Methods for the measurement of peptidylglycine α-amidate monooxygenase (PAM) and its use for diagnostic purposes.

Abstract

The present invention relates to a method for determining the levels of PAM and / or its isoforms and / or fragments in a body fluid or tissue sample using an assay comprising at least one binder for the structural epitope of PAM, and to its use for diagnostic purposes.

Description

[Technical Field]

[0001] The present invention relates to a method for determining the levels of PAM and / or its isoforms and / or fragments in a body fluid or tissue sample using an assay comprising at least one binder for the structural epitope of PAM, and to its use for diagnostic purposes. [Background technology]

[0002] Biologically active peptide hormones function as signaling molecules. Most bioactive peptide hormones are synthesized from larger, inactive precursor peptides. During biosynthesis, these peptides undergo several concurrent and posttranslational modifications, including cleavage of the signal peptide, intracellular proteolytic cleavage of the precursor propeptide by specific endopeptidases, mainly at pairs of basic residues, 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 neuroendocrine peptides require further modification steps, including the formation of a C-terminal α-amide group, to obtain full biological activity (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, in a two-step enzymatic reaction, cleaves the glyoxylate from the C-terminal glycine residue of the peptide, leading to the formation of a C-terminal α-amidated peptide hormone, where the resulting α-amide group originates from the cleaved C-terminal glycine (Prigge et al. 2004. Science 304(5672):864-67). This amidation reaction occurs in the lumen of the secretory granule before 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, amylin, calcitonin, and neurokinin A. However, it has been previously demonstrated that PAM can also catalyze the formation of α-amides from non-peptide glycinated substrates, such as N-lipid acylglycines, which are converted by PAM to primary fatty acid amides (PFAMs) such as oleamides.The identified and purified peptidyl-glycinamide activity was shown to be dependent on copper and ascorbate (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 160kb long and contains 25 known exons, located on chromosome 5q21.1 (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 is expressed at different levels in almost all mammalian cell types and has been found to be significantly expressed in airway epithelium, endothelial cells, brain ependymal cells, 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 human PAM activity was reported in the pituitary gland, stalk, and hypothalamus. Plasma amidation activity was significantly higher in healthy children under 15 years of age than in healthy adults (Wand et al. 1985 Metabolism 34(11):1044-52).

[0005] Figure 1 shows the precursor protein (1-973 amino acids) of the largest known PAM isoform 1 (SEQ ID NO: 1) encoded by PAM cDNA. The N-terminal signal sequence (amino acids 1-20) ensures the orientation of the nascent PAM polypeptide into the secretory lumen of the endoplasmic reticulum, where it is subsequently cleaved co-translationally. The PAM propeptide is then processed by the same mechanism used for the biosynthesis of endogenous membrane proteins and secretory proteins, including cleavage of the pro region (amino acids 21-30), 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 peptidyl-glycine 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-hydroxy-glycine α-amidate lyase (PAL; EC 4.3.2.5) and is an enzyme that can catalyze the conversion of α-hydroxyglycine to α-amide, and subsequently release the glyoxylate. The sequential action of these separate enzymatic activities results in the overall peptidyl-glycine α-amidate activity. The first enzymatic activity (PHM) is located immediately upstream of the pro region (within the range of amino acids 31-494 of isoform 1 (SEQ ID NO: 7)). The second catalytic activity (PAL) is located after exon 16 of isoform 1, within amino acids 495-817 (SEQ ID NO: 8).

[0007] As shown in Figure 1, both activities can be encoded within the range of a single polypeptide as a membrane-bound protein (isoforms 1, 2, 5, and 6; corresponding to SEQ ID NOs: 1, 2, 5, and 6) and within the range of a single polypeptide as a soluble protein lacking a transmembrane domain (isoforms 3 and 4; corresponding to SEQ ID NOs: 3 and 4). Isoforms 1, 2, 5, and 6 remain on the extraplasmic membrane after fusion of secretory vesicles with the plasma membrane, followed by endocytosis and recycling 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 the TMD during the secretory pathway (Bousquet-Moore et al. 2010. J Neurosci Res 88(12):2535-45). The PHM subunit can be cleaved from soluble or membrane-bound PAM in the secretory pathway by prohormone-converting enzymes that target a double basic cleavage site in the exon 16 region. Moreover, during endocytosis, full-length PAM proteins can also be converted to a soluble form by the action of α and γ secretases (Bousquet-Moore et al. 2010. J Neurosci Res 88(12):2535-45). Membrane-bound PAM from late endosomes can 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 isolated activity of PHM and PAL in soluble forms also results in the formation of C-terminal α-amidate products from C-terminal glycinated substrates, which has allowed their separate reactions to occur in the same compartment, body fluid, or in vitro experimental setting. How the transfer of PHM hydroxylation products to PAL occurs is not precisely understood to date. There is evidence that the hydroxylation products are released into solution and not directly transferred from PHM to PAL (Yin et al. 2011. PLoS One 6(12):e28679). Furthermore, the source of circulating PAM is also not yet clear.

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

[0010] Therefore, the terms “amidation activity,” “α-amidation activity,” “peptidyl-glycine α-amidation activity,” “PAM activity,” or “active PAM” refer to the sequential enzymatic activity of PHM and PAL resulting in the formation of peptide or non-peptide α-amidation products from peptide or non-peptide glycinated substrates, independently of the splice variants or mixtures thereof of the present invention, or post-translational modified PAM enzymes, or soluble isolated PHM or PAL activity, or soluble PHM and membrane-bound PAL, or any combination of the forms mentioned. In other words, the terms “amidation activity,” “α-amidation activity,” “peptidyl-glycine α-amidation activity,” or “PAM activity,” or “active PAM,” independently of the splice variants or mixtures thereof of the present invention, can be described as the sequential action of enzymatic activity located within amino acids 31-817 of the propeptide encoded by human PAM cDNA.

[0011] PAM activity was analyzed in several human tissues and bodily fluids from healthy specimens and specimens suffering from certain diseases. A summary of previous efforts is as follows: The detection of PAM activity in human body fluids is primarily done by, 125 ID-TyrValGly, 125This includes the use of radiolabeled synthetic tripeptides such as IN-acetyl-TyrValGly or equivalently modified tripeptides, and the quantification of amidation products by γ-scintillation (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, substance P-Gly or cleaved neuropeptide Y-Gly were used as substrates for PAM activity assays (Gether et al. 1991 Mol Cell Endocrinol 79(1-3):53-63; Hyyppae et al. 1990 Pain 43:163-68; Jeng et al. 1990 Analytical Biochemistry 185(2):213-19).

[0012] 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 some variation in PAM activity in specific disease states, although no sex differences were observed. Plasma PAM activity was increased in adults with hypothyroidism and in patients with medullary thyroid carcinoma. Increased PAM activity was shown in the tissues of medullary thyroid carcinoma, pheochromocytoma, and islet tumors, suggesting increased formation 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).

[0013] Patients with multiple endocrine neoplasia type 1 (MEN-1) and pernicious anemia showed reduced plasma PAM activity compared to healthy controls (Kapuscinski et al. 1993. Clin Endocrinol 39(1):51-58).

[0014] The presence of amidation activity in human cerebrospinal fluid (CSF) was demonstrated by Wand et al. (Wand et al. 1985 Neuroendocrinol 41:482-89). In patients with Alzheimer's disease (AD), plasma PAM activity was shown to be unchanged compared to healthy controls, but CSF PAM activity was significantly reduced compared to activity from normal samples (Wand et al. 1987 Neurology 37:1057-61). Furthermore, in International Publication No. 2015 / 103594, it was proposed that the presence of PAM protein in CSF detected by mass spectrometry in AD patients was reduced compared to healthy controls. In addition, ADM-NH2, one of the amidation products of PAM, was shown to be reduced in patients with prevalent and incident Alzheimer's disease (International Publication No. 2019 / 154900). However, to date, no direct association between circulating PAM activity and the prediction, diagnosis, or progression of Alzheimer's disease (AD) has been reported.

[0015] Amidation activity in CSF of patients with low back pain was analyzed using 1-12 substance P-Gly (SP-Gly) as a substrate (Hyyppae et al. 1990 Pain 43:163-68). PAM activity was shown to be increased in CSF and significantly decreased in serum in patients with multiple sclerosis (MS) (Tsukamoto et al. 1995. Internal Medicine 34(4):229-32; International Publication No. 2010 / 005387). The association between plasma PAM activity and type 2 diabetes was described in International Publication No. 2014 / 118634.

[0016] International Publication No. 2021 / 170752 describes a method for determining the level (including concentration or activity) of PAM in a body fluid sample and its use for diagnostic purposes. In particular, this patent application shows the determination of the level of PAM using a binder, such as an antibody, against a linear peptide epitope.

[0017] It is a surprising finding of the present invention to determine the activity level of PAM as the total amount (concentration) of PAM in a body fluid or tissue of a subject for the diagnosis, prognosis, prediction or monitoring of a disease or adverse event using a binder, such as an antibody, against a conformational epitope of PAM.

Mode for Carrying Out the Invention

[0018] The subject matter of this application is a method for determining the level of PAM and / or its isoforms and / or fragments in a sample of body fluid or tissue using an assay comprising at least one binder against a conformational epitope of peptidylglycine α-amidating monooxygenase (PAM).

[0019] One embodiment of this application relates to a method for determining the level of PAM and / or its isoforms and / or fragments in a sample of body fluid or tissue using an assay comprising at least one binder against a conformational epitope of PAM of at least 4 amino acids, preferably at least 5 amino acids.

[0020] In one embodiment of this application, at least one binder binds to a conformational epitope contained within the PHM subunit (SEQ ID NO: 7) of PAM or to a conformational epitope contained within the PAL subunit (SEQ ID NO: 8) of PAM.

[0021] In one embodiment of this application, at least one binder binds to a conformational epitope contained within the following PAM sequences: a PHM fragment containing amino acids 31 - 377 (SEQ ID NO: 25) of PAM, or a PAL fragment containing amino acids 495 - 817 (SEQ ID NO: 8) of PAM).

[0022] One embodiment of the present application relates to a method for determining the level of PAM and / or its isoforms and / or fragments in a sample of body fluid or tissue using an assay comprising two binders that bind to two different regions of PAM, wherein at least one of the two binders is a conformational epitope of PAM.

[0023] One embodiment of the present application relates to a method for determining the level of PAM and / or its isoforms and / or fragments in a sample of body fluid or tissue using an assay comprising two binders that bind to two different regions of PAM, wherein each of the two binders is a conformational epitope of PAM.

[0024] In one embodiment of this application, the first binder of the two binders binds to a stereochemical epitope contained within the PHM subunit (SEQ ID NO: 7) of PAM, and the second binder of the two binders binds to a stereochemical epitope contained within the PAL subunit (SEQ ID NO: 8) of PAM.

[0025] In one embodiment of this application, each of the two binders is for epitopes contained in the following PAM sequence: a PHM fragment containing amino acids 31-377 of PAM (SEQ ID NO: 25) and a PAL fragment containing amino acids 495-817 of PAM (SEQ ID NO: 8).

[0026] One embodiment of the present application relates to the diagnosis or prognosis of a disease in a subject, and / or the prediction of the risk of contracting a disease or adverse event in a subject, and / or the monitoring of a disease or adverse event in a subject, by using an assay to determine the levels of PAM and / or its isoforms and / or fragments in a sample of a subject's body fluid or tissue, wherein the assay comprises at least one binder to a structural epitope of PAM, the disease in the subject is selected from the group including dementia, cardiovascular disorders, renal diseases, cancer, inflammatory or infectious diseases and / or metabolic diseases, and the adverse event is selected from the group including cardiac events, cardiovascular events, cerebrovascular events, cancer, diabetes, infections, severe infections, septic-like systemic infections, sepsis and death from any cause.

[0027] One embodiment of the present application relates to the diagnosis or prognosis of a disease in a subject, and / or the prediction of the risk of contracting a disease or adverse event in a subject, and / or monitoring of a disease or adverse event in a subject, by using an assay to determine the levels of PAM and / or its isoforms and / or fragments in a sample of a subject's body fluid or tissue, wherein the assay comprises at least one binder to a structural epitope of PAM, the disease in the subject is selected from the group including dementia, cardiovascular disorders, renal diseases, inflammatory or infectious diseases and / or metabolic diseases, and the adverse event is selected from the group including cardiac events, cardiovascular events, cerebrovascular events, diabetes, infections, severe infections, septic-like systemic infections, sepsis and death from any cause.

[0028] One embodiment of this application is a method for diagnosing or prognosing a disease in a subject, and / or predicting the risk of contracting a disease or adverse event in a subject, and / or monitoring a disease or adverse event in a subject, by determining the levels of PAM and / or its isoforms and / or fragments thereof in a sample of a body fluid or tissue of a subject, The steps include: determining the level of PAM and / or its isoforms and / or fragments in a sample of the body fluid or tissue of the subject using an assay comprising at least one binder for the three-dimensional epitope of PAM; The steps include comparing the amount determined above with a predetermined threshold. Includes, • If the amount determined above falls below or exceeds the predetermined threshold above, the subject is diagnosed with the disease, or • If the amount determined above falls below or exceeds the predetermined threshold above, the disease outcome is predicted, or • If the amount determined above falls below or exceeds the predetermined threshold above, the risk of the patient developing the disease or adverse event is predicted, or The above-mentioned diseases or adverse events are monitored. Regarding the method.

[0029] One preferred embodiment of the above method for diagnosing or prognosticating a disease in a subject, and / or predicting the risk of developing a disease or adverse event in a subject, and / or monitoring a disease or adverse event in a subject, comprises determining the level of PAM and / or its isoform and / or its fragments in a sample of the subject's body fluid or tissue, wherein the level of PAM and / or its isoform and / or its fragments is the total concentration of PAM and / or its isoform and / or its fragments having at least 12 amino acids in a sample of the subject's body fluid or tissue, and an assay is used that includes at least one binder for the structural epitope of PAM.

[0030] Another embodiment of the present application is a method for diagnosing or prognosing a disease in a subject, and / or predicting the risk of a disease or adverse event in a subject, and / or monitoring a disease or adverse event in a subject, by using an assay to determine the level of PAM and / or its isoforms and / or its fragments in a sample of a body fluid or tissue of a subject, wherein the assay comprises at least one binder to a structural epitope of PAM, and the PAM and / or its isoforms and / or its fragments are selected from the group comprising 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.

[0031] Those skilled in the art will understand that the PAM isoform sequences (SEQ ID NOs. 1-6) shown in the sequence listing contain an N-terminal signal sequence (amino acids 1-20) that is cleaved before protein secretion. Therefore, in preferred embodiments, the PAM isoform sequences (SEQ ID NOs. 1-6) and / or fragments thereof do not contain an N-terminal signal sequence.

[0032] Another 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 in a subject, and / or monitoring a disease or adverse event in a subject, by determining the levels of PAM and / or its isoforms and / or its fragments in a sample of a body fluid or tissue of a subject, wherein the total concentration of PAM and / or its isoforms and / or its fragments having at least 12 amino acids is detected by an immunoassay, the immunoassay comprising at least one binder to the structural epitope of PAM.

[0033] 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 in a subject, and / or monitoring a disease or adverse event in a subject, by determining the level of PAM and / or its isoforms and / or its fragments in a sample of a body fluid or tissue of a subject, wherein the assay comprises at least one binder to a structural epitope of PAM, and the PAM and / or its isoforms and / or its fragments are selected from the group comprising SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 10.

[0034] Another 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 subject developing a disease or adverse event, and / or monitoring a subject developing a disease or adverse event, by using an assay to determine the levels of PAM and / or its isoforms and / or fragments in a sample of a subject's body fluid or tissue, wherein the assay comprises at least one binder to a structural epitope of PAM, the risk of a subject developing a disease is determined, and the subject is a healthy subject.

[0035] 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 in a subject, and / or monitoring a disease or adverse event in a subject, by using an assay to determine the levels of PAM and / or its isoforms and / or fragments in a sample of a body fluid or tissue of a subject, wherein the assay comprises at least one binder to a structural epitope of PAM, and the disease is • A group of dementias selected from the following: mild cognitive impairment (MCI), Alzheimer's disease, vascular dementia, mixed 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). Cardiovascular disorders may be selected from the group including atherosclerosis, hypertension, heart failure (including acute and acute decompensated heart failure), atrial fibrillation, cardiovascular ischemia, cerebral ischemic injury, cardiogenic shock, stroke (including ischemic and hemorrhagic stroke and transient ischemic attack), and myocardial infarction. • Renal diseases may be selected from a group including nephrotoxicity (drug-induced kidney disease), acute kidney injury (AKI), chronic kidney disease (CKD), diabetic nephropathy, and end-stage renal disease (ESRD). 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. Infectious diseases caused by infectious organisms such as bacteria, viruses, fungi, or parasites, selected from the group including SIRS, sepsis, and septic shock. • Metabolic disorders selected from the group including type 1 diabetes, type 2 diabetes, and metabolic syndrome. Selected from the group consisting of .

