Method for the diagnosis of acute pancreatitis (AP) by detection of glycoprotein 2 isoform alpha (GP2A)

Abstract

The invention relates to an in vitro method for the diagnosis of acute pancreatitis (AP) in a subject by detection of Glycoprotein 2 isoform alpha (GP2a) protein. In particular the invention pro-vides an in vitro method for the diagnosis of acute pancreatitis (AP) in a subject by detection of Glycoprotein 2 isoform alpha (GP2a) protein, comprising providing a sample of a human subject exhibiting symptoms of having pancreatic disease, wherein said sample is obtained from the subject within 72 hours of the appearance of said symptoms, providing an affinity reagent directed against GP2a, contacting said sample with said affinity reagent thereby capturing GP2a from said sample, and determining the concentration of GP2a from said sample, wherein determining a concentration of GP2a in said sample that is greater than the average concentration of GP2a in control samples, such as in a group of healthy individuals, indicates the presence of AP and the absence of one or more of chronic pancreatitis, pancreatic cancer, gastrointestinal cancer, liver cancer, neuroendocrine tumor, sarcoma, peptic ulcer or peritonitis. The invention further provides a kit and a system developed for carrying out the claimed method and determining the concentration of GP2a and performing an automated analysis of one or more samples.

Claims

1. An in vitro method for diagnosis and treatment of acute pancreatitis (AP) in a human subject exhibiting symptoms of pancreatic disease by detection of Glycoprotein 2 isoform alpha (GP2a) protein, comprising: providing a sample of the human subject exhibiting symptoms of pancreatic disease, wherein said sample is obtained from the subject within 72 hours of the appearance of said symptoms, providing an affinity reagent directed against GP2a, contacting said sample with said affinity reagent thereby capturing GP2a from said sample, and determining a concentration of GP2a greater than 0.7 ng/ml in said sample diagnosing, based on the concentration of GP2a, a presence of AP and an absence of chronic pancreatitis and pancreatic cancer, and administering an AP specific treatment of the patient.

2. The method according to claim 1, wherein the GP2a comprises or consists of a protein with an amino acid sequence according to SEQ ID NO: 1 or 2.

3. The method according to claim 1, wherein the affinity reagent specifically binds the GP2a with no binding or negligible binding to Glycoprotein 2 isoform beta (GP2b).

4. The method according to claim 3, wherein said affinity reagent is a monoclonal antibody.

5. The method according to claim 4, wherein said monoclonal antibody binds specifically the GP2a, with no binding or negligible binding to the GP2b in both native and denaturated sample conditions.

6. The method according to claim 1, wherein the method is conducted as an Enzyme Linked Immunosorbent Assay (ELISA), wherein said affinity reagent is immobilized on a solid surface before contacting said sample.

7. The method according to claim 6, wherein the determination of the GP2a concentration comprises: a) capturing the GP2a from the sample via the affinity reagent that is immobilized to the solid surface to create a captured GP2a, b) treating said captured GP2a with a labelled secondary affinity reagent directed to GP2, c) detecting a signal emitted from said labelled secondary affinity reagent directed to GP2, and d) comparing the signal obtained from said labelled secondary affinity reagent with the signal from one or more control samples of pre-determined GP2a concentration.

8. The method according to claim 7, wherein the signal is obtained from horseradish peroxidase conjugated to the secondary affinity reagent.

9. The method according to claim 1, wherein the sample is a blood sample, a plasma sample or a serum sample.

10. The method according to claim 3, wherein the affinity reagent specifically binds GP2a comprising or consisting of a protein with an amino acid sequence according to SEQ ID NO 1 or 2, with no binding or negligible binding to GP2b comprising or consisting of a protein with an amino acid sequence according to SEQ ID NO 3 or 4.

11. The method according to claim 5, wherein said antibody binds specifically GP2a comprising or consisting of a protein with an amino acid sequence according to SEQ ID NO 1 or 2, with no binding or negligible binding to GP2b comprising or consisting of a protein with an amino acid sequence according to SEQ ID NO 3 or 4.

12. A method for determining a concentration of GP2a, comprising: providing a sample having a complex comprising at least one affinity reagent directed against GP2a bound to said GP2a in a bodily fluid obtained from a human subject exhibiting symptoms of pancreatic disease, wherein said sample is obtained from the subject within 72 hours of appearance of said symptoms, wherein said sample shows that the concentration of GP2a of the human subject is greater than 0.7 ng/ml, wherein an acute pancreatitis (AP) specific treatment is administered to the subject whose sample has a concentration of GP2a that is greater than 0.7 ng/ml and the AP specific treatment or treatment regime differs from a treatment for chronic pancreatitis or pancreatic cancer.

