Method for determining peptidylglycine α-amidate monooxygenase (PAM) and its use for diagnostic purposes.

Abstract

The present invention is directed to methods for diagnosing or prognosing a disease in a subject, and / or predicting the risk of developing a disease or an adverse event in said subject, and / or monitoring a disease or an adverse event in said subject, by measuring the level of peptidylglycine alpha amidating monooxygenase (PAM) and / or its isoforms and / or fragments thereof in a sample of a body fluid from said subject.

Description

[Technical Field]

[0001] This invention relates to a method for determining the level of PAM and / or its isoforms and / or fragments in a bodily fluid sample, and its use for diagnostic purposes. [Background technology]

[0002] Bioactive peptide hormones function as signaling molecules. Most bioactive peptide hormones are synthesized from larger, inactive precursor peptides. During their biosynthesis, these peptides undergo several cotranslational and posttranslational modifications, including cleavage of the signal peptide, intracellular proteolytic cleavage of the precursor propeptide by specific endopeptidases, primarily on basic residue pairs, removal of basic residues by carboxypeptidases, formation of disulfide bonds, and N- and O-glycosylation (Eipper et al. 1993. Protein Science 2(4): 489-97). More than half of known neuropeptides and endocrine peptides require additional modification steps to achieve full bioactivity, including the formation of a C-terminal α-amide group (Guembe, et al. 1999. J Histochem Cytochem 47(5): 623-36). This final step in peptide hormone biosynthesis involves the action of the bifunctional enzyme peptidylglycine α-amidate monooxygenase (PAM). PAM specifically recognizes the C-terminal glycine residue in its substrate and cleaves glyoxylic acid from the C-terminal glycine residue of the peptide in a two-step enzymatic reaction to produce an α-amidated peptide hormone at the C-terminus. The resulting α-amide group is derived from the cleaved C-terminal glycine (Prigge et al. 2004. Science 304(5672): 864-67). This amidation reaction occurs in the lumen of secretory granules prior to the exocytosis of the amidation product (Martinez and Treston 1996. Molecular and Cellular Endocrinol 123: 113-17). Examples of α-amidated peptides include adrenomedullin, substance P, vasopressin, neuropeptide Y, amyrin, calcitonin, and neurokinin A. However, it had previously been demonstrated that PAMs also catalyze the formation of α-amides from glycinated substrates such as non-peptide-like N-fatty acid acylglycines, which are converted by PAMs to primary fatty acid amides (PFAMs) such as oleic acid amides.The amidation activity of identified and purified peptidylglycine has been shown to be copper and ascorbate-dependent (Emeson et al. 1984. Journal of Neuroscience: 2604-13; Kumar et al. 2016. J Mol Endocrinol 56(4):T63-76; Wand et al. 1985. Neuroendocrinology 41: 482-89).

[0003] In humans, the PAM gene is located on chromosome 5q21.1, a 160 kb long chromosome containing 25 known exons (Gaier et al. 2014. BMC Endocrine Disorders 14). At least six isoforms are known to be generated by alternative splicing (SEQ ID NOs: 1-6). The PAM enzyme has been found to be expressed at various levels in almost all mammalian cell types, with significant expression observed in airway epithelium, endothelial cells, epithelial cells in the brain, adult atria, brain, kidney, pituitary gland, gastrointestinal tract, and reproductive tissues (Chen et al. 2018. Diabetes Obes Metab 20 Suppl 2:64-76; Oldham et al. 1992. Biochem Biophys Res Commun 184(1): 323-29; Schafer et al. 1992. J Neurosci 12(1): 222-34).

[0004] However, the highest activity of human PAM was recorded in the pituitary gland, pituitary stalk, and hypothalamus. Amidation activity in the plasma of healthy children under 15 years of age was significantly higher than that of healthy adults (Wand et al. 1985 Metabolism 34(11): 1044-52).

[0005] Figure 1 shows the precursor protein (amino acids 1-973) of PAM isoform 1 (SEQ ID NO: 1), the largest known PAM isoform encoded by PAM cDNA. The N-terminal signal sequence (amino acids 1-20) ensures the orientation of the developing PAM polypeptide into the secretory lumen of the endoplasmic reticulum, followed by co-translational cleavage. The PAM propeptide is then processed by the same mechanism used for the biosynthesis of integrated membrane proteins and secretory proteins, including cleavage of the pro region (amino acids 21-30), thereby ensuring proper folding, disulfide bond formation, phosphorylation, and glycosylation. (Bousquet-Moore et al. 2010. J Neurosci Res 88(12):2535-45).

[0006] As shown in Figure 1, the PAM cDNA further encodes two different enzymatic activities. The first enzymatic activity is named peptidylglycine α-hydroxylated monooxygenase (PHM; EC 1.14.17.3) and is an enzyme that can catalyze the conversion of the C-terminal glycine residue to α-hydroxyglycine. The second activity is named peptidyl α-hydroxyglycine α-amidate lyase (PAL; EC 4.3.2.5) and is an enzyme that can catalyze the conversion of α-hydroxyglycine to α-amide, followed by the release of glyoxylic acid. The sequential action of these separate enzymatic activities results in the overall peptidylglycine α-amidate activity. The first enzymatic activity (PHM) is located directly upstream of the pro region (within amino acids 31-494 of isoform 1 (SEQ ID NO: 7)). The second catalytic activity (PAL) is located after exon 16 (within amino acids 495-817 of isoform 1 (SEQ ID NO: 8)).

[0007] As shown in Figure 2, both activities may be encoded together within one polypeptide as membrane-bound proteins (isoforms 1, 2, 5, and 6; corresponding to SEQ ID NOs: 1, 2, 5, and 6) and within the same polypeptide as soluble proteins lacking a transmembrane domain (isoforms 3 and 4; corresponding to SEQ ID NOs: 3 and 4). Isoforms 1, 2, 5, and 6 remain within the outer plasma membrane after fusion of secretory vesicles with the plasma membrane and subsequent intracellular uptake and regeneration or degradation, while the soluble PAM isoforms lacking a TMD (isoforms 3 and 4) (amino acids 864-887) are co-secreted with peptide hormones (Wand et al. 1985 Metabolism 34(11): 1044-52). Furthermore, prohormone-converting enzymes can convert membrane-bound PAM proteins to soluble PAM proteins by cleaving them within the flexible region (exons 25 / 26) that links PAL to TMD in the secretory pathway (Bousquet-Moore et al. 2010. J Neurosci Res 88(12):2535-45):2535-45). The PHM subunit may also be cleaved from soluble or membrane-bound PAM in the secretory pathway by prohormone-converting enzymes that process the double basal cleavage site in the exon 16 region. Furthermore, during intracellular uptake, full-length PAM proteins may also be converted to a soluble form by the action of α- and γ-secreting enzymes (Bousquet-Moore et al. 2010. J Neurosci Res 88(12):2535-45). Membrane-bound PAM derived from late endosomes may be further secreted in the form of exosomal vesicles.

[0008] The activity of PHM and PAL, as well as the activity of full-length PAM, has been determined in several human tissues and body fluids. However, the individual PHM and PAL activities in soluble forms lead to the formation of C-terminal α-amidate products from C-terminal glycinated substrates, provided that these individual reactions can be performed in the same compartment, body fluid, or in vitro experimental apparatus. How the transfer of PHM hydroxylated products to PAL occurs is not currently precisely understood. There is evidence that the hydroxylated products are released into the liquid and are not directly transferred from PHM to PAL (Yin et al. 2011. PLoS One 6(12):e28679). The source of circulating PAM is also currently unknown.

[0009] Figure 2 shows a partial reaction of PHM. PHM is a copper-dependent monooxygenase responsible for the stereospecific hydroxylation of C-terminal glycine at the α-carbon atom. In the hydroxylation reaction, ascorbate is considered to be a naturally occurring reducing agent, but the oxygen in the newly formed hydroxyl group has been shown to originate from molecular oxygen. Figure 2 shows a partial reaction of PAL. The catalytic activity of PAL involves the abstraction of a proton from hydroxyglycine forming PHM by a base derived from the protein backbone, and nucleophilic attack of the oxygen of the hydroxyl group on a divalent metal, resulting in the cleavage of glyoxylic acid and the formation of the C-terminal amide.

[0010] Therefore, the terms “amidation activity,” “α-amidation activity,” “peptidylglycine α-amidation activity,” or “PAM activity” refer to the sequential enzymatic activity of PHM and PAL, but are independent of the splice variants or mixtures of splice variants of the present invention, or the post-translational modified PAM enzymes or the individual soluble PHM or PAL activity, or membrane-bound PAL or combinations thereof in all descriptive forms leading to the formation of peptide or non-peptide-like α-amidation products from soluble PHM and peptide or non-peptide-like glycinated substrates. In other words, the terms “amidation activity,” “α-amidation activity,” “peptidylglycine α-amidation activity,” or “PAM activity” may be described as the sequential action of enzymatic activity located within amino acids 31-817 in the propeptide encoded by human PAM cDNA, independently of the splice variants or mixtures of splice variants of the present invention.

[0011] PAM activity has been analyzed in several human tissues and fluids from healthy specimens, as well as in human tissues from several diseased individuals. The results of previous studies are summarized below.

[0012] For detecting PAM activity in human body fluids, 125 ID-TyrValGly, 125This includes the use of radiolabeled synthetic tripeptides such as IN-acetyl-TyrValGly or equivalent modified tripeptides, and the quantification of amidation products by γ-flash emission (Kapuscinski et al. 1993. Clinical Endocrinology 39(1): 51-58; Wand et al. 1985 Metabolism 34(11): 1044-52; Tsukamoto et al. 1995. Internal Medicine 34(4): 229-32; Wand et al. 1987 Neurology 37: 1057-61; Wand et al. 1985 Neuroendocrinol 41: 482-89). Furthermore, P-Gly or truncated neuropeptide Y-Gly were used as substrates for PAM activity testing (Gether et al. 1991 Mol Cell Endocrinol 79 (1-3): 53-63; Hyyppa et al. 1990 Pain 43: 163-68; Jeng et al. 1990 Analytical Biochemistry 185(2): 213-19).

[0013] The presence of α-amidal activity in human circulation was first demonstrated by Wand et al. (Wand et al. 1985 Metabolism 34(11): 1044-52). They reported that there was no difference between sexes, but there was some variation in PAM activity in certain pathological conditions; for example, plasma PAM activity was increased in adults with thyroid dysfunction and in patients with medullary thyroid carcinoma. Increased PAM activity was shown in the tissues of medullary thyroid carcinoma, pheochromocytoma, and pancreatic islet tumors, suggesting increased production of amidated peptides in endocrine tumor tissues (Gether et al. 1991 Mol Cell Endocrinol 79 (1-3): 53-63; Wand et al. 1985 Neuroendocrinol 41: 482-89).

[0014] Patients with multiple endocrine neoplasms (MEN-1) and fatal anemia showed reduced plasma PAM activity compared to healthy controls (Kapuscinski et al. 1993. Clin Endocrinol 39(1): 51-58).

[0015] The presence of amidation activity in human cerebrospinal fluid (CSF) had been demonstrated by Wand and his collaborators (Wand et al. 1985 Neuroendocrinol 41: 482-89). In patients with Alzheimer's disease (AD), plasma PAM activity was not altered compared to healthy controls, but PAM activity in CSF was significantly reduced compared to activity from normal samples (Wand et al. 1987 Neurology 37: 1057-61). Furthermore, international patent WO2015 / 103594 showed that the amount of PAM protein in CSF of AD patients, as detected by mass spectrometry, was reduced compared to healthy controls. In addition, it was shown that ADM-NH2, one of the amidation products of PAM, is reduced in patients with progressive and incidental Alzheimer's disease (WO2019 / 154900). However, to date, no direct association has been reported between cyclic PAM activity and the prediction, diagnosis, or progression of Alzheimer's disease (AD).

[0016] The amidation activity in CSF of patients with low back pain was analyzed using 1 to 12 P-Gly (Sp-Gly) substrates (Hyyppa et al. 1990 Pain 43: 163-68). It was shown that PAM activity increased in CSF and significantly decreased in serum in patients with multiple sclerosis (MS) (Tsukamoto et al. 1995. Internal Medicine 34(4): 229-32; WO2010 / 005387). The association between plasma PAM activity and type 2 diabetes is described in (WO2014 / 118634). [Overview of the project]

[0017] Although several discoveries have been made regarding the relationship between PAM activity in human body fluids and diseases or disease progression, there is in particular no information relating to PAM concentrations measured by immunoassay in circulating human body fluids. Measuring the level of PAM as the total amount or activity of PAM in a subject's body fluid for the diagnosis, prognosis, prediction or monitoring of a disease or adverse event is an unexpected discovery according to the present invention.