[0036] Another specific embodiment of this application relates to a method for determining the levels of PAM and / or its isoforms and / or fragments in a body fluid or tissue sample using an assay, wherein the assay comprises two binders that bind to two different regions of PAM, the two binders being for structural epitopes of at least 5 amino acids, preferably at least 4 amino acids in length, and the two binders being for structural epitopes contained in the following PAM sequences:PHM fragment (SEQ ID NO: 25) and / or PAL fragment (SEQ ID NO: 8).

[0037] Another embodiment of this application relates to the use of an antibody for determining the levels of PAM and / or its isoforms and / or fragments, wherein the antibody specifically binds to a structural epitope of a sequence selected from the group of PHM fragments (SEQ ID NO: 25) and / or PAL fragments (SEQ ID NO: 8).

[0038] Another preferred embodiment of this application relates to a kit for determining the level of PAM, comprising one or more antibodies that bind to a structural epitope of a PAM sequence selected from the group comprising a PHM fragment (SEQ ID NO: 25) and / or a PAL fragment (SEQ ID NO: 8).

[0039] The object of the present invention is to provide a method for determining the levels of PAM and / or its isoforms and / or fragments in a sample of body fluid or tissue. The object of the present invention is to provide the respective assays and kits.

[0040] Another object of the present invention is to provide a method for diagnosing or prognosing a disease in a subject, and / or predicting the risk of a subject suffering from a disease or adverse event, and / or monitoring a disease or adverse event in a subject, by determining the levels of PAM and / or its isoforms and / or fragments thereof in a sample of a subject's body fluid or tissue using an assay, wherein the assay comprises at least one binder to a structural epitope of PAM.

[0041] 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 contracting a disease or adverse event in a subject, and / or monitoring a disease or adverse event in a subject, A step of determining the level of PAM and / or its isoforms and / or fragments in a sample of the body fluid or tissue of the subject using an assay, wherein the assay includes at least one binder for the structural epitope of PAM. The steps include comparing the amount determined above with a predetermined threshold. Includes, • If the amount determined above falls below or exceeds the predetermined threshold above, the subject is diagnosed with the disease, or • If the amount determined above falls below or exceeds the predetermined threshold above, the disease outcome is predicted, or • If the amount determined above falls below or exceeds the predetermined threshold above, the risk of the patient developing the disease or adverse event is predicted, or The above-mentioned diseases or adverse events are monitored. It is a method.

[0042] The threshold is predetermined by measuring the levels of PAM and / or its isoforms and / or its fragments in healthy controls and calculating, for example, the 75th percentile, more preferably the 90th percentile, and even more preferably the 95th percentile accordingly. The upper limits of the 75th percentile, more preferably the 90th percentile, and even more preferably the 95th percentile define the threshold for healthy versus affected patients, healthy versus affected subjects, or subjects without risk of adverse events versus subjects at risk of adverse events, if the level of affected subjects or subjects at risk of developing a disease or adverse event exceeds the threshold. The threshold is predetermined by measuring the levels of PAM and / or its isoforms and / or its fragments in healthy controls and calculating, for example, the 25th percentile, more preferably the 10th percentile, and even more preferably the 5th percentile accordingly. The lower limit of the 25th percentile, more preferably the 10th percentile, and even more preferably the 5th percentile, defines the threshold for healthy individuals versus affected individuals, healthy individuals versus individuals at risk of developing the disease, or individuals without risk of developing the adverse event versus individuals at risk of developing the adverse event, when the level of affected individuals or individuals at risk of developing the disease or adverse event falls below the threshold. The levels of PAM and / or its isoforms and / or fragments may be detected as total PAM concentrations. The given values ​​may vary among selected specific populations depending on specific factors such as sex, age, genetics, habits, and ethnicity.

[0043] Those skilled in the art know how to determine thresholds from previously conducted studies. Those skilled in the art know that a particular threshold may depend on the cohort used to calculate a given threshold that may later be routinely used. Those skilled in the art know that a particular threshold may depend on the calibration used in the assay. Those skilled in the art know that a particular threshold may depend on the sensitivity and / or specificity that the practitioner deems acceptable.

[0044] The sensitivity and specificity of a diagnostic test depend not only on the analytical "quality" of the test but also on the definition of what constitutes an abnormal outcome. In practice, the receiver operating characteristic curve (ROC curve) is typically calculated by plotting the values ​​of a variable against their relative frequencies in the "normal" (i.e., seemingly healthy) and "disease" populations (i.e., patients suffering from an infection). Depending on the specific diagnostic problem addressed, the reference group does not necessarily have to be "normal" and may be a group of patients suffering from another disease in which the disease group of interest is distinguished. For any given marker, the distribution of marker levels in subjects with and without the disease is likely to overlap. Under such conditions, the test will not be able to absolutely distinguish between normal and disease with 100% accuracy, and the overlapping region indicates when the test cannot distinguish between normal and disease. A threshold is chosen; above this threshold (or below it, depending on how the marker changes with the disease), the test is considered abnormal; below it, the test is considered normal. The area under the ROC curve is a measure of the probability that the recognized measurement allows for accurate identification of the disease. ROC curves can be used even when test results do not necessarily provide precise numerical values. ROC curves can be constructed as long as the results can be ranked. For example, test results for “disease” samples may be ranked according to degree (e.g., 1=low, 2=normal, and 3=high). This ranking can be correlated with results in the “normal” population, and an ROC curve can be constructed. These methods are well known in the field (see, e.g., Hartley et al, 1982). Preferably, the threshold is selected to provide an ROC curve area greater than about 0.5, more preferably greater than about 0.7. In this context, the term “about” refers to + / - 5% of a given measurement.

[0045] Once thresholds are determined by using previous research cohorts and taking all of the above points into consideration, a physician uses predetermined thresholds for methods of diagnosing or prognosing a disease and / or predicting the risk of a subject developing a disease or adverse event and / or monitoring a disease or adverse event, in accordance with the present invention, to determine whether a subject has a value above or below the predetermined threshold in order to perform appropriate diagnosis, prognosis, prediction, or monitoring.

[0046] The thresholds mentioned above may differ in other assays if those assays are calibrated in a different way than the assay system used in this invention. Therefore, the threshold(s) mentioned should be applied appropriately to such different calibration assays, taking into account the differences in calibration. One possibility for quantifying the differences in calibration is a method-comparative analysis (correlation) of the assay in question (e.g., the PAM assay) with each biomarker assay used in this invention by measuring the respective biomarker or its activity (e.g., PAM) in samples using both methods. Another possibility is to determine the median biomarker level in a representative normal population using the assay in question, assuming this test has sufficient analytical sensitivity, compare the result to the median biomarker level in another assay, and recalculate the calibration based on the difference obtained from this comparison. The calibrations used in this invention measure samples from normal (healthy) subjects. The median plasma PAM concentration was 78.6 ng / mL (interquartile range [IQR] 66.4–92.5 ng / mL).

[0047] As used herein, the term “diagnosis” means detecting a disease or determining the stage or degree of a disease. Typically, a diagnosis of a disease is based on an assessment of one or more factors and / or symptoms that indicate the disease. That is, a diagnosis may be made based on the presence, absence, or quantity of factors that indicate the presence or absence of a disease or disorder. Each factor or symptom considered to be an indicator of a particular disease does not have to be exclusively related to that particular disease; for example, there may be differential diagnoses that can be 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.

[0048] As used herein, the term “prognostic diagnosis” refers to the prediction of the possible course and outcome of a clinical condition or disease, such as sepsis. Prognosis is usually 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 course or outcome of a clinical condition or disease in a patient. The term “prognostic diagnosis” does not mean the ability to predict the course or outcome of a clinical condition or disease with 100% accuracy. Instead, a person skilled in the art will understand that the term “prognostic diagnosis” refers to an increased probability that a particular course or outcome will occur. That is, the course or outcome is more likely to occur in a patient exhibiting a given clinical condition or disease compared to an individual exhibiting no clinical condition or disease.

[0049] In a particular embodiment of the above method for using an assay to diagnose or predict the prognosis of a disease in a subject, and / or predict the risk of developing a disease or adverse event in a subject, and / or monitor a disease or adverse event in a subject, the assay comprises at least one binder to a structural epitope of PAM, and the disease is Dementia and / or dementia selected from the group including mild cognitive impairment (MCI), Alzheimer's disease, vascular dementia, mixed 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). Cardiovascular disorders, and / or a condition selected from the group including atherosclerosis, hypertension, heart failure (including acute and acute decompensated heart failure), atrial fibrillation, cardiovascular ischemia, cerebral ischemic injury, cardiogenic shock, stroke (including ischemic and hemorrhagic stroke and transient ischemic attack), and myocardial infarction. • Renal diseases, and / or, which may be selected from the group including nephrotoxicity (drug-induced kidney disease), acute kidney injury (AKI), chronic kidney disease (CKD), diabetic nephropathy, and end-stage renal disease (ESRD). Cancers and / or cancers that 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. • Infectious diseases caused by infectious organisms such as bacteria, viruses, fungi, or parasites, selected from the group including SIRS, sepsis, and septic shock, and / or • Metabolic disorders selected from the group including type 1 diabetes, type 2 diabetes, and metabolic syndrome. Selected from the group consisting of .

[0050] In one embodiment of this application, the disease is dementia, and the dementia is selected from the group including mild cognitive impairment (MCI), Alzheimer's disease, vascular dementia, mixed 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).

[0051] In certain embodiments, the dementia described above is Alzheimer's disease.

[0052] In one embodiment of this 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), gastric cancer, liver cancer, pancreatic cancer, leukemia, non-Hodgkin lymphoma, kidney cancer, esophageal cancer, and pharyngeal cancer.

[0053] In certain embodiments, the cancers are colorectal cancer and pancreatic cancer.

[0054] In one embodiment of this application, the disease is a cardiovascular disorder, which is selected from the group including atherosclerosis, hypertension, heart failure (including acute and acute decompensated heart failure), atrial fibrillation, cardiovascular ischemia, cerebral ischemic injury, cardiogenic shock, stroke (including ischemic and hemorrhagic stroke and transient ischemic attack), and myocardial infarction.

[0055] In certain embodiments, the cardiovascular disorder is heart failure (including acute and acute decompensated heart failure).

[0056] In another specific embodiment, the cardiovascular disorders are stroke (including ischemic and hemorrhagic strokes and transient ischemic attacks) and myocardial infarction.

[0057] In another specific embodiment, the cardiovascular disorder is atrial fibrillation (AF).

[0058] In another specific embodiment of this application, the disease is SIRS, sepsis, or septic shock.

[0059] In another specific embodiment of this application, the disease is type 1 diabetes, type 2 diabetes, or metabolic syndrome.

[0060] In the context of the method of the present invention, the body fluids and soluble tissue extracts 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 above method, the sample is selected from the group consisting of whole blood, serum, and plasma.

[0061] The term "tissue" refers to soluble components obtained by destroying organ structures using mechanical and ultrasonic forces, thereby releasing intracellular components into a liquid medium. In certain embodiments, the tissue is selected from the group including the liver, pituitary gland, and skin, which includes the whole brain, muscles, epidermis, dermis, and subcutaneous tissue.

[0062] The term "monitoring" refers to controlling (detecting any changes in) a patient's disease or pathophysiological state, such as the risk of developing a disease or adverse event, the severity of the disease, or the occurrence of a response to treatment.

[0063] The subject of the present invention is a method for performing the above-mentioned monitoring to evaluate changes in the risk of contracting a disease or adverse event, changes in the severity of a disease, or the response of a patient or subject to treatment.

[0064] A particular subject of the present invention is a method for performing the monitoring described above to evaluate the response of the subject to the preventive and / or therapeutic measures taken.

[0065] The subject of this invention is a method according to the present invention, used for stratifying the above-mentioned subjects into risk groups.

[0066] As used herein, the term “risk” refers to the probability of suffering an undesirable event or effect (e.g., disease or adverse event).

[0067] The term "elevated level" refers to a level that exceeds a certain threshold level.

[0068] The term "decreased level" refers to a level that is lower than a certain threshold level.

[0069] An "adverse event" is defined as an event that impairs the health of an individual. Adverse events may include, but are not limited to, cardiac events, cardiovascular events, cerebrovascular events, cancer, diabetes, and death from any cause. Adverse events include infections, severe infections, and septic-like systemic infections and sepsis. Adverse events are not events caused by acute exogenous substance-induced adverse events and / or exogenous substance-induced trauma. Exogenous substance-induced trauma includes those that can be induced by accidents, such as car accidents, and are therefore excluded from the group of adverse events.

[0070] In certain embodiments of the present invention, the adverse event is a cardiovascular event selected from the group including myocardial infarction, acute decompensated heart failure, stroke, and death associated with myocardial infarction, stroke, or acute heart failure.

[0071] The risk of contracting a disease or adverse event means the risk of contracting 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.

[0072] In certain embodiments of the present invention, "level of PAM and / or its isoform and / or its fragment" is the total concentration (preferably expressed as weight / volume; w / v) of the PAM and / or its isoform and / or its fragment having at least 12 amino acids, comprising 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, in a sample taken from a subject.

[0073] In this disclosure, the term "PAM" refers to the amino acid sequences of PAM isoforms 1-6 shown in SEQ ID NOs: 1-6. In some embodiments, the PAMs disclosed herein have 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% sequence identity with respect to the amino acid sequences of SEQ ID NOs: 1-6.

[0074] In some embodiments, the PAM is a functional fragment (i.e., PHM (SEQ ID NO: 7) or PAL (SEQ ID NO: 8), a PAM 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 some embodiments, the PAM is a variant or derivative of the PAM disclosed herein.

[0075] In a particular embodiment of the present invention, the above-mentioned peptidylglycine α-amidate monooxygenase is an active PAM.

[0076] The percentage of amino acid or nucleic acid sequence identity, or the term "% sequence identity," is defined herein as the percentage of residues in a candidate amino acid or nucleic acid sequence that are identical to residues in a reference sequence after two sequences have been aligned and gaps introduced, if necessary, to achieve the maximum identity percentage. In preferred embodiments, the calculation of at least the sequence identity percentage is performed without introducing gaps. Methods and computer programs for alignment are well known in the Art, such as "Align 2" or the BLAST service of the National Center for Biotechnology Information (NCBI).

[0077] In certain embodiments of the present invention, an assay is used to determine the levels of PAM and / or its isoforms and / or its fragments, wherein such assay is a sandwich assay, preferably a fully automated assay, wherein the assay comprises at least one binder to the structural epitope of PAM.

[0078] In one embodiment of the present invention, this may be a so-called POC test (point-of-care test), which is a testing technique that enables testing to be performed near the patient within one hour without requiring a fully automated assay system. An example of this technique is immunochromatography testing.

[0079] In one embodiment of the present invention, such an assay is a sandwich immunoassay using any type of detection technique, including but not limited to enzyme labeling, chemiluminescence labeling, and electrochemiluminescence labeling, and is preferably a fully automated assay. In one embodiment of the present invention, such an assay is an enzyme-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®, BiomerieuxVidas®, Alere Triage®, and Ortho Clinical Diagnostics Vitros®.

[0080] In a specific embodiment of this method, at least one of the two binders is labeled for detection.

[0081] Preferred detection methods include, for example, radioimmunoassays (RIA), homogeneous enzyme multiplication immunoassays (EMIT), chemiluminescent and fluorescent immunoassays, enzyme-linked immunoassays (ELISA), Luminex-based bead arrays, protein microarray assays, and various formats of immunoassays, such as rapid test formats like immunochromatography strip tests.

[0082] In a preferred embodiment, the label is selected from the group including chemiluminescent labels, enzyme labels, fluorescent labels, and radioactive iodine labels.

[0083] The assay may be homogeneous or heterogeneous, competitive or non-competitive. In one embodiment, the assay is a sandwich assay, which is a non-competitive immunoassay, in which the molecule to be detected and / or quantified is conjugated to a first antibody and a second antibody. The first antibody may be conjugated to a solid phase, e.g., beads, wells or the surface of another container, a tip or strip, and the second antibody may be labeled, e.g., with a dye, a radioisotope, or a reactive or catalytically active moiety. The amount of labeled antibody conjugated to the analyte is then measured by an appropriate method. The general compositions and procedures involved in a "sandwich assay" 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).

[0084] In another embodiment, the assay comprises two capture molecules, preferably antibodies, both present as dispersions in a liquid reaction mixture, wherein a first labeling component binds to the first capture molecule, the first labeling component being part of a labeling system based on fluorescence, chemiluminescence quenching, or amplification, and a second labeling component of the marking system binds to the second capture molecule, thereby generating a measurable signal that allows for the detection of a sandwich complex formed in a solution containing the sample when both capture molecules bind to the analyte.

