Methods and compositions for detecting and diagnosing renal and periodontal disease

Abstract

To provide methods for detecting renal disease at earlier stages so that disease progression can be halted.SOLUTION: The methods enable detection of grade 1, 2 or 3 acute kidney injury (AKI) in a human before creatinine assays can detect 1, 2 or 3 AKI in the human. Specifically, the invention provides methods for diagnosis by comparison of the amounts of cystatin B polypeptides between a sample and a control sample or control standard, where higher levels of cystatin B polypeptides in the sample than in the control sample or control standard is an indication of renal disease.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Priority This application is filed on March 2, 2016, which is incorporated herein by reference in its entirety. The benefit of US Patent Application No. 62 / 302,299 is claimed.

[0002] Sequence Listing This document is an electronic text file of the sequence listing submitted electronically via EFS-Web. This text file is incorporated by reference. nal.txt" is 7.10KB and was created on March 2, 2017. [Background technology]

[0003] Kidney disease is accompanied by increased water consumption, frequent urination, loss of appetite, weight loss, and muscle wasting. By the time clinical symptoms of kidney disease appear, irreversible kidney damage has occurred. Early detection leads to early treatment. Current treatments include dialysis, phosphorus and phosphate This includes a low-protein diet. , early detection is crucial.

[0004] Renal disease progression in mammals is classified into five levels: Renal disease in mammals, e.g., dogs Current methods for detecting urinary urinary protein / creatinine levels are renal ultrasound biopsy and Biopsy is invasive, and creatinine measurement is unlikely to result in significant tissue damage. It is only accurate until stage 3 of renal failure occurs after the disease progression. What is needed is a method to detect early-stage kidney disease so that renal disease can be stopped. Summary of the Invention

[0005] In one embodiment, a method for detecting cystatin B ("Cys B") polypeptide in a sample is The method comprises combining a sample with SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 2 3, 24, 25, 26, or 27 One or more antibodies may be used in combination with a cystatin B polypeptide and one ... , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, or 27. contacting the polypeptide under conditions suitable for the formation of a complex with one or more antibodies that specifically bind the polypeptide; Cystatin B polypeptide and a polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, or 27. Complexes with one or more antibodies that specifically bind the polypeptide are detected.

[0006] In another embodiment, a method of diagnosing kidney disease in a subject is provided, the method comprising: determining the amount of cystatin B polypeptide in a sample from the elephant, The amount of polypeptides is SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 2 6, or 27, or more polypeptides that specifically bind to The method includes determining the presence or absence of cystatin B polynucleotides in a sample using one or more antibodies against cystatin B polynucleotides. The amount of peptide is compared to a control sample or a control standard to determine the level of cystatin B polypeptide in the sample. A higher level compared to a control sample or control standard is indicative of kidney disease.

[0007] In yet another embodiment, a method of treating a disease condition in a subject is provided. The amount of cystatin B polypeptide in a sample from a subject is determined by comparing the amount of cystatin B polypeptide with the amount of SEQ ID NOs: 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 2 1 consisting of (or including) 0, 21, 22, 23, 24, 25, 26, or 27 Assays for determining the presence or absence of a polypeptide using one or more antibodies that specifically bind to the polypeptide and requesting a test that provides a result of determining whether the sample contains cystatin B polypeptide. If the amount is higher than a control sample or control standard for the disease state, the subject is at risk for renal impairment. The disease state is acute kidney injury in patients with chronic kidney disease or Active renal impairment, acute kidney injury, active renal impairment, progressive chronic kidney disease, periodontal disease, upper urinary tract infection or a combination thereof.

[0008] In yet another embodiment, a method of diagnosing periodontal disease in a subject is provided. The method comprises determining the amount of cystatin B polypeptide in a sample from a subject, The amount of Tatin B polypeptide is SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, Specific for one or more polypeptides consisting of (or including) 25, 26, or 27 The method includes determining the presence or absence of cystatin in a sample using one or more antibodies that bind to the cystatin. The amount of cystatin B polypeptide in the sample is compared with a control sample or control standard, and the amount of cystatin B polypeptide in the sample is determined. Higher peptide levels compared to control samples or control standards are indicative of periodontal disease in the subject. This becomes:

[0009] In another embodiment, a method for distinguishing between an upper and a lower urinary tract infection is provided. The method comprises determining the amount of cystatin B polypeptide in a sample from a subject. , the amount of cystatin B polypeptide is SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, to one or more polypeptides consisting of (or including) 24, 25, 26, or 27 The method includes determining the specificity of a specific antibody that specifically binds to a specific protein in a sample. The amount of cystatin B polypeptide in the sample is compared with a control sample or a control standard. and (b) determining whether the polypeptide level is elevated in a subject relative to a control sample or a control standard; It is a sign of infection.

[0010] In another embodiment, a method for distinguishing between acute kidney injury and a lower urinary tract infection is provided. The method comprises (a) determining the amount of cystatin B polypeptide in a sample from a subject. and the amount of cystatin B polypeptide is SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 1, which specifically binds to one or more polypeptides consisting of 24, 25, 26, or 27 (b) determining the presence or absence of cystatin B polynucleotides in the sample, the presence or absence of cystatin B polynucleotides being determined using one or more antibodies; and comparing the amount of cystatin B peptide in the sample with a control sample or a control standard. A higher polypeptide level compared to a control sample or control standard is indicative of acute kidney injury in a subject. This includes comparing, which is a sign of harm.

[0011] In one embodiment, the cause of the kidney disease may be chronic kidney disease, acute kidney injury, or a bacterial infection. In one embodiment, the kidney disease, chronic kidney disease, or acute kidney injury is not caused by cancer. Bacterial infections are caused by Anaplasma species, Ehrlichia species, Leptospira species, Escherichia species, or or Borrelia species. The amount of cystatin B polypeptide may be determined by measuring the amount of cystatin B polypeptide. and sequence numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 2 7. One or more polypeptides that specifically bind to (or include) The determination can be made by detecting complexes with the antibody.

[0012] In one embodiment, a cystatin B polypeptide and a polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, or 27. The complex with one or more antibodies that specifically bind to the polypeptide is contacted with an indicator reagent prior to detection. The one or more antibodies may be selected from SEQ ID NOs: 5, 6, 7, 11, or 13. The polypeptide may be capable of specifically binding to one or more polypeptides consisting of (or including):

[0013] In one embodiment, the subject can be a non-human animal and the sample can be serum, plasma, urine, saliva, It can be plaque, crevicular fluid, gingival biopsy, or tongue swab.

[0014] In one embodiment, the amount of cystatin B polypeptide or cystatin B polypeptide is Immunoassay, competitive immunoassay, sandwich immunoassay, enzyme-linked immunosorbent assay (E Lipoprotein Immunoassay (LIA), radioimmunoassay (RIA), immunoturbidimetry, particle-enhanced immunoturbidimetry, or wet immunoassay This can be determined by stain blot assay.

[0015] In yet another embodiment, SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 2 Specific to one or more polypeptides consisting of (or including) 5, 26, or 27 An isolated antibody that binds to the antibody is provided. The isolated antibody may be lyophilized and is conjugated to a label. or may be immobilized on a solid support, and , 9, 10, 11, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 2 Specific to one or more polypeptides consisting of (or including) 5, 26, or 27 may be bound to or immobilized on a solid support and are SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, 25, 26, or 27. It may be specifically bound to

[0016] In one embodiment, the antibody may be immobilized on a solid support and may also be associated with one or more labels. It may be bonded.

[0017] In yet another embodiment, the present invention relates to a method for treating kidney disease, acute kidney injury in patients with chronic kidney disease, or active Renal impairment, active renal impairment, progressive chronic kidney disease, acute kidney injury, upper urinary tract infection, or periodontal disease A kit for diagnosing a disease is provided, which kit comprises a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25, 26, or 27 One or more antibodies that specifically bind to the above polypeptides, and the one or more antibodies are targets. one or more reagents that promote binding to cystatin B polypeptides present in the sample It can include.

[0018] In another embodiment, SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27. The polypeptide may be lyophilized, conjugated to a label, or immobilized on a solid support. and SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 2 6, or 27, or more polypeptides that specifically bind to The antibody may be specifically bound to one or more antibodies.

[0019] In yet another embodiment, the present invention relates to a method for treating renal disease in a mammalian subject, e.g., a human, canine, or feline subject. A method for diagnosing a disease is provided, the method comprising: (a) collecting a sample (e.g., urine, blood, plasma, blood) from a patient; (b) determining the amount of cystatin B polypeptide in the sample (supernatant, cells, tissue); and and comparing the amount of cystatin B polypeptide in the sample to a control sample or control standard. Cystatin B polypeptide levels in the sample are higher than in the control sample or control standard. The amount of Cystatin B polypeptide is indicative of a disease. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 1 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 (or The method is determined using an isolated antibody that specifically binds to one or more polypeptides (including Renal disease in mammals can be, for example, acute kidney injury in patients with chronic kidney disease. or active renal impairment, chronic kidney disease, progressive chronic kidney disease, acute kidney injury, active renal impairment, In one embodiment, the kidney disease may be a urinary tract infection, or a bacterial infection of the kidney. It's not kidney cancer.

[0020] In one embodiment, the immunoconjugate comprises: (i) SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27. and (ii) one or more isolated antibodies that specifically bind to the one or more isolated antibodies. Immunoconjugates are provided that comprise one or more polypeptides. For example, SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, An immune complex is a complex formed between an antigen (e.g., a polypeptide) and an antibody. The immune complex may be immobilized on a solid support.

[0021] From the following more detailed description of certain preferred embodiments and claims, Various embodiments will become apparent. [Brief explanation of the drawings]

[0022] [Figure 1] FIG. 1 shows normal tubular epithelium, cell destruction, and cell death in epithelial cells of the proximal tubule. [Figure 2] FIG. 2 shows a Western blot analysis of cystatin B in the presence of cystatin C protein. [Figure 3] FIG. 3 shows the standard curve obtained in the Cys B ELISA assay using native canine cystatin B (MDCK lysate). [Figure 4] FIG. 4 shows Cys B ELISA analysis of serum and urine from the canine gentamicin model. [Figure 5] FIG. 5 shows Cys B ELISA analysis of urine from dogs presented to the clinic with active inflammation- or ischemia-induced renal damage. [Figure 6] FIG. 6 shows, in panels A and B, reference ranges for dogs and cats. [Figure 7] FIG. 7 shows, in panels A and B, that urinary and serum cystatin B levels were significantly increased in patients diagnosed with AKI compared to healthy dogs and CKD patients. [Figure 8] FIG. 8 shows cystatin B levels in healthy patients and in patients with lower urinary tract infections. [Figure 9] FIG. 9 shows detection of Cys B polypeptide in canine AKI samples, human CKD samples, and canine negative control samples. [Figure 10] Figure 10 shows Cys B levels in serum from healthy dogs. These and other objects and features will be better understood from the following detailed description taken in conjunction with the drawings. DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention will be described in more detail below, but the examples given herein are given by way of illustration only. It is intended only as a general rule, as many modifications and variations will be apparent to those skilled in the art. As used throughout this description and the following claims, "a (one) "one of," "an," and "the" are used depending on the context. Unless expressly stated otherwise by the SEC, "about" with a numerical value includes plural referents. The term means that the value can vary by 5% above or below. For example, a value of about 100 is 95- 105 (i.e., any value between 95 and 105).

[0024] Generally, the terms used herein are used within the context of the compositions and methods described herein. Each term has its ordinary meaning in the art in the specific context in which it is used. The terminology in this section is provided below to provide additional guidance to the practitioner regarding the composition and method description. It is more specifically defined below.

[0025] The compositions and methods described herein are useful for treating several diseases and conditions (e.g., kidney disease, chronic Acute kidney injury or active kidney failure in patients with renal disease, progressive chronic kidney disease, acute kidney injury, active kidney disease Prediction, diagnosis, and treatment of chronic kidney disease (including idiopathic kidney disease, active kidney disease, upper urinary tract infections, and periodontal disease) It can be used to monitor the progression of kidney disease. any disease that results in (2) a decrease in the amount of blood in the kidney, or (3) physical damage to the kidney, or both In one embodiment, kidney disease does not include cancer, and does not include kidney cancer. Markers of cancer, including kidney cancer, are important predictors of acute kidney injury or kidney failure in patients with chronic kidney disease. chronic kidney disease, progressive chronic kidney disease, active kidney injury, acute kidney injury, upper urinary tract infection or different from markers of periodontal disease. Renal impairment, progressive chronic kidney disease, active renal impairment, acute kidney injury, upper urinary tract infection, or periodontal disease Diseases do not include cancer, including kidney cancer.