13. The method according to claim 12, wherein the affinity reagent is bound to said GP2a and is not bound or negligibly bound to GP2b in the sample.

14. The method according to claim 12, wherein the affinity reagent is bound to said GP2a, wherein the GP2a comprises an amino acid sequence according to SEQ ID NO 1 or 2, and is not bound or negligibly bound to GP2b in the sample, wherein the GP2b comprises an amino acid sequence according to SEQ ID NO 3 or 4.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1:

(2) Solid phase ELISA analysis of recognition of soluble recombinant isoforms of GP2 (GP2a and GP2b) in their native (A) and denaturated (B) state using different monoclonal antibodies, which have been generated against GP2. The respective monoclonal antibodies were used as solid phase immobilized antibodies in ELISA.

(3) FIG. 2:

(4) Correlation of glycoprotein 2 isoform a (GP2a) and total GP2 (GP2t). Serum GP2a and GP2t levels were detected in 153 patients with acute pancreatitis (AP) and 752 controls by enzyme-linked immunosorbent assay. The regression equation of all 905 samples was y=−0.05+0.41x, coefficient of determination (R2) 0.49. In contrast to controls (y=0.02+0.27x), for the 153 patients with AP (y=−0.16+0.58x) a higher R2 was determined (0.72 vs 0.29).

(5) FIG. 3:

(6) Correlation of glycoprotein 2 isoform a (GP2a) (A) and total GP2 (GP2t) (B) levels with disease duration. Serum GP2a and GP2t were detected in patients with acute pancreatitis (AP) with different disease severity by enzyme-linked immunosorbent assay.

(7) I, mild AP; II, severe AP with local complications; Ill, severe AP with systemic complications; IV, severe AP with lethal outcome

(8) FIG. 4:

(9) Serum glycoprotein 2 isoform a (GP2a) (A) and total GP2 (GP2t) (B) levels in patients with acute pancreatitis (AP) and controls. GP2a and GP2t were analyzed in 153 patients with AP, 651 disease controls, 101 blood donors (BD) by enzyme-linked immunosorbent assay. Patients with AP were stratified into AP patients with up to three days of disease duration (n=12), between 4 and 10 days (n=47), and more than 10 days (n=94). As disease controls, 26 patients with chronic pancreatitis (CP), 125 with pancreatic cancer, 118 with liver cancer (LCa), 126 with gastrointestinal cancer (GICa), 40 with neuroendocrine tumor (NET), 40 with sarcoma (SA), 109 with benign liver and biliary diseases (bL/BD), 27 with peptic ulcer (PU), and 40 with peritonitis (PT) investigated. (Optimized cut-offs for GP2a and GP2t obtained by receiver-operating characteristic curve analysis were illustrated by dashed horizontal lines. Data are displayed in Box-and-Whisker plots with far out values, defined as values that are smaller than the lower quartile minus 3 times the interquartile range, or larger than the upper quartile plus 3 times the interquartile range, displayed as solid triangles.)

(10) P, Kruskal Wallis test, post-hoc analysis

(11) FIG. 5:

(12) Comparison of assay accuracy of glycoprotein 2 isoform a (GP2a) and total GP2 (GP2t) detection by enzyme-linked immunosorbent assay. GP2a and GP2t were analyzed in 12 patients with acute pancreatitis as disease criterion and 752 controls as non-disease criterion and subjected to receiver-operating characteristic curve analysis. Optimal cut-off levels for GP2a and GP2t were determined at 0.7 and 2.3 ng/mL, respectively.

(13) FIG. 6:

(14) Correlation of glycoprotein 2 isoform a (GP2a) (A,C) and total GP2 (GP2t) (B,D) levels with procalcitonin (PCT) (A,B) and c-reactive protein (CRP) concentrations in 153 patients with acute pancreatitis. Optimized cut-offs for GP2a and GP2t obtained by receiver-operating characteristic curve analysis and generally accepted ones for CRP and PCT were illustrated by dashed horizontal and vertical lines, respectively. (Optimized cut-offs for GP2a and GP2t obtained by receiver-operating characteristic curve analysis as well as established cut-off of procalcitonin and C-reactive protein were illustrated by dashed lines.)

(15) FIG. 7:

(16) Serum glycoprotein 2 isoform a (GP2a), procalcitonin (PCT), and c-reactive protein (CRP) levels in follow-up samples of patients with acute pancreatitis (AP).