[0018] The subject matter of the present application is a method for diagnosing or prognosing a disease in a subject, and / or a method for predicting the risk of developing a disease or adverse event in a subject, and / or a method for monitoring a disease or adverse event in a subject, by measuring the level of peptidylglycine α-amidating monooxygenase (PAM) and / or isoforms thereof and / or fragments thereof in a body fluid sample from the subject, said disease in the subject is selected from the group comprising dementia, cardiovascular disorders, kidney disease, cancer, inflammation or infection, and / or metabolic diseases, adverse events are selected from the group comprising cardiac events, cardiovascular events, cerebrovascular events, cancer, diabetes, infectious diseases, severe infectious diseases, sepsis-like systemic infections, sepsis, and death from any of these causes. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] [Figure 1] A schematic diagram of PAM isoform 1 is shown. The thick black arrow indicates the cleavage site at dibasic amino acids. [Figure 2] The enzymatic reaction catalyzed by PAM is shown. [Figure 3] A representative calibration curve of recombinant PAM (ADM maturation activity [AMA]) is shown. [Figure 4] The frequency distribution (histogram) of AMA in self-reported healthy subjects (n=120) is shown. [Figure 5] The correlation of AMA in dual substrates (Li-heparin and serum) from self-reported healthy subjects (n=20) is shown. [Figure 6A]A-L show typical calibration curves for the PAM sandwich immunoassay. The following (A)-(J) containing recombinant PAM were prepared as calibration materials: (A) Solid phase: antibody against peptide 10 (sequence number 20), tracer: antibody against peptide 9 (sequence number 19); (B) Solid phase: antibody against peptide 10 (sequence number 20), tracer: antibody against peptide 10 (sequence number 20); (C) Solid phase: antibody against peptide 9 (sequence number 19), tracer: antibody against peptide 10 (sequence number 20); (D) Solid phase: antibody against recombinant PAM (sequence number 10), tracer: antibody against recombinant PAM (sequence number 10); (E) Solid phase: antibody against peptide 10 (sequence number 20), tracer: antibody against recombinant PAM (sequence number 10); (F) Solid phase: Antibody against peptide 13 (SEQ ID NO: 23), tracer: Antibody against peptide 10 (SEQ ID NO: 20); (G) Solid phase: Antibody against peptide 14 (SEQ ID NO: 24), tracer: Antibody against peptide 13 (SEQ ID NO: 23); (H) Solid phase: Antibody against recombinant PAM (SEQ ID NO: 10), tracer: Antibody against peptide 13 (SEQ ID NO: 23); (I) Solid phase: Antibody against peptide 13 (SEQ ID NO: 23), tracer: Antibody against peptide 9 (SEQ ID NO: 19); (J) Solid phase: Antibody against peptide 10 (SEQ ID NO: 20), tracer: Antibody against peptide 13 (SEQ ID NO: 23). (K) and (L) containing natural PAM (EDTA-plasma) were prepared as calibration materials. (K) Solid phase: Antibody against peptide 14 (SEQ ID NO: 24), tracer: Antibody against peptide 13 (SEQ ID NO: 23); (L) Solid phase: Antibody against peptide 10 (SEQ ID NO: 20), tracer: Antibody against peptide 13 (SEQ ID NO: 23). [Figure 6B-C]A-L show typical calibration curves for the PAM sandwich immunoassay. The following (A)-(J) containing recombinant PAM were prepared as calibration materials: (A) Solid phase: antibody against peptide 10 (sequence number 20), tracer: antibody against peptide 9 (sequence number 19); (B) Solid phase: antibody against peptide 10 (sequence number 20), tracer: antibody against peptide 10 (sequence number 20); (C) Solid phase: antibody against peptide 9 (sequence number 19), tracer: antibody against peptide 10 (sequence number 20); (D) Solid phase: antibody against recombinant PAM (sequence number 10), tracer: antibody against recombinant PAM (sequence number 10); (E) Solid phase: antibody against peptide 10 (sequence number 20), tracer: antibody against recombinant PAM (sequence number 10); (F) Solid phase: Antibody against peptide 13 (SEQ ID NO: 23), tracer: Antibody against peptide 10 (SEQ ID NO: 20); (G) Solid phase: Antibody against peptide 14 (SEQ ID NO: 24), tracer: Antibody against peptide 13 (SEQ ID NO: 23); (H) Solid phase: Antibody against recombinant PAM (SEQ ID NO: 10), tracer: Antibody against peptide 13 (SEQ ID NO: 23); (I) Solid phase: Antibody against peptide 13 (SEQ ID NO: 23), tracer: Antibody against peptide 9 (SEQ ID NO: 19); (J) Solid phase: Antibody against peptide 10 (SEQ ID NO: 20), tracer: Antibody against peptide 13 (SEQ ID NO: 23). (K) and (L) containing natural PAM (EDTA-plasma) were prepared as calibration materials. (K) Solid phase: Antibody against peptide 14 (SEQ ID NO: 24), tracer: Antibody against peptide 13 (SEQ ID NO: 23); (L) Solid phase: Antibody against peptide 10 (SEQ ID NO: 20), tracer: Antibody against peptide 13 (SEQ ID NO: 23). [Figure 6D-E]A to L show typical calibration curves for the PAM sandwich immunoassay. The following (A) to (J) containing recombinant PAM as calibration materials were prepared. (A) Solid phase: antibody against peptide 10 (SEQ ID NO: 20), tracer: antibody against peptide 9 (SEQ ID NO: 19); (B) Solid phase: antibody against peptide 10 (SEQ ID NO: 20), tracer: antibody against peptide 10 (SEQ ID NO: 20); (C) Solid phase: antibody against peptide 9 (SEQ ID NO: 19), tracer: antibody against peptide 10 (SEQ ID NO: 20); (D) Solid phase: antibody against recombinant PAM (SEQ ID NO: 10), tracer: antibody against recombinant PAM (SEQ ID NO: 10); (E) Solid phase: antibody against peptide 10 (SEQ ID NO: 20), tracer: antibody against recombinant PAM (SEQ ID NO: 10); (F) Solid phase: antibody against peptide 13 (SEQ ID NO: 23), tracer: antibody against peptide 10 (SEQ ID NO: 20); (G) Solid phase: antibody against peptide 14 (SEQ ID NO: 24), tracer: antibody against peptide 13 (SEQ ID NO: 23); (H) Solid phase: antibody against recombinant PAM (SEQ ID NO: 10), tracer: antibody against peptide 13 (SEQ ID NO: 23); (I) Solid phase: antibody against peptide 13 (SEQ ID NO: 23), tracer: antibody against peptide 9 (SEQ ID NO: 19); (J) Solid phase: antibody against peptide 10 (SEQ ID NO: 20), tracer: antibody against peptide 13 (SEQ ID NO: 23). (K) and (L) containing natural PAM (EDTA-plasma) as calibration materials were prepared. (K) Solid phase: antibody against peptide 14 (SEQ ID NO: 24), tracer: antibody against peptide 13 (SEQ ID NO: 23); (L) Solid phase: antibody against peptide 10 (SEQ ID NO: 20), tracer: antibody against peptide 13 (SEQ ID NO: 23). [Figure 6F-G]A-L show typical calibration curves for the PAM sandwich immunoassay. The following (A)-(J) containing recombinant PAM were prepared as calibration materials: (A) Solid phase: antibody against peptide 10 (sequence number 20), tracer: antibody against peptide 9 (sequence number 19); (B) Solid phase: antibody against peptide 10 (sequence number 20), tracer: antibody against peptide 10 (sequence number 20); (C) Solid phase: antibody against peptide 9 (sequence number 19), tracer: antibody against peptide 10 (sequence number 20); (D) Solid phase: antibody against recombinant PAM (sequence number 10), tracer: antibody against recombinant PAM (sequence number 10); (E) Solid phase: antibody against peptide 10 (sequence number 20), tracer: antibody against recombinant PAM (sequence number 10); (F) Solid phase: Antibody against peptide 13 (SEQ ID NO: 23), tracer: Antibody against peptide 10 (SEQ ID NO: 20); (G) Solid phase: Antibody against peptide 14 (SEQ ID NO: 24), tracer: Antibody against peptide 13 (SEQ ID NO: 23); (H) Solid phase: Antibody against recombinant PAM (SEQ ID NO: 10), tracer: Antibody against peptide 13 (SEQ ID NO: 23); (I) Solid phase: Antibody against peptide 13 (SEQ ID NO: 23), tracer: Antibody against peptide 9 (SEQ ID NO: 19); (J) Solid phase: Antibody against peptide 10 (SEQ ID NO: 20), tracer: Antibody against peptide 13 (SEQ ID NO: 23). (K) and (L) containing natural PAM (EDTA-plasma) were prepared as calibration materials. (K) Solid phase: Antibody against peptide 14 (SEQ ID NO: 24), tracer: Antibody against peptide 13 (SEQ ID NO: 23); (L) Solid phase: Antibody against peptide 10 (SEQ ID NO: 20), tracer: Antibody against peptide 13 (SEQ ID NO: 23). [Figure 6H-I]A-L show typical calibration curves for the PAM sandwich immunoassay. The following (A)-(J) containing recombinant PAM were prepared as calibration materials: (A) Solid phase: antibody against peptide 10 (sequence number 20), tracer: antibody against peptide 9 (sequence number 19); (B) Solid phase: antibody against peptide 10 (sequence number 20), tracer: antibody against peptide 10 (sequence number 20); (C) Solid phase: antibody against peptide 9 (sequence number 19), tracer: antibody against peptide 10 (sequence number 20); (D) Solid phase: antibody against recombinant PAM (sequence number 10), tracer: antibody against recombinant PAM (sequence number 10); (E) Solid phase: antibody against peptide 10 (sequence number 20), tracer: antibody against recombinant PAM (sequence number 10); (F) Solid phase: Antibody against peptide 13 (SEQ ID NO: 23), tracer: Antibody against peptide 10 (SEQ ID NO: 20); (G) Solid phase: Antibody against peptide 14 (SEQ ID NO: 24), tracer: Antibody against peptide 13 (SEQ ID NO: 23); (H) Solid phase: Antibody against recombinant PAM (SEQ ID NO: 10), tracer: Antibody against peptide 13 (SEQ ID NO: 23); (I) Solid phase: Antibody against peptide 13 (SEQ ID NO: 23), tracer: Antibody against peptide 9 (SEQ ID NO: 19); (J) Solid phase: Antibody against peptide 10 (SEQ ID NO: 20), tracer: Antibody against peptide 13 (SEQ ID NO: 23). (K) and (L) containing natural PAM (EDTA-plasma) were prepared as calibration materials. (K) Solid phase: Antibody against peptide 14 (SEQ ID NO: 24), tracer: Antibody against peptide 13 (SEQ ID NO: 23); (L) Solid phase: Antibody against peptide 10 (SEQ ID NO: 20), tracer: Antibody against peptide 13 (SEQ ID NO: 23). [Figure 6J-K]A-L show typical calibration curves for the PAM sandwich immunoassay. The following (A)-(J) containing recombinant PAM were prepared as calibration materials: (A) Solid phase: antibody against peptide 10 (sequence number 20), tracer: antibody against peptide 9 (sequence number 19); (B) Solid phase: antibody against peptide 10 (sequence number 20), tracer: antibody against peptide 10 (sequence number 20); (C) Solid phase: antibody against peptide 9 (sequence number 19), tracer: antibody against peptide 10 (sequence number 20); (D) Solid phase: antibody against recombinant PAM (sequence number 10), tracer: antibody against recombinant PAM (sequence number 10); (E) Solid phase: antibody against peptide 10 (sequence number 20), tracer: antibody against recombinant PAM (sequence number 10); (F) Solid phase: Antibody against peptide 13 (SEQ ID NO: 23), tracer: Antibody against peptide 10 (SEQ ID NO: 20); (G) Solid phase: Antibody against peptide 14 (SEQ ID NO: 24), tracer: Antibody against peptide 13 (SEQ ID NO: 23); (H) Solid phase: Antibody against recombinant PAM (SEQ ID NO: 10), tracer: Antibody against peptide 13 (SEQ ID NO: 23); (I) Solid phase: Antibody against peptide 13 (SEQ ID NO: 23), tracer: Antibody against peptide 9 (SEQ ID NO: 19); (J) Solid phase: Antibody against peptide 10 (SEQ ID NO: 20), tracer: Antibody against peptide 13 (SEQ ID NO: 23). (K) and (L) containing natural PAM (EDTA-plasma) were prepared as calibration materials. (K) Solid phase: Antibody against peptide 14 (SEQ ID NO: 24), tracer: Antibody against peptide 13 (SEQ ID NO: 23); (L) Solid phase: Antibody against peptide 10 (SEQ ID NO: 20), tracer: Antibody against peptide 13 (SEQ ID NO: 23). [Figure 6L]A-L show typical calibration curves for the PAM sandwich immunoassay. The following (A)-(J) containing recombinant PAM were prepared as calibration materials: (A) Solid phase: antibody against peptide 10 (sequence number 20), tracer: antibody against peptide 9 (sequence number 19); (B) Solid phase: antibody against peptide 10 (sequence number 20), tracer: antibody against peptide 10 (sequence number 20); (C) Solid phase: antibody against peptide 9 (sequence number 19), tracer: antibody against peptide 10 (sequence number 20); (D) Solid phase: antibody against recombinant PAM (sequence number 10), tracer: antibody against recombinant PAM (sequence number 10); (E) Solid phase: antibody against peptide 10 (sequence number 20), tracer: antibody against recombinant PAM (sequence number 10); (F) Solid phase: Antibody against peptide 13 (SEQ ID NO: 23), tracer: Antibody against peptide 10 (SEQ ID NO: 20); (G) Solid phase: Antibody against peptide 14 (SEQ ID NO: 24), tracer: Antibody against peptide 13 (SEQ ID NO: 23); (H) Solid phase: Antibody against recombinant PAM (SEQ ID NO: 10), tracer: Antibody against peptide 13 (SEQ ID NO: 23); (I) Solid phase: Antibody against peptide 13 (SEQ ID NO: 23), tracer: Antibody against peptide 9 (SEQ ID NO: 19); (J) Solid phase: Antibody against peptide 10 (SEQ ID NO: 20), tracer: Antibody against peptide 13 (SEQ ID NO: 23). (K) and (L) containing natural PAM (EDTA-plasma) were prepared as calibration materials. (K) Solid phase: Antibody against peptide 14 (SEQ ID NO: 24), tracer: Antibody against peptide 13 (SEQ ID NO: 23); (L) Solid phase: Antibody against peptide 10 (SEQ ID NO: 20), tracer: Antibody against peptide 13 (SEQ ID NO: 23). [Figure 6M] M to O indicate the Enzyme Capture Assay (ECA). (M) Solid-phase antibody against peptide 10 (SEQ ID NO: 20); (N) Solid-phase antibody against full-length PAM (SEQ ID NO: 10); (O) Solid-phase antibodies against peptide 7 (SEQ ID NO: 17), peptide 8 (SEQ ID NO: 18), peptide 9 (SEQ ID NO: 19), peptide 13 (SEQ ID NO: 23), and peptide 14 (SEQ ID NO: 24) in recombinant PAM / heparinized plasma used as the sample. [Figure 6N-O]M to O indicate the Enzyme Capture Assay (ECA). (M) Solid-phase antibody against peptide 10 (SEQ ID NO: 20); (N) Solid-phase antibody against full-length PAM (SEQ ID NO: 10); (O) Solid-phase antibodies against peptide 7 (SEQ ID NO: 17), peptide 8 (SEQ ID NO: 18), peptide 9 (SEQ ID NO: 19), peptide 13 (SEQ ID NO: 23), and peptide 14 (SEQ ID NO: 24) in recombinant PAM / heparinized plasma used as the sample. [Figure 6P] P represents the correlation between PAM activity and PAM concentration in heparin samples from healthy volunteers (n=26; Spearman correlation r=0.49, p=0.0109). [Figure 7] This shows a typical ADM-Gly dose / signal curve. [Figure 8] This shows the ADM maturation activity (PAM activity) in MPP studies (prediction of Alzheimer's disease). [Figure 9] The Kaplan-Meier plot (prediction of Alzheimer's disease [AD] in MPP studies) is shown. [Figure 10] This shows ADM maturation activity (PAM activity) in MPP studies (prediction of colorectal cancer [CRC]). [Figure 11] This shows MR-proADM in the MPP study (prediction of colorectal cancer [CRC]). [Figure 12] The Kaplan-Meier plot (prediction of colorectal cancer [CRC] in the MPP study) is shown. [Figure 13] This shows the diagnosis of pancreatic cancer in the MPP study. [Figure 14] This shows the receiver response characteristic curve (ROC plot) of ADM maturation activity (PAM activity) for the diagnosis of pancreatic cancer (MPP study). [Figure 15] The Kaplan-Meier plot (prediction of all-cause mortality in the MPP study) is shown. [Figure 16] The Kaplan-Meier plot (prediction of cardiovascular mortality in the MPP study) is shown. [Figure 17] A Kaplan-Meier plot (prediction of heart failure in the MPP study) is shown. [Figure 18]The Kaplan-Meier plot (prediction of atrial fibrillation in the MPP study) is shown. [Figure 19] It exhibits ADM maturation activity (PAM activity) for the diagnosis of progressive Alzheimer's disease (AD). [Figure 20] This shows ADM maturation activity (PAM activity) for determining the outcome of sepsis / septic shock in the AdrenOSS-1 study (n=145 survivors; n=52 non-survivors). [Figure 21] The Kaplan-Meier plot of ADM maturation activity (PAM activity) against mortality over 28 days (AdrenOSS-1 study) is shown. [Figure 22] This shows the ADM maturation activity (PAM activity) in the saliva of healthy individuals (n=5). [Modes for carrying out the invention]

[0020] One embodiment of this application is a method for diagnosing or prognosing a disease in a subject, and / or predicting the risk of a disease or adverse event occurring in a subject, and / or monitoring a disease or adverse event in a subject, by measuring the levels of peptidylglycine α-amidate monooxygenase (PAM) and / or its isoform and / or fragment in a body fluid sample of the subject, the method being - A step of measuring PAM and / or its isoform and / or fragments in the target body fluid sample, and The process includes comparing the measured value with a predetermined threshold, • If the measured value falls below or exceeds the threshold, the subject is diagnosed with a disease, or • If the measured value falls below or exceeds the threshold, the outcome of the disease is judged as a prognosis, or • If the measured value falls below or exceeds the threshold, it is predicted that there is a risk of disease or adverse event occurring, or This includes monitoring for the aforementioned disease or adverse event.