[0026] Chronic kidney disease (CKD) is a condition characterized by the gradual loss of kidney function over time. CKD is a chronic kidney disease. CDKs are also known to be involved in kidney cancer, renal cell carcinoma, bladder cancer, and other cancers. As CKD worsens, waste products accumulate in the blood at high levels, causing high blood pressure, Anemia, weakened bones, malnutrition, and nerve damage can occur. CKD is a heart and vascular disease. CKD increases the risk of kidney failure and ultimately leads to kidney failure. , and other abnormalities. Early detection and treatment can often prevent the disease from worsening.

[0027] by the International Renal Interest Society The established stages of CKD in dogs are shown in Table 1.

[0028] [Table 1]

[0029] [Table 2]

[0030] Kidney disease in humans is staged according to the glomerular filtration rate (GFR). Calculate GFR using the formula below, using age, race, sex, and serum creatinine. The five stages of CKD and GFR for each stage are shown below.

[0031] Stage 1: Normal or high GFR (GFR>90mL / min) Stage 2 Mild CKD (GFR = 60-89 mL / min) Stage 3A Moderate CKD (GFR=45-59mL / min) Stage 3B Moderate CKD (GFR = 30-44 mL / min) Stage 4 Severe CKD (GFR = 15-29 mL / min) Stage 5 End-stage CKD (GFR<15mL / min) The methods described herein are directed to treating stage 1, 2, 3A, 3B, 4, or 5 CKD in humans. In one embodiment, the method can detect acute kidney injury or active kidney injury. The method is to determine if the creatinine assay or GFR value is in stage 1, 2, 3A, 3B, 4, or Before acute kidney injury or active kidney damage in CKD can be detected, the steroid hormone is required in humans. Acute kidney injury or active kidney damage in CKD stages 1, 2, 3A, 3B, 4, or 5 can be detected.

[0032] Chronic kidney disease or renal disease ase) can be a glomerular or tubular disease.

[0033] Acute kidney injury (AKI) is defined as a sudden or rapid decline in kidney filtration function. AKI can be caused by chronic kidney disease (CKD), kidney failure requiring dialysis (end-stage renal disease), or kidney disease. Even mild AKI or complete recovery from AKI may not be sufficient to AKI can be caused by any of a number of short-term and long-term health problems. Damage to kidney tissue due to reduced blood flow to the kidneys caused by hypotension and dehydration, and exposure to substances harmful to the kidneys exposure to inflammatory processes in the kidney, systemic diseases, crush injuries, antibiotics, sepsis, or urinary tract infections AKI can be caused by metabolic acidosis, high potassium levels, uremia, This can lead to changes in fluid balance and effects on other organ systems. I does not include cancer, including kidney cancer. International Rena AKI in cats and dogs established by the International Interest Society The grades are shown in Table 3.

[0034] [Table 3]

[0035] In one embodiment, the methods described herein are directed to treating grade 1, 2, 3, 4, or 5 AK. In one embodiment, the methods described herein can detect creatinine adenosine 1 (creatinine adenosine 1). Before the assay could detect grade 1, 2, 3, 4, or 5 AKI, , 3, 4, or 5 AKI can be detected.

[0036] AKI in humans can be staged as follows: [Table 4]

[0037] In one embodiment, the method includes detecting stage 1, 2, or 3 AKI in a human. In one embodiment, the method comprises determining whether a creatinine assay is suitable for human gray matter. Before grade 1, 2, or 3 AKI can be detected in humans, It is possible to detect AKI.

[0038] In one embodiment, the cause of the kidney disease, CKD, or AKI is a bacterial infection. So, the cause of bacterial infection is Anaplasma species, Ehrlichia species, Leptospira species, Escherichia A species or Borrelia species.

[0039] Active renal impairment is defined as ongoing or progressive renal damage, abnormalities, or pathology. Active renal injury results in cumulative damage to the kidney.

[0040] Polypeptides Cystatins A and B are family 1 members of the cystatin superfamily. These proteins are relatively small, measuring 11 kDa. The proteins are monomeric and approximately 11 kDa in size. They are not glycosylated. It does not have the disulfide bridges found in other members of the cystatin superfamily. Because they lack null sequences, they are generally intracellular proteins restricted to certain cells. Chieng and Chaudhuri, J Health Care Poor Und Served 2010, 21(1 Suppl):51. Some amount of cystatin B is present in extracellular fluid and has been purified from human urine. See n et al., J Biol Chem 1986, 261:11282-11289. Statin B is a member of the cathepsin family of lysosomal cysteine ​​proteinases. , specifically inhibiting cathepsins B, H, and L. Green et al. Biochem J 1984 218:939;D'Amico et al., J Transl. Med 2014, 12:350; Jarvinen and Rinne, Biochim See Biophys Acta 1982, 708:210-217.

[0041] Cystatin B polypeptides are described in detail in Example 1 and include: Included: QVKAQLEERENKKYTTFKAVTFRSQVVAGTPYFIKVQVDD DEFVHLRVFQSLPHENKPLALSSYQTNKAKHDELAYF(sequence Number 1) MMCGAPSASQPATADTQAIAD (SEQ ID NO: 2) MMCGAPSASQPATADTQAIADQVKAQLEERENKKYTTFKA VTFRSQVVAGTXYFIKVQVDDDEFVHLRVFQSLPHENKPL ALSSYQTNKAKHDELAYF (SEQ ID NO: 3) (where X is any amino acid) or X can be P or N) QTNKAKHDELAYF (SEQ ID NO: 4) Cystatin BC terminal "peptide 9" CGAPSASQPATADTQAIA (SEQ ID NO: 5) Cystatin B N-terminal "peptide 3-20" CGAPSASQ (SEQ ID NO: 6) Cystatin B N-terminus "peptide 3-10" CAIADQVKA (SEQ ID NO: 7) Cystatin B N-terminus "peptide 18-25" FQSLPHENKPLALSS (SEQ ID NO: 8) Cystatin B "peptide 2" SQVVAGTPYFIKVQVDDD (SEQ ID NO: 9) Cystatin B "Peptide 1" KHDELAYF (SEQ ID NO: 10) MMCGAPSASQPATADTQAIADQVKAQLEE (SEQ ID NO: 11) AIADQVKA (SEQ ID NO: 12) SQVVAGTNYFIKVQVDDD (SEQ ID NO: 13)

[0042] In one embodiment, a purified polypeptide comprising SEQ ID NO: 1-27 or a fragment thereof is provided. The polypeptide fragments of SEQ ID NOs: 1-27 are provided in the range of about 95, 90, 80, 70, 60, 50, 40, 35, 30, 25, 20, 15, 10 (or any value between about 10 and about 95) In one embodiment, the polypeptide fragment may consist of less than Approximately 10, 15, 20, 25, 30, 35, 40, 50, 60, 70 of SEQ ID NOs: 1-27 In one embodiment, the polypeptide comprises more than 80, 90, or 95 consecutive amino acids. The peptide or fragment thereof is non-naturally occurring.

[0043] SEQ ID NOs: 1-2, 4-13, and 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 polypeptides from the full-length Cys B polypeptide The fact that small polypeptides are also small is important because they are not as large as full-length polypeptides. This is because they may have higher specificity and / or sensitivity than peptide assays. Small polypeptides of this type may be less expensive to produce, and full-length polypeptides Furthermore, the level of low molecular weight fragments and low molecular weight fragments present in the sample can be determined. The level can be indicative of a disease state. Fragmented polypeptides (i.e., less than full-length) Increased levels of can be a marker for disease.

[0044] A polypeptide is a polymer of three or more amino acids covalently linked by amide bonds. Polypeptides may be post-translationally modified. Purified polypeptides are those that have been purified from cellular material, other Polypeptides of the type, chemical precursors, chemicals used in polypeptide synthesis, or A preparation of a polypeptide that is substantially free of cellular material, culture medium, chemical precursors, or any combination thereof. A polypeptide preparation that is substantially free of substances, chemicals used in polypeptide synthesis, is about Less than 30%, 20%, 10%, 5%, 1% or more of other polypeptides, media, It contains chemical precursors and / or other chemicals used in its synthesis. The purity of the polypeptide may be about 70%, 80%, 90%, 95%, 99% or more. do.

[0045] The term "polypeptide" refers to a type of polypeptide (e.g., a polypeptide A "polypeptide" can refer to one or more of two or more distinct polypeptides. A mixture of different types of polypeptides (i.e., including but not limited to, full-length polypeptides) a mixture of polypeptides containing a polypeptide fragment, a truncated polypeptide, or a polypeptide fragment The term "polypeptide" or "polypeptides" can also refer to The terms can also each mean "one or more polypeptides."

[0046] Polypeptide variants or polypeptides set forth in SEQ ID NOs: 1-27 or For example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids may be added to a fragment. The amino acid residues differ (e.g., amino acid additions, substitutions, or deletions). If necessary, align the sequences for maximum homology. It can occur at any position of the peptide.

[0047] Generally, variant polypeptides are those that modify one of the polypeptide sequences described herein. and evaluating the modified polypeptide to determine whether it is a biological equivalent. The biological equivalent of the variants can be confirmed by immunohistochemical assays. , enzyme-linked immunosorbent assay (ELISA), immunoturbidimetry, particle-enhanced immunoturbidimetry, radioimmunology In assays such as radioimmunoassays (RIA), or Western blot assays, If the polypeptide reacts substantially the same as the polypeptide described in the document, e.g., 90% of the original polypeptide, In one embodiment, the assay is performed in a biologically equivalent manner. A suitable polypeptide is one described herein that binds to a corresponding reactive antigen or antibody. It is a competitive assay that can reduce the Antibodies that specifically bind to the antibody polypeptide also specifically bind to the variant polypeptide. do.

[0048] Variant polypeptides have at least one amino acid sequence similar to that of the polypeptides set forth in SEQ ID NOs: 1-27. Both are approximately 80%, or approximately 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 , 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical. For example, , variant polypeptides of SEQ ID NOs: 1-27 have at least Approximately 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 90%, 87%, 8 A variant polypeptide may have one or more conservative amino acids identical to the amino acid sequence of the target amino acid. and retaining biological activity while having acid variations or other minor modifications, i.e. Biologically functional equivalents of SEQ ID NOs: 1-27. Biologically active equivalents has substantially equivalent functions to the corresponding polypeptide.

[0049] Methods for introducing mutations into amino acid sequences are well known to those skilled in the art. l (ed.), Current Protocols in Molecular Biol. ogy, John Wiley and Sons, Inc. (1994);Mania tis et al., Molecular Cloning: A Laboratory Manu al, Cold Spring Harbor laboratory, Cold Sp See Ring Harbor, NY (1989). Mutations are Commercially available mutagenesis kits, such as the "hange™ Site-Directed Mutagenesis Kit" (Stratagene), Those skilled in the art can easily introduce functionally active variant polypeptides into the host cell using a kit. The peptide is produced by replacing amino acids that do not affect the function of the polypeptide. It can be achieved.

[0050] The variant polypeptides may be conserved at one or more predicted non-essential amino acid residues. Conservative substitutions are those that are considered by those skilled in the art of peptide chemistry to be A shape that would predict that the secondary structure and hydropathic properties of the polypeptide will remain substantially unchanged. In this case, one amino acid is replaced with another amino acid with similar properties. Groups of amino acids correspond to conservative changes: (1) ala, pro, gly, glu; asp, gln, asn, ser, thr;(2)cys, ser, tyr, thr;( 3) val, ile, leu, met, ala, phe; (4) lys, arg, his and (5) phe, tyr, trp, his. In one embodiment, the polypeptide has about 1 , 2, 3, 4, 5, 10, 20, or fewer conservative amino acid substitutions.