(17) AP patients with available follow-up samples between the 2nd and 49th day of disease duration:

(18) A, male, 47 years, alcohol abuse, severe AP with local complications

(19) B, male, 31 years, alcohol abuse, severe AP with systemic complications

(20) C, male, 48 years, alcohol abuse, severe AP with local complications

(21) D, male, 31 years, alcohol abuse, severe AP with systemic complications

(22) AP patients with late increase of GP2a (>10th day)

(23) E, female, 65 years, biliary, severe AP with lethal outcome

(24) F, female, 83 years, biliary, severe AP with lethal outcome

(25) G, male, 59 years, alcohol abuse, severe AP with systemic complications

(26) H, male, 26 years, alcohol abuse, severe AP with systemic complications

EXAMPLES

(27) Without intending to be limiting, the invention will be explained in more detail with reference to an example.

(28) Materials:

(29) Subjects

(30) Characteristics of the 153 patients with acute pancreatitis and 752 controls including 26 patients with CP, 125 with PCa, 118 with LCa, 126 with GICa, 40 with neuroendocrine tumors (NET), 40 with sarcoma (SA), 109 with benign liver or biliary disease (bL/BD), 27 with peptic ulcer (PU), 40 with peritonitis (PT), and 101 healthy blood donors (BD) are given in Table 4. According to disease severity, patients with acute pancreatitis were stratified retrospectively into mild and severe AP with local as well as systemic complications or lethal cases using clinical and imaging data.[10; 21] Furthermore, the etiology of AP (biliary, alcohol abuse, idiopathic, post-endoscopic retrograde cholangiopancreatography (ERCP), drug induced) was determined in accordance with international guidelines.[10] Sera of patients with AP and disease controls were collected at the department of surgery of the Otto-von-Guericke University Magdeburg. For 30 of the 152 patients with acute pancreatitis, 3 or more consecutive samples could be obtained. Thus, 128 additional samples were included into the study which covered a median observation period of 28 days (interquartile range [IQR] 28 days). Of note, disease duration from clinical onset of AP until first examination at the intensive care unit of the department of surgery was determined for all patients. For 152 and 146 AP patients, CRP and procalcitonin (PCT) levels could be obtained, respectively.

(31) All clinical diagnoses were based upon standard clinical, imaging, endoscopic and histological criteria. The diagnosis of CP was established using a scoring system based on the presentation of calcifications or pancreatic duct abnormalities, evidence of pancreatic insufficiency, and abdominal pain, weight loss or glucose intolerance.[22]

(32) The study was approved by the local ethics committee and complies with the World Medical Association Declaration of Helsinki regarding ethical conduct of research involving human subjects and/or animals. Aliquots of the sera stored at −80° C. were used to detect serum GP2 levels.

(33) TABLE-US-00004 TABLE 4 Patients' and blood donors' (BD) characteristics disorder Number (%) Age (IQR) Gender (f/m) acute pancreatitis 153 50.0 (28.0) 58/95  etiology alcohol abuse 59 (38.6) 40.0 (12.0) 11/48  biliary disease 54 (35.3) 65.0 (24.0) 30/24  drug induced 1 (0.7) 50.0 0/1  idiopathic 21 (13.7) 61.0 (24.8) 11/10  post-trauma 11 (7.2)  57.0 (19.0) 2/9  post-ERCP 7 (4.6) 59.0 (39.5) 4/3  severity mild 22 (14.4) 57.5 (29.0) 14/8  local complications 74 (48.4) 45.5 (30.0) 27/47  systemic complications 41 (26.8) 54.0 (28.5) 11/30  lethal outcome 16 (10.5) 65.6 (29.0) 6/10 chronic pancreatitis 26 48.0 (19.0).sup.§ 13/13.sup.& pancreatic cancer 125 60.0 (16.0) 68/57  pancreatic carcinoma 25 60.5 (15.2).sup.§  8/17.sup.& papillary carcinoma 36 63.0 (17.5) 16/20.sup.& pancreatic cystic 34 61.0 (16.0) 27/7  neoplasm IPMN 18 64.0 (12.0) 10/8.sup.&  islet cell tumor 12 49.0 (14.5).sup.§ 7/5.sup.& liver cancer 118 64.0 (11.0) 59/59  hepatocellular cancer 40 64.0 (12.2) 20/20.sup.& cholangiocelluar cancer 38 63.0 (8.2) 19/19.sup.& cholangiocelluar 40 66.0 (9.0) 20/20.sup.& cystadenocarcinoma gastrointestinal cancer 126 63.3 (16.8) 54/72.sup.& Barrett's esophagus 26 63.5 (9.5)  6/20.sup.& gastric cancer 40 69.0 (14.2) 20/20.sup.& GIST 26 62.5 (16.5) 11/15.sup.& colon carcinoma 34 58.0 (24.2).sup.§ 17/17.sup.& neuroendocrine tumors 40 62.5 (19.5) 20/20.sup.& sarcoma 40 61.0 (16.2) 20/20.sup.& benign liver/biliary disease 109 56.0 (24.0) 78/31  benign liver disease 46 43.5 (23.8) 38/8  hepatic cyst 23 59.0 (9.0).sup.§ 20/3  benign biliary disease 40 61.0 (19.8) 20/20.sup.& peptic ulcer 27 63.0 (12.0)  7/20.sup.& peritonitis 40 66.0 (24.8) 20/20.sup.& blood donors 101 27.5 (15.0) 39/62.sup.& ERCP, endoscopic retrograde cholangiopancreatography, f, female; GIST, gastrointestinal stromal tumor; IPMN, intraductal papillary mucinous neoplasm; IQR, interquartile range; m, male .sup.§age not significantly different to the age of patients with acute pancreatitis (p > 0.05) .sup.&gender distribution not significantly different to the one of patients with acute pancreatitis (p > 0.05)
Anti-GP2 Antibody Production