[0021] A preferred embodiment of this application relates to a method for diagnosing or prognosing a disease in a subject, and / or predicting the risk of a disease or adverse event occurring in a subject, and / or monitoring a disease or adverse event in a subject, by measuring the level of peptidylglycine α-amidate monooxygenase (PAM) and / or its isoform and / or fragment in a body fluid sample of the subject, wherein the level of PAM and / or its isoform and / or fragment is the total concentration of PAM and / or its isoform and / or fragment having at least 12 amino acids in the body fluid sample of the subject, or the activity of PAM and / or its isoform and / or fragment.

[0022] Another embodiment of this application relates to a method for diagnosing or prognosing a disease in a subject, and / or predicting the risk of a disease or adverse event occurring in a subject, and / or monitoring a disease or adverse event in a subject, by measuring the level of peptidylglycine α-amidate monooxygenase (PAM) and / or its isoforms and / or fragments in a body fluid sample of the subject, wherein the activity of PAM and / or its isoforms and / or fragments is selected from the group including the sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 10.

[0023] As shown in the sequence number list, it is known to those skilled in the art that the PAM isoform sequences (sequence numbers 1-6) contain an N-terminal signal sequence (amino acids 1-20) that is cleaved before protein secretion. Therefore, in a preferred embodiment, the PAM isoform sequences (sequence numbers 1-6) and / or fragments thereof do not contain an N-terminal signal sequence.

[0024] Another embodiment of this application relates to a method for diagnosing or prognosing a disease in a subject, and / or predicting the risk of a disease or adverse event occurring in a subject, and / or monitoring a disease or adverse event in a subject, by measuring the level of peptidylglycine α-amidate monooxygenase (PAM) and / or its isoforms and / or fragments in a body fluid sample of the subject, wherein the total concentration of PAM and / or its isoforms and / or fragments having at least 12 amino acids is detected by an immunoassay.

[0025] Another specific embodiment of this application relates to a method for diagnosing or prognosing disease in a subject, and / or predicting the risk of disease or adverse events occurring in a subject, and / or monitoring disease or adverse events in a subject, by measuring the level of peptidylglycine α-amidate monooxygenase (PAM) and / or its isoforms and / or fragments in a body fluid sample of the subject, wherein the activity of PAM and / or its isoforms and / or fragments is detected using peptide-Gly as a substrate.

[0026] Another preferred embodiment of this application relates to a method for diagnosing or prognosing disease in a subject, and / or predicting the risk of disease or adverse events occurring in a subject, and / or monitoring disease or adverse events in a subject, by measuring the levels of peptidylglycine α-amidate monooxygenase (PAM) and / or its isoforms and / or fragments in a body fluid sample of the subject, wherein the peptide-Gly substrate is adrenomedullin (ADM), adrenomedullin-2, short intermedin, proadrenomedullin N-20 terminal peptide (PAMP), amylin, gastrin-releasing peptide, neuromedin C, neuromedin B, neuromedin S, neuromedin U, calcitonin, calcitonin gene-related peptide (CGRP) 1 and 2 The group includes pancreatic islet amyloid polypeptide, chromogranin A, insulin, pancreastatin, prolactin-releasing peptide (PrRP), cholecystokinin, big gastrin, gastrin, glucagon-like peptide 1 (GLP-1), pituitary adenylyl cyclase-activating polypeptide (PACAP), secretin, somatriverine, peptide histidine methionine (PHM), vasoactive intestinal peptide (VIP), gonadriverine, kisspeptin, MIF-1, metastine, neuropeptide K, neuropeptide γ, substance P, neurokinin A, neurokinin B, peptide YY, pancreatic hormone, deltorphin I, orexin A and B, melanotropin α (α-MSH), melanotropin γ, thyroid-stimulating-releasing hormone (TRH), oxytocin, and vasopressin.

[0027] One embodiment of the present application relates to a method for diagnosing or prognosing a disease in a subject, and / or predicting the risk of a disease or adverse event occurring in a subject, and / or monitoring a disease or adverse event in a subject, by measuring the level of PAM and / or its isoforms and / or fragments in a body fluid sample of the subject, wherein PAM and / or its isoforms and / or fragments are selected from the group including SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 10.

[0028] Another embodiment of the present application relates to a method for diagnosing or prognosing disease in a subject, and / or predicting the risk of disease or adverse events occurring in a subject, and / or monitoring disease or adverse events in a subject, by measuring the levels of PAM and / or its isoforms and / or fragments in a body fluid sample of the subject, wherein the risk of disease occurring in the subject is determined when the subject is a healthy subject.

[0029] Another embodiment of the present application relates to a method for diagnosing or prognosing a disease in a subject by measuring the levels of PAM and / or its isoforms and / or fragments in a subject's bodily fluid sample, and / or predicting the risk of a disease or adverse event occurring in a subject, and / or monitoring a disease or adverse event in a subject, wherein the disease is selected from the group of Alzheimer's disease, colorectal cancer, and pancreatic cancer.

[0030] Another specific embodiment of this application relates to a method for determining the level of PAM and / or its isoforms and / or fragments in a body fluid sample using a test method, wherein the test method comprises two binders that bind to two different regions of PAM, the two binders target epitopes of at least five amino acid lengths, preferably at least four amino acid lengths, and the two binders target epitopes contained within the following sequences of PAM: peptide 1 (sequence number 11), peptide 2 (sequence number 12), peptide 3 (sequence number 13), peptide 4 (sequence number 14), peptide 5 (sequence number 15), peptide 6 (sequence number 16), peptide 7 (sequence number 17), peptide 8 (sequence number 18), peptide 9 (sequence number 19), peptide 10 (sequence number 20), peptide 11 (sequence number 21), peptide 12 (sequence number 22), peptide 13 (sequence number 23), peptide 14 (sequence number 24), and recombinant PAM (sequence number 10).

[0031] Further embodiments of this application relate to a method for measuring the activity of PAM and / or its isoforms or fragments thereof in a body fluid sample of interest, the method being: - A step of contacting the sample with a scavenging agent that specifically binds to the active full-length PAM, its isoforms, and / or its active fragments. - A step of separating the PAM bound to the aforementioned scavenging agent, The steps of adding a PAM substrate to the separated PAM, This includes a step of quantifying PAM activity by measuring the substrate conversion rate of PAM.

[0032] Another embodiment of this application relates to a method for measuring the activity of PAM and / or its isoforms and / or fragments in a body fluid sample of interest, the method being: - A step of contacting the sample with the PAM substrate (peptide-Gly) at time intervals from t=0 minutes to t=n+1 minutes. The steps include: detecting the reaction product of PAM (α-amidate peptide) in the sample at t=0 min and t=n+1 min; The process includes a step of calculating the difference in reaction products between t=0 and t=n+1 minutes to quantify the activity of PAM.

[0033] A particular embodiment of this application relates to a method wherein the peptide-Gly substrate is adrenomedullin (ADM), adrenomedullin-2, short intermedin, proadrenomedullin N-20 terminal peptide (PAMP), amylin, gastrin-releasing peptide, neuromedin C, neuromedin B, neuromedin S, neuromedin U, calcitonin, calcitonin gene-related peptide (CGRP) 1 and 2, islet amyloid polypeptide, chromogranin A, insulin, pancreastatin, prolactin-releasing peptide (PrRP), cholecystokinin, big gastrin, gastrin The group includes phosphorus, glucagon-like peptide 1 (GLP-1), pituitary adenylate cyclase-activating polypeptide (PACAP), secretin, somatriverine, peptide histidine methionine (PHM), vasoactive intestinal peptide (VIP), gonadriverine, kisspeptin, MIF-1, metastine, neuropeptide K, neuropeptide γ, substance P, neurokinin A, neurokinin B, peptide YY, pancreatic hormone, deltorphin I, orexin A and B, melanotropin α (α-MSH), melanotropin γ, thyroid-stimulating-releasing hormone (TRH), oxytocin, and vasopressin.

[0034] Another embodiment of this application relates to the use of an antibody for measuring the level of PAM and / or its isoforms and / or fragments, wherein the antibody specifically binds to sequences selected from the group consisting of recombinant PAM (SEQ ID NO: 10), peptide 1 (SEQ ID NO: 11), peptide 2 (SEQ ID NO: 12), peptide 3 (SEQ ID NO: 13), peptide 4 (SEQ ID NO: 14), peptide 5 (SEQ ID NO: 15), peptide 6 (SEQ ID NO: 16), peptide 7 (SEQ ID NO: 17), peptide 8 (SEQ ID NO: 18), peptide 9 (SEQ ID NO: 19), peptide 10 (SEQ ID NO: 20), peptide 11 (SEQ ID NO: 21), peptide 12 (SEQ ID NO: 22), peptide 13 (SEQ ID NO: 23), and peptide 14 (SEQ ID NO: 24).

[0035] Another preferred embodiment of this application relates to a kit for measuring the level of PAM, comprising one or more antibodies that bind to a PAM sequence selected from the group including recombinant PAM (SEQ ID NO: 10), peptide 1 (SEQ ID NO: 11), peptide 2 (SEQ ID NO: 12), peptide 3 (SEQ ID NO: 13), peptide 4 (SEQ ID NO: 14), peptide 5 (SEQ ID NO: 15), peptide 6 (SEQ ID NO: 16), peptide 7 (SEQ ID NO: 17), peptide 8 (SEQ ID NO: 18), peptide 9 (SEQ ID NO: 19), peptide 10 (SEQ ID NO: 20), peptide 11 (SEQ ID NO: 21), peptide 12 (SEQ ID NO: 22), peptide 13 (SEQ ID NO: 23), and peptide 14 (SEQ ID NO: 24).

[0036] The object of the present invention is to provide a method for measuring the levels of PAM and / or its isoforms and / or fragments in a bodily fluid sample. Providing a test method and a kit is also an object of the present invention.

[0037] Another object of the present invention is to provide a method for diagnosing or prognosing disease in a subject by measuring the levels of PAM and / or its isoforms and / or fragments in a subject's body fluid sample, and / or a method for predicting the risk of disease or adverse events occurring in a subject, and / or a method for monitoring disease or adverse events in a subject.

[0038] Another important embodiment of the present invention is a method for diagnosing or prognosing a disease in a subject, and / or predicting the risk of a disease or adverse event occurring in a subject, and / or monitoring a disease or adverse event in a subject, the method being The steps include: measuring the level of PAM and / or its isoforms and / or fragments in the body fluid sample of the subject, and The process includes comparing the measured value with a predetermined threshold, Here, if the measured value falls below or exceeds the predetermined threshold, the subject is diagnosed with a disease, or Here, if the measured value falls below or exceeds the predetermined threshold, the outcome of the disease is judged as a prognosis, or Here, if the measured value falls below or exceeds the predetermined threshold, it is predicted that there is a risk of disease or adverse event occurring, or This includes monitoring for the aforementioned disease or adverse event.

[0039] Methods for measuring the level of PAM are known in the art. In the context of the methods for diagnosing or prognosing disease in subjects, and / or predicting the risk of disease or adverse events occurring in subjects, and / or monitoring disease or adverse events in subjects, any of the methods and tests of the current art may be used, or the aforementioned methods and tests for measuring the level of PAM may be used.

[0040] Thresholds are preset by measuring the levels of PAM and / or its isoforms and / or fragments in healthy controls and calculating, for example, the corresponding 75th percentile, more preferably the 90th percentile, and even more preferably the 95th percentile. The upper limit of the 75th percentile, more preferably the 90th percentile, and even more preferably the 95th percentile is set as a threshold for patients with disease compared to healthy patients, or a threshold for subjects at risk of developing disease compared to healthy subjects, or a threshold for subjects at risk of developing adverse events compared to subjects not at risk of developing adverse events, when the level of subjects with disease or subjects at risk of developing disease or adverse events exceeds a specific threshold. Thresholds are preset by measuring the levels of PAM and / or its isoforms and / or fragments in healthy controls and calculating, for example, the corresponding 25th percentile, more preferably the 10th percentile, and even more preferably the 5th percentile. The lower limit of the 25th percentile, more preferably the 10th percentile, and even more preferably the 5th percentile, is set as the threshold for patients with disease compared to healthy patients, or for subjects at risk of developing disease compared to healthy subjects, or for subjects at risk of developing adverse events compared to subjects that are not at risk of developing adverse events, when the level of subjects with disease or subjects at risk of developing disease or adverse events falls below a specific threshold. The levels of PAM and / or its isoforms and / or fragments may be detected as total PAM concentration and / or PAM activity.In relation to the aforementioned percentiles, the lower threshold for distinguishing between healthy patients and patients with disease, or between healthy subjects and subjects at risk of developing disease, or between subjects without risk of developing adverse events and subjects at risk of developing adverse events, by detecting PAM activity in plasma may be 15 to 8 μg / (L*h) or less, more preferably 13.5 to 8 μg / (L*h) or less, even more preferably 10.5 to 8 μg / (L*h) or less, and most preferably less than 8 μg / (L*h); the PAM activity in serum using the PAM activity assay method may be 10 to 5 μg / (L*h) or less, more preferably 8 to 5 μg / (L*h) or less, and most preferably less than 5 μg / (L*h). In relation to the aforementioned percentiles, the upper threshold for distinguishing between healthy patients and diseased patients, or between healthy subjects and subjects at risk of developing disease, or between subjects without risk of developing adverse events and subjects at risk of developing adverse events, by detecting PAM activity in plasma may be 20-40 μg / (L*h) or higher, more preferably 25-40 μg / (L*h) or higher, even more preferably 30-40 μg / (L*h) or higher, and most preferably greater than 40 μg / (L*h); the PAM activity in serum using the PAM activity assay method may be 10-25 μg / (L*h) or higher, more preferably 15-25 μg / (L*h) or higher, even more preferably 20-25 μg / (L*h), and most preferably greater than 25 μg / (L*h).

[0041] These pre-set values ​​may differ among specific groups selected based on certain factors such as gender, age, genetics, customs, and ethnicity.

[0042] A person skilled in the art knows how to set thresholds from past studies that have been conducted. A person skilled in the art knows that a particular threshold may depend on the population used to calculate pre-set thresholds that can be used later on a routine basis. A person skilled in the art knows that a particular threshold may depend on the calibration used in this test method. A person skilled in the art knows that a particular threshold may depend on the sensitivity and / or specificity that is deemed acceptable to healthcare professionals.

[0043] The sensitivity and specificity of a diagnostic test depend on factors beyond the analytical "quality" of the test itself, and these also depend on the definition of the factors that constitute abnormal outcomes. In practice, receiver response characteristic curves (ROC curves) are typically calculated by plotting the relative frequencies of the "normal" population (i.e., the seemingly healthy population) and the "disease" population (i.e., the population of patients with infectious diseases) against a variable value. Depending on the specific diagnostic problem to be addressed, the control group does not necessarily have to be the "normal" population; it could be a group of patients with a different disease, from which the target disease group should be differentiated. For any given marker, the distribution of marker levels in the subjects may overlap, regardless of the presence or absence of disease. Under such conditions, the test cannot absolutely distinguish the disease population from the normal population with 100% accuracy, and the overlapping region indicates the portion where the test cannot distinguish between the disease population and the normal population. A threshold is selected so that if the result exceeds this value (or falls below it; depending on how the marker changes with the disease), the test is considered abnormal, and if it falls below this value, the test is considered normal. The region under the ROC curve is a measure of the probability that the obtained measurement allows for accurate identification of the disease. The ROC curve can be used even when the test results do not necessarily show exact numerical values. An ROC curve may be constructed whenever possible to grade the results. For example, the test results of “disease” samples may be graded according to degree (e.g., 1=low, 2=normal, 3=high). This grading may correlate with the “normal” population and the results on the constructed ROC curve. These methods are well known in the art (see, for example, Hartley et al, 1982). Preferably, a threshold is selected to provide an ROC curve region greater than about 0.5, more preferably greater than about 0.7. The term “about” as used herein refers to ±5% of a given measurement.