[0051] As used herein, the percent identity of two amino acid sequences (or two nucleic acid sequences) is based on the algorithm of Karlin and Altschul (PNAS USA 87:22 64-2268, 1990) as amended by Karlin and Altschul (PN AS USA 90:5873-5877, 1993). The algorithm is based on that of Altschul et al. (J. Mol. Biol. 215:403-410, It is incorporated into the NBLAST and XBLAST programs of the BLAST (1990). Nucleotide searches were performed using the NBLAST program with a score of 100 and word count of 100. Length = 12. BLAST protein searches are performed using the XBLAST program. For comparison, the score is set to 50 and the word length is set to 3. To obtain a matched alignment, use Altschul et al. (Nucleic Acids Re GappedBLAST was used as described in (S. 25:3389-3402, 1997). When using BLAST or GappedBLAST, The default parameters for the programs (e.g., XBLAST and NBLAST) are as set forth herein. Used to obtain nucleotide sequences homologous to the described nucleic acid molecules.

[0052] Identity or identical refers to the similarity of amino acid sequences, and these terms are used in the art. Identifying sequences have the commonly recognized meaning. Sequence identity is the percentage of amino acids that are identical to the original amino acid sequence of the antibody. The sequences are aligned and gaps are inserted as needed to maximize sequence identity. Therefore, the amino acid sequence must share 85% amino acid sequence identity with the reference sequence. A candidate sequence having the following structure is one in which, after alignment of the candidate sequence with the reference sequence, 85% of the amino acids in the candidate sequence are Identical to the corresponding amino acid in the reference sequence and / or constitute conservative amino acid changes It is necessary.

[0053] A polypeptide or antibody is a molecule that is covalently linked to an amino acid sequence that is not normally associated with it. Additionally, the polypeptide or antibody may contain non-amino acid residues. It may be covalently or non-covalently bound to a compound or molecule, for example a polypeptide or The antibody or antibody may contain an indicator reagent, an amino acid spacer, an amino acid linker, a signal sequence, a transporter whether bound to a stop sequence, a transmembrane domain, a purified protein ligand, or a combination thereof In one embodiment, the protein purification ligand may be, for example, a polypeptide at the amino terminus or or one or more C amino acid residues at the carboxy terminus. is a sequence of amino acids not normally associated with a polypeptide or antibody The amino acid spacer can be approximately 1, 5, 10, 20, 100, or 1,000 amino acids. It can include.

[0054] The polypeptide may further contain a signal that directs the transport of the protein co- or post-translationally. The polypeptide may also include a nucleic acid sequence (or leader sequence). to facilitate synthesis, purification, or identification of (e.g., poly-His), or Inclusion of a linker or other sequence to enhance binding of the polypeptide to the solid support For example, the polypeptide may be a polypeptide derived from the Fc region of an immunoglobulin or bovine serum albumin. It may be bound to

[0055] A polypeptide is a sequence of amino acids that is not normally related in any way, i.e., a heterologous amino acid. The heterologous amino acid sequence may be covalently or non-covalently linked to the amino acid sequence. These sequences may be synthetic sequences, usually located at the carboxy terminus of the polypeptide. Furthermore, a polypeptide may be a sequence that is not located at the amino terminus. The antibody may be covalently or non-covalently bound to another compound or molecule, such as an indicator reagent. The polypeptide may contain an amino acid spacer, an amino acid linker, a signal sequence, a stop transport sequence, Covalently or non-covalently linked to a transmembrane domain, a protein purification ligand, or a combination thereof The polypeptide may also be combined with a moiety that enhances the immune response (i.e., a functional group which may be a peptide or other compound (e.g., a cytokine such as IL-2); Moieties that facilitate purification (e.g., 6-histidine tag, trpE, glutathione maltose affinity tags, such as binding proteins), or moieties that promote the stability of the polypeptide (e.g., polyethylene glycol; acetyl, propyl, succinyl, benzyl, amino-terminus protecting groups such as benzyloxycarbonyl, or t-butyloxycarbonyl; carbonyl-terminated protecting groups such as amide, methylamide, and ethylamide) In one embodiment, the protein purification ligand may be located at, for example, the amino terminus of the polypeptide. or one or more C amino acid residues at the carboxy terminus or at both termini. An amino acid spacer is a sequence of amino acids that is not associated with any polypeptide. The amino acid spacer can be approximately 1, 5, 10, 20, 100, or 1,000 amino acids. It can include.

[0056] If desired, the polypeptide may contain other amino acid sequences, such as amino acid linkers, amino acid Spacer, signal sequence, TMR stop sequence, transmembrane domain, and protein purification Ligands useful for the production of, e.g., glutathione-S-transferase, histidine tags and Staphylococcal protein A, or a combination thereof. A fusion protein is a protein that contains two or more different amino acid sequences operably linked together. Fusion protein constructs are synthesized by synthesizing individual polypeptides using organic synthesis techniques. They can be chemically synthesized by linking peptide fragments together into a fixed sequence. The fusion protein construct also contains a sequence of amino acid residues encoding the correct sequence. In vitro cultured cells carrying an expression vector containing a recombinant DNA sequence It can also be expressed by cultivated genetically modified host cells (e.g., E. coli). The polypeptide may be fused, for example, to the N-terminus or C-terminus of the polypeptide. There may be more than one polypeptide in a fusion protein. The fusion protein may comprise, for example, SEQ ID NOs: 1-27, fragments thereof, or The polypeptide may be in a multimeric form or a combination thereof. That is, the polypeptide may contain two or more copies of SEQ ID NOs: 1-27 or It may include combinations thereof.

[0057] In one embodiment, the polypeptide is from a human, rabbit, mouse, dog, cat, other mammal, or a combination thereof. The polypeptide may be purified using standard protein purification techniques. The polypeptides can be isolated from cells or tissue sources, or can be prepared by recombinant DNA techniques. Polypeptides can also be synthesized or produced chemically by conventional techniques. It can be synthesized using a peptide synthesizer of the type.

[0058] In one embodiment, the polypeptide is attached, covalently or non-covalently, to a solid phase or substrate. Cohesively fix.

[0059] The polypeptide may be recombinantly produced. The recombinant gene can be expressed in a suitable expression host system using techniques well known in the art. A variety of bacterial, yeast, mammalian, and insect expression systems are known in the art. Any such expression system available in the art can be used. Thus, the polynucleotide encoding the polypeptide is translated in a cell-free translation system. The polypeptide may be lyophilized, powdered, or dried, e.g., free It can also be dried.

[0060] Polynucleotides In one embodiment, an isolated polypeptide encoding one or more of the polypeptides disclosed herein is provided. The polynucleotides of the present invention include those containing less than an entire genome, Polynucleotides can be single-stranded or double-stranded nucleic acids. It can be human DNA, chemically synthesized RNA or DNA, or a combination thereof. Polynucleotides can be synthesized from other components, such as proteins, lipids, and other polynucleotides. For example, polynucleotides can be purified to be 50%, 75%, 90%, or less. %, 95%, 96%, 97%, 98%, 99%, or 100% purified. In one embodiment of the invention, the polynucleotide is a polypeptide set forth in SEQ ID NOs: 1-27. The gene encodes a gene or a fragment thereof.

[0061] The polynucleotides of the invention may contain other nucleotide sequences, such as sequences encoding linkers. Useful for sequences, signal sequences, TMR stop transport sequences, transmembrane domains, or protein purification Suitable ligands include glutathione-S-transferase, histidine tags, and It may contain staphylococcal protein A.

[0062] The polynucleotides of the present invention can be isolated. An isolated polynucleotide is one that is naturally occurring. is not immediately adjacent to one or both of the associated 5' and 3' flanking genomic sequences, An isolated polynucleotide is a polynucleotide that is naturally occurring. However, the recombinant DNA molecule may be a recombinant D The nucleic acid sequences found in nature immediately adjacent to the NA molecule are removed or absent. An isolated polynucleotide also includes non-naturally occurring nucleic acid molecules.

[0063] Degenerate nucleotide sequences encoding the polypeptides of the present invention are also used in the context of polynucleotide sequences of the present invention. Degenerate nucleotide sequences are sequences that encode the polypeptides of the invention or fragments thereof. polynucleotide, but due to the degeneracy of the genetic code, it is possible to distinguish between wild-type polynucleotide sequences and The polynucleotides of the invention that encode physiologically functional polypeptides differ in nucleic acid sequence. Complementary DNA (cDNA) molecules, species homologs, and variants of the nucleotides are also included in the polynucleotides of the present invention. It is leotide.

[0064] The polynucleotides of the present invention may comprise a coding sequence for a naturally occurring polypeptide. Alternatively, the polynucleotide may encode a modified sequence that does not occur in nature. The nucleotides may be part of expression control elements (e.g., origins of replication, promoters, enhancers, or or other regulatory elements that drive expression of the polynucleotide of the invention in a host cell. The expression vector can be cloned into an expression vector containing, for example, pBR 322, pUC, or ColE1 plasmids, or adenovirus type 2 vectors. The vector may be an adenoviral vector, such as a type 1 or type 5 vector. Other vectors may also be used, including but not limited to: Indo-Viral, Simian Virus 40, Alphavirus vector, Poxvirus viral vectors, as well as cytomegalovirus and retroviral vectors (e.g., mouse sarcoma virus, mouse mammary tumor virus, Moloney murine leukemia virus, and Routine Also included are minichromosomes (e.g., MC and MC1), bacteriophages (e.g., sarcoma viruses), and Phage, phagemid, yeast artificial chromosome, bacterial artificial chromosome, virus particle, virus rus-like particles, cosmids (plasmids into which the cos site of phage lambda has been inserted), and Precons (genetic elements capable of replicating under their own control within a cell) can also be used.

[0065] Preparing a polynucleotide operably linked to an expression control sequence and expressing it in a host cell Methods are well known in the art. See, for example, U.S. Pat. No. 4,366,246. The polynucleotides of the present invention may be used to direct the transcription and / or translation of the polynucleotide. operably linked when located adjacent to or near one or more expression control elements There are.

[0066] The polynucleotides of the present invention can be used, for example, as probes or primers (e.g., PCR primers). primers) to detect the presence of polynucleotides in a test sample (e.g., a biological sample). A probe is a molecule capable of detecting the presence of a target molecule, typically in a sequence-specific manner, e.g., a nucleic acid. A molecule capable of interacting with a target nucleic acid through hybridization. refers to a nucleic acid that is capable of supporting enzymatic manipulation and that hybridizes with a target nucleic acid so that the enzymatic manipulation occurs. A primer is a subset of a probe that can be hybridized with a nucleotide sequence. nucleotide or nucleotide derivatives, or similar available in the art that do not interfere with enzymatic manipulation. The antibody can be made from any combination of analogs.

[0067] The probe or primer may be selected from about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 39, 38, 39, 40, 41, 4 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 4 0, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, or It may be more than one consecutive nucleotide.

[0068] antibody Antibodies include those containing cystatin B polypeptides as described herein, variant cystatin B polypeptides as described herein, and variant cystatin B polypeptides as described herein. Antibodies that specifically bind to a statin B polypeptide or a fragment thereof are also included. The term "antibody" refers to an antibody that is capable of specifically binding to multiple polypeptides. An intact antibody, or an antigen-binding portion thereof that competes with the intact antibody for antigen binding, is also The term "antibody" also refers to one or more cystatin B polypeptides or fragments thereof. Any type of antibody molecule or specific molecule that specifically binds to (e.g., SEQ ID NOs: 1-27). Antibodies include naturally occurring, non-naturally occurring, synthetic binding molecules. As used herein, the "antigen-binding portion" of an antibody may be a fragment of an antibody that is specifically or genetically engineered. The terms "antigen-binding fragment" and "antigen-binding fragment" of an antibody and the like refer to a cystatin B polypeptide (e.g., A naturally occurring, enzymatically isolated antibody that specifically binds to and forms a complex with a target molecule (SEQ ID NO: 1-27). any obtainable, synthetic, or genetically engineered polypeptide, glycoprotein, or Contains immunoglobulins.

[0069] The antibody or fragment thereof binds to an epitope of a polypeptide described herein. They can be produced in vivo in suitable laboratory animals or by recombinant DNA techniques. Antibodies can also be produced in vitro using methods such as: It is well known in the art, see, e.g., Dean, Methods Mol. Biol. 80 :23-37(1998);Dean, Methods Mol.Biol.32:36 1-79(1994); Baileg, Methods Mol.Biol.32:38 1-88(1994);Gullick, Methods Mol.Biol.32:3 89-99(1994);Drenckhahn et al. Methods Cell.Bio l.37:7-56(1993);Morrison,Ann.Rev.Immunol .10:239-65(1992);Wright et al. Crit.Rev.Immuno 12:125-68 (1992). For example, polyclonal antibodies are described herein. The polypeptides described in administration to animals (cats, guinea pigs, goats, pigs, dogs, cattle, sheep, donkeys, or horses) Serum is collected from the immunized animal and antibodies can be generated by, for example, sulfonyltransferase. Precipitation with ammonium chloride followed by chromatographic techniques such as affinity chromatography Polyclonal antibodies are purified from plasma by chromatographic analysis. Techniques for processing are known in the art.