[0085] In another embodiment, the labeling system includes a rare earth cryptotate or rare earth chelate in combination with a fluorescent dye or chemiluminescent dye, particularly a cyanine-type dye.

[0086] In the context of the present invention, fluorescence-based assays include the use of dyes, such as FAM (5- or 6-carboxyfluorescein), VIC, NED, fluorescein, fluorescein isothiocyanate (FITC), IRD-700 / 800, cyanine dyes, such as CY3, CY5, CY3.5, CY5.5, Cy7, xanthene, 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), TET, 6-carboxy The following can be selected from the group including -4',5'-dichloro-2',7'-dimethodyfluorescein (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, e.g., BODIPY TMR, Oregon green, coumarin, e.g., umbelliferone, benzimide, e.g., Hoechst 33258; phenanthridine, e.g., Texas red, Yakima yellow, Alexa Fluor, PET, ethidium bromide, acridinium dyes, carbazole dyes, phenoxazine dyes, porphyrin dyes, polymethine dyes, etc.

[0087] In the context of the present invention, chemiluminescence-based assays include the use of dyes based on the physical principles described in Kirk-Othmer, Encyclopedia of chemical technology, 4th ed. 1993. John Wiley & Sons, Vol. 15: 518-562, which is incorporated herein by reference, including the citations on pages 551-562 for chemiluminescent materials. Preferred chemiluminescent dyes are acridinium esters.

[0088] As referred to herein, an "assay" or "diagnostic assay" can be of any type applied in the field of diagnosis. Such an assay can be based on the binding of an analyte to be detected to one or more capture probes having a specific affinity. The binding agent that can be used to determine the level of PAM and / or its isoforms and / or its fragments has an affinity constant for PAM and / or its isoforms and / or its fragments of at least 10 7 M -1 , preferably 10 8 M -1 , and the preferred affinity constant is greater than 10 9 M -1 , most preferably greater than 10 10 M -1 The person skilled in the art knows that it can be considered to compensate for a lower affinity by applying a higher dose of the compound, and in this measure, it does not fall outside the scope of the present invention.

[0089] In the context of the present invention, “binding molecule” is a molecule that can be used to bind a target molecule or object molecule derived from a sample, i.e., an analyte (i.e., in the context of the present invention, PAM and its isoforms and fragments). Therefore, the binding molecule needs to be appropriately shaped both spatially and in terms of surface characteristics such as surface charge, hydrophobicity, hydrophilicity, Lewis donors and / or acceptors, in order to specifically bind to the target molecule or object molecule. Thus, the binding may be mediated by, for example, ionic, van der Waals, π-π, sigma-π, hydrophobic or hydrogen bonding interactions, or a combination of two or more of the aforementioned interactions between the capture molecule and the target molecule or object molecule.

[0090] In the context of the present invention, the binder molecule may be selected from the group including, for example, nucleic acid molecules, carbohydrate molecules, PNA molecules, proteins, antibodies, peptides, or glycoproteins. Preferably, the binder molecule is an antibody and comprises an antibody fragment having sufficient affinity for the target or molecule of interest, and includes recombinant antibodies or recombinant antibody fragments, as well as chemically and / or biochemically modified derivatives of the antibody or mutant chain thereof.

[0091] In certain embodiments, the binder may be selected from the group consisting of antibodies, antibody fragments, or non-IgG scaffolds.

[0092] The basic structural unit of an antibody is generally a tetramer consisting of two identical pairs of immunoglobulin chains, each pair having one light chain and one heavy chain. In each pair, the light and heavy chain variable regions bind to the antigen, while the constant region mediates effector function. Immunoglobulins also exist in various other forms, including, for example, Fv, Fab, and (Fab')2, as well as bifunctional hybrid antibodies and single-chain antibodies (e.g., Lanzavecchia et al. 1987; Huston et al. 1988; Bird et al. 1988; Hood et al. 1984; Hunkapiller & Hood, 1986). The immunoglobulin light or heavy chain variable region contains a framework region interrupted by three hypervariable regions, also called complementarity determining regions (CDRs) (see Kabat et al. 1983). As mentioned above, the CDRs are primarily involved in the binding of antigens to epitopes. An immune complex is an antibody, such as a monoclonal antibody, chimeric antibody, humanized antibody, or human antibody, or a functional antibody fragment, that specifically binds to an antigen.

[0093] Chimeric antibodies are antibodies in which the light chain and heavy chain genes are constructed, typically by genetic engineering, from immunoglobulin variable region genes and constant region genes belonging to different species. For example, the variable segment of a gene derived from a mouse monoclonal antibody can be linked to a human constant segment, such as κ and γ1 or γ3. Thus, in one example, a therapeutic chimeric antibody is a hybrid protein composed of a variable domain or antigen-binding domain derived from a mouse antibody and a constant domain or effector domain derived from a human antibody, but other mammalian species may be used, or the variable region may be created by molecular technology. Methods for producing chimeric antibodies are well known in the art; see, for example, U.S. Patent No. 5,807,715. "Humanized" immunoglobulins are immunoglobulins that include a human framework region and one or more CDRs derived from non-human (e.g., mouse, rat, or synthetic) immunoglobulins. The non-human immunoglobulin providing the CDRs is called the "donor," and the human immunoglobulin providing the framework is called the "acceptor." In one embodiment, all CDRs in the humanized immunoglobulin are derived from the donor immunoglobulin. Constant regions are not required, but if present, they must be substantially identical to the human immunoglobulin constant regions, i.e., at least about 85-90%, e.g., about 95% or more identical. Therefore, in some cases, all parts of the humanized immunoglobulin, except for the CDRs, are substantially identical to the corresponding parts of the natural human immunoglobulin sequence. A "humanized antibody" is an antibody containing humanized light chain and humanized heavy chain immunoglobulins. The humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions with amino acids obtained from the donor framework. Humanized or other monoclonal antibodies may have further conserved amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions. Exemplary conserved substitutions include those of gly, ala;val, ile, leu;asp, glu;asn, gln;ser, thr;lys, arg; and phe, tyr.Humanized immunoglobulins can be constructed by genetic engineering (see, for example, U.S. Patent No. 5,585,089). Human antibodies are antibodies whose light and heavy chain genes are of human origin. Human antibodies can be produced using methods known in the art. Human antibodies can be produced by immortalizing human B cells that secrete the antibody of interest. Immortalization can be achieved, for example, by EBV infection, or by fusing human B cells with myeloma or hybridoma cells to produce trioma cells. Human antibodies can also be produced by phage display (see, for example, PCT International Publication No. 91 / 17271, PCT International Publication No. 92 / 001047, PCT International Publication No. 92 / 20791 (these are incorporated herein by reference)), or selected from a human combinatorial monoclonal antibody library (see Morphosys website). Human antibodies can also be prepared by using transgenic animals that possess human immunoglobulin genes (see, for example, PCT International Publication No. 93 / 12227 and PCT International Publication No. 91 / 10741, which are incorporated herein by reference).

[0094] Therefore, PAM antibodies may have formats known in the art. Examples include human antibodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, and CDR-transplanted antibodies. In preferred embodiments, the antibodies according to the present invention include, for example, antibodies recombinantly produced as IgG, a typical full-length immunoglobulin, or antibody fragments containing at least the F variable domain of the heavy and / or light chain as, for example, chemically bound antibodies (fragment antigen binding), and include, but are not limited to, Fab minibodies, single-chain Fab antibodies, monovalent Fab antibodies with epitope tags, such as Fab-V5Sx2; bivalent Fab (mini-antibodies) dimerized with a CH3 domain; bivalent or polyvalent Fab formed, for example, through multimerization using heterologous domains, such as dimerization of the dHLX domain, such as Fab-dHLX-FSx2; F(ab')2 fragments, scFv fragments, multimerized polyvalent and / or multispecific scFv fragments, bivalent and / or bispecific diabodies, BITE® (bispecific T-cell engager), trifunctional antibodies, such as polyvalent antibodies of a different class than G; single-domain antibodies, such as nanobodies derived from camelid or fish immunoglobulins, and many others.

[0095] In addition to anti-PAM antibodies, other biopolymer scaffolds are well known in the art to complex with target molecules and are used to produce highly target-specific biopolymers. Examples include aptamers, spiegelmers, anticharin, and conotoxins.

[0096] The non-Ig scaffold may also be a protein scaffold and can be used as an antibody mimetic because it can bind to a ligand or antigen. Non-Ig scaffolds include tetranectin-based non-Ig scaffolds (e.g., described in U.S. Patent Application Publication 2010 / 0028995), fibronectin scaffolds (e.g., described in European Patent No. 1266025); lipocalin-based scaffolds (e.g., described in International Publication 2011 / 154420); ubiquitin scaffolds (e.g., described in International Publication 2011 / 073214); translocation scaffolds (e.g., described in U.S. Patent Application Publication 2004 / 0023334); protein A scaffolds (e.g., described in European Patent No. 2231860); ankyrin repeat-based scaffolds (e.g., described in International Publication 2010 / 060748); microprotein (preferably microproteins forming cystine knots) scaffolds (e.g., described in European Patent No. 2314308); Fyn The scaffolds can be selected from a group including SH3 domain-based scaffolds (e.g., described in International Publication No. 2011 / 023685), EGFR-A domain-based scaffolds (e.g., described in International Publication No. 2005 / 040229), and Kunitz domain-based scaffolds (e.g., described in European Patent No. 1941867). Non-Ig scaffolds may be peptide or oligonucleotide aptamers. Aptamers are usually constructed by selecting them from a large random sequence pool and are short chains of oligonucleotides (DNA, RNA, or XNA; Xu et al. 2010, Deng et al. 2014), or short variable peptide domains conjugated to a protein scaffold (Li et al. 2011).

[0097] The chemiluminescent label may be an acridinium ester label, a steroid label accompanied by an isoluminol label, or the like.

[0098] Enzyme labels may include lactate dehydrogenase (LDH), creatine kinase (CPK), alkaline phosphatase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), acid phosphatase, glucose-6-phosphate dehydrogenase, and the like.

[0099] In one embodiment of the present invention, at least one of the two binders is bonded to the solid phase as magnetic particles and to the polystyrene surface.

[0100] The subject of the present invention is a method for determining the levels of PAM and / or its isoforms and / or fragments in a body fluid sample using an assay, wherein the assay comprises two binders that bind to two different epitopes of PAM, the two binders for epitopes with a length of at least 5 amino acids, preferably at least 4 amino acids.

[0101] Epitopes, also known as antigenic determinants, are parts of an antigen (e.g., a peptide or protein) that are recognized by the immune system, particularly by antibodies. For example, an epitope is a specific piece of an antigen to which an antibody binds. The antibody portion that binds to an epitope is called a paratope. Protein antigen epitopes are divided into two categories based on their structure and interaction with paratopes: structural epitopes and linear epitopes.

[0102] Linear or sequential epitopes are epitopes recognized by antibodies by their linear sequence or primary structure of amino acids and formed by a 3-D stereochemistry adopted through interactions between adjacent amino acid residues. Structural epitopes and linear epitopes interact with paratopes based on the 3-D stereochemistry adopted by the epitope, which is determined by the surface features of the epitope residues involved and the shape or tertiary structure of other segments of the antigen. In contrast, structural epitopes are formed by 3-D stereochemistry adopted through interactions that are continuously discontinuous but closely related in three-dimensional spatial amino acid residues.

[0103] Regarding structural epitopes, discontinuous stretches of amino acids come together during protein folding to form antibody binding sites, and antibody binding to such epitopes depends on the proper formation of the three-dimensional shape or tertiary structure of the protein antigen (Barlow et al. 1986. "Continuous and discontinuous protein antigenic determinants." Nature 322:747-748). When a protein (e.g., an enzyme) is denatured, the secondary, tertiary, and (in the case of subunits) quaternary structures change, while only the peptide bonds of the primary structure between amino acids remain intact. Since all structural levels of a protein determine its function, once a protein or enzyme is denatured, it can no longer perform its function. When a protein is denatured, its 3-D structure is lost, structural epitopes are no longer exposed, and binders specific to structural epitopes can no longer bind.

[0104] In one embodiment of the present invention, the binder for the structural epitope of PAM does not bind to modified PAM or modified subunits of PAM (e.g., PAL or PHM).

[0105] In certain embodiments, the binder for the structural epitope of PAM does not bind to the modified PAM or the modified subunit of PAM (e.g., PAL or PHM) using Western blotting techniques as described in Example 2.

[0106] In another embodiment of the present invention, the binder for the three-dimensional epitope of PAM binds to enzymatically active PAM or an enzymatically active subunit of PAM (e.g., PAL or PHM), but does not bind to enzymatically inactive PAM or an enzymatically inactive subunit of PAM (e.g., PAL or PHM).

[0107] Binding agents (e.g., antibodies) can be produced using different immunological strategies. Classical protein immunological strategies almost always rely on synthetic peptides, large fragments or full-length recombinant proteins of bacterial or mammalian cell origin, or purified native proteins as sources of immunogens. Peptides of 12-20 amino acid residues, the size conventionally used, rarely contain more than two epitopes and are likely to lack secondary and tertiary three-dimensional structure. Therefore, anti-peptide antibodies often lack the ability to bind to native proteins due to the unstructured nature of peptides. Full-length protein antigens address many of the limitations inherent in peptides. Essentially, they contain surface regions, multiple immunogenic epitopes, and are likely to fold to form (at least partially) a native structure, even when synthesized in prokaryotic systems. More innovative approaches, such as DNA (or "genetic") immunization, are emerging as alternative and / or complementary tools to classical antibody production strategies. DNA immunization immunizes animals using expression plasmids encoding a selected antigen. Transfected tissues of immunized animals express the antigen, which subsequently drives an antibody response. DNA immunization using sequences encoding polypeptide protein regions combines the advantages of both full-length proteins and peptides with an immunological approach, providing an immunogen that contains a relatively large region of the target protein with multiple potential epitopes and offers greater accessibility than full-length proteins (Brown et al. 2011. PLoS One. 6(12):e28718).

[0108] In one embodiment of the present invention, the binder is produced using a large fragment protein, a full-length protein, or DNA immunotherapy.

[0109] A large fragment protein is defined as a peptide sequence having at least 100 amino acids, more preferably at least 150 amino acids, even more preferably at least 200 amino acids, even more preferably at least 250 amino acids, and most preferably at least 300 amino acids.

[0110] In certain embodiments, the large fragment protein is SEQ ID NO: 7 and / or SEQ ID NO: 8 and / or SEQ ID NO: 25.

[0111] Another embodiment of the present invention relates to a method for producing antibodies against the three-dimensional structure epitopes of PAM.

[0112] A method for generating antibodies that target structural epitopes includes the following steps: • Synthesis of DNA encoding full-length PAM, PHM subunits, PAL subunits, or enzymatically active PAM protein fragments, and / or Cloning into an expression vector of DNA encoding full-length PAM, PHM subunit, PAL subunit, or enzymatically active PAM protein fragment, and / or • Transfection of expression vectors into appropriate cell lines, and / or • Purification of the expressed PAM construct (e.g., using nickel affinity chromatography or anion exchange chromatography for the polyhistidine C-terminal cleavage construct), and / or • Testing of the activity of the expressed PAM construct, and / or • Immunization of animals with enzymatically active full-length PAM, PHM subunit, PAL subunit, or enzymatically active PAM protein fragments (e.g., using fusion technology between immunized Balb / c mouse splenocytes and SP2 / 0 myeloma cells), and / or • Screening of hybridoma cell lines for the secretion ability of specific monoclonal antibodies against enzymatically active full-length PAM, enzymatically active PAM protein fragments, PHM subunits, or PAL subunits (e.g., using ELISA assay techniques or flow cytometry), and / or • Purification of antibodies from cell lines identified as positive (e.g., using protein A chromatography).

[0113] Another method for generating antibodies that target structural epitopes includes the following steps: • Synthesis of DNA encoding enzymatically active full-length PAM, enzymatically active PHM subunits, enzymatically active PAL subunits, or enzymatically active PAM protein fragments, and / or • Incorporation of synthetic DNA into plasmid vectors for DNA immunization, and / or • Immunization of host animals with plasmid DNA using delivery methods such as gene guns, electroporation, or intramuscular injection, and / or • Fusion of animal spleen cells and myeloma cells to create hybridoma cell lines, and / or • Screening of hybridoma cell lines for the secretion ability of specific monoclonal antibodies against enzymatically active full-length PAM, enzymatically active PHM subunits, enzymatically active PAL subunits, or enzymatically active PAM protein fragments (e.g., using ELISA assay techniques or flow cytometry), and / or • Purification of antibodies from cell lines identified as positive (e.g., using protein A chromatography).