[0044] Once thresholds are set using past research populations and taking all of the above-mentioned aspects into consideration, healthcare professionals can use the preset thresholds for the methods for diagnosing or prognosing a disease according to the present invention, and / or predicting the risk of a disease or adverse event occurring in a subject, and / or monitoring a disease or adverse event, and determine whether a subject has a value above or below the preset threshold in order to perform appropriate diagnosis, prognosis, prediction, or monitoring.

[0045] If the thresholds described above are calibrated differently from the assay system used in this invention, those thresholds may differ in other assay methods. Therefore, the thresholds described above should be applied to assay methods that have been calibrated differently, taking into account the differences in calibration. One possibility for quantifying the differences in calibration is to perform a comparative analysis (correlation analysis) by measuring each biomarker or its activity (e.g., PAM) in a sample using both the assay method in question (e.g., the PAM assay) and the respective biomarker assay methods used in this invention. Another possibility is to assume that this test has sufficient analytical sensitivity, measure the median biomarker level of a representative normal population using the assay method in question, compare the result with the median biomarker level using other assay methods, and recalculate the calibration based on the difference obtained from this comparison. In the calibration used in this invention, samples were measured from normal subjects (healthy subjects), and the median plasma PAM activity was 18.4 μg / (L*h) (interquartile range [IQR] was 13.5~21.9 μg / (L*h)), and the median serum PAM activity was 11.0 μg / (L*h) (interquartile range [IQR] was 8.1~13.1 μg / (L*H)).

[0046] As used herein, the term “diagnosis” means detecting a disease or determining the stage or degree of a disease. A diagnosis of a disease is usually based on an assessment of one or more factors and / or symptoms that indicate the disease. That is, a diagnosis can be made based on the presence or amount of factors indicating the presence or absence of a disease or disorder. Each factor or symptom considered to be presented for the diagnosis of a particular disease does not have to be associated only with that specific disease; for example, it may result in a different diagnosis inferred from the diagnostic factors or symptoms. Similarly, factors or symptoms that indicate a particular disease may be present in individuals who do not have that particular disease.

[0047] As used herein, the term “prognosis” refers to the prediction and outcome of the possible course of a clinical condition or disease, such as sepsis. Prognosis is typically made by evaluating disease factors or symptoms that indicate a favorable or unfavorable course or outcome of the disease. As used herein, the phrase “determine prognosis” refers to the process by which a person skilled in the art can predict the clinical condition or course or outcome of a patient’s disease. The term “prognosis” does not mean the ability to predict the clinical condition or course or outcome of a disease with 100% accuracy. Rather, a person skilled in the art will know that “prognosis” means an increased probability of a particular course or outcome occurring; that is, a particular course or outcome is more likely to occur in a patient exhibiting a particular clinical condition or disease compared to an individual exhibiting no clinical condition or disease.

[0048] In certain embodiments of methods for diagnosing or prognosing a disease in a subject, and / or predicting the risk of a disease or adverse event occurring in a subject, and / or monitoring a disease or adverse event in a subject, the disease is selected from the group including dementia, cardiovascular disorders, renal diseases, cancer, infectious diseases, and metabolic diseases. The aforementioned dementias are selected from the group that includes mild cognitive impairment (MCI), Alzheimer's disease, vascular dementia, mixed dementia of Alzheimer's disease and vascular dementia, Lewy body dementia, frontotemporal dementia, focal dementia (including progressive aphasia), subcortical dementia (including Parkinson's disease), and secondary causes of dementia syndromes (including intracranial lesions). The cardiovascular disorders may be selected from the group including atherosclerosis, hypertension, heart failure (including acute heart failure and acute uncompensated heart failure), atrial fibrillation, cardiovascular ischemia, cerebrovascular ischemic injury, cardiogenic shock, stroke (including ischemic stroke, hemorrhagic stroke, and transient ischemic attack), and myocardial infarction. The aforementioned kidney disease may be selected from a group that includes nephrotoxicity (drug-induced kidney disease), acute kidney disease (AKI), chronic kidney disease (CKD), diabetic nephropathy, and end-stage renal disease (ESRD). The aforementioned cancers may be selected from the group including prostate cancer, breast cancer, lung cancer, colorectal cancer, bladder cancer, ovarian cancer, cervical cancer, skin cancer (including melanoma), stomach cancer, liver cancer, pancreatic cancer, leukemia, non-Hodgkin lymphoma, kidney cancer, esophageal cancer, and pharyngeal cancer. The aforementioned infectious diseases are caused by infectious organisms such as bacteria, viruses, fungi, or parasites, and are selected from the group including SIRS, sepsis, and septic shock. The aforementioned metabolic disorders are selected from a group that includes type 1 diabetes, type 2 diabetes, and metabolic disorders.

[0049] In one embodiment of the present application, the disease is dementia, and the dementia is selected from the group including mild cognitive impairment (MCI), Alzheimer's disease, vascular dementia, mixed dementia of Alzheimer's disease and vascular dementia, Lewy body dementia, frontotemporal dementia, focal dementia (including progressive aphasia), subcortical dementia (including Parkinson's disease), and secondary causes of dementia syndromes (including intracranial lesions).

[0050] In certain embodiments, the dementia is Alzheimer's disease.

[0051] In one embodiment of the present application, the disease is cancer, and the cancer is selected from the group including prostate cancer, breast cancer, lung cancer, colorectal cancer, bladder cancer, ovarian cancer, cervical cancer, skin cancer (including melanoma), stomach cancer, liver cancer, pancreatic cancer, leukemia, non-Hodgkin lymphoma, kidney cancer, esophageal cancer, and pharyngeal cancer.

[0052] In certain embodiments, the cancers are colorectal cancer and pancreatic cancer.

[0053] In one embodiment of the present application, the disease is a cardiovascular disorder, which is selected from the group including atherosclerosis, hypertension, heart failure (including acute heart failure and acute uncompensated heart failure), atrial fibrillation, cardiovascular ischemia, cerebrovascular ischemic injury, cardiogenic shock, stroke (including ischemic stroke, hemorrhagic stroke and transient ischemic attack), and myocardial infarction.

[0054] In certain embodiments, the cardiovascular disorder is heart failure (including acute heart failure and acute decompensated heart failure).

[0055] In another specific embodiment, the cardiovascular disorder is a stroke (including ischemic stroke, hemorrhagic stroke, and transient ischemic attack) and a myocardial infarction.

[0056] In another specific embodiment, the cardiovascular disorder is atrial fibrillation (AF).

[0057] In another specific embodiment of this application, the diseases are SIRS, sepsis, and septic shock.

[0058] In another specific embodiment of this application, the disease is type 1 diabetes, type 2 diabetes, or metabolic disorder syndrome.

[0059] The body fluid in the context of the method of the present invention may be selected from the group consisting of blood, serum, plasma, cerebrospinal fluid (CSF), urine, saliva, sputum, and pleural fluid. In a particular embodiment of the method, the sample is selected from the group consisting of whole blood, serum, and plasma.

[0060] The term "monitoring" refers to managing the progression of a patient's disease and pathophysiological state, including the risk of developing a disease or adverse event, the severity of the disease, or the response to treatment (i.e., detecting any changes).

[0061] The present invention relates to a method, in which the aforementioned monitoring is performed to evaluate changes in the risk of developing a disease or adverse event, changes in the severity of the disease, or the patient's or subject's response to treatment.

[0062] A particular subject of the present invention is a method, in which the monitoring described above is performed to evaluate the subject's response to corresponding preventive and / or therapeutic measures.

[0063] The subject of this invention is a method according to the present invention, which is used to stratify the subject into risk groups.

[0064] As used herein, the term "risk" refers to the possibility of suffering from undesirable events or effects (e.g., illness or adverse events).

[0065] The term "high level" refers to a level that exceeds a specific threshold level.

[0066] The term "low level" refers to a level that falls below a specific threshold level.

[0067] An "adverse event" is defined as an event that impairs the health of an individual. Such adverse events may be selected from, but are not limited to, cardiac events, cardiovascular events, cerebrovascular events, cancer, diabetes, and death from all of these causes. Adverse events include infections, serious infections, and septic-like systemic infections and sepsis. An adverse event is not an acute exogenous adverse event and / or an event caused by exogenous trauma. Exogenous trauma includes events induced by accidents, such as car accidents, and is therefore excluded from the group of adverse events.

[0068] In certain embodiments of the present invention, the adverse event is a cardiovascular event selected from the group including myocardial infarction, acute uncompensated heart failure, stroke, and death associated with myocardial infarction, stroke, or acute heart failure.

[0069] "Risk of developing disease or adverse event" means the risk of developing the disease or event within a certain period of time. In a particular embodiment, the period may be within 10 years, or within 8 years, or within 5 years, or within 2.5 years, or within 1 year, or within 6 months, or within 3 months, or within 30 days, or within 28 days.

[0070] In certain embodiments of the present invention, “level of PAM and / or its isoform and / or fragment” means the total concentration (preferably expressed as w / v) of PAM and / or its isoform and / or fragment having at least 12 amino acids in a sample taken from a subject, or the activity of PAM and / or its isoform and / or fragment in a sample taken from a subject containing the sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 10.

[0071] In this disclosure, the term "PAM" refers to the amino acid sequences of PAM isoforms 1-6, as shown in SEQ ID NOs: 1-6. In some aspects, the PAMs disclosed herein have sequence identity of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% with respect to the amino acid sequences of SEQ ID NOs: 1-6.

[0072] In some aspects, the PAM is a functional fragment (i.e., PHM (SEQ ID NO: 7) or PAL (SEQ ID NO: 8)) that preserves at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least 70%, at least about 80%, or at least about 90% of the PAM activity of the corresponding full-length PAM. In other aspects, the PAM is a variant or derivative of the PAM disclosed herein.

[0073] The percentage of amino acid sequence or nucleic acid sequence identity, i.e., the term “sequence identity %”, is defined herein as the percentage of residues in a candidate amino acid sequence or nucleic acid sequence that are identical to residues in a reference sequence after two sequences have been aligned and gaps have been introduced as necessary to achieve maximum percentage identity. In preferred embodiments, the calculation of at least the percentage of sequence identity is performed without introducing gaps. Methods and computer programs for alignment, such as the “Align 2” or BLAST services from the National Center for Biotechnology Information (NCBI), are well known in the art.

[0074] In certain embodiments of the present invention, the test method is used to determine the level of PAM and / or its isoforms and / or fragments, and this test method is a sandwich test method, preferably a fully automated test method.

[0075] In one embodiment of the present invention, the testing method may be a so-called POC (point-of-care) test, which is a testing technique that does not require a fully automated testing system and can be performed in the presence of the patient in less than one hour. An example of this technique is immunochromatography.

[0076] In one embodiment of the present invention, the assay is a sandwich immunoassay using any of the following detection techniques, but not limited to, enzymatic labeling, chemiluminescence labeling, or electrochemiluminescence labeling, and is preferably a fully automated assay. In one embodiment of the present invention, the assay is an enzymatically labeled sandwich assay. Examples of automated or fully automated assays include assays that can be used in one of the following systems: Roche Elecsys®, Abbott Architect®, Siemens Centauer®, Brahms Kryptor®, Biomerieux Vidas®, Alere Triage®, and Ortho Clinical DiagnosticsVitros®.

[0077] In a particular embodiment of the present invention, at least one of the two binders is labeled and detectable.

[0078] Preferred detection methods include various forms of immunoassays, such as radioactive immunoassay (RIA), homogeneous enzyme amplification immunoassay (EMIT), chemiluminescence immunoassay and fluorescence immunoassay, enzyme-linked immunosorbent assay (ELISA), fluorescence bead array assay, protein microarray assay, and rapid assay methods such as immunochromatography strip assay.

[0079] In a preferred embodiment, the label is selected from the group including chemiluminescent labels, enzyme labels, fluorescent labels, and radioactive iodine labels.

[0080] This assay method may be homogeneous or heterogeneous, competitive or non-competitive. In one embodiment, the assay method is in the form of a sandwich assay, which is a non-competitive immunoassay, in which the molecule to be detected and / or quantified is bound to a first antibody and a second antibody. The first antibody may be bound to a solid phase, such as beads, wells or other container surfaces, fragments or small pieces, and the second antibody is an antibody labeled, for example, with a dye, a radioisotope, or with a reactive or catalytically active moiety. The amount of labeled antibody bound to the sample is then measured by an appropriate method. Standard compositions and procedures involved in the "sandwich assay method" are well established and known to those skilled in the art (The Immunoassay Handbook, Ed. David Wild, Elsevier LTD, Oxford; 3rd ed. (May 2005); Hultschig et al. 2006. Curr Opin Chem Biol. 10 (1):4-10).

[0081] In another embodiment, the assay method comprises two capture molecules, preferably both antibodies present as dispersants in a liquid reaction mixture, wherein a first labeling component is attached to the first capture molecule, the first labeling component being part of a labeling system based on fluorescence quenching, chemiluminescence quenching, or amplification, and a second labeling component of the marking system is attached to the second capture molecule, thereby generating a measurable signal when both capture molecules bind to a sample, enabling the detection of a sandwich complex formed in a solution containing the sample.

[0082] In another embodiment, the labeling system includes a rare earth cryptotate, or rare earth chelate, combined with a fluorescent dye or chemiluminescent dye, particularly a cyanine-type dye.

[0083] In the context of the present invention, a fluorescence-based assay method involves the use of a dye, which is, for example, cyanide dyes such as FAM (5- or 6-carboxyfluorescein), VIC, NED, fluorescein, fluorescein isothiocyanate (FITC), IRD-700 / 800, CY3, CY5, CY3.5, CY5.5, Cy7, xanthene, 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HE X), TET, 6-carboxy-4',5'-dichloro-2',7'-dimethodifluorescein (JOE), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 5-carboxyrhodamine-6G (R6G5), 6-carboxyrhodamine-6G (RG6), rhodamine, rhodamine green, rhodamine red, rhodamine 110, BODIPY dyes such as BODIPY TMR, coumarins such as Oregon green and umbelliferone, benzimides such as Hoechst 33258, phenanthridines such as Texas red, Yakima yellow, Alexa Fluor, PET, ethidium bromide, acridinium dyes, carbazole dyes, phenoxazine dyes, porphyrin dyes, polymethine dyes, etc. may be selected from the group.

[0084] In the context of the present invention, the chemiluminescence-based assay method involves the use of dyes based on physical principles as described with respect to chemiluminescent materials in the literature (Kirk-Othmer, Encyclopedia of chemical technology, 4th ed. 1993. John Wiley & Sons, Vol. 15: 518-562, incorporated herein by reference including the citation on pages 551-562). Preferred chemiluminescent dyes are acridinium esters.

[0085] The "assay" or "diagnostic assay" described in the present specification may be any type of assay applied in the field of diagnosis. Such a measurement method may be based on binding an analyte to be detected to one or more capture probes having specific affinity. The binder that may be used for measuring the level of PAM and / or its isoforms and / or fragments thereof may have at least 10 7 M -1 , preferably 10 8 M -1 affinity constant for PAM and / or its isoforms and / or fragments thereof, with a preferred affinity constant of 10 9 M -1 or higher, most preferably 10 10 M -1 or higher. Those skilled in the art will understand that lower affinity may be compensated by using higher doses of the compound, and that this measure shall not be considered outside the scope of the present invention.