(34) Human GP2a and GP2b were expressed in the baculovirus system as described elsewhere. Briefly, the plasmids pcDNA3.1+GP2-trunc-Thrombin-His (GP2a, CCS GmbH, Hamburg, Germany) and pBluescript-GP2 (GP2b, ThermoScientific, Braunschweig, Germany) were used which code the respective isoform amino-acid sequences. At the C-terminal end, the GPI anchor was replaced by a His6-Tag. The two isoforms were cloned into pVL1392, respectively and resulting constructs were verified by sequencing. Transfection into and culture of insect Spodoptera frugiperda (Sf) 9 cells was performed as described elsewhere.[23] GP2 isoforms were purified from harvested supernatants by Ni-chelate chromatography.

(35) Polyclonal antibodies to GP2 were produced immunising rabbits with recombinant human GP2 isoforms according to a standard immunisation protocol (three cycles of injection of 100 μg protein). They were purified by affinity chromatography employing recombinant GP2a immobilized to sephadex.

(36) Monoclonal antibodies recognizing GP2a and GP2b isoforms were developed by immunizing Balb/c mice with modified immunoconjugates. GP2 isoforms were coupled to the major capsid protein VP1 of hamster polyomavirus (HaPyV) via glutaraldehyde linking according to standard procedures. The expression of recombinant HaPyV-VP1 and the immunization procedure were performed as previously reported.[24] The selection of GP2-specific monoclonal antibodies was performed by ELISA as described elsewhere.[25]

(37) ELISA for the Detection of GP2a and GP2t

(38) GP2a and GP2t were assessed in serum samples of patients and controls by ELISAs. The monoclonal anti-GP2a antibody K9 or polyclonal rabbit anti-GP2 antibodies at a concentration of 1 μg/mL were coated onto Maxisorb microtiter plates (Nunc, Roskilde, Denmark) in coating buffer (pH=9.5) at 4° C. over night. After blocking with 1% (w/v) bovine serum albumin in 50 mM Tris-buffered saline (pH=7.4) (Sigma Co, Taufkirchen, Germany) at room temperature for one hour, plates were sealed for further use. For the detection of GPa and GPt, serum samples diluted 1 in 100 in 50 mM Tris-buffered saline (pH=7.4) with 0.2% BSA (w/v) were incubated at room temperature for 1 hour and washed with Tris-buffered saline containing 0.1% (v/v) Tween 20 (Sigma). Horseradish peroxidase (HRP)-labeled anti-human polyclonal GP2 antibodies were added and developed with ready-to-use hydrogen peroxide/tetramethylbenzidine substrate (Seramun, Heidesee, Germany). Conjugation of affinity purified polyclonal anti-GP2 antibodies to HRP was done by the sodium periodate technique as described elsewhere.[26]

(39) The reaction was stopped with 0.25 mol/I sulphuric acid after 15 min. The optical density of the samples was read using a microplate reader (SLT, Crailsheim, Germany) at a wavelength of 450 nm/620 nm. Purified recombinant GP2a was used as standard material and GP2 levels were expressed in ng/mL.

(40) For interference experiments, GP2 containing sera were spiked with hemoglobulin, triglycerides, bilirubin, and GP2's urinary homolog Tamm-Horsfall protein (uromodulin) (Sigma Co). Final concentrations were 0.0-2.5 g/L for hemoglobin, 30.0-100.0 mg/L for bilirubin, 5.7-25.0 g/L for triglycerides and 0.0-10.0 g/L for uromodulin.