[0114] A further embodiment of the present invention relates to a method for screening three-dimensional antibodies.

[0115] Methods for screening antibodies that bind to the structural epitopes of enzymatically active full-length PAM, enzymatically active PHM subunits, enzymatically active PAL subunits, or enzymatically active PAM protein fragments may include, but are not limited to, the following: • Western blot analysis, and / or • Non-denatured PAGE analysis, and / or • Surface plasmon resonance (SPR) and related technologies, and / or • Cocrystallization analysis, and / or • Enzyme-linked immunosorbent assay (ELISA), and / or ·Co-elution analysis.

[0116] In one embodiment, a method for screening for antibodies that bind to the structural epitopes of enzymatically active full-length PAM, enzymatically active PHM subunits, enzymatically active PAL subunits, or enzymatically active PAM protein fragments is Western blot analysis, which includes the following steps: • Prepare a protein sample containing enzymatically active full-length PAM, enzymatically active PHM subunit, enzymatically active PAL subunit, or enzymatically active PAM protein fragment in a denaturation sample buffer (e.g., containing sodium dodecyl sulfate (SDS)), and then heat it to over 70°C to ensure protein denaturation, and / or • Perform sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to separate proteins by molecular weight, transfer the proteins from the gel to a nitrocellulose membrane, and / or • Blocking of nitrocellulose membranes with albumin to prevent nonspecific binding, and / or • Incubation of nitrocellulose membranes with potential conformational antibody candidates, and / or • Washing of nitrocellulose membranes to remove unbound species, and / or • Addition of a secondary antibody conjugated to an enzyme or fluorescent tag, and / or • Signal detection.

[0117] The absence of signal in the Western blot indicates that the developed antibody recognizes the structural epitope of enzymatically active full-length PAM, enzymatically active PHM subunit, enzymatically active PAL subunit, or enzymatically active PAM protein fragment.

[0118] In one embodiment, a method for screening antibodies that bind to the structural epitopes of enzymatically active full-length PAM, enzymatically active PHM subunits, enzymatically active PAL subunits, or enzymatically active PAM protein fragments is non-denaturing PAGE analysis, which includes the following steps: • Preparation of samples without the use of denaturants and heating, and / or • Performing non-denaturing (natural) polyacrylamide gel electrophoresis (PAGE) to separate proteins by molecular weight transfer of proteins from the gel to a nitrocellulose membrane, and / or • Blocking of nitrocellulose membranes with albumin to prevent nonspecific binding, and / or • Incubation of nitrocellulose membranes with potential conformational antibody candidates, and / or • Washing of nitrocellulose membranes to remove unbound species, and / or • Addition of a secondary antibody conjugated to an enzyme or fluorescent tag, and / or • Signal detection.

[0119] The detection of the signal indicates that the developed antibody recognizes a structural epitope of enzymatically active full-length PAM, an enzymatically active PHM subunit, an enzymatically active PAL subunit, or an enzymatically active PAM protein fragment.

[0120] In one embodiment, a method for screening antibodies that bind to the structural epitopes of enzymatically active full-length PAM, enzymatically active PHM subunits, enzymatically active PAL subunits, or enzymatically active PAM protein fragments is surface plasmon resonance (SPR), which includes the following steps: • Immobilization of native and denatured forms of PAM protein (enzymatically active full-length PAM, enzymatically active PHM subunit, enzymatically active PAL subunit, or enzymatically active PAM protein fragment) on an SPR chip, and / or • Application of antibodies to the chip, and / or • Real-time measurement of bonding, and / or • Comparison of binding dynamics between native PAM proteins and denatured PAM proteins.

[0121] The binding of PAM proteins to their native form and their non-binding to their denatured form demonstrate the specificity of each antibody to its respective structural epitope.

[0122] In one embodiment, a method for screening antibodies that bind to the structural epitopes of enzymatically active full-length PAM, enzymatically active PHM subunits, enzymatically active PAL subunits, or enzymatically active PAM protein fragments is cocrystallization analysis, which includes the following steps: • A mixture of the antibody to be tested and the antigen (enzymatically active full-length PAM, enzymatically active PHM subunit, enzymatically active PAL subunit, or enzymatically active PAM protein fragment), and / or • Screening of crystallization conditions, and / or • X-ray diffraction after obtaining crystals for structural determination.

[0123] Structural analysis confirms the interaction between the antibody and the three-dimensional epitope on the antigen.

[0124] In one embodiment, a method for screening for antibodies that bind to the structural epitopes of enzymatically active full-length PAM, enzymatically active PHM subunits, enzymatically active PAL subunits, or enzymatically active PAM protein fragments is an enzyme-linked immunosorbent assay (ELISA), which includes the following steps: Coating of a solid phase (e.g., wells of a microtiter plate) with both natural antigens (enzymatically active full-length PAM, enzymatically active PHM subunits, enzymatically active PAL subunits, or enzymatically active PAM protein fragments) and denatured antigens, and / or • A blocking step to prevent nonspecific binding, and / or • Addition of the antibody to be tested, and / or • A washing step to remove unbound antibodies, and / or • Addition of enzyme-coupled secondary antibody and substrate, and / or • Measurement of enzyme reactions.

[0125] The enzymatic reaction is measured, and the difference in binding between the native antigen and the denatured antigen indicates the preference for the three-dimensional epitope.

[0126] In one embodiment, a method for screening antibodies that bind to the structural epitopes of enzymatically active full-length PAM, enzymatically active PHM subunits, enzymatically active PAL subunits, or enzymatically active PAM protein fragments is co-elution analysis, which includes the following steps: • Mixing of antibodies and antigens (enzymatically active full-length PAM, enzymatically active PHM subunit, enzymatically active PAL subunit, or enzymatically active PAM protein fragment) in either their natural state or a denatured state (e.g., by heating to over 70°C), and / or • Incubation of the mixture for a certain period of time, and / or • Application of HPLC-based size exclusion chromatography to mixtures, and analysis of elution profiles.

[0127] Antibody-antigen complexes elute more quickly from the SEC column due to their larger molecular weight, producing additional peaks in the chromatogram, while peaks for unreacted antigens and antibodies have lower intensity. When the antibody reacts with a linear epitope, the antibody-antigen complex elution peak is formed only when a denatured antigen is used. When the antibody recognizes a structural epitope, the antibody-antigen complex elution peak is formed only when a non-denatured antigen is used.

[0128] In one embodiment of the present invention, the three-dimensional epitope is related to the following PAM sequences: 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, SEQ ID NO: 10, and SEQ ID NO: 25.

[0129] The above three-dimensional epitope may contain at least six amino acids, preferably at least five amino acids, and most preferably at least four amino acids.

[0130] In one embodiment of the present invention, the first and second binders bind to the stereochemical epitopes contained in the following PAM sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 10.

[0131] In one embodiment of the present invention, the first and second binders bind to the stereochemical epitopes contained within the PAL subunit (SEQ ID NO: 8) of PAM.

[0132] In one embodiment of the present invention, the first and second binders bind to the stereochemical epitopes contained within the PHM subunit (SEQ ID NO: 7) of PAM.

[0133] In one specific embodiment of the present invention, the first binder binds to a stereochemical epitope contained within the PAL subunit of PAM (SEQ ID NO: 8), and the second binder binds to a stereochemical epitope contained within the PHM subunit of PAM (SEQ ID NO: 25).

[0134] The use of at least two binders for determining the levels of PAM and / or its isoforms and / or fragments thereof, wherein at least one of the binders is used for a stereochemical epitope contained within the following PAM sequences: SEQ ID NO: 8 and / or SEQ ID NO: 25.

[0135] One embodiment of the present application relates to a kit for performing a method for diagnosing or prognosing a disease in a subject, and / or predicting the risk of contracting a disease or adverse event in a subject, and / or monitoring a disease or adverse event in a subject, the kit comprising at least two conjugates to the stereochemical epitopes in the following PAM sequences: 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, SEQ ID NO: 10 and SEQ ID NO: 25.

[0136] A particular embodiment of this application relates to a kit for detecting the level of PAM, comprising one or more binders that bind to a stereochemical epitope in a PAM sequence 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, SEQ ID NO: 10, and SEQ ID NO: 25.

[0137] Another embodiment of the present invention relates to a method for obtaining monoclonal antibodies.

[0138] In all of the following embodiments, the term monoclonal antibody means monoclonal antibody and fragments of monoclonal antibodies, such as those detailed herein, and more particularly monoclonal antibodies.

[0139] Hybridoma In a further embodiment, the antibody according to the present invention is a monoclonal antibody that can be obtained by a method comprising: i) A step of fusing antibody-secreting cells derived from animals pre-immunized with an antigen with myeloma cells to obtain a large number of hybridomas, and / or ii) A step of isolating a hybridoma that produces a desired monoclonal antibody from the above-mentioned number of hybridomas.

[0140] In certain embodiments, the antibody according to the present invention is a monoclonal antibody that can be obtained by isolating a hybridoma that produces a desired monoclonal antibody from a large number of hybridomas, where the large number of hybridomas is produced by fusing antibody-secreting cells derived from animals that have been pre-immunized with an antigen with myeloma cells to obtain a large number of hybridomas.

[0141] In particular, the desired monoclonal antibody is at least 10 7 M -1 Preferably 10 8 M -1 It is a monoclonal antibody that binds to the antigen with a binding affinity of 10, and a more preferred affinity is 10 9 M -1Larger, most preferably 10 10 M -1 Larger.

[0142] To determine the affinity of an antibody for a target (e.g., PAM), the binding kinetics of the target to the immobilized antibody may be determined by label-free surface plasmon resonance using the Biacore2000 system (GE Healthcare Europe GmbH, Freiburg, Germany).

[0143] In a particular embodiment of the method for obtaining antibodies, in step i), the animal is a mammal, more specifically a rabbit, mouse or rat, more specifically a mouse, and more specifically a Balb / c mouse.

[0144] In a particular embodiment of the method for obtaining antibodies, in step i), the antibody-secreting cells are splenic cells, more specifically activated B cells.

[0145] In a particular embodiment of the method for obtaining antibodies, step i) involves the use of polyethylene glycol.

[0146] In certain embodiments of the method for obtaining antibodies, in step i), the myeloma cells are of a mammalian origin, and in certain embodiments, they are of the same species of mammal from which a large number of antibody-secreting cells are obtained. In certain specific embodiments of the method for obtaining antibodies, in step i), the myeloma cells are of the cell line SP2 / 0.

[0147] In certain embodiments of the method for obtaining antibodies, the fusion in step i) includes PEG-mediated fusion, Sendai virus-mediated fusion, or electric current-mediated fusion.

[0148] In certain embodiments of the method for obtaining antibodies, the isolation in step ii) includes performing an antibody capture assay, an antigen capture assay, and / or functional screening.

[0149] In certain embodiments of the method for obtaining antibodies, in step ii), isolating a hybridoma that produces the desired monoclonal antibody may involve cloning and recloning the hybridoma using the limiting dilution method.

[0150] In one embodiment, the antigen capture assay includes the following: a) Binding the produced antibody to a substrate, particularly a solid substrate, and / or b) Binding the antigen to the antibody, and / or c) Removing unbound antigens by washing, and / or d) Detecting the bound antigen; Alternatively, the antigen capture assay described above includes: a) Binding the antigen to the produced antibody to form an antibody-antigen complex, and / or b) Binding the antibody-antigen complex to a substrate, particularly a solid substrate, and / or c) Removing unbound antigens by washing, and / or d) Detect the bound antigen.

[0151] In one embodiment, the isolation described in step ii) includes performing an enzyme-linked immunosorbent assay, fluorescence-activated cell sorting, cell staining, immunoprecipitation, and / or Western blotting.

[0152] In one embodiment, the detection of the antibody or antigen is achieved using an immunoassay.

[0153] In one embodiment, the animal is a transgenic animal, particularly a transgenic mouse (in particular, in which the mouse immunoglobulin (Ig) locus in the transgenic animal genome is replaced with a human locus), such as HuMabMouse or XenoMouse.

[0154] In one embodiment, the antigen comprises a peptide listed in Table 1 herein, which in certain embodiments (particularly for immunology) may be conjugated to a protein, in particular a serum protein, more specifically serum albumin, and more specifically BSA.

[0155] In a preferred embodiment, the antibody according to the present invention is a monoclonal antibody that can be obtained by a method comprising the following: i) Using polyethylene glycol, fusion of Balb / c mouse-derived splenocytes pre-immunized with the peptides listed in Table 1 herein with SP2 / 0 myeloma cells to obtain a large number of hybridomas, and / or ii) Isolating hybridomas that produce the desired monoclonal antibody from the above-mentioned number of hybridomas; More preferably, the method includes: 1) Growing hybridomas in HAT medium [RPMI1640 culture medium supplemented with 20% fetal bovine serum and HAT supplements] for a first period (especially 2 weeks), and / or 2) Subsequently, replace the HAT medium with HT medium for multiple (especially 3) passagings, and / or 3) Subsequently, during the second period, particularly until three weeks have elapsed since fusion, return to normal cell culture medium and / or 4) Perform primary screening of cell culture supernatant for antigen-specific IgG antibodies, and / or 5) Propagating microcultures of cells that tested positive in 4), and / or 6) Retest the cell culture supernatant of the microculture for antigen-specific IgG antibodies, and / or 7) Cloning and recloning the cultures that were positive in 6) using the limiting dilution method, and / or 8) Optionally, determine the isotype of the clone obtained from 7), and / or 9) Optionally, purify the antibody via protein A. Phage display In a further embodiment, the antibody according to the present invention is a monoclonal antibody that can be obtained by a method comprising: i) Isolating at least one antibody having affinity for the antigen from an antibody gene library, and / or ii) To generate at least one cell line expressing at least one of the above antibodies, and / or iii) Isolate at least one antibody from the culture of at least one cell line obtained in step ii).

[0156] Antibodies that have affinity for an antigen are, in particular, at least 10 7 M -1 Preferably 10 8 M -1 The antibody has a binding affinity of 10, with a more preferred affinity being 10. 9 M -1 Larger, most preferably 10 10 M -1 Larger.

[0157] In a particular embodiment, the antibody according to the present invention is a monoclonal antibody that can be obtained by isolating at least one antibody from a culture derived from at least one cell line expressing at least one antibody having affinity for an antigen from an antibody gene library.

[0158] In one embodiment, the antigen comprises a peptide listed in Table 1 herein, which may be bound to a solid phase in certain embodiments.

[0159] In a particular embodiment of the method for obtaining antibodies, in step i), the antibody gene library is a naive antibody gene library, more specifically a human naive antibody gene library, and more specifically in the library, the antibodies are presented via phage display, i.e., on a phage containing the nucleotide sequence encoding each such antibody; more specifically, the antibody gene library is a library comprising libraries HAL7, HAL8, or HAL9, more specifically the human naive antibody gene library HAL7 / 8.

[0160] In certain embodiments of the method for obtaining antibodies, in step i), screening includes the use of an antigen, more particularly an antigen containing a tag linked to it via two different spacers, and more particularly a biotin tag. In certain embodiments, such a panning strategy includes a mixture of panning rounds with nonspecifically bound antigens and antigens specifically bound via a tag (in the case of a biotin tag, bound to streptavidin). In this way, background from nonspecific binders can be minimized.

[0161] In certain embodiments of the method for obtaining an antibody, in step i), if the library is a phage display library, the antibody is isolated by isolating a phage that displays the antibody (and contains a nucleotide sequence encoding the antibody).

[0162] In a particular embodiment of the method for obtaining antibodies, in step ii), the cell line is generated by introducing a nucleotide sequence encoding the antibody, and in an embodiment where the library in step i) is a phage display library, the phages isolated from step i) can be used to produce an antibody-expressing bacterial strain, such as an Escherichia coli (E. coli) strain.

[0163] In certain embodiments of the method for obtaining antibodies, in step iv), the library in step i) is a phage display library, and in embodiments where the bacterial strain is prepared in step ii), the antibody can be isolated from the culture supernatant.

[0164] When used to describe methods for obtaining antibodies, the term "one antibody" in the expression "at least one antibody" is understood to include two or more antibody molecules, particularly those having the same amino acid sequence. This understanding applies to the term "one cell line," with any necessary modifications.

[0165] In a particular embodiment of the method for obtaining antibodies, two or more antibodies (referring to a number of antibodies, each having a distinct amino acid sequence) are isolated in step i), and thus two or more cell lines are generated in step ii). Such a method may involve, for example, selection of clones that are positive for binding to an antigen via a binding assay, such as an ELISA assay with an antigen, and the cells that are positive for binding to the antigen can be isolated to produce monoclonal cell lines.