[0086] A "binding molecule" in the context of the present invention is a molecule used to bind a target molecule from a sample, that is, a molecule of interest, i.e., an analyte (in other words, PAM and its isoforms and fragments thereof in the context of the present invention). Therefore, to allow specific binding to the target molecule or molecule of interest, the binding molecule needs to be appropriately configured in terms of the positions of both molecules, and surface properties such as surface charge, hydrophobicity, hydrophilicity, and the presence or absence of Lewis donors and / or acceptors. Accordingly, the binding between the capture molecule and the target molecule, i.e., the molecule of interest, may be mediated, for example, by ionic interactions, van der Waals interactions, π-π interactions, σ-π interactions, hydrophobic interactions, or hydrogen bonding interactions, or a combination of two or more of the foregoing interactions. In the context of the present invention, the binding molecule may be selected from the group comprising, for example, nucleic acid molecules, carbohydrate molecules, PNA molecules, proteins, antibodies, peptides, or glycoproteins. Preferably, the binding molecule is an antibody, said antibody comprising fragments thereof having sufficient affinity for the target or molecule of interest, including recombinant antibodies or recombinant antibody fragments thereof, as well as chemically and / or biochemically modified derivatives of said antibodies or fragments derived from mutated chains.

[0087] In certain embodiments, the binder may be selected from the group consisting of an antibody, an antibody fragment, or a non-IgG scaffold.

[0088] The chemiluminescent label may be an acridinium ester label, such as an isoluminol label, or a steroid label.

[0089] The enzyme label may be lactate dehydrogenase (LDH), creatine kinase (CPK), alkaline phosphatase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), acidic phosphatase, glucose-6-phosphate dehydrogenase, etc.

[0090] In one embodiment of the present invention, at least one of the two binders is bonded to the magnetic particles and the solid phase as a polystyrene surface.

[0091] The present invention relates to a method for measuring the level of PAM and / or its isoforms and / or fragments in a body fluid sample using a assay method, the assay method comprising two binders that bind to two different epitopes of PAM, the two binders being directed to epitopes having a length of at least 5 amino acids, preferably at least 4 amino acids.

[0092] Epitopes, also known as antigenic determinants, are parts of antigens (e.g., peptides or proteins) that are recognized by the immune system, particularly antibodies. For example, an epitope is a specific portion of an antigen to which an antibody binds. The portion of the antibody that binds to an epitope is called a paratope. Protein antigen epitopes are classified into two types based on their structure and interaction with paratopes: three-dimensional epitopes and linear epitopes.

[0093] A linear, or continuous, epitope is an epitope recognized by an antibody based on its linear sequence of amino acids or primary structure, and is formed by the three-dimensional structure adopted by the interactions of consecutive amino acid residues. Structural epitopes and linear epitopes interact with paratopes based on the three-dimensional structure adopted by the epitope, which is determined by the surface shape of the epitope residues involved and the shape or tertiary structure of other compartments of the antigen. Structural epitopes are formed by the three-dimensional structure adopted by the interactions of discontinuous amino acid residues.

[0094] In one embodiment of the present invention, the chain-like epitopes are related to the following sequences of the immunizing peptides of PAM: peptide 1 (sequence number 11), peptide 2 (sequence number 12), peptide 3 (sequence number 13), peptide 4 (sequence number 14), peptide 5 (sequence number 15), peptide 6 (sequence number 16), peptide 7 (sequence number 17), peptide 8 (sequence number 18), peptide 9 (sequence number 19), peptide 10 (sequence number 20), peptide 11 (sequence number 21), peptide 12 (sequence number 22), peptide 13 (sequence number 23), and peptide 14 (sequence number 24).

[0095] In one embodiment of the present invention, the chain-like and / or three-dimensional epitopes are related to the following sequences of PAM: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 10.

[0096] The epitope may contain at least six amino acids, preferably at least five amino acids, and most preferably at least four amino acids.

[0097] In one embodiment of the present invention, the first and second binders bind to epitopes contained within the following sequences of PAM: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

[0098] In one embodiment of the present invention, the first and second binders bind to epitopes contained within the PAL subunit of PAM (SEQ ID NO: 8).

[0099] In one embodiment of the present invention, the first and second binders bind to epitopes contained within the PHM subunit of PAM (SEQ ID NO: 7).

[0100] In a particular embodiment of the present invention, the first binder binds to an epitope contained within the PAL subunit of PAM (SEQ ID NO: 8), and the second binder binds to an epitope contained within the PHM subunit of PAM (SEQ ID NO: 7).

[0101] In a particular embodiment of the present invention, the first and second binders bind to epitopes contained within the following sequences of PAM: peptide 1 (sequence number 11), peptide 2 (sequence number 12), peptide 3 (sequence number 13), peptide 4 (sequence number 14), peptide 5 (sequence number 15), peptide 6 (sequence number 16), peptide 7 (sequence number 17), peptide 8 (sequence number 18), peptide 9 (sequence number 19), peptide 10 (sequence number 20), peptide 11 (sequence number 21), peptide 12 (sequence number 22), peptide 13 (sequence number 23), peptide 14 (sequence number 24), and recombinant PAM (sequence number 10).

[0102] The use of at least two binders for measuring the level of PAM and / or its isoforms and / or fragments, wherein at least one binder is directed to an epitope contained within the following sequences of PAM: peptide 1 (sequence number 11), peptide 2 (sequence number 12), peptide 3 (sequence number 13), peptide 4 (sequence number 14), peptide 5 (sequence number 15), peptide 6 (sequence number 16), peptide 7 (sequence number 17), peptide 8 (sequence number 18), peptide 9 (sequence number 19), peptide 10 (sequence number 20), peptide 11 (sequence number 21), peptide 12 (sequence number 22), peptide 13 (sequence number 23), peptide 14 (sequence number 24), and recombinant PAM (sequence number 10).

[0103] The subject of this invention is a method for measuring the activity of PAM and / or its isoforms and / or fragments in a sample of a body fluid, and this method is - A step of contacting the sample with a scavenging agent that specifically binds to the active full-length PAM, its isoform, and / or its active fragments. - A step of separating the PAM bound to the aforementioned scavenging agent, The steps of adding a PAM substrate to the separated PAM, This includes a step of quantifying the activity of PAM by measuring the substrate conversion of PAM.

[0104] In certain embodiments of the present invention, the method is an enzyme capture assay (ECA, see, for example, U.S. Patents US5612186A and US5601986A).

[0105] In a particular embodiment of the method for measuring PAM activity in a target bodily fluid sample, the separation step is a washing step to remove sample components not bound to the capture binder from the captured PAM and / or its isoforms and / or fragments. The separation step may be any other step to separate the PAM bound to the capture binder from the components of the bodily fluid sample.

[0106] One embodiment of the present invention includes a chemical assay method for PAM. This assay method uses a peptide substrate that reacts with PAM and / or its isoforms and / or fragments to form a detectable reaction product. Alternatively, the reaction rate of the substrate can be monitored to determine the level of PAM and / or its isoforms and / or fragments in the test sample.

[0107] Such reagents and reaction-based assay methods may be carried out in any suitable reaction vessel, for example, in a test tube or a well of a microtiter plate. Alternatively, assay devices may be developed in disposable forms, such as measuring rod systems or test piece systems, which are well known to those skilled in the art and are easy to manufacture and use. Such disposable assay devices may be packaged in the form of a kit containing all the necessary materials, reagents, and instructions for use.

[0108] In another embodiment of the assay method, the rate at which the reaction occurs may be detected as an indicator of the level of PAM and / or its isoforms and / or fragments present in the test sample. For example, the rate at which the substrate reacts can be used to indicate the level of PAM and / or its isoforms and / or fragments present in the test sample. Alternatively, the rate at which the reaction product is formed can be used to indicate the level of PAM and / or its isoforms and / or fragments present in the test sample.

[0109] In yet another embodiment, the activity of PAM and / or its isoforms and / or fragments can be measured by performing a capture assay or a binding assay. For example, an antibody that reacts with the PAM protein but does not interfere with its enzymatic activity may be immobilized on a solid phase surface. The test sample passes over the immobilized antibody surface, and PAM and / or its isoforms and / or fragments, if present, bind to the antibody and are immobilized for detection. A substrate may then be added, and the reaction product can be detected to indicate the level of PAM and / or its isoforms and / or fragments in the test sample. The term “solid phase” as used herein may be used to include, but is not limited to, any material or container on which the assay can be performed, including, but is not limited to, porous materials, non-porous materials, test tubes, wells, slides, etc.

[0110] In certain embodiments of the method for diagnosing or prognosing disease in a subject, and / or predicting the risk of disease or adverse events occurring in a subject, and / or monitoring disease or adverse events in a subject, by measuring the levels of peptidylglycine α-amidate monooxygenase (PAM) and / or its isoforms and / or fragments in a body fluid sample of the subject, the scavenging binder is immobilized on a surface. For the measurement of PAM activity, a binder that reacts with PAM and / or its isoforms and / or fragments but does not inhibit enzyme activity by more than 50%, preferably less than 40%, and preferably less than 30%, may be immobilized on the solid-phase surface. To prevent inhibition of PAM, the scavenging binder should not bind to PAM in the region around the active site and substrate-binding region.

[0111] In a particular embodiment of the method for measuring the level of PAM and / or its isoforms and / or fragments in a body fluid sample of interest, the binder may be selected from the group consisting of antibodies, antibody fragments, non-Ig scaffolds, or aptamers.

[0112] Another subject of the present invention is a method for measuring the activity of PAM and / or its isoforms and / or fragments in a sample of a body fluid, the method being - A step of contacting the sample with the PAM substrate (peptide-Gly) at time intervals from t=0 minutes to t=n+1 minutes. The steps include: detecting the reaction product of PAM (α-amidate peptide) in the sample at t=0 min and t=n+1 min; The process includes a step of calculating the difference in reaction products between t=0 and t=n+1 to quantify the activity of PAM.

[0113] Another subject of the present invention is a method for measuring PAM activity in a body fluid sample, and this method is - A step of contacting the sample with ADM-Gly, which is the substrate of PAM, at time intervals from t=0 minutes to t=n+1 minutes. - A step of detecting ADM-NH2, which is the reaction product of PAM in the sample, at t=0 min and t=n+1 min using an immunoassay method, and The process includes a step of calculating the difference in the reaction product ADM-NH2 between t=0 and t=n+1 minutes to quantify the activity of PAM. The term "t=n+1 minutes" represents a time interval, where n is defined as 0 minutes or greater.

[0114] One embodiment of the present application relates to a kit for carrying out a method for diagnosing or prognosing a disease in a subject, and / or predicting the risk of a disease or adverse event occurring in a subject, and / or monitoring a disease or adverse event in a subject, the kit comprising at least two binders directed to recombinant PAM (SEQ ID NO: 10), peptide 1 (SEQ ID NO: 11), peptide 2 (SEQ ID NO: 12), peptide 3 (SEQ ID NO: 13), peptide 4 (SEQ ID NO: 14), peptide 5 (SEQ ID NO: 15), peptide 6 (SEQ ID NO: 16), peptide 7 (SEQ ID NO: 17), peptide 8 (SEQ ID NO: 18), peptide 9 (SEQ ID NO: 19), peptide 10 (SEQ ID NO: 20), peptide 11 (SEQ ID NO: 21), peptide 12 (SEQ ID NO: 22), peptide 13 (SEQ ID NO: 23), and peptide 14 (SEQ ID NO: 24).

[0115] A particular embodiment of this application relates to a kit for detecting levels of PAM, the kit comprising one or more binders that bind to a PAM sequence selected from the group including recombinant PAM (SEQ ID NO: 10), peptide 1 (SEQ ID NO: 11), peptide 2 (SEQ ID NO: 12), peptide 3 (SEQ ID NO: 13), peptide 4 (SEQ ID NO: 14), peptide 5 (SEQ ID NO: 15), peptide 6 (SEQ ID NO: 16), peptide 7 (SEQ ID NO: 17), peptide 8 (SEQ ID NO: 18), peptide 9 (SEQ ID NO: 19), peptide 10 (SEQ ID NO: 20), peptide 11 (SEQ ID NO: 21), peptide 12 (SEQ ID NO: 22), peptide 13 (SEQ ID NO: 23), and peptide 14 (SEQ ID NO: 24).

[0116] Another embodiment of the present application relates to a kit for carrying out a method for measuring the activity of PAM and / or its isoforms and / or fragments in a sample of bodily fluids of interest, wherein the kit comprises peptide-Gly PAM as a substrate, and the peptide-Gly is ADM-Gly.

[0117] PAM activity can be measured by detecting the α-amidated peptide (peptide-amide) from the glycinated precursor peptide substrate (peptide-Gly). Nearly half of bioactive peptides terminate with an α-amide at the C-terminus (Vishvanatha et al. 2014. J Biol Chem 289(18):12404-20).

[0118] The glycination precursor peptide substrates include adrenomedullin (ADM), adrenomedullin-2, short intermedin, proadrenomedullin N-20 terminal peptide (PAMP), amyrin, gastrin-releasing peptide, neuromedin C, neuromedin B, neuromedin S, neuromedin U, calcitonin, calcitonin gene-related peptide (CGRP) 1 and 2, islet amyloid polypeptide, chromogranin A, insulin, pancreastatin, prolactin-releasing peptide (PrRP), cholecystokinin, big gastrin, gastrin, and glucagon-like peptide. The group may be selected from the following: GLP-1, pituitary adenylyl cyclase-activating polypeptide (PACAP), secretin, somatriverine, peptide histidine methionine (PHM), vasoactive intestinal peptide (VIP), gonadriverine, kisspeptin, MIF-1, metastine, neuropeptide K, neuropeptide γ, substance P, neurokinin A, neurokinin B, peptide YY, pancreatic hormone, deltorphin I, orexin A and B, melanotropin α (α-MSH), melanotropin γ, thyroid-stimulating-releasing hormone (TRH), oxytocin, and vasopressin.

[0119] In a preferred embodiment, the peptide-Gly is adremedullin-Gly (ADM-Gly), and the peptide-amide is adremedullin-amide (ADM-NH2).

[0120] Other non-peptide substrates may include N-fatty acid acylglycines that are converted by PAM to primary fatty acid amides (PFAMs) such as oleic acid amides.