(41) Statistical Analysis

(42) A Kolmogorov-Smirnov test was used to reject the normal distribution of data. Thus, measured values were expressed as medians with IQR. The two-tailed, non-parametric Kruskal-Wallis test was used to test for statistically significant differences of independent samples in 2 or more groups. Comparison of independent samples between two groups was performed by two-tailed Mann-Whitney test.

(43) Spearman's rank correlation test was applied for within group comparison. Comparison of prevalence rates between groups was performed by two-tailed Fisher's exact test. P values of less than 0.05 were considered significant. Assay performance and the comparison thereof were analyzed by receiver-operating characteristics (ROC) curve analysis. All calculations were performed using Medcalc statistical software (Medcalc, Mariakerke, Belgium).

Example 1: Development of Anti-GP2 ELISA

(44) For the development of anti-GP2 ELISA, one monoclonal antibody to GP2a, coded K9, could be generated with a special immunization protocol which was needed due to several unsuccessful attempts to immunize mice with human recombinant GP2a. K9 recognized soluble recombinant GP2a readily in its native and denaturated state, but not GP2b when used as solid-phase immobilized antibody in ELISA (FIG. 1). In contrast, the sole monoclonal antibody P102 generated successfully to the shorter isoform GP2b interacted with both immobilized isoforms in ELISA but with soluble GP2 at a low binding strength only which did not allow to develop a sensitive ELISA for GP2b (FIG. 1). Thus, K9 was employed as a solid-phase antibody for the development of an ELISA for the analysis of GP2a and affinity-purified rabbit polyclonal anti-GP2 antibodies for the assessment of GP2t, respectively. Subsequently, bound GP2a and GP2t were detected by HRP-labelled polyclonal affinity purified anti-GP2 antibodies.

Example 2: Regression of GP2a and GP2t Levels

(45) As expected, GP2a and GP2t levels were linearly related to each other (y=−0.05+0.41x, coefficient of determination [R.sup.2]=0.49) when samples of all AP patients and controls are analyzed (FIG. 2). However, regression analysis in AP patients alone revealed a higher R.sup.2 of 0.72 compared to controls (R.sup.2=0.29) and a significantly different slope and intercept (p<0.0001, =0.0001, respectively). This indicates a poorer association of GP2a and GP2t levels in controls compared to AP patients.

Example 3: Correlation GP2a and GP2t Levels with Disease Duration

(46) Interestingly, both GP2a and GP2t demonstrated a significantly negative correlation with disease duration in AP (FIG. 3) (Spearmen's coefficient of rank correlation [rho]=−0.22, 95% confidence interval [Cl]−0.37-−0.07, p=0.0053; rho=−0.41, 95% Cl −0.54-−0.27, p<0.0001; respectively). For further evaluation, patients with AP were stratified into 3 groups covering samples of AP patients with disease duration of <=3 days (n=12), with disease duration from 4 to 10 days (n=47) and greater than 10 days (n=94) (Table 5).