[0166] In a preferred embodiment, the antibody according to the present invention is a monoclonal antibody that can be obtained by a method comprising the following: i) Isolating at least one antibody having affinity for an antigen from an antibody gene library containing the human naive antibody gene library HAL7 / 8 by eluting a phage possessing the antibody from the library, and / or ii) To generate at least one Escherichia coli cell line expressing at least one of the above antibodies, and / or iii) Isolate at least one antibody from the supernatant of the culture of at least one Escherichia coli cell line obtained in step ii).

[0167] In a further embodiment, the antibody fragment according to the present invention is prepared by a method involving enzymatic digestion of the antibody. In a particular embodiment, this method prepares, for example, Fab or F(ab)2 antibody fragments. In a particular embodiment, this method involves digestion with pepsin or papain immobilized on the surface, which is optional.

[0168] In certain embodiments, antibodies can be humanized by CDR transplantation, particularly by a method comprising the following steps: -A step of extracting RNA from a hybridoma expressing the antibody of interest (for example, obtained by a method described herein), and / or - The steps of amplifying the extracted RNA via RT-PCR, using a primer set particularly specific to the heavy and light chains of the antibody of interest, to obtain a DNA product, and / or -A step of further amplifying the DNA product via PCR, particularly using a seminested primer set specific to the antibody variable region, and / or - A step of determining the sequence of the DNA product, and / or - A step of determining the humanized sequence for the variable heavy chain and variable light chain sequences (of the desired antibody) by aligning the above sequence with a homologous human framework sequence.

[0169] In certain embodiments, antibodies can be humanized by aligning the sequence of a DNA product obtained by amplifying RNA extracted from a hybridoma expressing the antibody of interest via RT-PCR using primer sets particularly specific to the heavy and light chains of the antibody of interest, and further by amplifying the DNA obtained therefrom using homologous human framework sequences via PCR using semi-nested primer sets particularly specific to the antibody variable region, thereby determining the humanized sequence for the variable heavy and variable light chain sequences (of the desired antibody).

[0170] In a particular embodiment, the antibody may be humanized by: - Determining the complementary determinant region (CDR), which can be achieved by analyzing the structural interaction between the framework region (FR), the complementary determinant region (CDR), and the antigen, and / or - Transplanting the above CDR sequence into a human framework region.

[0171] In certain embodiments, antibodies can be humanized by transplanting a CDR sequence into a human framework region, which can be determined by analyzing the structural interaction between the framework region (FR), the complementary determination region (CDR), and the antigen.

[0172] In certain embodiments, mutations in the amino acid sequences of CDR or FR can be introduced, for example, by a random approach using a phage display library, or by a targeted approach guided by molecular modeling, to maintain a structural interaction with the antigen (this structural interaction can otherwise be lost by introducing a human FR sequence).

[0173] The DNA sequence encoding the antibody, as detailed herein, may be introduced into a cell by known genetic engineering techniques and used for antibody production.

[0174] Antibody production In a further embodiment, the antibody according to the present invention is a monoclonal antibody that can be obtained by the method described herein, which is prepared by a method comprising: -Culturing cell lines containing nucleotide sequences encoding antibodies, - Isolate the antibody from the culture.

[0175] In a further specific embodiment, the antibody according to the present invention is a monoclonal antibody that can be obtained by the method described herein, which is produced by isolating the antibody from a culture of a cell line containing a nucleotide sequence encoding the antibody.

[0176] In certain embodiments of the method, the cell line is prepared as described above herein and includes bacterial cells such as Gram-negative bacteria, e.g., Escherichia coli, Proteus mirabilis, or Pseudomonas putidas; Gram-positive bacteria, e.g., Lactobacillus species such as Bacillus brevis, Bacillus subtilis, Bacillus megaterium, Lactobacillus zeae / casei, or Lactobacillus paracasei; or Streptomyces species such as Streptomyces lividans; and yeast, e.g., Pichia pastris. Eukaryotic cells such as *Trichoderma*, *A. niger* (e.g., the subgenus *A. awamori*) and *Aspergillus oryzae*, and *Trichoderma*. Fungi such as Chrysosporium species (e.g., reesei), C. lucknowense; Leishmania, for example, L. talentlae (L.This may include protozoa such as *Tarentolae*; insect cells transfected with baculoviruses, such as AcNPV, such as insect cell lines derived from the fall armyworm (*Spodoptera frugiperda*), such as Sf-9 or Sf-21; insect cell lines derived from *Drosophila melanogaster*, such as DS2; or insect cell lines derived from the nettle moth (*Trichopulsia ni*), such as High Five cells (BTI-TN-5B1-4); hamster cells, such as Chinese hamster ovary cells, such as K1-, DukX B11-, DG44, Lec13, or BHK cells; mouse cells, such as mouse myeloma cells, such as NS0 cells; and mammalian cells, such as Homo sapiens cells, such as Per.C6 cells, AGE1.HN cells, HEK293 cells, etc.

[0177] In certain embodiments of the method, the cells may be hybridoma cells, such as those described herein.

[0178] In certain embodiments of the method, culturing may be carried out in static suspension culture, agitated suspension culture, membrane-based culture, matrix-based culture, or in a high-cell-density bioreactor. Vessels for such cultures may be selected from the group including T flasks, roller cultures, spinner cultures, agitated tank bioreactors, air-lift bioreactors, static membrane-based or matrix-based culture systems, suspension bioreactors, fluidized bed bioreactors, ceramic bioreactors, perfusion systems, and hollow fiber bioreactors.

[0179] In certain embodiments of the method, cells can be immobilized on a matrix.

[0180] High cell density bioreactors are particularly important. 8 This culture system can achieve cell densities exceeding cells / ml.

[0181] In a further embodiment, the antibody according to the present invention is a monoclonal antibody that can be obtained by the method described herein, which is prepared by a method comprising: - To produce transgenic plants or animals containing nucleotide sequences encoding antibodies, and / or - Isolating antibodies from the above-mentioned plants or animals, or from the secretions or products of said plants or animals.

[0182] In a particular further embodiment, the antibody according to the present invention is a monoclonal antibody that can be obtained by the method herein, which is produced by isolating the antibody from a transgenic plant or transgenic animal having a nucleotide sequence encoding the antibody, or from a secretion or product of a transgenic plant or transgenic animal.

[0183] The animals mentioned above may be selected from, for example, chickens, mice, rats, rabbits, cows, goats, sheep, and pigs. The secretions or products mentioned above may be, for example, milk or eggs. The plants mentioned above may be selected from, for example, tobacco (N. tabacum or N. benthamiana), duckweed (Lemna minor), Chlamydomonas reinhardtii, rice, Arabidopsis thaliana, alfalfa (Medicago sativa), lettuce, and maize.

[0184] Antibodies can be isolated in certain embodiments by physicochemical fractionation, e.g., size exclusion chromatography, precipitation using ammonium sulfate, ion exchange chromatography, immobilized metal chelate chromatography, gel filtration, and zone electrophoresis; by classification, e.g., based on binding to bacterial proteins A, G, or L, or jacarin; and by antigen-specific affinity purification with immobilized ligands / antigens. If necessary, low molecular weight components can be removed by methods such as dialysis, desalting, and diafiltration.

[0185] In some embodiments, the antibody is encoded by a nucleotide sequence, which is a reverse transcription of an amino acid sequence derived from the antibody, prepared by one of the methods described herein.

[0186] Based on the above context, the following sequentially numbered embodiments provide further specific aspects of the present invention. 1. A method for determining the levels of PAM and / or its isoforms and / or fragments in a body fluid or tissue sample using an assay comprising at least one binder to the structural epitope of PAM.

[0187] 2. The assay comprising two binders that bind to two different regions of peptidylglycine α-amidate monooxygenase (PAM), each of the two binders being a method for determining the level of PAM and / or its isoforms and / or fragments in a sample of body fluid or tissue according to Embodiment 1, relative to the three-dimensional epitope of PAM.

[0188] 3. A method for determining the level of PAM and / or its isoforms and / or fragments in a sample of body fluid or tissue according to Embodiment 2, wherein the first binder of the two binders described above binds to a stereochemical epitope contained in the PHM subunit (SEQ ID NO: 7) of peptidylglycine α-amidate monooxygenase (PAM), and the second binder of the two binders described above binds to a stereochemical epitope contained in the PAL subunit (SEQ ID NO: 8) of PAM.

[0189] 4. A method for determining the level of PAM and / or its isoforms and / or fragments in a sample of body fluid or tissue according to Embodiment 2 or 3, with respect to epitopes contained in the following PAM sequences of human peptidylglycine α-amidate monooxygenase (PAM): SEQ ID NO: 25 (PHM, amino acids 31-377 of SEQ ID NO: 1) and SEQ ID NO: 8 (PAL, amino acids 495-817 of SEQ ID NO: 1), where each of the two binders described above is a human peptidylglycine α-amidate monooxygenase (PAM).

[0190] 5. A method for determining the level of peptidylglycine α-amidate monooxygenase (PAM) and / or its isoform and / or fragment in a sample of body fluid or tissue according to any one of Embodiments 1 to 4, wherein the above-mentioned three-dimensional epitope consists of at least four amino acids, preferably at least five amino acids.

[0191] 6. A method for determining the level of PAM and / or its isoforms and / or fragments in a sample of body fluid or tissue according to any one of Embodiments 1 to 5, wherein the binder does not bind to denatured peptidylglycine α-amidate monooxygenase (PAM) or a denatured subunit of PAM (e.g., PAL or PHM).

[0192] 7. A method for determining the level of PAM and / or its isoforms and / or fragments in a body fluid or tissue sample according to Embodiments 1 to 6, wherein the binder does not bind to denatured peptidylglycine α-amidate monooxygenase (PAM) or a denatured subunit of PAM (e.g., PAL or PHM) using Western blotting techniques.

[0193] 8. A method for determining the level of PAM and / or its isoforms and / or fragments in a sample of body fluid or tissue according to any one of Embodiments 1 to 7, wherein the binder to the three-dimensional epitope of peptidylglycine α-amidate monooxygenase (PAM) binds to enzymatically active PAM or an enzymatically active subunit of PAM (e.g., PAL or PHM), but does not bind to enzymatically inactive PAM or an enzymatically inactive subunit of PAM (e.g., PAL or PHM).

[0194] 9. A method for determining the level of peptidylglycine α-amidate monooxygenase (PAM) and / or its isoform and / or fragment in a sample of body fluid or tissue according to any one of Embodiments 1 to 8, wherein at least one binder is selected from the group consisting of an antibody, an antibody fragment, or a non-IgG scaffold.

[0195] 10. A method for determining the level of peptidylglycine α-amidate monooxygenase (PAM) and / or its isoform and / or fragment in a sample of body fluid or tissue according to any one of Embodiments 1 to 9, wherein at least one of the binders is produced using a large fragment protein, a full-length protein, or DNA immunotherapy.

[0196] 11. A method for diagnosing or predicting the prognosis of a disease in a patient, and / or predicting the risk of a patient suffering from a disease or adverse event, and / or monitoring a patient suffering from a disease or adverse event, by determining the levels of peptidylglycine α-amidate monooxygenase (PAM) and / or its isoform and / or its fragments in a sample of a patient's body fluid or tissue according to any of Embodiments 1 to 10, The disease in the above patients is selected from the group including dementia, cardiovascular disease, renal disease, cancer, inflammatory or infectious disease and / or metabolic disease. Adverse events are selected from a group that includes cardiac events, cardiovascular events, cerebrovascular events, cancer, diabetes, infections, severe infections, septic-like systemic infections, sepsis, and death from any cause. method.

[0197] 12. A method for diagnosing or predicting the prognosis of a disease in a patient, and / or predicting the risk of a patient suffering from a disease or adverse event, and / or monitoring a patient suffering from a disease or adverse event, by determining the levels of peptidylglycine α-amidate monooxygenase (PAM) and / or its isoform and / or its fragments in a sample of a patient's body fluid or tissue according to any of Embodiments 1 to 11, The steps include: determining the level of PAM and / or its isoforms and / or fragments in a sample of the patient's bodily fluids or tissues; The steps include comparing the amount determined above with a predetermined threshold. Includes, • If the amount determined above falls below or exceeds the predetermined threshold above, the patient is diagnosed with the disease, or • If the amount determined above falls below or exceeds the predetermined threshold above, the disease outcome is predicted, or • If the amount determined above falls below or exceeds the predetermined threshold above, the risk of the patient developing the disease or adverse event is predicted, or • The patient's disease or adverse events are monitored. method.

[0198] 13. The method according to Embodiments 1 and 12, 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 sample of the patient's body fluid or tissue.

[0199] 14. The method according to any one of Embodiments 1 to 13, wherein the total concentration of PAM and / or its isoforms and / or fragments having at least 12 amino acids is detected by immunoassay.

[0200] 15. A method for diagnosing or prognosing a disease in a patient, and / or predicting the risk of a patient suffering from a disease or adverse event, and / or monitoring a patient suffering from a disease or adverse event, by determining the level of PAM and / or its isoform and / or its fragments in a sample of a patient's body fluid or tissue according to any of Embodiments 1 to 14, wherein the PAM and / or its isoform and / or its 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.

[0201] 16. A method for diagnosing or prognosing a disease in a patient, and / or predicting the risk of a disease or adverse event in a patient, and / or monitoring a disease or adverse event in a patient, by determining the levels of PAM and / or its isoforms and / or fragments in a sample of a patient's bodily fluids or tissue according to any of Embodiments 1 to 15, wherein the risk of the patient contracting a disease is determined and the patient is a healthy patient.

[0202] 17. The method according to Embodiment 16, wherein the disease is selected from the group consisting of Alzheimer's disease, colorectal cancer, and pancreatic cancer.

[0203] 18. A method by any one of Embodiments 1 to 17 for determining the level of peptidylglycine α-amidate monooxygenase (PAM) and / or its isoform and / or fragment in a sample of body fluid or tissue, wherein the level is the level of active PAM.

[0204] 19. Use of an antibody to determine the level of PAM and / or its isoforms and / or its fragments, wherein the antibody is used against a stereochemical epitope contained in the following PAM sequence:PHM fragment (amino acids 31-377 of PAM) (SEQ ID NO: 25) and / or PAL fragment (amino acids 495-817 of PAM) (SEQ ID NO: 8).

[0205] 20. Use of the antibody described in Embodiment 19 to determine the level of PAM and / or its isoform and / or its fragment, wherein the antibody does not bind to denatured PAM or a denatured subunit of PAM (e.g., PAL or PHM).

[0206] 21. Use of an antibody for determining the levels of PAM and / or its isoforms and / or fragments as described in Embodiments 19 and 20, wherein the antibody against the conformational epitope of PAM binds to enzymatically active PAM or a subunit of enzymatically active PAM (e.g., PAL or PHM) but not to enzymatically inactive PAM or a subunit of enzymatically inactive PAM (e.g., PAL or PHM).

[0207] 22. A kit for determining the levels of PAM and / or its isoforms and / or its fragments, comprising one or more antibodies that bind to PAM and are against the following PAM sequences: PHM fragment (amino acids 31-377 of PAM) (SEQ ID NO: 25) and / or PAL fragment (amino acids 495-817 of PAM) (SEQ ID NO: 8).

[0208] 23. A kit for determining the level of PAM and / or its isoform and / or fragment thereof, comprising one or more antibodies that bind to PAM as described in Embodiment 22, wherein the antibodies do not bind to denatured PAM or denatured subunits of PAM (e.g., PAL or PHM).

[0209] 24. A kit for determining the level of PAM and / or its isoform and / or fragment thereof, comprising one or more antibodies that bind to PAM as described in Embodiments 22 and 23, wherein the antibodies against the structural epitope of PAM bind to enzymatically active PAM or a subunit of enzymatically active PAM (e.g., PAL or PHM), but not to enzymatically inactive PAM or a subunit of enzymatically inactive PAM (e.g., PAL or PHM). [Brief explanation of the drawing]

[0210] [Figure 1] Schematic diagram of PAM isoform 1. The thick black arrows indicate cleavage sites in bibasic amino acids.

[0211] [Figure 2] Enzyme reactions catalyzed by PAM.

[0212] [Figure 3] Structural basis for antibody production in mice against human PAM protein (Uniprot ID: P19021) as predicted by Alpha Fold. A: Full-length structure of the protein with PHM and PAL domains highlighted in dark gray, and unstructured and transmembrane domains shown in light gray. Constructed antibodies were produced through immunization with a stable, well-structured protein construct (B), while immunization with an unstructured synthetic peptide (mapped in black, C) generated antibodies against linearized epitopes.