[0070] Antibodies include IgG (IgG1, IgG2, IgG2a, Ig2b, IgG3, IgG4) any isoform, including IgM, IgA (IgA1 and IgA2), IgD, and IgE It can be a type.

[0071] The antibody may be a monoclonal antibody, a polyclonal antibody, a chimeric antibody, or an antigen-binding A monoclonal antibody is a antibody obtained from a population of substantially homogeneous antibodies. A substantially homogenous antibody population may contain minor amounts of mutants or variants. Monoclonal antibodies are highly specific and interact with a single antigenic site. Monoclonal antibodies typically target a single epitope, whereas polyclonal antibodies A population of antibodies typically contains a variety of antibodies that target a diverse set of epitopes. Clonal antibodies can be obtained, for example, by the hybridoma method (Kohler and Milstein, N ature 256:495, 1975), recombinant methods (U.S. Pat. No. 4,816,567 ), and isolation from a phage antibody library (Clackson et al., Nature 3 52:624-628, 1991; Marks et al., J. Mol. Biol. 222:58 It can be produced by a number of methods, including (1991, 1-597).

[0072] Chimeric antibodies or antigen-binding portions thereof may be derived from a particular species or a particular class or subclass of antibodies. have a portion of the heavy and / or light chains derived from a subclass, with the remainder of the chains derived from another species, or are from different antibody classes or subclasses. For example, Morrison n,Science 229:1202(1985);Oi et al., BioTechniqu es 4:214(1986);Gillies et al., J.Immunol.Method s 125:191-202 (1989); U.S. Patent No. 5,807,715; U.S. Patent No. 4, See No. 816,567; No. 4,816,397.

[0073] Chimeric antibodies can be produced, for example, by CDR grafting (EP 239,400; PCT Publication WO 2004 / 023106). 91 / 09967; U.S. Patent Nos. 5,225,539; 5,530,101; and No. 5,585,089), veneering or resurfacing Resufacing (EP592,106; EP519,596; Padl an, Molecular Immunology 28:489-498(1991) ;Studnicka et al., Protein Engineering 7(6):805 -814(1994);Roguska et al., PNAS 96:969-973(1994) )), and chain shuffling (U.S. Patent No. 5,565,332). It can be generated using

[0074] In one embodiment, a chimeric antibody may comprise variable and constant regions of different species. For example, an antibody can be constructed by combining the heavy and light chain variable regions of one mammal with those of a different animal (e.g., The constant regions of the heavy and light chains of a human (mouse, rabbit, dog, cat, or human) can be included. Chimeric antibodies contain neither CDR nor framework sequences that are introduced into the recipient antibody. It can contain additional amino acids that are not part of the lactic acid bacteria. Such amino acids are introduced to better optimize the antibody's ability to recognize and bind to antigens. For example, amino acids in the framework regions of the variable regions of an antibody can be modified as needed. The CDRs of the reshaped antibody can be replaced to form a suitable antigen-binding site. See Sato et al., Cancer Res. (1993) 53:851-856. .

[0075] Non-limiting examples of antigen-binding portions or fragments of antibodies include: Fab fragment; Fab' fragment; Fab'-SH fragment; F(ab')2 fragment; Fd fragment; Fv fragments; single-chain Fv (scFv) molecules; sdAb fragments (nanobodies); Fab-like antibodies ( Contains the variable regions of the heavy and light chains equivalent to the Fab fragment obtained by papain digestion Antigen-binding fragment; F(ab')2-like antibody (F(ab')2 obtained by pepsin digestion) an antigen-binding fragment containing two antigen-binding domains equivalent to an antigen fragment; Multispecific antibodies, diabodies, bispecific antibodies, multifunctional antibodies, humanized antibodies, caninized antibodies Antibodies, human antibodies, dog antibodies, cat antibodies, mouse antibodies, rabbit antibodies, synthetic antibodies, CDR graphs and minimal recognition units consisting of amino acid residues that mimic the hypervariable regions of antibodies (e.g., , an isolated complementarity determining region (CDR) such as a CDR3 peptide, or a restricted FR3-CDR 3-FR4 peptides, as well as other engineered molecules such as domain-specific antibodies, Single domain antibodies, domain deleted antibodies, CDR grafted antibodies, diabodies, tribodies antibodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies) di), single chain (Fv)2 (sc(Fv)2); bivalent (scFv)2); tetravalent ([sc( Fv)2]2) scFV antibodies, small modular immunotherapeutics (SMIPs), and small Variable IgNAR domains are also considered "antigen-binding fragments or portions" herein. do. "specifically binds" "(specifically bind)" or "specific to" means to bind to a first antigen For example, the polypeptides of SEQ ID NOs: 1-27 have a higher affinity than other non-specific molecules. The term "specific binding" refers to the ability of a molecule to recognize and bind to an antibody described herein. "specifically binds" and "specifically binds" "specific for" refers to a first antibody, e.g., SEQ ID NO: 1 Antibodies raised against -27 bind to SEQ ID NO: 1 with higher affinity than other non-specific molecules. The term "non-specific molecule" also refers to a molecule that recognizes and binds to the first antigen and the second antigen. Specific binding is demonstrated by, for example, enzyme-linked immunosorbent assay (ELISA). ISA), radioimmunoassay (RIA), immunoturbidimetry, particle-enhanced immunoturbidimetry, or Wes These can be tested using a tandem blot assay using methodology well known in the art. do.

[0076] In one embodiment, the antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 2 1, 22, 23, 24, 25, 26, 27, or 28. In one embodiment, the epitope specifically binds to AYFLAYF (SEQ ID NO: 20), ELAYF (SEQ ID NO: 21), DELAYF (SEQ ID NO: 22), HDELAYF (SEQ ID NO: 23), KHDELAYF (SEQ ID NO: 10), AKHDELAYF (SEQ ID NO: 24), KAKHDELAYF (SEQ ID NO: 25), NKAKHDELAYF (SEQ ID NO: 2 6), TNKAKHDELAYF (SEQ ID NO: 27), or QTNKAKHDELAYF (SEQ ID NO: 4).

[0077] In one embodiment, the antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NOs: 1-27 or fragments thereof. The antibody competes with a second or reference antibody for binding to the antibody. Any competitive binding assay can be used. can be used to measure competition between two antibodies against the same antigen. A cross-ELISA assay can be used for this purpose. The means for doing so are well known to those skilled in the art (see, for example, Dowbenko et al. (1988) J. Virol. .62:4703-4711).

[0078] Using any assay used to assess competitive binding, the first antibody is compared with the first antibody in the presence of the second antibody. 2. The binding of the antibody to the antigen is at least about 30%, 40%, 50%, 60%, 75%, If the binding is reduced by 90% or more, the first antibody competitively inhibits the binding of the second antibody. It is thought that this is the case.

[0079] Competitive binding may involve binding of one or more isolated polypeptides set forth in SEQ ID NOS: 1-27 attached to a solid support. and determining the ability of the antibody to bind to or interact with the polypeptide. by assaying the ability of the antibody to compete with the antibodies described herein for binding of It is possible.

[0080] The antibody includes: (a) a reference antibody in binding to SEQ ID NO: 1-27 or an antigen-binding fragment thereof; (b) antibodies and antigen-binding fragments thereof that compete with the reference antibody; (c) antibodies and antigen-binding fragments thereof that compete with the reference antibody; (d) antibodies and antigen-binding fragments thereof that compete with the reference antibody; (e) antibodies and antigen-binding fragments thereof that compete with the reference antibody; (f) antibodies and antigen-binding fragments thereof that compete with the reference antibody; (g ... (c) an antibody that binds to an epitope of the antigen-binding fragment thereof and an antigen-binding fragment thereof; and (c) a reference antibody and Virtually the same K d Antibodies that bind to SEQ ID NOs: 1-27 or antigen-binding fragments thereof and their antigens and / or (d) a binding fragment of SEQ ID NO: 1- with substantially the same off-rate as the reference antibody. 27 or an antigen-binding fragment thereof, wherein The reference antibody is 10 7 Binding affinity K l / mol or higher a SEQ ID NO: 1- Antibodies that specifically bind to the polypeptides of 27 or their antigen-binding fragments, or antigen-binding fragments thereof It is a fragment.

[0081] Affinity of an antibody or antigen-binding fragment thereof for a polypeptide partner can be expressed by the dissociation constant (Kd). The equilibrium dissociation constant (Kd) is k off / k onIt is calculated by quantification. Chen, Y. et al., 1999, J. Mol. Biol. 29 See 3:865 - 881. Various methods for measuring affinity constants are known in the art In certain embodiments, the reference antibody has a binding affinity for the polypeptide of SEQ ID NOs: 1 - 27 at a specific K on rate / association rate or K or K off rate. In one embodiment, the antibody is an antibody or an antigen - binding fragment thereof that specifically binds to the polypeptide of SEQ ID NOs: 1 - 27 with a K of 6×10 5 M -1 s - 1 or better, and the antibody specifically binds with a K on of 5×10 -6 s -1 or better, or the antibody specifically binds with a binding affinity of 50 pM, 40 pM, 30 pM, 20 pM, 100 pM, 50 pM, 40 pM, 30 pM, 20 pM, or better off Specifically binds to SEQ ID NOs: 1 - 27. Antibodies that specifically bind to SEQ ID NOs: 1 - 27 are particularly useful for detecting the presence of cystatin B polypeptides and fragments thereof present in a sample (e.g., serum, blood, plasma, cells, tissues, saliva, plaque, gingival crevicular fluid, gingival biopsy, tongue swab, or urine sample) in Immunoassays can utilize one antibody or several antibodies. Immunoassays

[0082] For example, a monoclonal antibody specific for one epitope, a combination of monoclonal antibodies specific for multiple epitopes of one polypeptide, monoclonal antibodies specific for epitopes of different polypeptides, polyclonal antibodies specific for the same antigen, different <000 && 860>

[0083]

[0084]

[0085] [[ID=7 #Antigen-specific polyclonal antibodies, or monoclonal and polyclonal antibodies Immunoassay protocols can be used, for example, competitive, direct reaction, or combinations thereof. can be based on assays of the ELISA type, or sandwich type, e.g., using labeled antibodies The antibody can be labeled with any type of label known in the art, and such labels can be used to Examples of such identification include fluorescence, chemiluminescence, radioactivity, enzymes, colloidal metals, radioisotopes, and bioluminescent labels.

[0083] The antibodies or antigen-binding fragments thereof are bound to a support and are detected in samples for certain diseases and conditions. It can be used to detect the presence or amount of polypeptide present. For example, glass, polystyrene, polypropylene, polyethylene, dextran, nylon Amylases, natural and modified celluloses, polyacrylamides, agarose, and magnesium Magletite. Antibodies or antigen-binding fragments thereof can be freeze-dried. It may be dried, powdered or desiccated, for example freeze-dried.

[0084] The antibodies may also be used to isolate polypeptides by immunoaffinity columns. Antibodies can also be modified, for example by absorption or covalent coupling, so as to retain their immunoselective activity. , can be immobilized on a solid support. Optionally, the accessibility of the antigen-binding site of the antibody can be improved. Spacer groups can be included to ensure that the nucleotide sequence is maintained. , biological samples (including but not limited to saliva, plaque, gingival crevicular fluid, gingival biopsy) , tongue swabs, serum, blood, and urine) It can be effectively bonded.