(47) TABLE-US-00005 TABLE 5 Prevalences of total GP2 (GP2t) and isoform alpha (GP2a) positives detected by enzyme-linked immunosorbent assay (ELISA) in 153 patients with acute pancreatitis and 752 controls employing 2.3 and 0.7 ng/mL as optimized cut-offs, respectively. disorder n GP2a (%) GP2t (%) acute pancreatitis 153 40 (26.1) 39 (25.5) disease duration <=third disease day 12 11 (91.7).sup.§§§ 11 (91.7).sup.§§§ <=fourth disease day 30 16 (53.3).sup.§§,& 14 (46.7).sup.§,& <=tenth disease day 59 26 (44.1).sup.§,&& 25 (42.4).sup.§,&& etiology alcohol abuse 59 19 (32.2) 17 (28.8) biliary disease 54 9 (16.7) 8 (14.8) drug induced 1 1 (100.0) 1 (100.0) idiopathic 21 8 (38.1) 9 (42.9) post-trauma 11 3 (27.3) 4 (36.4) post-ERCP 7 0 (0.0) 0 (0.0) severity mild 22 2 (9.1) 2 (9.1) local complications 74 24 (32.4) 25 (33.8) systemic complications 41 7 (17.1) 6 (14.6) lethal outcome 16 7 (43.8) 6 (37.5) disease controls and 752 28 (3.7).sup.§§§, &&& 56 (7.4).sup.§§§, &&& blood donors chronic pancreatitis 24 3 (12.5) .sup.&&& 3 (12.5) .sup.&&& pancreatic cancer 125 11 (8.8).sup.§§§, &&& 14 (11.2).sup.§§, &&& pancreatic carcinoma 25 2 (8.0).sup.§, &&& 4 (16.0) .sup.&&& papillary carcinoma 36 4 (11.1) .sup.&&& 5 (13.9) .sup.&&& pancreatic cystic 34 1 (2.9).sup.§§, &&& 3 (8.8).sup.§, &&& neoplasm IPMN 18 3 (16.7) .sup.&&& 2 (11.1) .sup.&&& islet cell tumor 12 1 (8.3).sup.&& 0 (0.0) .sup.&&& liver cancer 118 5 (4.2).sup.§§§, &&& 18 (15.2) .sup.&&& hepatocellular cancer 40 1 (2.5).sup.§§§, &&& 9 (22.5).sup.&& cholangiocelluar cancer 38 2 (5.3).sup.§§, &&& 5 (13.2) .sup.&&&& cholangiocelluar 40 2 (5.0).sup.§§, &&& 4 (10.0) .sup.&&& cystadenocarcinoma gastrointestinal cancer 126 2 (1.6).sup.§§§, &&& 6 (4.8).sup.§§§, &&& Barrett's esophagus 26 0 (0.0).sup.§§, &&& 0 (0.0).sup.§, &&& gastric cancer 40 1 (2.5).sup.§§, &&& 3 (7.5).sup.§, &&& GIST 26 0 (0.0).sup.§§, &&& 1 (3.8).sup.§, &&& colon carcinoma 34 1 (2.9).sup.§§, &&& 2 (5.9).sup.§, &&& neuroendocrine tumors 40 1 (2.5).sup.§§, &&& 3 (7.5).sup.§, &&& Sarcoma 40 1 (2.5).sup.§§, &&& 1 (2.5).sup.§§, &&& benign liver/bilebladder 109 2 (1.8).sup.§§§, &&& 2 (1.8).sup.§§§, &&& disease benign liver disease 46 0 (0.0).sup.§§§, &&& 0 (0.0).sup.§§§, &&& hepatic cyst 23 1 (4.3).sup.§, &&& 1 (4.3).sup.§, &&& benign bilebladder 40 1 (2.5).sup.§§, &&& 1 (2.5).sup.§§§, &&&& disease peptic ulcer 27 1 (3.7).sup.§§, &&& 2 (7.4).sup.§, &&& peritonitis 40 2 (5.0).sup.§§, &&& 5 (12.5) .sup.&&& blood donors 101 0 (0.0).sup.§§§, &&& 2 (2.0).sup.§§§, &&& GIST, gastrointestinal stromal tumor; IPMN, intraductal papillary mucinous neoplasm; .sup.§p < 0.05; .sup.§§p < 0.01; .sup.§§§ p < 0.0001; comparison of GP2 prevalence with the one in all acute pancreatitis cases by Fisher's test: .sup.&p < 0.05; .sup.&&p < 0.01; .sup.&&& p < 0.0001; comparison of GP2 prevalence with the one in acute pancreatitis <= third disease day by Fisher's test:

Example 4: Comparison of GP2a and GP2t Levels in AP and Control Groups

(48) Patients with AP were age and gender matched with patients suffering from CP, PCa, islet cellular tumor, and colon carcinoma (p>0.05, Kruskal Wallis test and Post hoc analysis, Fisher's test, respectively).

(49) Serum GP2a and GP2t determined by ELISAs demonstrated significantly different levels in 153 patients with AP and 752 controls (p<0.0001, Kruskal Wallis test) (FIG. 4A,B). Thus, median levels of GP2a and GP2t in AP patients with disease duration <=3 days, with disease duration from 4 to 10 days, and with disease duration >10 days from the first day of occurrence of clinical symptoms demonstrated a significant decline from time period to time period (Post hoc analysis, p>0.05, respectively). The median GP2a and GP2t levels in AP patients until the 3rd day of disease duration were significantly elevated in comparison with all control groups including patients with chronic pancreatitis (Post hoc analysis, p<0.05, respectively). The median GP2t level of AP patients with >10 days disease duration was additionally not different in patients with GICa, NET, SA and even CP (Post hoc analysis, p<0.05, respectively).