[0213] [Figure 4] Antibody characterization in PAM-LIA assay: Samples were prepared using EDTA-spiked human plasma and recombinant full-length PAM at 15 ng / mL in 1× PBS, with a total of 200 ng per load. The control included antibodies against linearized PHM (Pep 14, SEQ ID NO: 24) and PAL (Pep 4, SEQ ID NO: 14) peptides. The predicted molecular weight of full-length PAM is approximately 90 kDa.

[0214] [Figure 5] Reactivity of antibody cell lines tested in ELISA assays against recombinant PAL(A) and PHM(C) subunits, and against full-length PAM(B, D).

[0215] [Figure 6A]Typical calibration curves for PAM sandwich immunoassays. A-J use recombinant PAM as the calibration material in settings utilizing antibodies against linear peptides. (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 used undenatured PAM (EDTA-plasma) as the calibration material. (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). M used recombinant PAM as the calibration material in a setting that utilized antibodies against three-dimensional peptides. Solid phase: Antibody against the PAL subunit; Tracer: Antibody against the PHM subunit. [Figure 6B]Typical calibration curves for PAM sandwich immunoassays. A-J use recombinant PAM as the calibration material in settings utilizing antibodies against linear peptides. (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 used undenatured PAM (EDTA-plasma) as the calibration material. (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). M used recombinant PAM as the calibration material in a setting that utilized antibodies against three-dimensional peptides. Solid phase: Antibody against the PAL subunit; Tracer: Antibody against the PHM subunit. [Figure 6C]Typical calibration curves for PAM sandwich immunoassays. A-J use recombinant PAM as the calibration material in settings utilizing antibodies against linear peptides. (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 used undenatured PAM (EDTA-plasma) as the calibration material. (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). M used recombinant PAM as the calibration material in a setting that utilized antibodies against three-dimensional peptides. Solid phase: Antibody against the PAL subunit; Tracer: Antibody against the PHM subunit. [Figure 6D]Typical calibration curves for PAM sandwich immunoassays. A-J use recombinant PAM as the calibration material in settings utilizing antibodies against linear peptides. (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 used undenatured PAM (EDTA-plasma) as the calibration material. (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). M used recombinant PAM as the calibration material in a setting that utilized antibodies against three-dimensional peptides. Solid phase: Antibody against the PAL subunit; Tracer: Antibody against the PHM subunit. [Figure 6E]Typical calibration curves for PAM sandwich immunoassays. A-J use recombinant PAM as the calibration material in settings utilizing antibodies against linear peptides. (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 used undenatured PAM (EDTA-plasma) as the calibration material. (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). M used recombinant PAM as the calibration material in a setting that utilized antibodies against three-dimensional peptides. Solid phase: Antibody against the PAL subunit; Tracer: Antibody against the PHM subunit. [Figure 6F]Typical calibration curves for PAM sandwich immunoassays. A-J use recombinant PAM as the calibration material in settings utilizing antibodies against linear peptides. (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 used undenatured PAM (EDTA-plasma) as the calibration material. (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). M used recombinant PAM as the calibration material in a setting that utilized antibodies against three-dimensional peptides. Solid phase: Antibody against the PAL subunit; Tracer: Antibody against the PHM subunit. [Figure 6G]Typical calibration curves for PAM sandwich immunoassays. A-J use recombinant PAM as the calibration material in settings utilizing antibodies against linear peptides. (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 used undenatured PAM (EDTA-plasma) as the calibration material. (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). M used recombinant PAM as the calibration material in a setting that utilized antibodies against three-dimensional peptides. Solid phase: Antibody against the PAL subunit; Tracer: Antibody against the PHM subunit. [Figure 6H]Typical calibration curves for PAM sandwich immunoassays. A-J use recombinant PAM as the calibration material in settings utilizing antibodies against linear peptides. (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 used undenatured PAM (EDTA-plasma) as the calibration material. (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). M used recombinant PAM as the calibration material in a setting that utilized antibodies against three-dimensional peptides. Solid phase: Antibody against the PAL subunit; Tracer: Antibody against the PHM subunit. [Figure 6I]Typical calibration curves for PAM sandwich immunoassays. A-J use recombinant PAM as the calibration material in settings utilizing antibodies against linear peptides. (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 used undenatured PAM (EDTA-plasma) as the calibration material. (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). M used recombinant PAM as the calibration material in a setting that utilized antibodies against three-dimensional peptides. Solid phase: Antibody against the PAL subunit; Tracer: Antibody against the PHM subunit. [Figure 6J]Typical calibration curves for PAM sandwich immunoassays. A-J use recombinant PAM as the calibration material in settings utilizing antibodies against linear peptides. (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 used undenatured PAM (EDTA-plasma) as the calibration material. (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). M used recombinant PAM as the calibration material in a setting that utilized antibodies against three-dimensional peptides. Solid phase: Antibody against the PAL subunit; Tracer: Antibody against the PHM subunit. [Figure 6K]Typical calibration curves for PAM sandwich immunoassays. A-J use recombinant PAM as the calibration material in settings utilizing antibodies against linear peptides. (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 used undenatured PAM (EDTA-plasma) as the calibration material. (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). M used recombinant PAM as the calibration material in a setting that utilized antibodies against three-dimensional peptides. Solid phase: Antibody against the PAL subunit; Tracer: Antibody against the PHM subunit. [Figure 6L]Typical calibration curves for PAM sandwich immunoassays. A-J use recombinant PAM as the calibration material in settings utilizing antibodies against linear peptides. (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 used undenatured PAM (EDTA-plasma) as the calibration material. (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). M used recombinant PAM as the calibration material in a setting that utilized antibodies against three-dimensional peptides. Solid phase: Antibody against the PAL subunit; Tracer: Antibody against the PHM subunit. [Figure 6M]Typical calibration curves for PAM sandwich immunoassays. A-J use recombinant PAM as the calibration material in settings utilizing antibodies against linear peptides. (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 used undenatured PAM (EDTA-plasma) as the calibration material. (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). M used recombinant PAM as the calibration material in a setting that utilized antibodies against three-dimensional peptides. Solid phase: Antibody against the PAL subunit; Tracer: Antibody against the PHM subunit.

[0216] [Figure 7] Frequency distribution (histogram) of PAM concentration (three-dimensional structure PAM-LIA) in healthy individuals (n=4106).

[0217] [Figure 8] Correlation between PAM concentration (RLU) and α-amidation activity (AMA) (ng / L*h) in corresponding EDTA- and Li-heparinized plasma, using a three-dimensional structure antibody (A) and an antibody against linearized peptides (B). n is the number of participants, and r is the Spearman correlation coefficient.

[0218] [Figure 9] Correlation between PAM concentration (ng / mL) and α-amidation activity (AMA) (μg / L*h) in corresponding EDTA and Li-heparin plasmas. n is the number of participants, and r is the Spearman correlation coefficient.

[0219] [Figure 10] A typical calibration curve for recombinant PAM (AMA).

[0220] [Figure 11] Frequency distribution of AMA in self-reported healthy individuals (n=120).

[0221] [Figure 12] The incidence of Alzheimer's disease measured in prospective large-scale cohorts in PAM-LIA and PAM-AMA was calculated using cutoff values ​​of 93.2 ng / mL and 14.4 μg / L*h, respectively. Significance was calculated using the Gehan-Breslow-Wilcoxon test.

[0222] [Figure 13] PAM concentrations (ng / mL) in sepsis and healthy control cohorts, as measured by PAM-LIA. Significance was calculated using the two-sided Mann-Whitney U test.

[0223] [Figure 14](A) Recombinantly produced active PAL subunit, (B) anti-PAL binding conformational antibody, (C) complex of anti-PAL binding conformational antibody and PAL subunit, (D) recombinantly produced active PHM subunit, (E) anti-PHM binding conformational antibody, and (F) HPLC elution profile showing the complex of anti-PHM binding conformational antibody and PHM subunit.

[0224] [Figure 15] SDS-PAGE analysis of HPLC elution fractions. Lane 1: Recombinantly produced active PAL subunit. Lane 2: Recombinantly produced active PHM subunit. Lane 3: Conformational antibody (Note: Anti-PHM and anti-PAL antibodies yielded the same pattern. Anti-PAL antibody is not shown). Lane 4: Pre-stained protein marker as molecular weight standard. Lane 5: Complex of anti-PAL binding conformational antibody and PAL subunit. Lane 6: Complex of anti-PHM binding conformational antibody and PHM subunit.

Example

[0225] Example 1 - Preparation of Recombinant PAM 1.1. Generation of Full-Length PAM for Immunization Variant A: A PAM cDNA encoding amino acids 21 - 834 of the PAM protein with codon optimization for expression in mammalian cells was synthesized according to Uniprot accession number P19021. The signal sequence of PAM was replaced with the human serum albumin signal sequence (MKWVTFISLLFLFSSAYSFR [SEQ ID NO: 9]). A hexahistidine tag linked to PAM via a GS linker was added to the C-terminus of PAM. The sequence of the recombinant PAM (amino acids 21 - 834 of PAM without 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 having the cDNA for PAM expression was replicated in Escherichia coli and prepared from Escherichia coli as a low endotoxin preparation.

[0226] HEK-INV cells were transfected with an expression vector using the INVect transfection reagent in serum-free suspension culture. Transfection rates were controlled by co-transfection with a GFP (green fluorescent protein)-containing expression vector. Cell culture was performed at 37°C and 5% CO2 in the presence of valproic acid and penicillin-streptomycin. Cells were harvested by centrifugation when viability reached <60% (>2000g, 30-45 minutes, 2-8°C). The cell culture supernatant (CCS) was washed five times with 100 mM Tris / HCl, pH 8.0 by tangential flow filtration (TFF, 30 kDa cutoff).

[0227] Purification of recombinant PAM included the application of buffer exchange CCS on Q-Sepharose fast-flow resin (GE Healthcare) using NaCl gradient elution (up to 2M). Fractions containing amidation activity were pooled and applied to a Superdex 200pg (GE Healthcare) size exclusion chromatography column using elution buffer of 100 mM Tris / HCl, 200 mM NaCl, pH 8.0. Fractions containing amidation activity were pooled, dialyzed against 100 mM TrisHCl, 200 mM NaCl, pH 8.0, and filtered sterile (0.2 μm). Endotoxin load was determined to be less than 5 EU / mL by the Charles River PTS Endosafe system.

[0228] Mutant B: A second construct of the full-length PAM for immunization was commercially available from SinoBiological, which contained human PAM (UniProtKB:P19021-1, SEQ ID NO: 1) with 31–973 residues, lacking amino acid sequences 388–494, and was C-terminated with a decahistidine tag.

[0229] 1.2. Generation of a soluble catalytic core of a single PAM subunit for immunity Soluble PHM and PAL subunits containing residues 31-377 (SEQ ID NO: 25) and 495-817 (SEQ ID NO: 8) of human PAM (UniProtKB: P19021-1), respectively, were synthesized and separately expressed in transiently transfected human kidney 293 (HEK293) cells. Both constructs contained a TEV cleavage site isolated by N-terminal excision, decahistidine tagging, and subsequent GS linker. The signal sequences of the PHM and PAL constructs were replaced with the human serum albumin signal sequence (MKWVTFISLLFLFSSAYSFR [SEQ ID NO: 9]). The cDNA was cloned into expression vectors (plasmid DNA) using 5'-NotI and 3'HindIII restriction sites. Expression vectors containing cDNA for PHM and PAL expression were separately replicated in E. coli and prepared from E. coli as low-endotoxin preparations.

[0230] Transfection with PAL or PHM constructs and culture of transfected HEK-INV cells were performed as described for full-length PAM in mutant A (see above).

[0231] Overexpressed recombinant constructs PHM and PAL were purified by cobalt affinity chromatography to a sample purity of over 95%, as measured by capillary gel electrophoresis. The eluted fractions obtained from the protein purification process were subjected to analysis by Western blotting. Specifically, the eluted fractions were loaded onto a sodium dodecyl sulfate (SDS) gel and transferred to a nitrocellulose membrane. The membrane was probed with an anti-His antibody to detect the target protein. The fractions containing PHM or PAL protein were pooled, dialyzed against 50 mM TrisHCl, 150 mL NaCl, pH 8.0, and filtered sterile (0.2 μm). The endotoxin load was determined to be less than 5 EU / mL using the Charles River PTS Endosafe system.

[0232] Example 2 - Production of antibodies against linear epitopes and structural epitopes Two types of antibodies, namely antibodies against linearized epitopes and antibodies against structural epitopes, can be robustly produced in mice based on the procedure described in Figure 3. First, the structure of the protein must be known or predicted. The program AlphaFold, which requires only the protein sequence and not preliminary knowledge of the structure already elucidated by X-ray crystallography, is used to predict the protein structure in this particular case. This enables unbiased structure prediction. Once the structure is predicted, a solid fragment with minimal unstructured regions is required for the production of structural antibodies. For antibodies against linearized epitopes, unstructured and disordered regions are required because they are linear even in vivo.

[0233] The unstructured regions are identified and produced ex vivo, and then expressed, for example, synthetically or recombinantly. Finally, the solid fragments and synthetic peptides are used for immunization. Through this process, mice can produce antibodies against both linearized and conformational epitopes, which can be used for a variety of applications.

[0234] Anti-PAM antibodies against linear epitopes were synthesized as follows. PAM peptides for immunization were synthesized using an additional C-terminal cysteine ​​residue (if cysteine ​​was not present in the selected PAM sequence) for peptide conjugate to bovine serum albumin (BSA) (see Table 1) (Peptides & Elephants, Hennigsdorf, Germany). The peptides were covalently bonded to BSA using a Sulfolink coupling gel (Perbio-science, Bonn, Germany). The coupling procedure was performed according to the Perbio manual.

[0235] [Table 1] * According to Sequence ID No. 1; amino acid (aa) Balb / c mice were intraperitoneally (ip) injected with 100 μg of PAM-peptide-BSA conjugate (emulsified in TiterMax Gold Adjuvant) on day 0, 100 μg and 100 μg (emulsified in complete Freund's adjuvant) on day 14, and 50 μg and 50 μg (emulsified in incomplete Freund's adjuvant) on days 21 and 28. On day 45, the animals were intravenously (iv) injected with 50 μg of PAM-peptide-BSA conjugate dissolved in physiological saline. Three days later, the mice were sacrificed and immunocellular fusion was performed.

[0236] Anti-PAM antibodies against the three-dimensional epitope according to the present invention were synthesized as follows. Constructions of both soluble PHM and PAL proteins for immunization (SEQ ID NOs. 25 and 8, respectively), and recombinant full-length PAM (mutant A (SEQ ID NOs. 10) and B) were prepared as described in Example 1.

[0237] Balb / c mice were administered 100 μg of PAL, PHM, or full-length PAM protein via intravenous injection on day 0, followed by another 100 μg on day 14, and 50 μg on days 21 and 28. A single intravenous injection of 50 μg of recombinant protein was administered on day 45. Three days later, the mice were sacrificed and immunocellular fusion was performed.

[0238] Splenocytes from immunosuppressed 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 into 96-well cell culture plates. Hybrid clones were selected by growing them in HAT medium (RPMI 1640 culture medium supplemented with 20% fetal bovine serum and HAT supplement). 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.

[0239] The cell culture supernatant was subjected to primary screening for recombinant PHM, PAL, and full-length PAM-binding IgG antibodies two weeks after fusion. Thus, recombinant PAM (SEQ ID NO: 10), PHM (SEQ ID NO: 25), and PAL (SEQ ID NO: 8) were immobilized in 96-well plates (100 ng / well) and incubated with 50 μl of cell culture supernatant per well for 2 hours at room temperature. After washing the plates, 50 μl / well of POD-rabbit anti-mouse IgG was added and incubated for 1 hour at room temperature.

[0240] After the next washing step, 50 μl of chromogen solution (3.7 mM o-phenylenediamine, 0.012% H2O2 in citrate / phosphate buffer) was added to each well, incubated for 15 minutes at room temperature, and the color reaction was stopped by the addition of 50 μl of 4N sulfuric acid. Absorbance at 490 nm was detected.

[0241] Microcultures with positive test results were transferred to 24-well plates for growth. After retesting, the selected cultures were cloned and recloned using the limiting dilution method, and the isotype was determined.

[0242] Antibodies generated against recombinant human PAM, PHM, or PAL or PAM peptides were prepared by standard antibody production methods (Marx et al. 1997) and purified by protein A. The antibody purity was over 90% based on SDS gel electrophoresis analysis.