[0121] With the above background in mind, further specific aspects of the present invention are provided by the following sequentially numbered embodiments. 1. A method for diagnosing or prognosing disease in a subject by measuring the levels of peptidylglycine α-amidate monooxygenase (PAM) and / or its isoform and / or fragment in a subject's body fluid sample, and / or a method for predicting the risk of disease or adverse events occurring in a subject, and / or a method for monitoring disease or adverse events in a subject. The aforementioned target diseases are selected from the group including dementia, cardiovascular disorders, kidney disease, cancer, inflammation or infection, and / or metabolic diseases. Adverse events are selected from a group that includes cardiac events, cardiovascular events, cerebrovascular events, cancer, diabetes, infections, serious infections, septic-like systemic infections, sepsis, and death from all of these causes. 2. A method for diagnosing or determining the prognosis of a disease in a subject by measuring the levels of peptidylglycine α-amidate monooxygenase (PAM) and / or its isoform and / or fragment in a subject's body fluid sample, and / or a method for predicting the risk of disease or adverse events occurring in a subject, and / or a method for monitoring disease or adverse events in a subject, wherein this method is - A step of measuring PAM and / or its isoform and / or fragments in the target body fluid sample, and The process includes comparing the measured value with a predetermined threshold, If the measured value falls below or exceeds the threshold, the subject is diagnosed with the disease, or • If the measured value falls below or exceeds the threshold, the outcome of the disease is determined to be prognostic, or • If the measured value falls below or exceeds the threshold, the risk of disease or adverse event occurring is predicted, or The aforementioned disease or adverse event will be monitored. 3. The method according to Embodiments 1 and 2, wherein the level of PAM and / or its isoform and / or fragment is the total concentration of PAM and / or its isoform and / or fragment having at least 12 amino acids in the body fluid sample of the subject, or the activity of PAM and / or its isoform and / or fragment. 4. The method according to embodiments 1 to 3, wherein the activity of the PAM and / or its isoform and / or fragment is selected from the group including the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 10. 5. The method according to embodiments 1 to 3, wherein the total concentration of the PAM having at least 12 amino acids and / or its isoforms and / or fragments is detected by immunoassay. 6. The method according to Embodiments 3-4, wherein the activity of the PAM and / or its isoform and / or fragment is detected using peptide-Gly as a substrate. 7. The peptide-Gly substrates include adrenomedullin (ADM), adrenomedullin-2, short intermedin, proadrenomedullin N-20 terminal peptide (PAMP), amyrin, gastrin-releasing peptide, neuromedin C, neuromedin B, neuromedin S, neuromedin U, calcitonin, calcitonin gene-related peptide (CGRP) 1 and 2, islet amyloid polypeptide, chromogranin A, insulin, pancreastatin, prolactin-releasing peptide (PrRP), cholecystokinin, big gastrin, gastrin, glucagon-like peptide 1 (G The method according to Embodiment 6, selected from the group comprising LP-1), pituitary adenylyl cyclase-activating polypeptide (PACAP), secretin, somatriverine, peptide histidine methionine (PHM), vasoactive intestinal peptide (VIP), gonadriverine, kisspeptin, MIF-1, metastine, neuropeptide K, neuropeptide γ, substance P, neurokinin A, neurokinin B, peptide YY, pancreatic hormone, deltorphin I, orexin A and B, melanotropin α (α-MSH), melanotropin γ, thyroid-stimulating-releasing hormone (TRH), oxytocin, and vasopressin. 8. A method for diagnosing or determining the prognosis of a disease in a subject, and / or predicting the risk of a disease or adverse event occurring in a subject, and / or monitoring a disease or adverse event in a subject, by measuring the level of peptidylglycine α-amidate monooxygenase (PAM) and / or its isoform and / or fragment in a body fluid sample of a subject according to Embodiments 1 to 7, wherein the PAM and / or its isoform and / or fragment is selected from the group including SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 10. 9. A method for diagnosing or prognosing a disease in a subject, and / or predicting the risk of a disease or adverse event occurring in a subject, and / or monitoring a disease or adverse event in a subject, by measuring the level of peptidylglycine α-amidate monooxygenase (PAM) and / or its isoform and / or fragment in a body fluid sample of a subject according to Embodiments 1 to 8, wherein the risk of a disease occurring in the subject is determined when the subject is a healthy subject. 10. The method according to Embodiment 9, wherein the disease is selected from the group consisting of Alzheimer's disease, colorectal cancer, and pancreatic cancer. 11. A method for determining the level of PAM and / or its isoforms and / or fragments in a body fluid sample using a assay method, the assay method comprising two binders that bind to two different regions of PAM, the two binders being directed to epitopes of at least five amino acid lengths, preferably at least four amino acid lengths, and the two binders being directed to epitopes contained within the following sequences of PAM: peptide 1 (sequence number 11), peptide 2 (sequence number 12), peptide 3 (sequence number 13), peptide 4 (sequence number 14), peptide 5 (sequence number 15), peptide 6 (sequence number 16), peptide 7 (sequence number 17), peptide 8 (sequence number 18), peptide 9 (sequence number 19), peptide 10 (sequence number 20), peptide 11 (sequence number 21), peptide 12 (sequence number 22), peptide 13 (sequence number 23), peptide 14 (sequence number 24), and recombinant PAM (sequence number 10). 12. A method for measuring the activity of PAM and / or its isoform or fragments in a body fluid sample, wherein the method comprises: - A step of contacting the sample with a scavenging agent that specifically binds to the active full-length PAM, its isoforms, and / or its active fragments. - A step of separating the PAM bound to the aforementioned scavenging agent, The steps of adding a PAM substrate to the separated PAM, This includes a step of quantifying PAM activity by measuring the substrate conversion rate of PAM. 13. A method for measuring the activity of PAM and / or its isoforms and / or fragments in a body fluid sample, wherein the method is: - A step of contacting the sample with the PAM substrate (peptide-Gly) at time intervals from t=0 minutes to t=n+1 minutes. The steps include: detecting the reaction product of PAM (α-amidate peptide) in the sample at t=0 min and t=n+1 min; The process includes a step of calculating the difference between the reaction products at t=0 and t=n+1 minutes to quantify the activity of PAM. 14. The peptide-Gly substrates include adrenomedullin (ADM), adrenomedullin-2, short intermedin, proadrenomedullin N-20 terminal peptide (PAMP), amyrin, gastrin-releasing peptide, neuromedin C, neuromedin B, neuromedin S, neuromedin U, calcitonin, calcitonin gene-related peptide (CGRP) 1 and 2, islet amyloid polypeptide, chromogranin A, insulin, pancreastatin, prolactin-releasing peptide (PrRP), cholecystokinin, big gastrin, gastrin, glucagon-like peptide 1 (G The method according to Embodiment 13, selected from the group comprising LP-1), pituitary adenylyl cyclase-activating polypeptide (PACAP), secretin, somatriverine, peptide histidine methionine (PHM), vasoactive intestinal peptide (VIP), gonadriverine, kisspeptin, MIF-1, metastine, neuropeptide K, neuropeptide γ, substance P, neurokinin A, neurokinin B, peptide YY, pancreatic hormone, deltorphin I, orexin A and B, melanotropin α (α-MSH), melanotropin γ, thyroid-stimulating-releasing hormone (TRH), oxytocin, and vasopressin. 15. Use of an antibody to measure the level of PAM and / or its isoforms and / or fragments, wherein the antibody specifically binds to sequences selected from the group consisting of recombinant PAM (SEQ ID NO: 10), peptide 1 (SEQ ID NO: 11), peptide 2 (SEQ ID NO: 12), peptide 3 (SEQ ID NO: 13), peptide 4 (SEQ ID NO: 14), peptide 5 (SEQ ID NO: 15), peptide 6 (SEQ ID NO: 16), peptide 7 (SEQ ID NO: 17), peptide 8 (SEQ ID NO: 18), peptide 9 (SEQ ID NO: 19), peptide 10 (SEQ ID NO: 20), peptide 11 (SEQ ID NO: 21), peptide 12 (SEQ ID NO: 22), peptide 13 (SEQ ID NO: 23), and peptide 14 (SEQ ID NO: 24). 16. A kit for measuring the level of PAM and / or its isoforms and / or fragments, the kit comprising one or more antibodies that bind to PAM sequences selected from the group including recombinant PAM (SEQ ID NO: 10), peptide 1 (SEQ ID NO: 11), peptide 2 (SEQ ID NO: 12), peptide 3 (SEQ ID NO: 13), peptide 4 (SEQ ID NO: 14), peptide 5 (SEQ ID NO: 15), peptide 6 (SEQ ID NO: 16), peptide 7 (SEQ ID NO: 17), peptide 8 (SEQ ID NO: 18), peptide 9 (SEQ ID NO: 19), peptide 10 (SEQ ID NO: 20), peptide 11 (SEQ ID NO: 21), peptide 12 (SEQ ID NO: 22), peptide 13 (SEQ ID NO: 23), and peptide 14 (SEQ ID NO: 24). [Examples]

[0122] Example 1 - Production of recombinant PAM PAM cDNA was synthesized according to Uniprot accession number P19021, which encodes amino acids 21-834 of the PAM protein, including codon optimization for expression in mammalian cells. The signal sequence of PAM was replaced with the human serum albumin signal sequence (MKWVTFISLLFLFSSAYSFR [SEQ ID NO: 9]). A hexahistidine tag was added to the C-terminus of PAM and ligated to PAM via a GS linker. The sequence of recombinant PAM (amino acids 21-834 of PAM, excluding the signal sequence and hexahistidine tag) is shown in SEQ ID NO: 10. The cDNA was cloned into an expression vector (plasmid DNA) using 5'-NotI and 3' HindIII restriction sites. The expression vector containing the cDNA for PAM expression was replicated in E. coli and prepared from E. coli as a low-endotoxin preparation.

[0123] HEK-INV cells were transfused using an expression vector with the INVect gene transduction reagent in serum-free suspension culture medium. The gene transduction rate was controlled by co-transduction with GFP (green fluorescent protein) containing the expression vector. Cell culture was performed at 37°C in 5% CO2 in the presence of valproic acid and penicillin-streptomycin. When the viability reached less than 60%, cells were harvested by centrifugation (over 2000 g, 30-45 minutes, 2-8°C). The cell culture supernatant (CCS) was washed five times with 100 mM Tris / HCl solution at pH 8.0 by tangential flow filtration (TFF, 30 kDa cutoff value)

[0124] For the purification of recombinant PAM, buffer-exchanged CCS was applied to the surface of Q-Sepharose high-performance fluid (GE Healthcare) resin, including elution under a NaCl gradient (maximum 2 M). The amidation activity-containing fraction was accumulated and subjected to Superdex 200pg (GE Healthcare) size exclusion chromatography column using a pH 8.0 elution buffer containing 100 mM Tris / HCl and 200 mM NaCl. The amidation activity-containing fraction was accumulated and dialyzed against a sterile filtered (0.2 μm) pH 8.0 solution containing 100 mM Tris / HCl and 200 mM NaCl. Endotoxin loading was measured using a Charles River PTS Endosafe system and was less than 5 EU / mL.

[0125] Example 2 - Antibody Production The anti-PAM antibody according to the present invention may be synthesized as follows. Table 1 shows the PAM peptides for immunization (Peptides & Elephants, Hennigsdorf, Germany), synthesized with an additional C-terminal cysteine ​​residue for peptide binding to bovine serum albumin (BSA) (if cysteine ​​was not present in the selected PAM sequence). These peptides were covalently bound to BSA using a Sulfolink-binding gel (Perbio-science, Bonn, Germany). The binding procedure was carried out according to Perbio's instructions. Recombinant PAMs were prepared by InVivo Biotech Services, Hennigsdorf, as described in Example 1. [Table 1]

[0126] Balb / c mice were intraperitoneally (ip) injected with 100 μg of recombinant PAM (emulsified in TiterMax Gold activator) or 100 μg of PAM-peptide-BSA conjugate on day 0, 100 μg and 100 μg (emulsified in complete Freund's activator) on day 14, and 50 μg and 50 μg (emulsified in incomplete Freund's activator) on days 21 and 28. These animals received intravenous (iv) injection of 50 μg of recombinant PAM on day 40, or 50 μg of PAM-peptide-BSA conjugate dissolved in saline on day 45. Three days later, these mice were killed and immunocellular fusion was performed.

[0127] Splenocytes from immunized mice and cells from the myeloma cell line SP2 / 0 were fused with 1 mL of 50% polyethylene glycol at 37°C for 30 seconds. After washing, the cells were seeded in 96-well cell culture plates. They were grown in HAT medium (RPMI1640 culture medium supplemented with 20% fetal bovine serum and HAT adjuvants), and hybrid clones were selected. After one week, the HAT medium was replaced with HT medium, and the cells were passed three times before being returned to standard cell culture medium.

[0128] First, the cell culture supernatant was sorted against recombinant PAM-conjugated IgG antibody two weeks after fusion. Accordingly, recombinant PAM (SEQ ID NO: 10) was immobilized in a 96-well plate (100 ng / well) and cultured at room temperature for 2 hours with 50 μL / well of cell culture supernatant. After washing the plate, 50 μL / well of POD-rabbit anti-mouse IgG was added and cultured at room temperature for 1 hour.

[0129] Following the subsequent washing step, 50 μL of the color-developing stock solution (citric acid / hydrogen phosphate buffer, 3.7 mM o-phenylenediamine in 0.012% H2O2) was added to each well, and the mixture was incubated at room temperature for 15 minutes. The color reaction was then stopped by adding 50 μL of 4N sulfuric acid. Absorption was detected at 490 mm.

[0130] The tested positive microcultures were transferred to 24-well plates for propagation. After retesting, selected cultures were cloned and re-cloned using the limiting dilution method to determine their isotypes.

[0131] Antibodies grown against recombinant human PAM or PAM peptide were produced using a standard antibody production method (Marx et al. 1997) and purified with protein A. Antibody purity was over 90% based on SDS gel electrophoresis analysis.

[0132] Example 3 - PAM activity assay method Human serum or Li-heparinized plasma from self-reported healthy volunteers was used as the source of natural human PAM. Each sample (20 μL) was diluted 2-fold with 100 mM Tris-HCl and replicated. The amidation reaction was initiated by adding 160 μL of PAM reaction buffer (containing 100 mM Tris-HCl, pH 7.5, 6.25 μM CuSO4, 2.5 mM L-ascorbic acid, 125 μg / mL catalase, 62.5 μM astatin, 250 μM leupeptin, 36 ng / mL synthetic ADM-Gly, and 375 μg / mL NT-ADM antibody). Subsequently, 100 μL of each reaction mixture from the replicated samples were mixed and transferred to 20 μL of 200 mM EDTA to terminate the amidation reaction and produce the reaction product at t=0 min, followed by incubation at 37°C for 40 minutes. The unconcluded reaction was then stopped with 10 μL of 200 mM EDTA. To measure PAM activity, bio-ADM as a reaction product was quantified for each sample using the sphingotest® bio-ADM immunoassay (Weber et al. 2017). The amidation assay was calibrated using a 6-point calibration curve generated with human recombinant PAM of known activity. The sample and calibration sample were processed in the same manner. The relative light units (RLU: t=40 min to t=0 min) measured by the sphingotest® bio-ADM assay for each sample were matched to the RLU (t=40 min to t=0 min) of the calibration sample to measure the PAM activity in the sample. PAM activity is expressed as "adrenomedullin maturation activity" (AMA), and is expressed in units of μg of bio-ADM produced per hour and per L of sample.

[0133] A typical PAM calibration curve is shown in Figure 3. Figure 4 shows the distribution of AMA in Li-heparin samples from 120 self-reported healthy volunteers. The median IQR of AMA in Li-heparin was 18.4 μg / (L*h) [13.5~21.9]. The 10th and 90th percentiles were 10.5 and 24.2 μg / (L*h), respectively. The 2.5th, 97.5th, and 99th percentiles were 8.1, 31.6, and 40.8 μg / (L*h). Furthermore, when comparable serum samples were measured from 20 subjects, serum AMA levels were approximately 40% lower than in Li-heparin, but a very significant correlation (r=0.89; p<0.0001) was found between the two (Figure 5).

[0134] Example 4 - Immunoassay of PAM Antibodies against recombinant PAM (SEQ ID NO: 10) and PAM peptides (SEQ ID NOs: 11-24) were grown as described in Example 1.

[0135] The technique used was a sandwich luminescence immunoassay based on acridinium ester labeling.

