuring the absorbance at 280 nm. Ringing gave the conjugate SDMA-SBAP-SH-G6PDH (see FIG. 7).

[0239] Hapten SDMA-SBAP was synthesized using a conjugation procedure similar to that previously described. The conjugate was SDMA-SBAP-SH-BSA (Figure 9). B-cells and monoclonal antibodies were screened by indirect ELISA as described in Example 6. I leaned.

[0240] Example 5 Preparation of polyclonal antibodies against SDMA SDMA-M-SH- prepared in Example 3 emulsified with complete Freund's adjuvant KLH immunogen, 200 μg / rabbit, was intraperitoneally injected into 24 female New Zealand White rabbits. Rabbits were immunized with the same immunization medium emulsified in incomplete Freund's adjuvant. The initial immunization was with 100 μg of the antigen per rabbit, followed by boosts every 4 weeks. After 2 days, a bleed containing polyclonal antibody was taken from the middle ear artery of each rabbit. Antisera from these bleeds containing SH-KLH antibodies were identified using the enzyme conjugate SDMA-SBAP- Maximum antibody inhibition of SH-G6PDH and symmetric dimethylarginine as described in Examples 7 and 8 The results were assessed in a homogeneous assay format (Figure 10) by measuring the regulation in the presence of nin.

[0241] In these experiments, rabbits 21342 and 26494 were selected for SDMA-M-SH-KL Isolate PBMC from rabbits immunized with H immunogen as a source of B-cells for replication (Example 6) 24 rabbits immunized with SDMA-M-SH-KLH were polyclonal. The initial screening results of the central antisera are shown in Table 1 below.

[0242] Table 1 JPEG2025032213000016.jpg4081

[0243] Example 6 Preparation of rabbit monoclonal antibodies Rabbit recombinant monoclonal antibodies efficiently amplify the natural antibody repertoire of immunized rabbits. The technique was developed to efficiently sample B-cells using a single B-cell screening method. The present application provides a method for the preparation of a monoclonal antibody against symmetric dimethylarginine, comprising: It can be applied to produce

[0244] Rabbit peripheral blood mononuclear cells (PBMC´) from immunized animals #21342 and #26494 s) were used as a source of B cells. Approximately 100% of each rabbit was isolated using conventional density gradient centrifugation. PBMCs were isolated from 40 mL of whole blood. The PBMCs were diluted in PBS and theoretically 1000 cells / well were cultured. Single cells were then distributed onto 40 96-well plates and cultured on the same day.

[0245] The supernatants generated in each well were indirectly incubated with the SDMA-SBAP-SH-BSA antigen. Tested by ELISA (Figure 9). Forty 96-well microtiter plates 0.1 μg / well SDMA-SBAP-SH- The plate was coated with BSA and stored at 4°C overnight. After removing the plate, it was shaken at room temperature for 1 hour. The plates were blocked with 3% nonfat dry milk in PBST while shaking. After removing the blocking solution, the plates were Rinse with PBST. 25 μL of PBST was added to each well, and then all wells were rinsed with 2 Add 5 μL of cell supernatant and incubate at 37°C for 1 hour. The plate was washed five times with PBST for a total wash time of 30 min. 100 μL / well secondary antibody 1:10,000 (v / v) Goat anti-Rabbit IgGFc-H RP conjugate was added and the plate was incubated for 1 hour at 37°C with constant shaking. The plate was washed 5 times with PBST for a total wash time of 30 min. 50 uL of TMB substrate was added. 1 / well of plate. Incubate in the dark for 5 minutes to allow color development. After that, 50 μL of 1N HCl was added to terminate the reaction. The color development is read through a colorimeter and the data is transferred to a computer for assay. The supernatant (color produced in the wells) bound to the SDMA-M-SH-BSA conjugate was A total of 32 cells were selected for cloning and expression.

[0246] For each ELISA well with antigen-specific antibody, the corresponding PBMC culture well was incubated with mRNA A was isolated and cDNA was separately synthesized from the variable regions of rabbit genes, IgH and IgK. After two rounds of PCR for amplification, the cDNA was divided into two parts, one for the heavy chain constant IgG region and the other for the Seamlessly ligated into a separate mammalian expression vector carrying the light chain constant IgK region .