[0085] Antibodies can also be used in immunolocalization studies to identify specific proteins in various cellular events or physiological states. The presence and distribution of the polypeptides described herein can also be analyzed using antibodies. can be used to identify molecules involved in passive immunity or involved in the biosynthesis of non-protein antigens. It is also possible to identify molecules that are useful for vaccine development. Antibodies (including, for example, monoclonal antibodies and single-chain antibodies) can be used to detect certain specific The progression of a particular disease or condition can be monitored by measuring the sequence number in a test sample from an animal. The aim is to improve the disorder by measuring the increase or decrease in the amount of No. 1-27 or a fragment thereof. It can be used to determine whether a particular targeted therapeutic regimen is effective. Direct binding assays, such as RIA, ELISA, or Western blot assays can be detected and / or quantified using

[0086] Methods of detection In one embodiment, a method for detecting a cystatin B polypeptide in a sample comprises the steps of: One or more antibodies specific for SEQ ID NOs: 1-27 are combined with a cystatin B polypeptide and SEQ ID NOs: and contacting the antibody with one or more antibodies specific for 1-27 under conditions suitable for the formation of a complex. The present invention provides a method for the preparation of a cystatin B polypeptide and one or more polypeptides specific for SEQ ID NOs: 1-27. Complexes with the above antibody are detected.

[0087] In one embodiment, the method for detecting polypeptides comprising SEQ ID NOs: 1-27 and fragments thereof comprises: Optionally, detecting the amount of polypeptides comprising SEQ ID NOs: 1-27 in a sample. The relative levels of polypeptides can be used to diagnose or treat a number of diseases or conditions. Any method for detecting a polypeptide known in the art can be used in this invention. It can be used in the manner described in the specification.

[0088] Assay methods for use with the antibodies described herein include direct and indirect labeling techniques. , immunoaffinity columns, immunomagnetic beads, fluorescence activated cell sorting (FAC) S), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), agglutination assay Nephelometric assay, quantitative nephelometric assay, and primary antibody A labeled secondary antibody that detects the

[0089] The antibody may be fluorescently labeled, for example, with a fluorescent label having excitation and emission wavelengths suitable for detection. The fluorescently labeled fragments can be detectably labeled using commercially available equipment such as fluorescent cell sorters. Examples of anti-inflammatory agents include phycoerythrin (PE), fluorescein isothiocyanate (FI) and TC), Rhodamine (RH), Texas Red (TX), Cy3, Hoechst 33258, and 4'6-diamino-2-phenylindole (DAPI). Such labels can be attached to the antibody using standard techniques (Maino et al. , 1995, Cytometry 20:127-133).

[0090] The methods described herein can be used to detect SEQ ID NOs: 1-27 or fragments thereof. and the antibody or antigen-binding antibody fragment specifically binds to SEQ ID NO: 1-27. Examples of biological samples include serum, blood from animals (e.g., dogs, cats, or humans). Examples of such samples include cells, plasma, saliva, plaque, gingival crevicular fluid, gingival biopsy, tongue swab, or urine. Test samples may be untreated or may have been precipitated, fractionated, separated, diluted, concentrated, or purified. stomach.

[0091] As used herein, a "patient" or "subject" refers to a human, Or it may refer to a non-human animal, including a cat, cow, pig, horse, or dog.

[0092] As used herein, the terms "sample," "test sample," "patient sample," or "subject sample" The term includes, but is not limited to, blood, serum, plasma, saliva, etc. obtained from a subject. , plaque, gingival crevicular fluid, gingival biopsy, tongue swab, or urine sample.

[0093] Assays include, but are not limited to, competitive, direct reaction, or sandwich assays. These include, but are not limited to, enzyme-linked immunosorbent assays based on ELISA. Adsorption assay (ELISA), Western blot, IFA, radioimmunoassay (RIA), Hemagglutination (HA), immunoturbidimetry, particle-enhanced immunoturbidimetry, fluorescence polarization immunoassay (FPIA) and microtiter plate assays (one or more wells of a microtiter plate) Some assays are performed using reversible flow chromatographs. Reversible flow chromatography binding assay phic binding assay), including, for example, the SNAP® assay See, for example, U.S. Patent No. 5,726,010.

[0094] The assay may use a solid phase or substrate, or may involve immunoprecipitation or solid phase The method can be carried out by any other method that does not utilize a solid phase or substrate. In some cases, one or more polypeptides or antibodies may be attached to a solid support or substrate (e.g., a microsphere). Titer wells, magnetic beads, non-magnetic beads, columns, matrices, membranes, synthetic or Natural fibers (e.g., glass or cellulose-based materials, or polyethylene, poly Fiber mats made of thermoplastic polymers such as polyethylene terephthalate (propylene or polyester) , sintered structures composed of particulate materials (e.g., glass or various thermoplastic polymers), or or nitrocellulose, nylon, polysulfone, etc. (generally synthetic in nature) In one embodiment, the substrate is Sintered polyethylene particles, commonly known as porous polyethylene, e.g., Ch 10-15 manufactured by romex Corporation (Albuquerque, NM) All of these substrate materials are available in film, sheet, or It can be used in any suitable shape such as a glass or plate, or can be made of paper, glass, plastic, etc. coated or bonded to a suitable inert carrier such as a plastic film or fabric Suitable methods for immobilizing antibodies on a solid phase include ionic and hydrophobic methods. Examples of such interactions include molecular and covalent interactions.

[0095] In one embodiment, the method comprises the step of: connecting the test sample to one or more antibodies that specifically bind to SEQ ID NOs: 1-27. allows the formation of polypeptide / antibody complexes, i.e., immune complexes, with multiple antibodies. That is, the antibody is contacted under conditions in which the antibody binds to SEQ ID NOS: 1-27 located in the sample. Those skilled in the art will appreciate that antibody / polypeptide complexes specifically bind to one or more of the polypeptides of interest. Familiar with the assays and conditions used to detect binding of conjugates. The formation of an antibody / polypeptide complex is detected. This indicates that the nucleotide sequence is present in the patient sample.

[0096] In one embodiment, an indicator reagent, such as an enzyme conjugate attached to the antibody, catalyzes a detectable reaction. If the polypeptide / antibody complex is detected, the polypeptide / antibody complex is detected. Optionally, a signal generating compound is added. an indicator reagent containing the compound under conditions that allow the formation of a polypeptide / antibody / indicator complex; This may be applied to polypeptide / antibody complexes. Optionally, the polypeptide or antibody is ligated prior to formation of the polypeptide / antibody complex. The subject may be labeled with an indicator reagent. The method optionally includes positive or negative controls. The positive control is a test that specifically binds to Cys B and produces a positive result. A negative control may contain one or more polypeptides that are thought to affect the activity of the antibody. Any Cys B polypeptide may also specifically bind or cross-link with an antibody specific for Cys B. It does not contain any polypeptides or other components that may react.

[0097] In one embodiment, one or more antibodies are attached, either covalently or non-covalently, to a solid phase or substrate. A sample that may contain Cys B polypeptide is added to the substrate. One or more antibodies specific for Cys B are added to the substrate. The antibodies are used as a solid phase. may be the same antibody as the antibody of interest, or may be from a different source or species. The antibody may be conjugated to an indicator reagent, such as an enzyme conjugate. A washing step is carried out before each addition. A chromophore or enzyme substrate is added to develop color. The color reaction is stopped, e.g. Color development can be quantified, for example, using a spectrophotometer.

[0098] In another embodiment, one or more antibodies are immobilized on a solid phase or substrate. A test sample that may contain the Cys B polypeptide is added to the substrate. Add a second anti-species antibody that binds differently This second anti-species antibody is from a different species than the antibody immobilized on the solid phase. a third antibody that specifically binds to the first anti-species antibody but does not specifically bind to the antibody immobilized on the solid phase; A species antibody is added. The third anti-species antibody may contain an indicator reagent, such as an enzyme conjugate. A washing step may be performed before each addition. A chromophore or enzyme substrate is added to develop color. The color reaction is stopped and the color development can be quantified, for example, using a spectrophotometer. Cut.

[0099] In one embodiment, the one or more capture antibodies are directed to one or more of the polypeptides described herein. The one or more capture antibodies can specifically bind to the epitope(s) represented by SEQ ID NOs: 1-27. One or more of the polypeptides can be used to immobilize, for example, a solid support. The one or more detection antibodies may target the same epitope or epitopes of the polypeptide or different epitopes. The detection antibody can specifically bind to the above epitope. This embodiment can be used to detect or visualize fixation to the body. It is more specific and sensitive than assays that use a single antibody for both capture and detection functions. It is advantageous because of its high

[0100] In one type of assay format, one or more antibodies are coated onto a solid phase or substrate. Any polypeptides suspected of containing SEQ ID NOs: 1-27 or fragments thereof may be used. A test sample containing the polypeptide is subjected to a signal generating antibody or antibody fragment specific for the polypeptide. A solid phase antibody or polypeptide directed against the test sample polypeptide is applied along with an indicator reagent containing a compound. conditions sufficient for the formation of an antigen / antibody complex of an indicator reagent compound bound to a peptide-specific antibody The indicator reagent bound to the anti-polypeptide antibody is incubated for a certain period of time. Binding can be measured quantitatively. The signal generated from a control sample or control standard can be measured. A measurable change in signal compared to the control is indicative of a polypeptide comprising SEQ ID NO: 1-27 or This type of assay is useful for detecting the presence of polypeptides or fragments thereof in a test sample. The amount of tide can be quantified.

[0101] Another type of assay format involves coating one or more antibodies to a carrier or substrate. The antibody is conjugated to an indicator reagent and added to the test sample. This mixture is applied to a carrier or substrate. If Cys B polypeptide is present in the test sample, the Cys B polypeptide will react with the Binding to one or more antibodies bound to a labeling agent or to one or more antibodies immobilized on a support The polypeptide / antibody / indicator complex can then be detected. Such assays can quantify the amount of polypeptide in a test sample.

[0102] Another type of assay format involves coating one or more antibodies to a carrier or substrate. The test sample is applied to the support or substrate and incubated. The solid support is washed with a wash solution. By washing away unbound components from the sample, If a polypeptide containing the polypeptide or a fragment thereof is present in the test sample, the polypeptide coated on the solid phase is This polypeptide / antibody complex binds to a second antibody bound to an indicator reagent. and can be detected using an anti-species specific antibody of the polypeptide / antibody / anti-species antibody. This type of assay is useful for detecting polypeptides in a test sample. The amount of peptide can be quantified.

[0103] The formation of a polypeptide / antibody complex or a polypeptide / antibody / indicator reagent complex can be achieved by, e.g. Detection may be by radiometric, colorimetric, fluorometric, size separation, or precipitation methods. Optionally, detection of the polypeptide / antibody complex can be achieved by the use of a signal generating compound. This is done by adding a secondary antibody conjugated to an indicator reagent containing The indicator reagent containing the signal-generating compound (label) associated with the antibody complex can be prepared using the methods described above. These can be detected using colorants, catalysts such as enzyme conjugates, fluoresceins and rhodontics. Fluorescent compounds such as dammine, dioxetane, acridinium, phenanthridinium, ruthenium chemiluminescent compounds such as fluorescein, ... Cofactors, inhibitors, magnetic particles, etc. Examples of enzyme binding pairs include alkaline phosphatase (ALPHA) and Examples include α-galactosidase, horseradish peroxidase, and beta-galactosidase. The choice of a particular sign is not critical, but choosing a particular sign can be and capable of generating a signal by itself or together with one or more further substances. It is thought that this will be possible.

[0104] As used herein, the phrase "determining the amount" refers to determining the amount of one or more polypeptides in a sample. This refers to measuring or distinguishing the level of a polypeptide, as is known in the art. This can be done by a methodology for detecting the presence of IgG, for example, enzyme-linked immunosorbent assay (ELISA). SA), radioimmunoassay (RIA), immunoturbidimetry, particle-enhanced immunoturbidimetry, or Western Alternatively, methods such as immunoblot assays or immunohistochemistry may be used. The identity of the 27 polypeptides may be determined by mass spectrometry or similar methods known to those skilled in the art. Determining the amount of polypeptide present in a sample is possible using such in vitro analysis. This is done through analysis and experimental manipulation.

[0105] Diagnostic methods In one embodiment, a method of diagnosing kidney disease in a subject is provided. The method comprises: determining the amount of cystatin B polypeptide in the sample, The amount of peptide is determined using one or more antibodies that specifically bind to SEQ ID NOs: 1-27 and determining the amount of cystatin B polypeptide in the sample relative to a control sample or control group. The level of cystatin B polypeptide in the sample is higher than that in the control sample or control standard. High levels are a sign of kidney disease.

[0106] Other methods can diagnose AKI or AKI in CKD patients. In an embodiment, the method comprises treating a patient with a disease caused by cancer or kidney cancer, such as a patient with a reduced kidney function or a patient with a physical injury to the kidney. In another embodiment, the method can be used to diagnose kidney disease caused by bacterial infection. , can diagnose decreased kidney function or physical damage to the kidney. Bacterial infections can be the cause, e.g. For example, Anaplasma species, Ehrlichia species, Leptospira species, Escherichia species, or Borrelia species. It can be a type.