[0243] Results: Antibodies produced using the technique described in Figure 3 were subjected to Western blotting to distinguish between antibodies against the conformational peptide and antibodies against the linearized peptide. Recombinant full-length PAM was linearized by spiking it into EDTA plasma or by dissolving it in 1× PBS and diluting it in SDS loading dye supplemented with β-mercaptoethanol, and then heated at 95°C for at least 10 minutes. The samples were then loaded onto SDS PAGE (200 ng of recombinant PAM per load) and transferred to Western blotting. Either a 2 μg / mL conformational antibody or an antibody against the linearized epitope was used as the primary antibody, while the secondary antibody was a rabbit anti-mouse antibody conjugated with horseradish peroxidase. When linearized antibodies were used for detection, strong signals between 70 and 100 kDa were observed in both EDTA-spiked and PBS-soluble PAM samples, which is expected given that the full-length protein has a molecular weight of 90 kDa (Figure 4). However, when antibodies against the three-dimensional epitopes were used for detection, no signal was observed in any of the samples (Figure 4). When subjected to SDS analysis, PAM does not exhibit any tertiary structure, and therefore antibodies should not be able to bind to it, thus demonstrating the three-dimensional structural properties of these antibodies.

[0244] To evaluate the ability of hybridoma cell lines to bind to target antigens, cell culture supernatants were subjected to dilution tests. Constructural antibodies generated against PAL or PHM subunits showed significantly high affinity binding to their respective protein targets, as well as to full-length PAM (Figure 5). No signal was detected when using common His-tagged proteins as targets.

[0245] Example 3 - PAM Immunoassay Antibodies against recombinant PAM (mutant A (SEQ ID NO: 10) and mutant B, Example 1), as well as antibodies against their recombinant subunits (SEQ ID NOs: 25 and 8, Example 1) and linear PAM peptides (SEQ ID NOs: 11-24), were produced as described in Example 2.

[0246] We used a sandwich luminescence immunoassay technique based on acridinium ester labeling.

[0247] 3.1. Labeled compounds (tracers) Purified antibody (0.2 g / L) was labeled in 10% labeled buffer (500 mmol / L sodium phosphate, pH 8.0) with MACN-acridinium-NHS ester (1 g / L, InVent GmbH) in a 1:5 mol / L ratio by incubation at 22°C for 20 minutes. After adding 5% 1 mol / L Tris-HCl, pH 8.0 for 10 minutes, each antibody was separated from the free-labeled antibody via a CentriPure P10 column (emp Biotech GmbH). The purified labeled antibody was 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 was approximately 20 ng of labeled antibody per 150 μL.

[0248] 3.2.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) (at 20°C for 18 hours). After blocking with 30 g / L Karion, 5 g / L BSA (protease-free), 6.5 mmol / L monopotassium phosphate, and 3.5 mmol / L sodium dihydrogen phosphate (pH 6.5), the plates were vacuum-dried.

[0249] 3.3. Calibration The assay was calibrated using commercially available recombinant PAM (Example 1, mutant B) dilutions. The typical concentration range was 1–1,000 ng / mL.

[0250] 3.4. PAM Immunoassay: 3.4.1. PAM-LIA One-step version: A minimum of 10 μL of sample / calibrator was pipetteed into a pre-coated microtiter plate. 200 μL of labeled antibody in buffer (300 mmol / L potassium phosphate, 100 mmol / L NaCl, 10 mmol / L Na-EDTA, 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 (20°C) for at least 3 hours with stirring at 600 rpm. Unbound tracers were removed by washing five times (350 μL each) with washing solution (20 mmol / L PBS, 1 g / L Triton X-100, pH 7.4).

[0251] Using a Centro LB 960 microtiter plate chemiluminescence reader (Berthold Technologies), chemiluminescence bound to the wells was measured for 1 second per well.

[0252] Two-step version: A minimum of 10 μL of sample / calibrator was pipetteed into a pre-coated microtiter plate. After adding 200 μL of buffer (as described in the one-step version), 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 each), then adding 200 μL of tracer material, and incubating the microtiter plate at room temperature (20°C) for 2 hours. Unbound tracer was removed by washing four times with washing solution (350 μL each). Chemiluminescence bound to the wells was measured for 1 second per well using a Centro LB 960 microtiter plate emission reader (Berthold Technologies).

[0253] Results: The exemplary antibody production process described in Example 2 (Figure 3) provides a robust approach for generating antibodies suitable for immunoassay applications. These antibodies can be used in various combinations, both as solid antibodies and tracer antibodies, enabling versatile assay design. Table 2 shows the analysis of signal-to-noise ratios measured in various bodily fluids and tissue extracts.

[0254] [Table 2] ELISA based on structural antibodies showed high signal linearity in the range of 1–1000 ng / mL and was found to be suitable for measuring protein targets in serum, plasma, and tissue extracts, such as the pituitary gland. Samples were not matched. The background signal of the immunoassay using structural antibodies was found to be approximately 192 RLU.

[0255] The mean intra-assay CV was 2.2% [1.3%–3.8%], and the mean inter-assay CV was 6.7% [2.8%–12.9%]. The LOD and LOQ were 189 pg / mL and 250 pg / mL, respectively. The precision of the PAM-LIA assay was determined by spiking analyte-depleted EDTA plasma with recombinant PAM of known concentrations, and ranged from 90.3% to 99.2%. The linearity of the assay was evaluated by dilution and mixing. In the first case, the mean deviations between the measured concentration and the target concentration for samples with starting PAM concentrations of 91.2 ng / mL, 323.5 ng / mL, and 684.7 ng / mL were 13.2% [8.9%–17.9%], 1.2% [3.7%–8.2%], and 5.2% [0.4%–8.8%], respectively. In the second case, the determined concentration of PAM deviated by 4.9% [0.7% to 10.2%] on average from the predicted concentration.

[0256] Typical calibration curves for LIA using linear and stereochemical antibodies are shown in Figures 6A-6L and 6M, respectively. Figure 7 shows the distribution of PAM concentrations (PAM-LIA) in serum samples from n=4106 individuals (a prospective study based on a Swedish population, a subcohort of randomly selected individuals without a history of cardiovascular disease from the Malmöe Prevention Project (MPP)). The mean PAM-LIA was 77.8 ng / mL [SD=19.0]. The median plasma PAM concentration was 78.6 ng / mL (interquartile range [IQR] 66.4-92.5 ng / mL). The 10th and 90th percentiles were 56.3 and 106.6 ng / mL, respectively. The 2.5th, 97.5th, and 99th percentiles were 45, 123.5, and 135.5 ng / mL, respectively.

[0257] PAM amidation activity (ng / mL) * The correlation between AMA (at h) and PAM concentration (LIA at ng / mL) was investigated using both structural and linear peptide antibodies in different immunoassay settings. Significant correlations were observed when both antibodies were used (Figure 8). However, the correlation between AMA and PAM concentration (LIA) was significantly higher when using structural antibodies to measure PAM levels in EDTA plasma samples (r=0.809, p<0.0001) compared to when using linear antibodies in the immunoassay setting (r=0.431, p=0.014). Statistical analysis of the data reveals that the higher correlation coefficients and lower p-values ​​achieved for comparable size cohorts in the ELISA setting using structural antibodies compared to antibodies against linear epitopes indicate more stable and significant relationships between variables, enhancing the reliability and validity of the results. For immunoassays using antibodies against three-dimensional epitopes, the correlation remained highly significant (r=0.71, p<0.0001) when determined in a large subcohort of n=4850 individuals with plasma MPP (Figure 9).

[0258] Example 4 - PAM Activity Assay 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 in 100 mM Tris-HCl (two-component dilution). The amidation reaction was initiated by adding 160 μl of PAM reaction buffer (100 mM Tris-HCl, pH 7.5, 6.25 μM CuSO4, 2.5 mM L-ascorbate, 125 μg / mL catalase, 62.5 μM amastatin, 250 μM leupeptin, 36 ng / mL synthetic ADM-Gly, and 375 μg / mL NT-ADM antibody). Subsequently, 100 μl of each individual reactant from the two-component samples were combined and transferred to 20 μl of 200 mM EDTA to terminate the amidation reaction and create a reaction time point t=0 min, followed by incubation at 37°C for 40 minutes. Subsequently, the unterminating reaction was stopped using 10 μl of 200 mM EDTA. To determine PAM activity, bio-ADM was quantified as a reaction product in each sample using the sphingotest® bio-ADM immunoassay (Weber et al. 2017). The amidation assay was calibrated using a 6-check curve prepared with human recombinant PAM of known activity. Samples and calibrators were processed in the same manner. For each sample, the relative luminescence (RLU, t40 min-t0 min) determined by the sphingotest® bio-ADM immunoassay was fitted to the RLU (t40 min-t0 min) of the calibrator to determine the PAM activity in the sample. PAM activity is described as "adrenomedullin maturation activity" (AMA) in μg of bio-ADM formed per hour and per L of sample.

[0259] A typical PAM calibration curve is shown in Figure 10. The distribution of AMA in Li-heparin samples from n=120 self-reported healthy volunteers is shown in Figure 11. The median Li-heparin AMA [IQR] was 18.4 μg / (L). * h)[13.5~21.9]. The 10th and 90th percentiles were 10.5 and 24.2 μg / (L), respectively. * h) was the result. The 2.5th, 97.5th, and 99th percentiles were 8.1, 31.6, and 40.8 μg / (L). *h) was the result.

[0260] Example 5 - Prediction of disease in healthy subjects According to Example 3, the concentration of PAM showed a strong correlation with its activity. As a result, this can be used as an additional, simpler method for determining PAM levels in a high-throughput manner for clinical application.

[0261] 5.1. Research Cohort The Malmoe Prevention Project (MPP) was funded in the mid-1970s to explore cardiovascular disease risk factors in the general population and enrolled 33,346 individuals residing in Malmoe (Fedorowski et al. 2010. Eur Heart J 31:85-91). Between 2002 and 2006, a total of 18,240 original participants responded to invitations (participation rate, 70.5%) and were screened, including comprehensive physical examinations and blood sample collection (Fava et al. 2013. Hypertension 2013;61:319-26). Re-examination in the MPP is considered baseline in this study. Subjects with a prior history of CVD at baseline were excluded. Informed consent was obtained from all participants, and the study protocol was approved by the Ethical Committee of Lund University, Lund, Sweden (see Tables 3 and 4 for detailed patient characteristics).

[0262] [Table 3] N / A: Not applicable [Table 4] Statistical Analysis: Values ​​are expressed as mean and standard deviation, median and interquartile range (IQR), or, where necessary, as counts and percentages. Group comparisons of continuous variables were performed using the Kruskal-Wallis test. Biomarker data were logarithmically transformed. The effects of risk factors on survival in univariate and multivariate analyses were analyzed using Cox proportional hazards regression. The proportional hazards assumption was tested for all variables. For continuous variables, hazard ratios (HRs) were standardized, and the HR for a single IQR biomarker change was stated. Significance levels for 95% confidence intervals (CIs) and chi-squared (Wald test) for risk factors are shown. Predictions for each model were evaluated using the model likelihood ratio chi-squared statistic. The coincident index (C-index) is shown as a measure of effect. This is equivalent to the AUC concept adopted for binomial results. For multivariate models, a bootstrap-corrected version of the C-index is given. Survival curves plotted using the Kaplan-Meier method are used for illustrative purposes. To test the independence of PAM from clinical variables, the inventors used a likelihood ratio chi-squared test for nested models. All statistical tests were two-tailed tests, and a two-tailed p-value of 0.05 was considered significant.

[0263] 5.2. Prediction of Alzheimer's disease 3716 samples with information on dementia diagnosis were selected (n=169 with onset of Alzheimer's disease). Information on dementia diagnosis was requested from the Swedish National Patient Registry (SNPR). Registered diagnoses were collected according to different revisions of the International Classification of Diseases (ICD) codes 290, 293 (ICD-8), 290, 331 (ICD-9) or F00, F01, F03, G30 (ICD-10). Since 1987, the SNPR includes all inpatient care in Sweden, and further includes outpatient visit data, including day surgeries and psychiatric care by both private and public caregivers, recorded since 2000. All causes of dementia were diagnosed according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders (DSM)-III Revised Edition, while the DSM-IV criteria were applied to the diagnosis of Alzheimer's disease and vascular dementia. Diagnoses were verified through thorough review of medical records and, where available, neuroimaging data. Research physicians assigned a final diagnosis to each patient, and for unresolved cases, geriatric specialists specializing in cognitive impairment were consulted. PAM activity (AMA) and PAM concentration (LIA) were determined as described in Examples 4 and 3, respectively. When both measurement methods were applied, PAM activity and PAM concentration in the MPP cohort were significantly lower in the AD-affected group (n=169) compared to the non-AD group (p=0.01). For analysis, LIA was measured at 93.2 ng / mL (groups 1 and 2 included 2779 and 937 individuals, respectively), and AMA at 14.4 μg / L. * Based on the cutoff value of h (groups 1 and 2 contained 2779 and 937 individuals, respectively), the data were divided into two groups. As shown in Figure 12, the PAM-LIA assay showed higher predictive ability for AD development with an HR of 0.58 (0.41–0.82, p=0.003) compared to AMA with an HR of 0.73 (0.51–1.02, p=0.03). Example 6 - Prognostic diagnosis and monitoring 6.1. Research Cohort AdrenOSS-1 on AMA Measurement AdrenOSS-1 was a prospective observational study conducted in Europe. Twenty-four centers in five countries (France, Belgium, the Netherlands, Italy, and Germany) contributed to the completion of the trial with 583 enrolled patients (recruitment took place from June 2015 to May 2016). The study protocol was approved by the regional ethics committee 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 for sepsis or septic shock, or (2) were transferred from another ICU in a state of sepsis and septic shock within 24 hours of admission. Inclusion of patients was stratified by severe sepsis and septic shock based on the 2001 definition 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 local practices, and treatments and procedures were registered. The primary endpoint was 28-day mortality. Secondary endpoints included organ failure (defined by the Sequential Organ Failure Assessment [SOFA] score) and organ support, use of vasopressors / inotropes, fluid balance, and use of renal replacement therapy (RRT).

[0264] Upon admission, demographic data (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 comorbidities (i.e., treated within the previous 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 registration, the following data were collected daily during the first week: SOFA score, antibiotic therapy, fluid balance, ventilation status, Glasgow Coma Scale score, central venous pressure, need for RRT, invasive procedures for sepsis control, and vasopressor / inotropic agent administration. Furthermore, discharge status and mortality were recorded 28 days after ICU admission.

[0265] 6.2. Sepsis study cohort for LIA measurement The sepsis cohort included 12 individuals, whose plasma was collected upon admission to the ICU, accompanied by clear signs of sepsis.

[0266] 6.3. Self-reported healthy cohort For both assay measurements, the healthy cohort included n=98 (AMA) and n=12 (LIA) individuals of various ages and sexes, none of whom had a history of overt disease.

[0267] 6.4. Outcomes and prognosis in sepsis In the AdrenOSS-I subcohort (n=98), the AMA level was 17.0 μg / L * Compared to healthy individuals with AMA h[SD=30.8], the sepsis group had a 27.7 μg / L *We identified a significantly higher AMA (hypo-aminobutyric acid) level in h[SD=55.8] (p<0.0001). Similarly, PAM concentration (LIA) was significantly lower in healthy individuals compared to the sepsis cohort (174.5 ng / mL[SD=28.8], p<0.001) (96.6 ng / mL[SD=11.5], n=12) (Figure 13). In sepsis, PAM concentration (LIA) was found to increase 1.8 times, while in AMA, the increase was only 1.6 times compared to the respective healthy cohorts. These results suggest that PAM-LIA is an appropriate and convenient method for predicting the likelihood of sepsis.

[0268] Example 7: Protocol for extracting soluble PAM from human tissue samples Tissue samples were extracted by first obtaining 1 gram of liver or pituitary tissue. The frozen tissue samples were then ground into a fine powder using a cryogenic grinder or mortar and pestle, while maintaining the powder on dry ice throughout the process. The ground powder was dissolved in a lysis buffer (50 mM Tris / HCl pH 7.4, 0.5% Triton, complete protease inhibitor cocktail) using a homogenizer or sonicator (0.5 cycles, 60% amplitude, 60 seconds). The mixture was centrifuged twice at 5000 × g at 4°C for 5 minutes to separate the soluble and insoluble fractions. After each centrifugation, the pellet was discarded, and the supernatant was collected and subjected to a final centrifugation at 20,000 × g at 4°C for 60 minutes. The supernatant containing the extracted liver or pituitary protein was transferred to a new container such as a 1.5 mL Eppendorf tube. The supernatant was stored at -80°C until use, and the container was appropriately labeled with the sample name, extraction date, and storage temperature.

[0269] Example 8 - Antibody against the structural epitope of active PAM or the active subunit of PAM. Antibodies against full-length PAM and its subunits PHM and PAL were prepared as described in Example 2. The antibodies were tested to determine whether they bound to the structural epitopes or linear epitopes, respectively. The epitopes of the antibodies were characterized using Western blotting (as described in Example 2), ELISA, and co-elution HPLC (see Table 5).