[0247] The ligation mixture was transformed into E. coli to select for the correct expression construct and cultured. The plasmids were isolated by co-transfection of the expression constructs into HEK293 cells. The infected cells were cultured for 2 days to secrete the recombinant antibody. The binding properties were determined by indirect ELISA against the SDMA-SBAP-BSA antigen as described above. The selected clones were evaluated by homogeneous enzyme synthesis as described in Examples 7 and 8. The selected rabbit monoclonal antibodies were further evaluated in a primary immunoassay format. The antibody is subjected to DNA sequencing and translated into conventional code to encode all the heavy and The protein sequence data for the monoclonal antibody VL1 chain variable region sequences are provided in the following table. The following summarizes the development steps to produce a cloned antibody.

[0248] Table 2 JPEG2025032213000017.jpg4683

[0249] Example 7 Preparation of SDMA standards and calibration protocol Symmetric dimethylarginine di(p-hydroxyazobenzene-p´-sulfonate) Salts (Sigma) were dissolved in DMF:HO (1:1) and 4 μg / mL in synthetic matrix. The SDMA stock solution was diluted to a final concentration of 0, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500 of synthetic matrices to achieve levels of 50, 100, 150, and 200 μg / dL. The synthetic matrix was prepared by mixing HEPES (6.5 mM, pH 6.7) and EDT. A (0.25 mM), from a proteinaceous solution buffered with surfactants, antifoams and preservatives The standards were verified by LC / MS (see Figure 17).

[0250] Quantify symmetrically dimethylated arginines in samples using a six-point calibration curve. Each calibrator level was measured in duplicate using a Beckman Coulter AU4 A calibration curve was generated on the 80 automated clinical chemistry analyzer (see Examples 7 and 8). .

[0251] Nα-acetyl-dimethylarginine (Ac-SDMA) calibrator was used as previously described. Nα-acetyl-dimethylarginine was prepared by a method similar to that used in Synthesized by Hesis Med Chem (Australia).

[0252] Example 8 Reagents and Assays The symmetric dimethylarginine antibody and enzyme conjugates can be used to detect symmetric dimethylarginine in a sample according to the present disclosure. Antibodies that are advantageously used in homogeneous assay formats to detect carboxylated arginine analytes include Known features such as binding inhibition, conjugate control, calibration, cross-reactivity and small spike recovery For this purpose, antibodies (polyclonal rabbit #21 342, rabbit #26494, or rabbit #27420, or cloned anti- Body 1H4 / 1K4, 8H1 / 8K3, 18H1 / 18K2, 1H2 / 1K4, 3H1 / 3 K3 or 5H1 / 5K1) is added to the antibody diluent to prepare the antibody reagent. As described above, the antibody, buffer, salt, stabilizer, preservative, NAD + and Gluco The enzyme conjugate SDMA-SBAP-SH-G6PDH is The enzyme conjugate reagent is prepared by adding the conjugate, buffer, Contains stabilizers, salts and preservatives.

[0253] Clinical chemistry analyzer useful for evaluating antibodies and enzyme conjugates in a homogeneous enzyme immunoassay format. Beckman Coulter AU480 (Beckman Coulter, The Beckman AU480 is used in medical laboratories to measure urine, cerebrospinal fluid, oral fluid, and An automated biochemistry system used to process biological fluid specimens such as cavities, plasma, and serum. It is a spectrophotometer analyzer. The analyzer maintains a constant temperature, pipets samples, mixes reagents, It is possible to accurately measure absorbance and reaction time.

[0254] Homogeneous enzyme immunoassays, as previously mentioned, are ready-to-use liquid two-reagent assays. Typically, 2-15 μL of symmetrically dimethylated arginine analyte is used. The sample was incubated with 75-150 μL of antibody reagent, followed by 50-100 μL of enzyme conjugate reagent. Add the medicine.