[0107] In one embodiment of the present invention, the present invention is directed to treating kidney disease, decreased kidney function, or physical damage to the kidney caused by bacterial infection. A method for diagnosing or detecting damage is provided, the method comprising: detecting cystatin in a sample from a subject; determining the amount of cystatin B polypeptide, wherein the amount of cystatin B polypeptide is SEQ ID NO: No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, One or more of 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 determining the presence or absence of a polypeptide of interest using one or more antibodies that specifically bind to the polypeptide of interest; The amount of cystatin B polypeptide in the sample is compared to a control sample or a control standard, and the sample A higher level of cystatin B polypeptide in the cell culture medium compared to a control sample or control standard is It can be a sign of kidney disease caused by a bacterial infection, decreased kidney function, or physical damage to the kidney.

[0108] The polypeptides disclosed herein are capable of detecting the activity of a non-disease subject in a sample from a diseased subject. found in amounts or at levels greater than those in control subject samples. The relative levels of these polypeptides in a subject's sample may indicate progression of the disease and disease severity. That is, in some cases, a higher amount or higher levels of cystatin B Polypeptides indicate a more severe disease state.

[0109] A high level of cystatin B polypeptide is about 10 times higher than a control sample or control standard. 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, A 500% or higher level of cystatin B polypeptide. , about 10-500% or more, about 20-500% or more than the control sample or control standard or more, about 30-500% or more, about 40-500% or more, about 50-500% or more, about 60-500% or more, about 100-500% Or at a higher level.

[0110] Also, a high level of cystatin B polypeptide refers to a level of cystatin B polypeptide that is higher than a control sample or control standard. It may also be a level at which the amount is increased statistically significantly.

[0111] Also, a high level of cystatin B polypeptide is about 10, 20, 50, 100, 20 00, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,5 00, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8 It can be 1,000, 9,000, 10,000 ng / ml or more. A control level or reference for a phospholipid B polypeptide is about 400, 350, 300, 250, or , 200, 150, 100, 50, 20, 10 ng / ml or less.

[0112] High levels of cystatin B polypeptide are associated with any type of kidney disease or condition. Control samples or samples determined using normal control subjects who do not have periodontal disease, bacterial infection, or periodontal disease. can be compared to a control standard.

[0113] In some embodiments, the level of cystatin B polypeptide in the test sample is determined by measuring the level of cystatin B polypeptide in one or more individuals. The level of cystatin B is compared to the level in a control sample from a normal control subject. The control level measured in the control sample is then compared to the cystatin B polypeptide level measured in the test sample. Alternatively, the level of cystatin B polypeptide in the test sample is compared to It is compared to a predetermined or predefined control level ("control standard"). A reference standard for the IL-16 polypeptide can be derived from the data, e.g., multiple normal or healthy control subjects. Calculating from data including the level of cystatin B polypeptide in a control sample from The normal or healthy control subjects and test subjects being evaluated can be of the same species.

[0114] In particular embodiments, in mammals, more particularly in dogs, cats, and humans: Reagents and methods are provided for identifying certain diseases or conditions. In one embodiment, a method for providing a diagnosis and prognosis for a patient is provided. Identification of these peptides may be an independent predictor of renal disease or a predictor of chronic kidney disease stage (e.g., stage The method may be an identifier of acute kidney injury or active kidney injury in any of the following categories: Advantageously, it enables the diagnosis and differentiation of acute kidney injury or active kidney damage in chronic kidney disease, and The test is not affected or confounded by the age or weight of the subject. and determining appropriate renal therapy using the aforementioned renal patient predictions determined using polypeptides. It is directed towards choosing.

[0115] Identification of Cys B polypeptide in subject samples may be used to determine AKI grade (e.g., grade 1). The methods and compositions described herein may be an independent predictor of grade 3 or 5. Advantageously, this allows for earlier diagnosis and differentiation of AKI stages, and is less sensitive to patient age or weight. Not subject to influence or confounding.

[0116] The antibodies are obtained from an animal, e.g., a human, cat, or dog, suspected of having kidney disease. Obtaining a test sample can be used in a method for diagnosing kidney disease. and the antibody-antigen complex (i.e., immune complex) are contacted under conditions that allow the formation of the antibody-antigen complex. Those skilled in the art will be aware of the conditions that allow and are suitable for the formation of antigen / antibody complexes. The presence or amount of antibody-antigen complexes can be determined by methodologies known in the art. This can be done.

[0117] Embodiments further include methods for identifying levels of specific polypeptides in patient samples. predicting a patient's health status, monitoring disease progression, and / or assessing / monitoring treatment effectiveness In one aspect, the method includes a method for assessing disease progression or treatment efficacy. In certain embodiments, the method is performed in a diagnostic setting. and then post-treatment identification of when a particular therapy should result in a reduction or amelioration of disease progression. This can be done at the time of

[0118] The methods described herein also provide methods for determining the amount of polypeptide comprising SEQ ID NOs: 1-27. The amount (nt) or quantity may also be indicated. The amount or quantity of a particular polypeptide is an indicator of the stage of disease (i.e., stages 1-4). ), disease progression, and / or prognostic indicators. Many indicator reagents, such as enzyme conjugates, are used. If the amount of polypeptide present is proportional to the signal produced, Depending on the situation, it may be diluted with a suitable buffer reagent, concentrated, or contacted with the solid phase without any manipulation. For example, pre-diluted serum or plasma samples, or concentrated samples such as urine, The condensed sample is used to determine the presence or amount of polypeptide present. It is possible.

[0119] The polypeptides and assays described herein may be combined with other polypeptides or assays. In combination, the polypeptides and assays can be used to detect the presence of kidney disease. , in combination with suitable reagents for detecting or measuring creatinine or general protein levels. It can be combined.

[0120] In one embodiment, a method for distinguishing between an upper and a lower urinary tract infection is also provided. A UTI is an infection of the kidneys (pyelonephritis). A lower UTI is an infection of the bladder. These conditions can be difficult to distinguish. Their treatments may vary. Therefore, it is advantageous for healthcare professionals to know the difference between upper and lower urinary tract infections. There is a need in the art for methods to distinguish between these infections.

[0121] The method comprises measuring the amount of cystatin B polypeptide in a sample from a subject, the amount of cystatin B polypeptide comprising SEQ ID NOs: 1-27 The method includes determining the presence or absence of cystatin B in a sample using one or more antibodies that specifically bind to cystatin B. The amount of polypeptide is compared to a control sample or control standard to determine the amount of cystatin B polypeptide in the sample. Higher levels of urinary acid compared to control samples or control standards are a sign of an upper urinary tract infection in the subject. He becomes a candidate.

[0122] In one embodiment, a method is provided for distinguishing between acute kidney injury and a lower urinary tract infection. These conditions can be difficult to distinguish. Treatment for these conditions can vary, making it difficult for healthcare professionals to It would be advantageous to be able to distinguish between acute kidney injury and a lower urinary tract infection. determining the amount of cystatin B polypeptide in a sample from the and an amount of a polypeptide that specifically binds to one or more polypeptides consisting of SEQ ID NOs: 1-27. The method includes determining the presence or absence of cystatin B polypeptide in a sample using one or more antibodies. The amount of cystatin B polypeptide in the sample is compared to a control sample or control standard to determine the cystatin B polypeptide level. A higher level of uric acid compared to a control sample or control standard is indicative of acute kidney injury in the subject.

[0123] In one embodiment, a method for diagnosing periodontal disease in a subject is provided. These include gum inflammation and periodontal disease, which can lead to attachment loss and alveolar bone damage. It is a disease of the periodontal tissues that leads to destruction. The clinical diagnosis of periodontal disease is made by examining the periodontal tissues that show disease. This is done by recognizing various signs and symptoms in the body. The appearance of signs and symptoms usually This occurs some time after the onset of the disease and after significant damage has occurred to the supporting bones and tissues. Furthermore, periodontal disease cannot be adequately evaluated or treated in veterinary patients without general anesthesia. There is a need in the art for methods to detect periodontal disease early. The method comprises determining the amount of cystatin B polypeptide in a sample from a subject. The amount of cystatin B polypeptide comprises one or more antibodies specific for SEQ ID NOs: 1-27. The amount of cystatin B polypeptide in the sample is determined using a control sample or control group. The level of cystatin B polypeptide in the sample is higher than that in the control sample or control standard. High levels are indicative of periodontal disease in a subject.

[0124] Treatment method In certain embodiments, a method of treating a disease condition in a subject is provided. The amount of cystatin B polypeptide in a sample from a subject is measured using one of the antibodies specific for Nos. 1-13. This includes requiring a test that provides analytical results for the determination using the above antibodies. The amount of cystatin B polypeptide contained in the sample is used as a reference sample or control for the disease state. If the IL-10 level is higher than IL-10, the subject is administered treatment for the disease state.

[0125] Disease conditions include AKI, periodontal disease, upper urinary tract infections, and renal disease. In this case, the disease state is neither cancer nor kidney cancer.

[0126] Treatment of CKD, AKI, and kidney disease includes, for example, treatment of obstructed nephron / ureteral stones. Surgery, chemotherapy for renal neoplasia, dietary management, intestinal phosphate binders, antiproteinuric drugs (e.g. , angiotensin-converting enzyme inhibitors (ACEIs), omega-3 fatty acids), antihypertensive drugs (e.g., ACEIs, calcium channel blockers (CCAs), fluid therapy to correct dehydration, Management of cirrhosis, administration of diuretics, dialysis, correction of electrolyte abnormalities, antiemetics and antacids, recombinant Upper urinary tract infections can be treated with antibiotics. Periodontal disease can be managed with thorough cleaning, scaling, and root planning. ning, gum graft surgery, laser treatment, regenerative procedures (membranes in pockets (fill tar), bone graft, or use of tissue stimulating proteins), dental implants, pockets Reduction procedure (removal of disease-causing bacteria before folding back the gum tissue and fixing it back in place) ) can be treated.

[0127] In an alternative embodiment, the methods described herein are used to prepare compositions or treatment regimes. The effectiveness of the (composition or diet) in ameliorating disease progression can be evaluated. Using the methods described herein, patient levels of polypeptides comprising SEQ ID NOs: 1-27 may be determined. The activity of a composition or treatment regimen against a rhesus macular degeneration can be evaluated.

[0128] kit In one embodiment, kits are provided for carrying out the methods described herein. In one embodiment, the kit comprises one or more polypeptides comprising SEQ ID NOs: 1-27. Optionally, in certain embodiments, the antibody may comprise one or more antibodies specific for In addition to instructions for use, a secondary antibody useful in, for example, a sandwich assay may be included. A distinctive labeled antibody, as well as reagents for labeling the antibody, may also be present in the kit.

[0129] In a further embodiment, the kit comprises one or more polypeptides each comprising SEQ ID NOs: 1-27. In certain embodiments, the antibody or antibodies specifically bind to the peptide. The body may be a solid support or a microarray, including but not limited to a chip, a microarray, a bead, etc. provided on a substrate.

[0130] The kit (e.g., article of manufacture) may include a kit for detecting a polypeptide or polypeptides described herein in a patient sample. The kit may be for detecting a protein fragment thereof. Determining the binding of the antibody to the full-length protein or protein fragment described herein The kit or article of manufacture also includes a composition comprising one or more antibodies or antibody fragments and Also included is a composition for determining the binding of the antibody or antibody fragment to a polypeptide in a sample. The kit may comprise a device containing one or more polypeptides or antibodies; Instructions for use of the one or more polypeptides or antibodies may be provided, for example, for the treatment of renal disease in a mammal. The kit may also include one or more polypeptides of the kit. or a label indicating that the antibody can be used to identify renal dysfunction. Packaging materials containing other components known to those skilled in the art, such as buffers, controls, etc. Components that may be included in such a test kit include the polypeptides, antibodies, The assays and kits are useful, for example, for the individual diagnosis of kidney disease in patients.

[0131] The kits are useful for diagnosing, predicting, or treating kidney disease, particularly kidney disease in dogs, cats, and humans. is useful to monitor.