[0136] 4.1. Labeled compounds (tracers) Purified antibody (0.2 g / L) was labeled with MACN-acridinium-NHS-ester (1 g / L, InVent GmbH) in a 1:5 mol / L ratio in 10% labeled buffer (500 mmol / L sodium phosphate, pH 8.0) at 22°C for 20 minutes. After adding 5% 1 mol / L Tris-HCl, pH 8.0 over 10 minutes, each antibody was separated from the free labeled product using a CentriPure P10 column (emp Biotech GmbH). The purified labeled antibodies were diluted in 300 mmol / L potassium phosphate, 100 mmol / L NaCl, 10 mmol / L Na-EDTA, and 5 g / L bovine serum albumin (pH 7.0). The final concentration of the labeled antibody was approximately 20 ng per 150 μL.

[0137] 4.2.Solid phase White polystyrene microtiter plates (Greiner Bio-One International AG) were coated with each antibody (2 μg / 0.2 mL per well, 50 mmol / L Tris-HCl, 100 mmol / L NaCl, pH 7.8) over 18 hours at 20°C. After blocking with 30 g / L Kalyon, 5 g / L BSA (proteinase-free), 6.5 mmol / L monopotassium phosphate, and 3.5 mmol / L sodium dihydrogen phosphate (pH 6.5), the plates were vacuum-dried.

[0138] 4.3. Calibration The assay method was calibrated using the recombinant PAM dilution described in Example 1. The typical concentration range was within the range of 5 to 5,000 ng / mL.

[0139] 4.4. PAM Immunotherapy 4.4.1. PAM-LIA One-step procedure: 50 μL of sample / calibration sample was pipetteed into a pre-coated microtiter plate. 200 μL of labeled antibody was added to a buffer (300 mmol / L potassium phosphate, 100 mmol / L NaCl, 10 mmol / L Na-EDTA, 50 μmol / L amastamin, 100 μmol / L leupeptin, 0.1% bovine IgG, 0.02% mouse IgG, 0.5% BSA, pH 7.0), and the microtiter plate was incubated at 2-8°C for 20 hours with stirring at 600 rpm. Unbound tracers were removed by washing five times with a washing solution (20 mmol / L PBS, 1 g / L Triton X-100, pH 7.4) (350 μL per well). The amount of chemiluminescence bound to the wells was measured per well for 1 second using a Centro LB 960 microtiter plate luminescence reader (Berthold Technologies).

[0140] Two-step procedure: 50 μL of sample / calibration sample was pipetteed into a pre-coated microtiter plate. After adding 200 μL of buffer (as described in the one-step procedure), the microtiter plate was incubated at 2–8°C for 15–20 hours with stirring at 600 rpm. Unbound sample was removed by washing four times with washing solution (350 μL per well), and then 200 μL of tracer material was added and the microtiter plate was incubated at room temperature for 2 hours. Unbound tracer was removed by washing four times with washing solution (350 μL per well). The amount of chemiluminescence bound to the wells was measured per well for 1 second using a Centro LB 960 microtiter plate emission reader (Berthold Technologies).

[0141] Results: Antibodies bound to the solid phase, and labeled antibodies against various PAM immunized peptides and full-length (recombinant) PAM (see Example 2) were tested with recombinant PAM and blood samples. Examples of standard curves for various antibody combinations are shown in Figure 6 (A-L). In Figure 6 (A-J), the following recombinant PAMs were used as calibration materials: (A) Solid phase: antibody against peptide 10 (SEQ ID NO: 20), tracer: antibody against peptide 9 (SEQ ID NO: 19); (B) Solid phase: antibody against peptide 10 (SEQ ID NO: 20), tracer: antibody against peptide 10 (SEQ ID NO: 20); (C) Solid phase: antibody against peptide 9 (SEQ ID NO: 19), tracer: antibody against peptide 10 (SEQ ID NO: 20); (D) Solid phase: antibody against recombinant PAM (SEQ ID NO: 10), tracer: antibody against recombinant PAM (SEQ ID NO: 10); (E) Solid phase: antibody against peptide 10 (SEQ ID NO: 20), tracer: recombinant PA (F) Solid phase: Antibody against M (SEQ ID NO: 10); (G) Solid phase: Antibody against peptide 14 (SEQ ID NO: 24); Tracer: Antibody against peptide 13 (SEQ ID NO: 23); (H) Solid phase: Antibody against recombinant PAM (SEQ ID NO: 10); Tracer: Antibody against peptide 13 (SEQ ID NO: 23); (I) Solid phase: Antibody against peptide 13 (SEQ ID NO: 23); Tracer: Antibody against peptide 9 (SEQ ID NO: 19); (J) Solid phase: Antibody against peptide 10 (SEQ ID NO: 20); Tracer: Antibody against peptide 13 (SEQ ID NO: 23). In Figure 6 (K and L), the following natural PAMs (EDTA-plasma) were used as calibration materials: (K) Solid phase: antibody against peptide 14 (SEQ ID NO: 24), tracer: antibody against peptide 13 (SEQ ID NO: 23); (L) Solid phase: antibody against peptide 10 (SEQ ID NO: 20), tracer: antibody against peptide 13 (SEQ ID NO: 23). In all antibody combinations, PAMs were also detectable in human plasma and human serum samples. 4.4.2. Enzyme Capture Analysis (ECA) for the Detection of PAM Activity

[0142] An enzyme capture assay was developed to detect PAM activity. 50 μL of sample / calibration sample was pipetteed into a pre-coated microtiter plate (as described in 4.2). 200 μL of buffer (300 mmol / L potassium phosphate, 100 mmol / L NaCl, 50 μmol / L amastatin, 100 μmol / L leupeptin, 0.1% bovine IgG, 0.02% mouse IgG, 0.5% BSA, pH 7.0) was added, and the microtiter plate was incubated at room temperature for 1 hour with stirring at 600 rpm. Unbound samples were removed by washing four times with washing solution (350 μL per well), followed by adding 200 μL of reaction buffer per well and incubation at 37°C. The reaction buffer containing all components and the final concentrations were the same as in Example 3, except that 100 μg / mL of NT-ADM antibody and 288 ng / mL of ADM-Gly were used. 10 μL of each reaction solution was transferred to 190 μL of EDTA-containing buffer (300 mmol / L potassium phosphate, 100 mmol / L NaCl, 10 mmol / L Na-EDTA, 50 μmol / L astatin, 100 μmol / L leupeptin, 0.1% bovine IgG, 0.02% mouse IgG, 0.5% BSA, pH 7.0), and the reaction was stopped at several stages. The stopped reaction solutions were subjected to the sphingotest® bio-ADM immunoassay to quantify the bio-ADM produced. A typical standard curve using an antibody against PAM immunosorbent peptide 10 (SEQ ID NO: 20) as the solid phase is shown in Figure 6, M. Figure 6, N, shows a typical standard curve using an antibody against full-length recombinant PAM (SEQ ID NO: 10). Further antibodies against peptides 7 (SEQ ID NO: 17), 8 (SEQ ID NO: 18), 9 (SEQ ID NO: 19), 13 (SEQ ID NO: 23), and 14 (SEQ ID NO: 24) were used as solid phases for enzyme capture assays to measure PAM activity in recombinant PAM or heparinized plasma samples (250 μL) (Figure 6, O). These results demonstrate that PAM activity in human samples can be detected using ECA technology by employing antibodies. PAM was also detectable in plasma and serum samples.

[0143] In a further step, PAM activity and PAM concentration (as described in Example 3) were measured in heparin samples from healthy volunteers (n=26) using PAM-LIA (solid-phase antibody against full-length PAM, tracer antibody against peptide 13 [SEQ ID NO: 23]). PAM activity and PAM concentration were significantly correlated, as shown in P in Figure 6 (Spearman r=0.49, p=0.0109).

[0144] Example 5 - ADM-Gly immunoassay ADM-Gly was quantified using bioactive ADM modified as described by Weber et al. (Weber et al. 2017. JALM 2(2): 222-233). The modification involved directing the tracer antibody used to detect MACN-acridinium-NHS-labeled ADM-Gly towards the C-terminal glycine of ADM-Gly. This assay was calibrated with synthetic ADM-Gly. The limit of detection (LOD) was 10 pg / mL of ADM-Gly. Cross-reactivity of the antibody against the C-terminal glycine of ADM with bio-ADM was concentration-dependent, ranging from 6% to 50%. All measured ADM-Gly concentrations were corrected for cross-reactivity as follows. For each ADM-Gly quantification, further quantification of bio-ADM in the corresponding sample was performed using the sphingotest® bio-ADM immunoassay. Using the corresponding bio-ADM values, the signal (RLU) generated by the antibody against the C-terminal glycine of ADM on the bio-ADM calibration curve was measured. The measured signal (RLU) was used to calculate the false-positive ADM-Gly concentration (pg / mL) using the ADM-Gly calibration curve. This concentration was subtracted from the initially measured ADM-Gly concentration. A typical standard curve is shown in Figure 7.

[0145] Example 6 - Disease prediction in healthy individuals 6.1. Research Group The Malmö Preventive Project (MPP) received funding in the mid-1970s to investigate cardiovascular risk factors in a general population, enrolling 33,346 residents of Malmö (Fedorowski et al. 2010. Eur Heart J 31: 85-91). Between 2002 and 2006, a total of 18,240 basic participants responded to invitations (participation rate; 70.5%) and underwent review including comprehensive health examinations and blood sampling (Fava et al. 2013. Hypertension 2013; 61: 319-26). The MPP review was used as the criterion for this study. As a criterion, subjects with a history of cardiovascular disease were excluded. Informed consent was obtained from all participants, and the research plan was approved by the Ethical Committee of Lund University, Lund, Sweden.

[0146] MR-proADM in plasma was measured using a commercially available, fully automated, homogeneously time-resolved immunofluorescence assay (BRAHMS MR-proADM KRYPTOR; BRAHMS GmbH, Hennigsdorf, Germany) (Caruhel et al. 2009. Clin Biochem. 42 (7-8):725-8).

[0147] Bio-ADM was measured as described by Weber et al. in 2017 (Weber et al. 2017. JAMA 2(2): 222-233). AMA was measured using 4942 serum samples from MPP as described in Example 3. Each sample was measured twice. Samples, reference samples, and calibration samples were processed in the same manner. Table 2 shows the baseline clinical characteristics of AMA after stratification by quartile. [Table 2]

[0148] Statistical Analysis: Numerical values ​​are presented as mean and standard deviation, median and interquartile range (IQR), or count and percentage values, as needed. Group comparisons of continuous variables were performed using the Kruskal-Wallis test. Biomarker data were logarithmically transformed. The effects of risk factors on survival rates in univariate and multivariate analyses were analyzed using Cox proportional hazards regression. The assumptions in proportional hazards were tested for all variables. For continuous variables, hazard ratios (HRs) were standardized, and the HR for biomarker changes at a single IQR was recorded. 95% confidence intervals (CIs) and chi-squared (χ²) values ​​were given for risk factors. 2 The significance level (Wald test) is provided. The predicted values ​​for each model were evaluated using the model likelihood ratio chi-squared statistic. The coincident index (C-index) is provided as a measure of influence. This corresponds to the concept of AUC (Area Under the Curve) adopted for two-variable outcomes. For multivariate models, a corrected form of the automatic formula for the C-index is provided. Survival curves plotted by the Kaplan-Meier method were used for explanatory purposes. The likelihood ratio chi-squared test for nested models was used to test the independence of PAM from clinical variables. All statistical tests were two-tailed tests, and a two-tailed p-value of 0.05 was considered significant.

[0149] 6.2. Prediction of Alzheimer's disease (AD) 3,954 samples were selected (from 174 individuals with incidental AD) that included information on resupply efficiency in patients diagnosed with dementia and those undergoing hemodialysis for chronic kidney disease (CKD). Information on dementia diagnoses was requested from the Swedish National Patient Register (SNPR). Diagnoses in the register were collected according to various revisions of the International Classification of Diseases (ICD) codes 290, 293 (ICD-8), 290, 331 (ICD-9) or F00, F01, F03, G30 (ICD-10). The SNPR includes all patient care in Sweden since 1987, and also includes data on outpatient visits, including day surgeries and psychiatric care, recorded since 2000 from private and public caregivers. All-factor dementia was diagnosed according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders (DSM) Rev. III, while the diagnoses of Alzheimer's disease and vascular dementia followed the criteria of the DSM Rev. IV. Diagnoses were verified by thorough review of medical records and, where available, neuroimaging data. Research physicians were assigned the final diagnosis for each patient, and geriatric specialists specializing in cognitive impairment were consulted for unresolved cases. PAM activity (AMA) was measured as described in Example 3. In the MPP population, AMA was significantly lower in patients who developed AD over time (174 individuals with incidental AD) compared to the group that did not develop AD, as shown in Figure 8 (p=0.01).

[0150] A decrease in serum AMA strongly predicts Alzheimer's disease, with a hazard ratio (HR) of 0.74 (CI 0.6–0.88; p<0.001), and an age-adjusted HR of 0.72 (CI 0.6–0.85) (Table 3). Figure 9 shows a Kaplan-Meier plot for predicting Alzheimer's disease using AMA (cases of advanced AD were excluded from this analysis). The lowest tertile is associated with the highest risk of developing AD.

[0151] Furthermore, AMA as a predictor of AD was independent of bio-ADM concentration. Both markers contributed to the prediction of AD. The C index for AMA alone was 0.571 (CI 0.525~0.616;χ²). 2 10.97) However, the C index of both the combined marker, namely AMA and bio-ADM, is 0.595(χ 2 18.96; p<0.0001). Furthermore, AMA combined with concentrations of bio-ADM and MR-proADM further improves the prediction of Alzheimer's disease onset. MR-proADM alone does not predict AD, but the combination of AMA, bio-ADM, and MR-proADM shows a predictive value of 0.622(χ²). 2 The C exponent was shown as 26.73 (p=0.00001). [Table 3]

[0152] 6.3. Prediction of Colorectal Cancer (CRC) Figure 10 shows the AMA for subjects with and without incidental CRC. AMA in patients who developed CRC over time (n=93) was significantly lower than in the group that did not develop CRC (p=0.0008; Kruskal-Wallis test). In contrast, as shown in Figure 11, MR-proADM concentrations were higher in patients who developed CRC over time compared to the group that did not develop CRC (p=0.023).

[0153] Table 3 shows the results for single and combined markers. A decrease in serum AMA (after age adjustment) strongly predicts the development of CRC, with a hazard ratio (HR) of 0.68 (p<0.0001). Figure 12 shows the Kaplan-Meier plot for the prediction of CRC by AMA (progressive cases were excluded from the analysis). The lowest tertile is associated with the highest risk of developing CRC (p<0.005).

[0154] Increased MR-proADM concentrations predict the development of CRC, with a risk factor of 1.36 (p<0.05). The highest quartile is associated with the highest risk of CRC development (p=0.051).

[0155] While bio-ADM concentration alone could not predict the onset of CRC, the combination of bio-ADM and AMA showed improved CRC prediction (see Table 4). Furthermore, the combination of AMA and MR-proADM further improved the prediction of CRC onset.

[0156] In summary, decreased AMA levels predict the onset of CRC. Increased MR-proADM concentrations also predict the onset of CRC. Combinations of AMA with bio-ADM or MR-proADM improve the predictive value of CRC. [Table 4]

[0157] 6.4. Prediction of Pancreatic Cancer Furthermore, AMA is increased in subjects with incidental pancreatic cancer compared to subjects without pancreatic cancer (p<0.005) (Figure 13). AMA strongly predicts pancreatic cancer, with an odds ratio (OR) of 0.44 (CI 0.33~0.58). Figure 14 shows the receiver response characteristic curves (ROC plots) for each AMA, revealing an AUC of 0.71.