Example 5: Comparison of GP2a and GP2t Assay Performance

(50) To take into consideration the declining GP2 levels over time, respective optimized GP2 cut-offs for the discrimination of AP patients from controls were obtained by ROC curve analysis employing AP patients till the 3rd disease day as positive criterion and all 752 disease controls and BD as negative criterion (FIG. 5). The comparison of ROC curves revealed a significantly higher area under the curve (AUC) for GP2a in the first 3 disease days in contrast to GP2t (FIG. 5) (0.93, 95% Cl: 0.91-0.95 vs 0.90, 95% Cl: 0.88-0.92, p=0.0427). Thus, the GP2a ELISA possessed a significantly better assay accuracy with a sensitivity of 91.7% (95% Cl: 61.5%-99.8%) and a specificity of 96.7% (95% Cl: 95.1%-97.8%). The GP2t ELISA did demonstrate the same sensitivity but a poorer specificity of 92.6% (95% Cl: 90.4%-94.3%). Altogether, these data resulted in a positive likelihood ratio (LR) of 24.6 (95% Cl: 16.5-36.8) and a −LR of 0.09 (95% Cl: 0.01-0.57) for GP2a whereas the +LR of GP2t reached 12.3 (95% Cl: 9.1-16.7) at the same −LR like GP2a only.

Example 6: Comparison of GP2a and GP2t Positivity in AP Patients and Controls

(51) The cut-offs obtained by ROC curve analysis of 0.7 ng/mL for GP2a and 2.3 ng/mL for GP2t were used to define the prevalence of positives in AP patients and controls (Table 5). There was a remarkable decrease in the prevalence of GP2a and GP2t positives after the 3rd disease day (53.3% and 46.7%, respectively). Within the first 10 days of disease duration the prevalence dropped to 44.1% and 42.4%, respectively.

(52) The GP2a ELISA revealed significantly less false positives compared with the GP2t assay (28/752 vs 56/752, p=0.0022). This was basically due to the significantly higher prevalence of false positive GP2t findings in contrast to GP2a ones in patients with liver cancer (18/118 vs 5/118, p=0.0073) and here in particular in patients with hepatocellular cancer (9/40 vs 1/40, p=0.0143).

Example 7: GP2a and GP2t Levels and Positivity in Etiological AP Variants

(53) GP2a and GP2t levels were significantly different in the 153 AP patients with varying etiology (Kruskal-Wallis test, p=0.0144 and 0.0199, respectively). In accordance with Post-hoc analysis, patients with idiopathic and alcoholic AP demonstrated significantly elevated GP2a and GP2t levels in contrast to AP patients with biliary disease and post-trauma AP (p<0.05, respectively). The respective differences in the prevalence of GP2a positivity, however, did not reach significance (p=0.0803, 0.0657). In contrast, there was a significantly higher prevalence of GP2t in AP patients with idiopathic disease compared with biliary AP patients (9/21 vs 8/54, p=0.0143). Of note, disease duration was not significantly different in the AP patient groups with various etiology (Kuskal-Wallis test, p=0.0997).

Example 8: Correlation of GP2a and GP2t with CRP and PCT

(54) GP2a and GP2t levels were significantly correlated with PCT (rho=0.21, 95% Cl: 0.05-0.36 and 0.26, 95% Cl: 0.11-0.41; p=0.0110 and 0.0012; respectively) as well as CRP values (rho=0.37, 95% CI: 0.22-0.50 and 0.40, 95% Cl: 0.26-0.53; p<0.0001; respectively) in 152 and 146 follow-up samples of AP patients, respectively (FIG. 6).

(55) In general, GP2a and GP2t levels declined rapidly after the 3rd disease day. However, patients with severe AP demonstrated elevated GP2a levels until the 21st day of disease duration with a steady decline over time (FIG. 7A). In particular CRP levels demonstrated a similar modulation which corresponded with the therapy success (FIGs. 7A,B,C,D). In severe cases with lethal outcome, there was a strikingly similar behavior of GP2a and PCT levels at time points beyond the 10th disease duration day (FIG. 7E,F). In these two cases, a combined elevation of GP2a and PCT shortly before death of the patients could be observed. Furthermore, the AP patient illustrated in FIG. 7E showed an interestingly simultaneous increase and subsequent decrease of GP2a and PCT levels in response to major intestinal surgery on disease day 34. Of note, increases in GP2a levels could be determined even later than the 50th disease duration day (FIG. 7G,H). GP2t levels showed similar alterations like GP2a (data not shown).

Example 9: Prediction of AP Severity by GP2a and GP2t Levels

(56) Given the established association of GP2a and GP2t levels with the disease severity markers PCT and CRP in follow-up samples, AP patients were stratified in 4 groups according to disease severity: I mild AP, II severe disease with local complications, III severe disease with systemic complications, and IV severe disease with lethal outcome, retrospectively. There was a tendency for a higher prevalence of GP2a and GP2t positivity in the 131 patients with severe AP (11, III, IV) compared to the 22 patients with mild AP (p=0.0650 and 0.0660, respectively). Of note, there was a significant difference of GP2a positivity with a lower cut-off of 0.4 ng/mL obtained by ROC curve analysis with all 153 AP patients included (45/131 vs 2/22, p=0.0226). Similar lowering of the cut-off for GP2t did not result in a significant differentiation of mild and severe AP.