[0132] The embodiments illustratively described herein may be used in conjunction with any method not specifically disclosed herein. The present invention may be suitably practiced in the absence of one or more of the elements, one or more of the limitations. For example, in each instance herein, the terms "comprising," "consisting essentially of," and and "consisting of" may be used interchangeably with the other two terms while retaining their ordinary meanings. The terms and expressions used are for explanatory purposes only. and the use of such terms and expressions is without limitation as to the It is not intended to exclude equivalents of the features or portions thereof, and the claimed embodiments It is recognized that various modifications are possible within the scope of the present invention. Although specifically disclosed by the embodiments, the present invention is not limited to the above-described embodiments. It is understood that the features, modifications, and variations described herein may be used by those skilled in the art and that such modifications and variations may be utilized. Such modifications and variations are intended to be included within the spirit and scope of the present invention as defined by this specification and the appended claims. It should be understood that this is considered within the scope of the embodiments.

[0133] Method embodiments incorporating the above-described features are intended to fall within the scope of this disclosure. [Example]

[0134] The following examples illustrate specific embodiments of the present disclosure and various applications thereof. These examples are given for illustrative purposes only and should not be construed as limiting. .

[0135] Example 1: Protein sequence of canine Cys B Previously, canine Cys B had a 77aa protein sequence as shown in SEQ ID NO:1 It was thought that... QVKAQLEERENKKYTTFKAVTFRSQVVAGTPYFIKVQVDD DEFVHLRVFQSLPHENKPLALSSYQTNKAKHDELAYF(sequence Number 1) However, this idea is incorrect. MDCK cell lines are grown to confluence and adherent. The cells were harvested and lysed in a physiological buffer containing a detergent. After lysis, the cells were The supernatant was then spun at 1000 rpm for 30 minutes to pellet the cell debris. The supernatant was used as a source of cystatin B. The 21aa deletion of canine Cys B was used. The N-terminus was sequenced using trypsin digestion and LC-MS identification. MMCGAPSASQPATADTQAIAD (SEQ ID NO: 2) Therefore, the complete amino acid sequence of canine Cys B (FL-Cys B) is as follows: It has been decided. MMCGAPSASQPATADTQAIADQVKAQLEERENKKYTTFKA VTFRSQVVAGTNYFIKVQVDDDEFVHLRVFQSLPHENKPL ALSSYQTNKAKHDELAYF (SEQ ID NO: 3)

[0136] Example 2: Antibodies against canine cystatin B A. Antibody production against recombinant proteins Recombinant IgG1 and IgG2 sequences of SEQ ID NO: 1 and SEQ ID NO: 3 were used as immunogens according to standard methodologies in rabbits. Polyclonal antibodies were generated using the same proteins. Antibodies were generated that specifically bound to each recombinant immunogen in an ELISA assay. .

[0137] Recombinant protein with SEQ ID NO:3 as immunogen was cultured according to standard methodology in mice. The protein is used with the adjuvant CPG to produce monoclonal antibodies. The seven antibodies were shown to specifically bind to their respective recombinant immunogens in an ELISA assay. Monoclonal antibodies were generated using HRP-goat anti-mouse IgG heavy and light chain secondary antibodies. Null binding was detected. All clones tested were resistant to 5 μg / ml of recombinant FL cystatin. Table 4 shows the activity of three of the clones. Illustrative binding data for one of the cases is shown.

[0138] [Table 5]

[0139] B: Antibody production against synthetic peptides The following peptides derived from canine cystatin B were conjugated to KLH. The conjugates were then incubated with rabbit antibodies. It was used as an immunogen for antibody production.

[0140] Cystatin BC terminal "peptide 9" QTNKAKHDELAYF (SEQ ID NO: 4) Cystatin B N-terminal "peptide 3-20" CGAPSASQPATADTQAIA( SEQ ID NO: 5) Cystatin B N-terminal "peptide 3-10" CGAPSASQ (SEQ ID NO: 6) Cystatin B N-terminal "peptide 18-25" CAIADQVKA (SEQ ID NO: 7) Cystatin B "Peptide 2" FQSLPHENKPLALSS (SEQ ID NO: 8) Cystatin B "Peptide 1" SQVVAGTPYFIKVQVDDD (SEQ ID NO: 9)

[0141] Additionally, SEQ ID NO:2 (N-terminus) was used as an immunogen according to standard methodologies in mice. and peptide of SEQ ID NO: 5 (peptide 3-20) with Freund's adjuvant. By doing so, polyclonal and monoclonal antibodies were produced. Antibodies were generated that specifically bound to each recombinant immunogen in an ELISA assay. Ta.

[0142] Thus, each antibody was generated to specifically bind to its respective peptide immunogen. For example, Table 5 below shows the results of the rabbit anti-peptide polyclonal antibody binding to peptides 1, 2, Typical binding curves of 9 and 9 with each immunogen are shown. All of the antibodies bound to the target with high affinity and were then used in cystatin B ELISA. SA can be used to form sandwiches.

[0143] [Table 6]

[0144] Furthermore, anti-peptide 9 antibody and anti-peptide 1 antibody showed no stimulatory activity in ELISA assay. Recombinant FL-Cys B (SEQ ID NO: 3) and Ne in the lysate of stimulated MDCK cells. Anti-peptide 9 antibody specifically bound to a native intracellular protein. Specific binding to the peptide KHDELAYF (SEQ ID NO: 10) in the assay was shown.

[0145] SEQ ID NOs: 11, 12, and 13 were used to immunize rabbits according to standard methodology. Each antibody specifically bound to its respective antigen in an ELISA assay.

[0146] Example 3: Immunoassay for the detection of canine cystatin B A cystatin B ELISA was developed as follows. Solid phase and capture antibody: A 96-well 4BX microtiter plate was filled with 10 μg / ml of affinity purified water. Plates are coated with Heron Anti-Peptide 9 overnight at 4° C. Plates are resuspended in 0.05% TWEE Wash three times with 1x PBS, pH 7.4, containing N® (Polysorbate) 20. The plates were then blocked with 1% BSA in 1x PBS, pH 7.4 for 2 hours. After washing as above, the plates are dried under vacuum at 37°C for 4 hours. The plates are stored dry at 4°C.

[0147] Preparation of detection antibody: From mice immunized with full-length recombinant cystatin B (rFL-Cys B) (SEQ ID NO: 3) Seven monoclonal antibodies were generated to evaluate their binding performance against rFL Cys B. One clone (3H4) was selected based on its activity and purified using protein G to give a 1.0 1 mg of purified antibody was purified using the SMCC method with horseradish peroxidase (HR The antibody was labeled with 0.25-2.0 P and desalted to remove excess HRP. ug / ml was used in the ELISA assay.

[0148] Cystatin B sandwich ELISA protocol: serum and urine Cystatin B is an intracellular protein and generally does not circulate freely in high concentrations. This was confirmed by the discovery of proteins in the supernatants collected from stressed canine kidney cells. However, cystatin B was not detected in the destroyed dogs. It was purified from kidney cells. Therefore, any cystatin B detected in serum is not a result of this breakdown. This may be due to necrosis or cell death of epithelial cells in the proximal tubules in active renal injury. Apoptosis and necrosis may result in increased serum and urinary cystatin B. There is (Figure 1).

[0149] Cystatin B has not been associated with kidney disease in companion animals. We produced a monoclonal antibody against recombinant cystatin B and confirmed its specificity by comparing it with that against cystatin C. The presence of these proteins was confirmed using Western blot analysis (Figure 2). A sandwich ELISA was also developed using this method.

[0150] Standards for sandwich ELISA were rFL-Cys B or MDCK cell pellets. Both standard preparations were quantified by LCMS. Urine samples were diluted in buffer A (0.1 M PO4; pH 7.4, containing at least 0.1% sarcosine). Serum samples were diluted at least 1:10 in buffer A (containing 100% ethanol). Both were diluted 1:100 and 100 ul was added to duplicate wells as above. 100 microliters (100 ul) was added in duplicate to the polyclonal antibody capture wells. The plate was then incubated at room temperature with shaking for 1 hour. Detergent (IDEXX Laboratories, Inc., Maine, US) Wash 6 times with A) and add 100ul of 0.25-2ug / ml HRP monoclonal detection antibody The detection antibody was added and incubated with shaking for 30 minutes, then washed six times. 100ul of the substrate TMB was added to the wells. The color was developed for 5 minutes, followed by 100ul of stopping Stopping solution (1N HCl) was added. The plate was then placed on a VMAX® Microplate Reader. The unknowns were read using a 4PL parameter fit in the Sigma plot. was quantified.

[0151] Native canine cystatin B (MDCK lysate) was used to detect Cy2+ as described above. A standard curve was obtained with the s B ELISA assay (Fig. 3 ).

[0152] Detection of canine cystatin B in patient specimens Cystatin B in dog urine using the cystatin B ELISA assay as described above The levels were determined and are shown in Table 6.

[0153] [Table 7]

[0154] As shown in Table 6, healthy dogs were treated with acute (active) kidney injury (AKI) and chronic kidney disease ( Urinary cystatin B levels were lower in AKI samples than in CKD samples. showed higher cystatin B levels than control.

[0155] Cystatin B ( Figure 4), and urinary system data from dogs presented to the clinic with active inflammatory or ischemia-induced renal damage. In a model system, serum creatinine was measured in dogs at 1.5 mJ / kg / day. Gentamicin was administered at 10 mg / kg every 8 hours until the blood pressure reached 100 g / dL. In dogs, this point was reached on day 8, while serum cystatin B was at baseline on day 1. These preliminary results suggest that cystatin B may be a key predictor of active renal damage. It is suggested that creatinine is an earlier marker than creatinine. A clear separation was observed between patients diagnosed with active renal disease.

[0156] Example 4: Cystatin B detection by oral swab Cotton swabs were used to sample the gums of dogs undergoing dental examinations. showed gingivitis, gum disease, and even severe tooth decay. The swabs were equilibrated to room temperature for 30 minutes and then added with a surfactant. The swabs were placed in 0.5 ml of cystatin B assay buffer for 30 minutes. A control swab without sample was used in the cystatin B assay described above. Control swabs were below the limit of detection (LOD) with an average OD of 0.04. In contrast, the OD of the swab from the dog that underwent extensive dental procedures was 1.04. This gives a signal to noise ratio of 26. See Table 7 below.

[0157] [Table 8]

[0158] The teeth of two dogs with periodontal disease were swabbed with cotton swabs and cystatin B ELI was analyzed. Cystatin B was extracted in SA buffer and the samples were subjected to the cystatin B assay. The positive control was a reference material spiked with 0.00 ng / ml of recombinant full-length canine cystatin B protein. The signal (OD at 450 nM) was measured using recombinant full-length canine cysteine. 0.322 for the tin B protein positive control, 1.8485 for dog 1, and 1.8485 for dog 2. The values ​​for the two dogs with periodontal disease were 1000 ng / ml of canine FL-Cys B reference material. It is a marker for periodontal disease in mammals such as dogs.

[0159] Example 5: Modified ELISA 96-well 4BX microtiter plates were filled with 5 μg / ml affinity-purified rabbit Plates were coated with anti-peptide 9 overnight at 4°C. Wash three times with 1x PBS containing TWEEN® (Polysorbate) 20. The plates were then blocked for 2 hours with 1x PBS, pH 7.4, containing 1% BSA. After washing as above, the plates are dried under vacuum at 37°C for 4 hours. Store the container dry at 4°C.

[0160] Standards for sandwich ELISA were rFL-Cys B or MDCK cell pellets. Both standard preparations were quantified by LCMS. The urine samples were diluted in buffer A (0.1 M phosphate, pH 7.2, containing at least 1.0% N-dodecahydronaphthalene). containing methyl-N-methylglycine sodium salt (Sarkosyl, Sigma) Serum samples were diluted at least 1:50 in buffer A and diluted 1:20. 100 microliters (100 μl) was added to the wells in duplicate as above. ) was added in duplicate to the polyclonal antibody capture wells and incubated at room temperature with shaking for 1 hour. The plates were then washed with PETCHEK® detergent (IDEXX L Wash six times with a 1000-ml syringe (Biolaboratories, Inc., Westbrook, Maine, USA). Wash and add 100ul of 0.25-2ug / ml HRP-conjugated monoclonal detection antibody The detection antibody was incubated for 30 minutes with shaking, then washed six times and 100 μl 1 μl of the substrate TMB was added to the wells. Color was developed for 5 minutes, followed by 100 μl of stop solution ( 1N HCl) was added. The plate was read using a VMAX® microplate reader. Read. Quantify the unknowns using 4PL parameter fits in a Sigma plot. did.