[0158] 6.5. Prediction of all-cause mortality and cardiovascular mortality Mortality analyses were performed on 4942 samples using information on death and cardiovascular events from the MPP population. Information on cardiovascular events and their diagnoses was requested from the Swedish National Patient Registry (SNPR). Diagnoses in the registry were collected according to various revisions of the International Classification of Diseases (ICD) code. The SNPR includes all patient care in Sweden since 1987, and also includes data on outpatient visits, including day surgeries and psychiatric care, recorded since 2000 by private and public caregivers. PAM activity (AMA) was measured as described in Example 3. Among the total serum samples from 4942 individuals in the MPP study population, 1361 subjects died (all causes of death) during a 12.8-year follow-up period. From the total number of death events of 1361 individuals, 480 events were counted as cardiovascular deaths.

[0159] Elevated serum AMA strongly predicts all-cause mortality, with a hazard ratio (HR) of 1.354 (CI 1.197–1.531; p<0.0001) (Table 5). The predictive value of AMA was independent of common cardiovascular risk factors (age, sex, blood pressure, body mass index, antihypertensive medications, low-density and high-density lipoproteins, and history of diabetes). Figure 15 shows a Kaplan-Meier plot for predicting all-cause mortality using AMA. Higher AMA levels are associated with an increased risk of death.

[0160] Elevated serum AMA strongly predicts cardiovascular mortality, with a hazard ratio (HR) of 1.6 (CI 1.3–1.969; p<0.0001) (Table 5). The predictive value of AMA was independent of common cardiovascular risk factors (age, sex, blood pressure, body mass index, antihypertensive medication, low-density and high-density lipoprotein levels, and history of diabetes). Figure 16 shows a Kaplan-Meier plot for predicting cardiovascular mortality using AMA. Higher AMA levels are associated with an increased risk of cardiovascular death. [Table 5]

[0161] 6.6. Prediction of cardiovascular events Cardiovascular events were analyzed in 4942 samples using information on mortality and cardiovascular events from the MPP population. Information on cardiovascular events and their diagnoses was requested from the Swedish National Patient Registry (SNPR). Diagnoses in the registry were collected according to various revisions of the International Classification of Diseases (ICD) code. The SNPR includes all patient care in Sweden since 1987, and also includes data on outpatient visits, including day surgeries and psychiatric care, recorded since 2000 by private and public caregivers. PAM activity (AMA) was measured as described in Example 3. Among the total serum samples from 4942 individuals in the MPP study population, 278 subjects developed heart failure (incidental heart failure) and 633 subjects developed atrial fibrillation (incidental atrial fibrillation) during a 12.8-year follow-up period.

[0162] Elevated serum AMA strongly predicts incidental heart failure, with a hazard ratio (HR) of 1.537 (CI 1.169–2.021; p<0.0007) (83 cases of advanced HF were excluded from the analysis) (Table 6). Figure 17 shows a Kaplan-Meier plot for predicting all-cause mortality using AMA. Higher AMA is associated with a higher risk of developing heart failure.

[0163] Elevated serum AMA strongly predicts incidental atrial fibrillation, with a hazard ratio (HR) of 1.459 (CI 1.214–1.752; p<0.0001) (267 cases of advanced AF were excluded from the analysis) (Table 6). Figure 18 shows a Kaplan-Meier plot for predicting all-cause mortality using AMA. Higher AMA is associated with a higher risk of developing heart failure. [Table 6]

[0164] Example 7 - Diagnosis of Disease 7.1. Diagnosis of Alzheimer's disease Serum samples from 27 individuals diagnosed with Alzheimer's disease were obtained from InVent Diagnostica GmbH. AD diagnosis was based on cognitive tests (CERAD, DemTec, MMST, and clock drawing test), as well as MRI (magnetic resonance imaging) and CT scans. Sixty-seven self-reported serum samples from healthy volunteers were used as controls. AMA was detected as described in Example 3.

[0165] As shown in Figure 19, patients from the AD population showed significantly lower serum AMA compared to the control population (n=67; p<0.0001).

[0166] 7.2. Diagnosis of cardiovascular and metabolic disorders The 4,942 total serum samples from the MPP study population included 267 cases of progressive atrial fibrillation, 83 cases of progressive chronic heart failure, and 533 cases of progressive diabetes mellitus. Compared to individuals without progressive atrial fibrillation (mean AMA: 12.8 AMA units, n=4675), individuals with progressive atrial fibrillation showed a significant increase in serum AMA (p<0.0001) (mean AMA: 13.92 AMA units, n=267). Compared to individuals without progressive heart failure (mean AMA: 12.84 AMA units, n=4859), individuals with progressive chronic heart failure showed a significant increase in serum AMA (p=0.0019) (mean AMA: 14.31 AMA units, n=83). Compared to individuals without progressive diabetes (mean AMA: 12.89 AMA units, n=4409), individuals with progressive diabetes showed a significant decrease in serum AMA (p=0.0035) (mean AMA: 12.69 AMA units, n=533).

[0167] Example 8 - Prognosis and Monitoring 8.1. The research group AdrenOSS-1 AdrenOSS-1 is a promising observational study in Europe. Twenty-four research sites in five countries (France, Belgium, the Netherlands, Italy, and Germany) contributed to the completion of the trial with 583 enrolled patients (recruited from June 2015 to May 2016). The study plan was approved by local ethics committees and conducted in accordance with the Declaration of Helsinki. This study enrolled patients aged 18 years or older who (1) were admitted to the ICU with sepsis or septic shock, or (2) were transferred from another ICU in a state of sepsis or septic shock within 24 hours of admission. Participating patients were stratified into severe sepsis and septic shock based on the 2001 definitions of sepsis and organ failure (Levy et al. 2003. 2001 SCCM / ESICM / ACCP / ATS / SIS International Sepsis Definitions Conference. Crit Care Med. 31(4):1250-6). The term "sepsis" refers to the latest definition in "Sepsis-3" (Singer et al. 2016 The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA. 315(8):801-10). Patients were treated according to clinical practice, and the treatment and procedures were documented. The primary outcome measure was mortality at day 28. Secondary outcomes included organ failure and organ support (defined by the Continuing Organ Failure Assessment [SOFA] score), use of vasopressors / cardiotonic agents, fluid balance, and use of renal replacement therapy (RRT).

[0168] Upon admission, personal statistics (age, sex), body mass index, presence of septic shock, type of ICU admission, organ dysfunction scores (SOFA, Acute Physiological Assessment and Chronic Health Assessment II [APACHE II]), origin of sepsis, pre-existing complications (i.e., cases treated within the past year), medical history, laboratory values, and organ support were recorded, and blood samples were taken for the measurement of bio-ADM and other markers. After patient enrollment, the following data were collected daily during the first week: SOFA score, antibiotic therapy, fluid balance, respiratory status, Glasgow Coma Scale score, central venous pressure, need for RRT, invasive procedures for sepsis control, and vasopressor / inotropic agent therapy. In addition, discharge status and mortality were recorded on day 28 after ICU admission.

[0169] Blood samples for the central laboratory were collected within 24 hours of ICU admission and again 2 days after the initial sample (mean 47 hours, standard deviation 9 hours). The samples were then processed and stored at -80°C. PAM activity (AMA) was measured in n=197 plasma samples randomly selected from the AdrenOSS-1 population, as described in Example 3. 8.2. Outcomes and prognosis in sepsis

[0170] Figure 20 shows that in the AdrenOSS-1 population, AMA was significantly higher in the non-survivor group compared to the survivor group (p<0.05). High plasma AMA strongly predicted mortality at day 28, with a hazard ratio (HR) of 1.41 (p<0.05). Figure 21 shows Kaplan-Meier plots for predicting mortality at day 28 in patients with sepsis and septic shock.

[0171] Furthermore, ADM-Gly concentrations in the AdrenOSS-1 population were also significantly higher in non-survivors compared to survivors (p<0.0001). High ADM-Gly concentrations strongly predicted 28-day mortality with a hazard ratio of 2.29 (p<0.005).

[0172] Table 7 shows the mortality outcomes at day 28 for single biomarkers AMA and ADM-Gly. Cutoff values ​​for AMA and ADM-Gly concentrations were selected to achieve equal sensitivity of 80.4%, while specificity was 21.7% for AMA and 38.5% for ADM-Gly, respectively. Combining both markers, i.e., in a predetermined ratio, increased the specificity of 28-day survival to 43.4%. Furthermore, the odds ratios (OR) for PAM and ADM-Gly were 1.13 and 2.56, respectively, but the OR increased to 3.14 when both markers were combined. [Table 7]

[0173] Example 9 - Measurement of PAM activity in human saliva Saliva was collected in separate sterile test tubes from five self-reporting healthy subjects. PAM activity in the human saliva samples was tested as described in Example 3. PAM activity in the saliva samples could be measured in the range of approximately 700 to 2000 ng / (L*h) (Figure 22), which was about one-tenth lower than that in plasma samples.

[0174] array Sequence ID 1-Prepro-PAM isoform 1 AS 1~973 [ka] [ka] Sequence ID 2-Prepro-PAM isoform 2 AS 1~868 [ka] [ka] SEQ ID NO: 3-Prepro-PAM isoform 3 AS (deletion of amino acids 829-896 of SEQ ID NO: 1) [ka] [ka] SEQ ID NO: 4-Prepro-PAM isoform 4 (deletion of amino acids 829-914 of SEQ ID NO: 1) [ka] [ka] SEQ ID NO: 5-Prepro-PAM isoform 5 (isoform 1 having an additional amino acid at position 896) [ka] [ka] SEQ ID NO: 6-Prepro-PAM isoform 6 (deletion of amino acids 897-914 of SEQ ID NO: 1) [ka] [ka] PHM subunit of sequence number 7-PAM [ka] PAL subunit of Sequence ID 8-PAM [ka] Sequence ID 9 - Signal Sequence Human Serum Albumin [ka] Sequence ID 10 - Sequence of recombinant human PAM [ka] Sequence ID 11-Peptide-1 (Amino acids 42-56 of PAM Sequence ID 1) [ka] SEQ ID NO: 12-Peptide-2 (Amino acids 109-128 of PAM SEQ ID NO: 1) [ka] SEQ ID NO: 13-Peptide-3 (Amino acids 168-180 of PAM SEQ ID NO: 1) [ka] SEQ ID NO: 14 - Peptide-4 (amino acids 204-216 of PAM SEQ ID NO: 1) [ka] SEQ ID NO: 15 - Peptide 5 (amino acids 329-342 of PAM SEQ ID NO: 1) [ka] SEQ ID NO: 16 - Peptide-6 (Amino acids 291-310 of PAM SEQ ID NO: 1) [ka] SEQ ID NO: 17-Peptide 7 (Amino acids 234-244 of PAM SEQ ID NO: 1) [ka] SEQ ID NO: 18-Peptide-8 (Amino acids 261-276 of PAM SEQ ID NO: 1) [ka] SEQ ID NO: 19 - Peptide 9 (amino acids 530-557 of PAM SEQ ID NO: 1) [ka] SEQ ID NO: 20 - Peptide 10 (Amino acids 611-631 of PAM SEQ ID NO: 1) [ka] Sequence ID 21 - Peptide 11 (amino acids 562-579 of PAM Sequence ID 1) [ka] SEQ ID NO: 22 - Peptide-12 (amino acids 745-758 of PAM SEQ ID NO: 1) [ka] SEQ ID NO: 23 - Peptide-13 (amino acids 669-687 of PAM SEQ ID NO: 1) [ka] SEQ ID NO: 24 - Peptide-14 (amino acids 710-725 of PAM SEQ ID NO: 1) [ka]

Claims

1. A method for assisting in the diagnosis or prognosis of a patient's disease, and / or for assisting in the prediction of the risk of disease or adverse events occurring in said patient, and / or for assisting in the monitoring of disease or adverse events in said patient, the method comprising measuring the concentration or activity of peptidylglycine α-amidate monooxygenase (PAM) and / or its isoform and / or fragment in a sample of the patient's blood, serum, or plasma, The diseases of the aforementioned patients were selected from the group consisting of Alzheimer's disease, colorectal cancer, pancreatic cancer, cardiovascular disease, and diabetes. The aforementioned adverse events are selected from the group consisting of cardiovascular events, sepsis, septic shock, and death from any cause. Herein, the PAM and / or its isoform and / or fragment is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 10, in a method.

2. The method according to claim 1, wherein the cardiovascular event is heart failure or atrial fibrillation.

3. A method for assisting in the diagnosis or prognosis of a disease in a patient, and / or a method for assisting in the prediction of the risk of the occurrence of a disease or adverse event in said patient, and / or a method for assisting in the monitoring of a disease or adverse event in said patient, the method comprising measuring the concentration or activity of peptidylglycine α-amidate monooxygenase (PAM) and / or its isoform and / or fragment in a blood, serum, or plasma sample of said patient, - A step of measuring the concentration or activity of PAM and / or its isoforms and / or fragments in the patient's blood, serum, or plasma sample, and - Includes a step of comparing the measured value with a predetermined threshold, - If the measured value falls below or exceeds the threshold, the patient is diagnosed with the disease, or - If the measured value falls below or exceeds the threshold, the outcome of the disease is determined to be prognostic, or - If the measured value falls below or exceeds the threshold, the risk of disease or adverse event occurring is predicted, or • The patient's illness or adverse events are monitored here. The diseases of the aforementioned patients were selected from the group consisting of Alzheimer's disease, colorectal cancer, pancreatic cancer, cardiovascular disease, and diabetes. The aforementioned adverse events are selected from a group consisting of cardiovascular events, sepsis, septic shock, and death from all causes. Herein, the PAM and / or its isoform and / or fragment is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 10, in a method.

4. The method according to claim 3, wherein the cardiovascular event is heart failure or atrial fibrillation.

5. The method according to any one of claims 1 to 4, wherein the level of PAM and / or its isoform and / or its fragment is the total concentration of PAM and / or its isoform and / or its fragment having at least 12 amino acids in the patient's blood, serum, or plasma sample, or the activity of PAM and / or its isoform and / or its fragment.

6. The method according to claim 5, wherein the total concentration of the PAM having at least 12 amino acids and / or its isoforms and / or fragments is detected by immunoassay.

7. The method according to claims 5 to 6, wherein the activity of the PAM and / or its isoform and / or fragment is detected using peptide-Gly as a substrate.

8. The peptide-Gly substrates include adrenomedullin (ADM), adrenomedullin-2, short intermedin, proadrenomedullin N-20 terminal peptide (PAMP), amyrin, gastrin-releasing peptide, neuromedin C, neuromedin B, neuromedin S, neuromedin U, calcitonin, calcitonin gene-related peptides (CGRP) 1 and 2, islet amyloid polypeptide, chromogranin A, insulin, pancreastatin, prolactin-releasing peptide (PrRP), cholecystokinin, big gastrin, gastrin, and glucagon-like peptide 1 (GL). The method according to claim 7, selected from the group comprising P-1), pituitary adenylyl cyclase-activating polypeptide (PACAP), secretin, somatriverine, peptide histidine methionine (PHM), vasoactive intestinal peptide (VIP), gonadriverine, kisspeptin, MIF-1, metastine, neuropeptide K, neuropeptide γ, substance P, neurokinin A, neurokinin B, peptide YY, pancreatic hormone, deltorphin I, orexin A and B, melanotropin α (α-MSH), melanotropin γ, thyroid-stimulating-releasing hormone (TRH), oxytocin, and vasopressin.

9. The method according to claims 1 to 8, wherein the risk of the disease occurring in the patient is determined as if the patient were a healthy patient.

10. The method according to claim 9, wherein the disease is selected from the group consisting of Alzheimer's disease and colorectal cancer.

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