[0270] To confirm that the antibody binds to the structural epitope using an ELISA assay, the following experiment was performed: Transformation 1 Solid-phase antibody coating: Polystyrene microtiter plates were coated with antibodies against either the three-dimensional structure of the PAL or PHM subunit or a linear epitope, and then blocked with 30 g / L Karion, 5 g / L BSA (protease-free), 6.5 mmol / L monopotassium phosphate, and 3.5 mmol / L sodium dihydrogen phosphate (pH 6.5), as detailed in Example 3.

[0271] Protein linearization: To linearize full-length PAM, it was denatured by incubation with 5% SDS solution and heating at 80°C for 10 minutes, or by treatment with 3M urea solution at room temperature for 1 hour. Subsequently, the linearized protein solution was diluted 200-fold with 1×PBS and incubated with solid-phase antibody. To confirm that the diluted concentrations of urea and SDS did not interfere with protein binding in its native three-dimensional structure, concentrations of 0.025% SDS and 15 mM urea were added to the native full-length PAM. The binding efficiency in the PAM immunoassay was then evaluated under standard conditions, as outlined in Example 4.

[0272] Testing of natural three-dimensional structure: The natural three-dimensional structure of PAM was confirmed by subjecting 200 μg of purified full-length PAM protein to HPLC and evaluating its elution profile and activity, as described in Example 4.

[0273] PAM immunoassay: Linear full-length PAM or its native structural PAM was incubated with solid-phase antibodies, including their respective tracer antibodies, for 3 hours at room temperature under standard assay conditions, as described in Example 3.

[0274] Results: The presence of 0.025% SDS and 15 mM urea in the dilution did not affect the binding of full-length PAM in its native conformation under standard conditions. When antibodies against the conformational epitope were used, no chemiluminescent signal was detected for the linearized full-length protein, indicating specific detection of the protein in its native conformation rather than in its linearized form. The chemiluminescent signal of the linearized protein was detected when antibodies against the linearized epitope were used, but not when conformational antibodies were used. As expected, the chemiluminescent signal was detected for the linearized protein when antibodies against the linearized epitope were used, but not when conformational antibodies were used.

[0275] Transformation 2: Protein coating and blocking: Polystyrene microtiter plates were coated with 2.5 μg of full-length PAM, PAL, or PHM subunits of the native three-dimensional structure per well, followed by blocking as described for the antibody in Example 3, with vacuum drying omitted. To confirm that the coated proteins maintained their native three-dimensional structure, the amidation activity of full-length PAM was measured as described in Example 4, and for PHM-coated proteins, 2.5 μg of PAL subunits were added to the reaction buffer, and for PAL-coated proteins, 2.5 μg of PHM subunits were added to the reaction buffer.

[0276] Protein linearization: Full-length PAM, PAL, or PHM subunit proteins were linearized as described in Modification 1 and coated onto polystyrene microtiter plates as outlined for the proteins in their native three-dimensional structures above.

[0277] PAM immunoassay: Either an anti-PAL or anti-PHM tracer antibody was applied to a pre-coated polystyrene microtiter plate as detailed in Example 3 and incubated under standard PAM immunoassay conditions.

[0278] Results: Similar to variation 1, no chemiluminescent signal was detected for the linearized full-length protein, confirming the antibody's specificity for the structural epitope of the protein, which is more precisely folded than its linearized form.

[0279] As expected, chemiluminescent signals were detected for linearized proteins when antibodies against linearized epitopes were used, but not when antibodies against three-dimensional structures were used.

[0280] To confirm that antibodies bind to structural epitopes, the following experiments were performed by analyzing the elution HPLC profiles of antibody-antigen complexes: A total of 78 μg of full-length PAM, either in its native three-dimensional structure or linearized form (linearization was performed as described above), was incubated with 122 μg of antibody recognizing the structural epitope of either the PAL or PHM subunit. This incubation was carried out at room temperature for 1 hour in 500 μL of 1×PBS solution. Similarly, 45 μg of either the PAL or PHM subunit, in its native or linearized form, was incubated with 155 μg of the corresponding antibody against their structural epitope under the same buffer and conditions. After incubation, the mixture was applied to an HPLC column (Protein KW-803, Fa Shodex) at a flow rate of 0.5 mL / min for 30 minutes. The elution process was fractionated, and the eluted fractions were then analyzed by SDS-PAGE. As a control, individual antibodies, as well as single PAL and PHM subunits, were subjected to HPLC separation and SDS-PAGE under the same conditions.

[0281] Results: For all tested antibodies targeting the structural epitopes, stable antibody-antigen complex formation was confirmed by elution HPLC profiles and SDS-PAGE of the eluted fractions; however, this was not the case for the linearized form. As shown in Figure 14, chromatographic profiling of both the PAL and PHM subunits, as well as their specific structural antibodies, was performed using size exclusion chromatography to evaluate the molecular weight of the subunit-antibody complexes. The PAL subunit with a molecular weight of 42 kDa showed a major peak at 16.56 min (Figure 14A). The antibody against the structural PAL epitope with a predicted molecular weight of 150 kDa showed a major elution peak at 11.91 min (Figure 14B). When the PAL subunit was mixed with its corresponding antibody, the elution profile showed a shifted peak at 11.02 min corresponding to the PAL-antibody complex, and an additional peak at 16.96 min representing free antibodies not involved in complex formation (Figure 14C).

[0282] Similarly, the 41 kDa PHM subunit alone was characterized by a peak at 16.80 minutes (Figure 14D). Antibodies against the three-dimensional PHM epitope eluted with a peak at 11.93 minutes (Figure 14E).

[0283] When the PHM subunit was mixed with its corresponding antibody, the elution profile showed a major shifted peak at 11.49 minutes and another peak at 16.85 minutes for excess unbound antibody (Figure 14F).

[0284] The eluted fractions were analyzed by SDS-PAGE, as shown in Figure 15. The presence of bands in the antigen-antibody complex fractions corresponding to individual PAL or PHM subunits and the heavy and light chains of the antibody indicates successful complex formation, as demonstrated in lanes 5 and 6.

[0285] [Table 5] array Sequence ID 1 - PreproPAM Isoform 1 AS 1-973 [Table 6] Sequence ID No. 2 - PreproPAM Isoform 2 AS 1-868 [Table 7] SEQ ID NO: 3 - PreproPAM isoform 3 AS (with amino acids 829-896 of SEQ ID NO: 1 deleted) [Table 8] SEQ ID NO: 4 - PreproPAM isoform 4 (with amino acids 829-914 of SEQ ID NO: 1 deleted) [Table 9] Sequence ID 5 - PreproPAM isoform 5 (Isoform 1 with an additional aa at position 896) [Table 10] SEQ ID NO: 6 - PreproPAM isoform 6 (with amino acids 897-914 of SEQ ID NO: 1 deleted) [Table 11] PHM subunit of sequence number 7-PAM [Table 12] PAL subunit of Sequence ID 8-PAM [Table 13] Sequence ID 9 - Human serum albumin signaling sequence [Table 14] Sequence ID 10 - Sequence of recombinant human PAM [Table 15] Sequence ID 11 - Peptide-1 (aa 42-56 of PAM Sequence ID 1) [Table 16] Sequence ID 12 - Peptide-2 (aa 109-128 of PAM Sequence ID 1) [Table 17] SEQ ID NO: 13-Peptide-3 (aa 168-180 of PAM SEQ ID NO: 1) [Table 18] Sequence ID 14 - Peptide-4 (aa 204~216 of PAM Sequence ID 1) [Table 19] Sequence ID 15 - Peptide 5 (aa 329~342 of PAM Sequence ID 1) [Table 20] SEQ ID NO: 16 - Peptide-6 (aa 291-310 of PAM SEQ ID NO: 1) [Table 21] Sequence ID 17 - Peptide 7 (aa 234~244 of PAM Sequence ID 1) [Table 22] Sequence ID 18 - Peptide-8 (aa 261-276 of PAM Sequence ID 1) [Table 23] Sequence ID 19 - Peptide 9 (aa 530~557 of PAM Sequence ID 1) [Table 24] Sequence ID 20 - Peptide 10 (aa 611~631 of PAM Sequence ID 1) [Table 25] Sequence ID 21 - Peptide-11 (aa 562~579 of PAM Sequence ID 1) [Table 26] Sequence ID 22 - Peptide-12 (PAM Sequence ID 1 aa 745~758) [Table 27] Sequence ID 23 - Peptide-13 (aa 669~687 of PAM Sequence ID 1) [Table 28] Sequence ID 24 - Peptide-14 (aa 710~725 of PAM Sequence ID 1) [Table 29] SEQ ID NO: 25 - PHM fragment (aa 31-377 of PAM SEQ ID NO: 1) [Table 30]

Claims

1. A method for determining the levels of PAM and / or its isoforms and / or fragments in a body fluid or tissue sample using an assay comprising at least one binder to the structural epitope of PAM.

2. The assay comprises two binders that bind to two different regions of peptidylglycine α-amidate monooxygenase (PAM), each of the two binders being a method for determining the level of PAM and / or its isoforms and / or fragments in a sample of body fluid or tissue according to claim 1, relative to the three-dimensional epitope of PAM.

3. A method for determining the level of PAM and / or its isoforms and / or fragments in a sample of body fluid or tissue according to claim 2, wherein the first binder of the two binders binds to a structural epitope contained in the PHM subunit (SEQ ID NO: 7) of peptidylglycine α-amidate monooxygenase (PAM), and the second binder of the two binders binds to a structural epitope contained in the PAL subunit (SEQ ID NO: 8) of PAM.

4. A method for determining the level of PAM and / or its isoforms and / or fragments in a sample of body fluid or tissue according to claim 2 or 3, with respect to epitopes contained in the following PAM sequences of human peptidylglycine α-amidate monooxygenase (PAM): SEQ ID NO: 25 (PHM, amino acids 31-377 of SEQ ID NO: 1) and SEQ ID NO: 8 (PAL, amino acids 495-817 of SEQ ID NO: 1), each of the two binders.

5. A method for determining the level of peptidylglycine α-amidate monooxygenase (PAM) and / or its isoform and / or fragment in a sample of body fluid or tissue according to any one of claims 1 to 4, wherein the three-dimensional epitope consists of at least four amino acids, preferably at least five amino acids.

6. A method for determining the level of PAM and / or its isoforms and / or fragments in a sample of body fluid or tissue according to any one of claims 1 to 5, wherein the binder does not bind to denatured peptidylglycine α-amidate monooxygenase (PAM) or a denatured subunit of PAM (e.g., PAL or PHM).

7. A method for determining the level of PAM and / or its isoforms and / or fragments in a sample of body fluid or tissue according to claims 1 to 6, wherein the binder does not bind to denatured peptidylglycine α-amidate monooxygenase (PAM) or a denatured subunit of PAM (e.g., PAL or PHM) by using Western blotting techniques.

8. A method for determining the level of PAM and / or its isoforms and / or fragments in a sample of body fluid or tissue according to any one of claims 1 to 7, wherein the binder to the three-dimensional epitope of peptidylglycine α-amidate monooxygenase (PAM) binds to enzymatically active PAM or an enzymatically active subunit of PAM (e.g., PAL or PHM), but does not bind to enzymatically inactive PAM or an enzymatically inactive subunit of PAM (e.g., PAL or PHM).

9. A method for determining the level of peptidylglycine α-amidate monooxygenase (PAM) and / or its isoform and / or fragment in a sample of body fluid or tissue according to any one of claims 1 to 8, wherein the at least one binder is selected from the group consisting of an antibody, an antibody fragment, or a non-IgG scaffold.

10. A method for determining the level of peptidylglycine α-amidate monooxygenase (PAM) and / or its isoform and / or fragment in a sample of body fluid or tissue according to any one of claims 1 to 9, wherein the at least one binder is produced using a large fragment protein, a full-length protein, or DNA immunotherapy.

11. A method for diagnosing or predicting a disease in a patient, and / or predicting the risk of contracting a disease or adverse event in a patient, and / or monitoring a disease or adverse event in a patient, by determining the level of peptidylglycine α-amidate monooxygenase (PAM) and / or its isoform and / or fragment thereof in a sample of the patient's bodily fluids or tissue according to any one of claims 1 to 10, The disease in the patient is selected from the group including dementia, cardiovascular disease, renal disease, cancer, inflammatory or infectious disease and / or metabolic disease. The aforementioned adverse events are selected from the group including cardiac events, cardiovascular events, cerebrovascular events, cancer, diabetes, infections, severe infections, septic-like systemic infections, sepsis, and death from any cause. method.

12. A method for diagnosing or prognosing a disease in a patient, and / or predicting the risk of contracting a disease or adverse event in a patient, and / or monitoring a disease or adverse event in a patient, by determining the level of peptidylglycine α-amidate monooxygenase (PAM) and / or its isoform and / or fragment thereof in a sample of the patient's bodily fluids or tissue according to any one of claims 1 to 11, - A step of determining the level of PAM and / or its isoform and / or its fragments in a sample of the patient's bodily fluids or tissue, - A step of comparing the determined amount with a predetermined threshold. Includes, - If the determined amount falls below or exceeds the predetermined threshold, the patient is diagnosed with the disease, or - If the determined amount falls below or exceeds the predetermined threshold, the disease outcome is predicted, or - If the determined amount falls below or exceeds the predetermined threshold, the risk of the patient developing a disease or adverse event is predicted, or - The patient's disease or adverse events are monitored. method.

13. The method according to claims 1 and 12, wherein the level of PAM and / or its isoform and / or its fragment is equal to the total concentration of PAM and / or its isoform and / or its fragment having at least 12 amino acids in a sample of the patient's body fluid or tissue.

14. The method according to any one of claims 1 to 13, wherein the total concentration of PAM and / or its isoforms and / or fragments having at least 12 amino acids is detected by immunoassay.

15. A method for diagnosing or predicting the prognosis of a disease in a patient, and / or predicting the risk of the patient suffering from a disease or adverse event, and / or monitoring a disease or adverse event in a patient, by determining the level of PAM and / or its isoform and / or its fragments in a sample of the body fluid or tissue of a patient according to any one of claims 1 to 14, wherein the PAM and / or its isoform and / or its 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.

16. A method for diagnosing or predicting the prognosis of a disease in a patient, and / or predicting the risk of contracting a disease or adverse event in the patient, and / or monitoring a disease or adverse event in the patient, by determining the level of PAM and / or its isoform and / or fragments thereof in a sample of the patient's bodily fluids or tissue according to any one of claims 1 to 15, wherein the risk of contracting a disease in the patient is determined and the patient is a healthy patient.

17. The method according to claim 16, wherein the disease is selected from the group consisting of Alzheimer's disease, colorectal cancer, and pancreatic cancer.

18. A method according to any one of claims 1 to 17 for determining the level of peptidylglycine α-amidate monooxygenase (PAM) and / or its isoform and / or fragment in a sample of body fluid or tissue, wherein the level is the level of active PAM.

19. The use of an antibody to determine the level of PAM and / or its isoforms and / or fragments thereof, wherein the antibody is used against a stereochemical epitope contained in the following PAM sequence: PHM fragment (amino acids 31-377 of PAM) (SEQ ID NO: 25) and / or PAL fragment (amino acids 495-817 of PAM) (SEQ ID NO: 8).

20. The use of an antibody for determining the level of PAM and / or its isoform and / or its fragment according to claim 19, wherein the antibody does not bind to denatured PAM or denatured subunits of PAM (e.g., PAL or PHM).

21. Use of an antibody for determining the levels of PAM and / or its isoforms and / or fragments according to claims 19 and 20, wherein the antibody against the conformational epitope of PAM binds to enzymatically active PAM or an enzymatically active subunit of PAM (e.g., PAL or PHM) but not to enzymatically inactive PAM or an enzymatically inactive subunit of PAM (e.g., PAL or PHM).

22. A kit for determining the levels of PAM and / or its isoforms and / or its fragments, comprising one or more antibodies that bind to PAM and are against the conformational epitopes contained in the following PAM sequences: PHM fragment (amino acids 31-377 of PAM) (SEQ ID NO: 25) and / or PAL fragment (amino acids 495-817 of PAM) (SEQ ID NO: 8).

23. A kit for determining the level of PAM and / or its isoform and / or fragment thereof, comprising one or more antibodies that bind to PAM as described in claim 22, wherein the antibodies do not bind to denatured PAM or denatured subunits of PAM (e.g., PAL or PHM).

24. A kit for determining the level of PAM and / or its isoform and / or fragment thereof, comprising one or more antibodies that bind to PAM according to claims 22 and 23, wherein the antibodies against the structural epitope of PAM bind to enzymatically active PAM or an enzymatically active subunit of PAM (e.g., PAL or PHM), but not to enzymatically inactive PAM or an enzymatically inactive subunit of PAM (e.g., PAL or PHM).

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