[0161] Example 6: Detection of human Cys-B In this example, an ELISA was performed to detect human Cys B using an anti-canine Cys B antibody. Demonstrate the construction of SA. Recombinant human cystatin B (rHFL Cys B) protein (SEQ ID NO: 14) was obtained from Genscript, USA. >sp|P04080|CYTB_Human Cystatin-B OS=Homo sapiens GN=CSTB PE=1 SV=2 MMCGAPSATQPATAETQHIADQVRSQLEEKENKKFPVFKA VSFKSQVVAGTNYFIKVHVG DEDFVHLRVFQSLPHENKPLTLSNYQTNKAKHDELTYF [ SEQ ID NO: 14]

[0162] Cross-reactivity of rH FL Cys B protein with antibodies raised against canine cystatin B Reactivity was assessed by sandwich ELISA. A mouse monoclonal antibody raised against a cystatin B sequence (rC FL cystatin B). was used as a capture reagent to bind multiple recombinant FL Cys B antigens in ELISA. The detection reagents screened were rabbit polyclonal N-terminal Anti-canine cystatin B antibody and horseradish peroxidase anti-species IgG (H&L) A series of reagent pairs were found that produced dose-dependent curves in ELISA. As shown in Table 8 below, the canine C-terminal "peptide 9" (QTNKAKH) Three monoclonal antibodies (IF10, 2B) were raised against DELAYF (SEQ ID NO: 4). 5, and 9A10) were incubated with anti-dog N-terminal cystatin B polyclonal antibody 327 (rabbit 328 (raised against CAIADQVKA (SEQ ID NO: 7) in rabbits), 328 (raised against CGA PSASQPATADTQAIA (SEQ ID NO: 5)), or 329 (raised in rabbits CGAPSASQ (SEQ ID NO: 6) in the sandwich E These pairs were then compared with rat and mouse rFl cystatin B. bound to the protein.

[0163] [Table 9]

[0164] Canine cystatin B was isolated using N-terminal rabbit polyclonal antibody 327 as a solid-phase capture. Monoclonal antibody generated against C-terminal "peptide 9" QTNKAKHDELAYF (SEQ ID NO: 4) Clonal mouse antibodies were analyzed for binding to rHFL Cystatin B. See Table 9. As shown in Table 10 below, a C-terminal monoclonal antibody (9A10) was used to whereas another C-terminal monoclonal antibody (2B5) formed a sandwich. The specificity of these monoclonal antibodies was mapped, and the 9A10 monoclonal antibody NAL recognized the homologous sequence of both human and canine cystatin B. rFL canine cystatin B The 3H4 monoclonal antibody was raised against and paired with the solid-phase-bound polyclonal antibody 327. The antibody showed binding to both human and canine rFL Cystatin B (Table 11).

[0165] [Table 10]

[0166] [Table 11]

[0167] [Table 12]

[0168] Antibodies specific for canine Cys B can be used to detect human Cys B. In particular, AYF, LAYF (SEQ ID NO: 20), ELAYF (SEQ ID NO: 21), DELAYF (SEQ ID NO: 22), HDELAYF (SEQ ID NO: 23), KHDELAYF (SEQ ID NO: 10 ), AKHDELAYF (SEQ ID NO: 24), KAKHDELAYF (SEQ ID NO: 25), N KAKHDELAYF (SEQ ID NO: 26), TNKAKHDELAYF (SEQ ID NO: 27), and QTNKAKHDELAYF (SEQ ID NO: 4), or a portion thereof. The antibody or its specific binding fragment can be used to detect Cys B in humans and dogs. can be done.

[0169] Example 7: Determination of reference ranges in dog and cat urine Urine samples from dogs and cats were collected at a local veterinary clinic over a two-year time frame. Urine was aliquoted and frozen until use. The ELISA assay described in Example 3 was used to identify the frozen Cystatin B was measured in frozen urine samples. kidney problems, such as attenuated or SDMA levels, history or symptoms of urinary stones or urinary tract infections Reference ranges were established from healthy animals with no signs of harm. Healthy dogs and 42 healthy cats were used, and the mean values ​​for each species were +3 standard deviations (Std. v) was used to determine the reference range of 257 ng / ml (see Figure 6). A healthy range is from about 0 ng / ml to about 257 ng / ml (e.g., about 0, 5, 10, 20, 50, 75, 100 to about 200, 210, 225, 250, or 257 ng / ml) Values ​​above 257 ng / ml (e.g., about 257, 260, 270, 280, 2 90, 300 ng / ml and above) are signs of kidney disease.

[0170] Example 8: Cystatin B in canine AKI populations 25 matched urine samples from dogs with clinically confirmed AKI and healthy dogs Serum samples were subjected to Cystatin B ELISA (Example 5). The etiology of AKI patients includes: Examples include nephrotoxic drugs, snakebites, sunstroke, ethylene glycol exposure, and infections. As shown in Figures 7A-B and Tables 12-13, the urinary and Serum cystatin B levels were significantly increased compared to healthy dogs and CKD patients.

[0171] [Table 13]

[0172] [Table 14]

[0173] Example 9: Cystatin B in infectious diseases The most common infectious diseases of the urinary system in animals are aerobic bacterial infections. Organisms include E. coli, staphylococci, enterococci, and streptococci. Organisms that cause bur infections include Klebsiella, Proteus, and Pseudomonas. Mycoplasma is a rare cause of urinary tract infections and is usually associated with bacteria. Leptospirosis is a zoonotic disease found worldwide. It is caused by filamentous Leptospira bacteria that infect the kidneys and many other organs. Rickettsiosis (rickettsioses) and related diseases (Anap The cause of scrub typhus (ehrlichiosis, Q fever, scrub typhus) is a gram-negative obligate intracellular rod-shaped bacterium. Babesia, a tick-borne disease, has also been implicated in kidney disease.

[0174] ELISA, PCR, and microscopic agglutination test (MAT) titers >1:800 (IDEXX 20 confirmed as Leptospira species positive by the National Institute of Allergy and Infectious Diseases (NIA) Laboratories, Inc. Serum samples from the canine patients were subjected to cystatin B ELISA. Dog serum samples were also subjected to ELISA to determine the mean serum cystatin B levels. As shown in Table 14, nine leptospirosis-positive samples (45%) were from healthy dogs. The serum relative cutoff (mean + 3SD) value (149.1 ng / ml) was exceeded (Figure 10 The data show that 45% of leptospirosis patients tested had renal impairment. Therefore, high serum levels of cystatin B are associated with Leptospira-infected patients. This indicates the presence of kidney damage in livestock.

[0175] [Table 15]

[0176] Example 10: Cystatin B in urinary tract infections Urine samples from 10 dogs with clinically confirmed urinary tract infection (UTI) and 1 A healthy cohort of 0 animals was subjected to cystatin B assay. UTI was defined as positive culture and clinical The interference from UTI significantly impacted the specificity of known AKI markers. Figure 8 shows the relationship between cystatin B levels in healthy animals and animals with urinary tract infections. Therefore, the cystatin B marker did not show any significant difference. can be used to distinguish between AKI and UTI.

[0177] Example 11: Cystatin B in feline kidney disease Urine samples from four cats diagnosed with kidney disease and three healthy cats were obtained from a local veterinary clinic. These were subjected to a cystatin B assay. The urinary cystatin B concentrations in the three healthy dogs were above the reference range of 257 ng / ml. Urinary cystatin B concentrations in each cat were within the reference range. See Table 15. Statin B is a marker of renal disease in cats.

[0178] [Table 16]

[0179] Example 12: Anti-cystatin B antibodies in sheep Two sheep were used to generate sheep polyclonal antibodies against SEQ ID NO: 4. The peptide was conjugated to KLH and emulsified in Freund's complete adjuvant. The antibody response in sheep serum was compared with that in rabbit serum. Similar titers were seen in sheep, even though it was early in the immunization protocol. Antibodies produced in rhesus can be used to detect Cys B polypeptides.

[0180] [Table 17]

[0181] Example 13: Detection of human urinary cystatin B in CKD patients Urine was collected from a human patient diagnosed with stage 4 CKD. The urine was subjected to cystatin B ELISA. Two anti-cystatin B monoclonal antibodies (3H4 and 3H5) were used. As shown in Figure 9, human Urine samples produced higher levels of cystatin B than canine AKI samples and lower levels than canine negative controls. Furthermore, different anti-cystatin B monoclonal antibodies were probably showed different responses due to the binding efficacies of the epitopes. Renal disease can be diagnosed in humans using antibodies specific for cystatin B.

Claims

1. 1. A method for diagnosing periodontal disease in a non-human subject, comprising: (a) determining the amount of cystatin B polypeptide in a sample from the subject, wherein the amount of cystatin B polypeptide is determined using one or more antibodies that specifically bind to one or more polypeptides consisting of SEQ ID NOs: 8, 9, 10, and 13; and (b) comparing the amount of cystatin B polypeptide in the sample with a control sample or control standard, wherein a higher level of cystatin B polypeptide in the sample compared to the control sample or control standard is indicative of periodontal disease in the subject.

2. 1. A method for distinguishing between an upper urinary tract infection and a lower urinary tract infection, comprising: (a) determining the amount of cystatin B polypeptide in a sample from a non-human subject, wherein the amount of cystatin B polypeptide is determined using one or more antibodies that specifically bind to one or more polypeptides consisting of SEQ ID NOs: 8, 9, 10, and 13; and (b) comparing the amount of cystatin B polypeptide in the sample with a control sample or control standard, wherein a higher level of cystatin B polypeptide in the sample compared to the control sample or control standard is indicative of an upper urinary tract infection in the subject.

3. 1. A method for distinguishing between acute kidney injury and a lower urinary tract infection, comprising: (a) determining the amount of cystatin B polypeptide in a sample from a non-human subject, wherein the amount of cystatin B polypeptide is determined using one or more antibodies that specifically bind to one or more polypeptides consisting of SEQ ID NOs: 8, 9, 10, and 13; and (b) comparing the amount of cystatin B polypeptide in the sample with a control sample or control standard, wherein a higher level of cystatin B polypeptide in the sample compared to the control sample or control standard is indicative of acute kidney injury in the subject.

4. The method of any one of claims 1, 2, or 3, wherein the amount of cystatin B polypeptide is determined by detecting a complex between cystatin B polypeptide and one or more antibodies that specifically bind to polypeptides consisting of or more of SEQ ID NOs: 8, 9, 10, and 13.

5. 5. The method of claim 1, wherein the antibody is immobilized on a solid support.

6. 6. The method of claim 1, 2, 3, 4, or 5, wherein the antibody is conjugated to one or more labels.

7. 5. The method of claim 4, further comprising contacting the complex of cystatin B polypeptide and one or more antibodies specific for one or more polypeptides of SEQ ID NOs: 3, 8, 9, 10, and 13 with an indicator reagent.

8. 8. The method of any of claims 2, 3, 4, 5, 6, or 7, wherein the sample is blood, serum, plasma, or urine.

9. The method of claim 1 , wherein the sample is saliva, plaque, gingival crevicular fluid, a gingival biopsy, or a tongue swab.

10. The method of any one of claims 1, 2, 3, 4, 5, 6, 7, 8, or 9, wherein the amount of the cystatin B polypeptide or cystatin B polypeptide is determined or detected by an immunoassay, a competitive immunoassay, a sandwich immunoassay, an enzyme-linked immunosorbent assay (ELISA), an immunoturbidimetric assay, a particle-enhanced immunoturbidimetric assay, a radioimmunoassay (RIA), or a Western blot assay.

11. A kit for diagnosing an upper urinary tract infection or periodontal disease, comprising: (a) one or more antibodies that specifically bind to one or more polypeptides consisting of SEQ ID NOs: 8, 9, 10, and 13; and (b) a kit comprising one or more reagents that promote binding of said one or more antibodies to a cystatin B polypeptide present in a sample from a non-human subject.

12. An immunoconjugate comprising: (i) one or more isolated antibodies that specifically bind to one or more polypeptides consisting of SEQ ID NOs: 8, 9, 10, and 13; and (ii) an immunoconjugate comprising one or more polypeptides that specifically bind to said one or more isolated antibodies.

13. The immune complex of claim 12, which is immobilized on a solid support.

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