[0255] The assay is a homogeneous enzyme immunoassay technique used to analyze SDMA in biological fluids. The assay involves binding of the enzyme glucose-6-phosphate to the antibody binding site on the sample and SDMA. Nα-acylated SDMA or Nα-alkylated SDMA labeled with dehydrogenase (G6PDH) The reaction is based on the competition between ELISA and SDMA. Binding to the antibody reduces the enzyme activity, so the sample The concentration of drugs can be measured in the blood by measuring the enzyme activity. Nucleotides (NAD + ) was converted to NADH and measured by a spectrophotometer at 340 nm. Endogenous serum G6PDH is converted to coenzyme NAD + was used in the assay. The enzyme and mechanism of the bacteria (Leuconostoc mesenteroides) The change in absorbance at 340 nm can be measured by a spectrophotometer. , is proportional to the activity of the enzyme conjugate, which in turn is related to the analyte concentration (see FIG. 10).

[0256] Example 9 Antibodies and Calibration with Symmetric Dimethylarginine Analytes Symmetrically Dimethylated Arginine Analyte Antibody and Enzyme Conjugate (SDMA-SBAP-SH- G6PDH) in a homogeneous assay format, as described in Example 7. A calibration curve was generated using antibody standards. The antibody reagents were as described in Example 8. Symmetrically dimethylated arginine analyte polyclonal antibody rabbit #2134 2. Rabbit #26494, rabbit #27420 and cloned antibody 1H4 / 1K 4, 8H1 / 8K3, 18H1 / 18K2, 1H2 / 1K4, 3H1 / 3K3 or 5H The enzyme conjugate SDMA-SBAP-SH-G6PDH was used. Conjugate reagents were prepared using a Beckman AU4 as described in Examples 7 and 8. A 6-point calibration curve was generated on 80 clinical chemistry analyzers. The ablation curves are shown in the table below, and the dose-response curves are shown in Figures 11 and 12. , polyclonal antibodies rabbit #21342, rabbit #26494, rabbit #27420 and and the cloned monoclonal antibodies 1H4 / 1K4, 8H1 / 8K3, and 18H1 / 1 8K2, 1H2 / 1K4, 3H1 / 3K3 or 5H1 / 5K etc. show dose-response relationship It was demonstrated that the antibody binds in response to symmetric dimethylarginine.

[0257] Table 3 JPEG2025032213000018.jpg6282

[0258] Example 10 Nα-Acetyl-Symmetric Dimethylarginine An Antibodies and calibration with alytes Symmetrically Dimethylated Arginine Analyte Antibody and Enzyme Conjugate (SDMA-SBAP-SH- G6PDH) in a homogeneous assay format as described in Example 7. A calibration curve was generated using standards prepared with dimethylarginine. The antibody reagent was prepared as described in Example 8. In Example 8, symmetrically dimethylated arginine Analyte Polyclonal Antibody Rabbit #21342, Rabbit #26494, Rabbit #2742 0 and cloned antibodies 1H4 / 1K4, 8H1 / 8K3, 18H1 / 18K2, 1H2 / 1K4, 3H1 / 3H1 / 5, or enzyme-conjugated SDMA-SBAP-SH-G Conjugate reagents were prepared using 6PDH. Beckman et al. as described in Example 8. A 6-point calibration curve was generated on an AU480 clinical chemistry analyzer. The calibration curve is shown in the table below, and the dose-response curves are shown in Figures 13 and 14. The study was performed using polyclonal antibodies rabbit #21342, rabbit #26494, and rabbit #2742. 0 and cloned monoclonal antibodies 1H4 / 1K4, 8H1 / 8K3, 18H 1 / 18K2, 1H2 / 1K4, 3H1 / 3K3 or 5H1 / 5K, etc. have a dose-response relationship The researchers demonstrated that they had antibodies that reacted with Nα-symmetric dimethylarginine. Ta.