(57) Remarkably, AP patients with lethal outcome during the observation period did demonstrate a significantly higher prevalence of GP2a and GP2t compared with mild cases on the day of admission to the intensive care surgery unit (7/16 vs 2/22, 6/16 vs 2/22, p=0.0211, 0.0497; respectively). This resulted in an odds ratio of 7.8 (95% Cl: 1.3-45.1, p=0.0222) for GP2a and 6.0 (95% Cl: 1.0-35.3, p=0.0474) for GP2t positivity regarding the prognosis for a lethal outcome in AP on the day of admission. When AP patients admitted until the 10th disease day were considered only, there was also a significantly higher GP2a positivity in AP patients with lethal outcome compared to patients with mild disease (5/6 vs 1/7, p=0.0291).

Example 10: Assay Performance of the GP2a ELISA

(58) Since GP2a testing proofed to be superior in terms of diagnostic accuracy, assay performance of this ELISA was analyzed for routine use. The limit of detection of GP2a was determined at 0.2 ng/mL by using recombinant GP2a.

(59) Linearity was evaluated by diluting a sample with a high GP2a concentration with increasing amounts (from 0% to 100% in increments of 20%) of a sample that did not contain GP2a from 0.5 ng/mL to 6 ng/mL. There was good linearity with a R.sup.2 values of 0.99 for GP2a.

(60) The intra-assay and inter-assay coefficients of variation (CV) were analyzed using sera with varying concentrations of GP2a in accordance with the CLSI protocol EP15-A2. The intra-assay CVs ranged from 6.4% to 15.0% and inter-assay CV from 7.7% to 30.0% for GP2a levels from 3.9-0.2 ng/mL, respectively (Table 6).

(61) Recovery experiments for the assessment of GP2a were conducted by spiking human serum devoid of GP2a with recombinant GP2a. Recovery of GP2a and GP2t ranged from 92.3%-113.6% for spiked GP2a and GP2t levels of 0.25-1.25 ng/mL.

(62) For interference experiments, GP2 containing sera were spiked with hemoglobulin, triglycerides, bilirubin and GP2's urinary homolog Tamm-Horsfall protein (uromodulin) which is synthesized in the tubular cells of the thick ascending limb and the early distal tubule in the kidneys. Final concentrations of 1.0 g/L hemoglobin, 30.0 mg/L bilirubin, 25.0 g/L triglycerides, and 10.0 g/L uromodulin did not interfere with the measurement of GP2a levels.

(63) TABLE-US-00006 TABLE 6 Intra and inter-assay variation of the enzyme-linked immunosorbent assay for the detection of the alpha isoform of glycoprotein 2 (GP2a). The intra-assay and inter-assay coefficients of variation (CV) were analyzed using sera with varying concentrations of GP2a. Intra-assay CV was determined by eight measurements for each serum while inter-assay CV was assessed by analyzing eight determinations for each serum on five different days in accordance with the CLSI protocol EP15-A2. Intra-assay variance GP2a (ng/mL) 3.9 0.8 0.2 n = 8 SD 0.25 0.08 0.03 CV % 6.4 10.0 15.0 Inter-assay variance GP2a (ng/mL) 3.9 0.8 0.2 5 days SD 0.30 0.08 0.06 CV % 7.7 10.0 30.0 SD, standard deviation

Abbreviations

(64) AP, acute pancreatitis; AUC, area under the curve; BMI; body mass index; CP, chronic pancreatitis, BD, blood donor; CI, confidence interval; CV, coefficient of variation; ERCP, endoscopic retrograde cholangiopancreatography; ELISA, enzyme-linked immunosorbent assay; GICa, gastrointestinal cancer; GIST, gastrointestinal stromal tumor; GP2, zymogen granule membrane glycoprotein 2; GPI, glycosyl phosphatidylinositol; IPMN, intraductal papillary mucinous neoplasm; IQR, interquartile range; LCa, liver cancer; LR, likelihood ratio; NET, neuroendocrine tumor; PCa, pancreatic cancer; PT, peritonitis; PU, peptic ulcer; rho, Spearman's rank coefficient of correlation; ROC, receiver-operating characteristics; SA, sarcoma; SD, standard deviation; ZG, zymogen granules.

LITERATURE

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