[0259] Table 4 JPEG2025032213000019.jpg6379

[0260] Example 11 Small amount added recovery rate A known amount of symmetrically methylated arginine analytical stock solution (4 pg / mL) was prepared as described in Example 7. Spiked into synthetic matrix as described to obtain 0, 15, 50, 100, 1 These samples achieved concentrations of 50, 200 pg / dL. To confirm the recovery rate of a small spike on the Beckman AU480, Quantification was performed in triplicate by enzyme immunoassay. Samples were run using a 6-point calibration. The concentration of the analyte in the samples was quantified using a set of standards prepared separately to generate a linear curve. Calibration using standards prepared with the analyte to be quantified in The curve was generated. The enzyme conjugate reagent contained the conjugate SDMA-SBAP-SH-G6PDH. The antibody reagents were rabbit #21342, rabbit #26494, rabbit #27420, and Somatic clones 1H4 / 1K4, 8H1 / 8K3, 18H1 / 18K2, / 1K4, 3H1 / 3K3 or 5H1 / 5K1. Symmetric methyl groups recovered in small spike recovery experiments The arginine analyte concentrations were plotted relative to the known concentrations (Figures 15-18). Polyclonal antibodies rabbit #21342, rabbit #26494, rabbit #27420 and cloned antibodies 1H4 / 1K4, 8H1 / 8K3, 18H1 / 18K2, 1 H2 / 1K4, 3H1 / 3K3 or 5H1 / 5K1 are symmetric dimethylarginine and N They demonstrated that they had an antibody response to α-acetyl-SDMA.

[0261] In another experiment, the same samples were analyzed by homogeneous enzyme immunoassay (see Example 8) and LC-MS- The internal standard, deuterated asymmetric dimethylarginine, was quantified in duplicate by LCMS. -MS-MS method was used. Antibody reagent clone 3H1 / 3K3 and enzyme conjugate reagent enzyme A homogeneous enzyme immunoassay was prepared using the conjugate SDMA-SBAP-SH-G6PDH. Results show that homogeneous enzyme immunoassay quantifies SDMA levels in agreement with LC-MS-MS. This indicates that.

[0262] In other experiments, known amounts of SDMA-Gly-Gly dipeptide were added as described in Example 8. To confirm the concentration (recovery rate) of a small amount of sample added to the Beckman AU480, The samples were analyzed by homogeneous enzyme immunoassay. A conversion curve was generated. The analyte is the analyte to be quantified in the sample under test. The conjugate reagent contains the conjugate SDMA-SBAP-SH-G6PDH, and the antibody reagent contains the antibody cloning The samples were prepared containing either 8H1 / 8K3 or 3H1 / 3K3. The detected SDMA-Gly-Gly dipeptide analyte concentrations were plotted against the known concentrations. (Figure 19).

[0263] Example 12 For symmetric dimethylarginine and Nα-acetyl-symmetric dimethylarginine analytes Antibodies and calibration Polyclonal from rabbit #27410 and SDMA-SBAP-SH-G6PDH The antibody was used to prepare the reagent, and symmetrically dimethylated arginines and It was used in a homogeneous assay format to detect Nα-Ac-SDMA analytes. Nα-Ac-SDMA samples were prepared as described in Example 7 and were prepared using SDMA. Quantification was performed from a calibration curve generated from standard samples. A small spike recovery experiment was performed as described in Example 11 using the polyclonal antibody Figure 20 shows that rabbit polyclonal antibody #27410 inhibited SDMA and Nα-Ac-S. The rabbit polyclonal antibody #27410 shows substantial binding to both DMA and DMA. The maximum inhibition (%) and regulation (%) for are given in Table 1 above.

[0264] The foregoing merely illustrates the principles of the presently disclosed embodiments. Any person skilled in the art will be entitled to practice the principles of the embodiments expressly described or not illustrated herein and to use the teachings and It should be understood that a variety of sequences falling within the range can be devised. In addition, all examples and conditional phrases etc. cited in this specification are intended to be illustrative and not restrictive. The purpose of the present invention is to facilitate understanding of the principles of the embodiments and the concept of the present invention's contribution to technological advancement. However, the present invention should not be construed as being limited to such specifically recited examples and conditions. Moreover, reference should be made to principles, aspects, and embodiments of the present disclosure as well as to specific examples thereof. All statements herein encompass all structural and functional equivalents thereof. The term "commercially available products" refers to products that have the same functionality regardless of structure, both currently known equivalents and future equivalents. It includes all the elements that have been developed to fulfill all of these functions.

Claims

[Claim 1] The compound of formula 1, wherein the compound of formula 1 is: During the ceremony: R １ HA-Y-Z; Y is a linking group; and Z is a compound represented by a labeling enzyme.

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