Method and kit for the detection of pancreatic dysfunction

Abstract

The purpose of the present invention is to provide a simple and highly accurate method for detecting pancreatic exocrine dysfunction with minimal invasiveness to a subject. The method comprising in vitro measurement of two APOA2 protein variants, mutants thereof and/or fragments thereof in a body fluid sample derived from the subject and detection of the presence or absence of pancreatic exocrine dysfunction on the basis of the measured amounts, and a detection kit for pancreatic exocrine dysfunction including antibodies that can specifically bind to said proteins are provided.

Claims

1. A kit for the detection of pancreatic exocrine dysfunction, comprising one or more of a monoclonal antibody or a fragment thereof selected from the group consisting of an anti-Apolipoprotein A2-ATQ (anti-APOA2-ATQ) terminus monoclonal antibody or a fragment thereof and an anti-APOA2 non-terminus monoclonal antibody or a fragment thereof, wherein the ATQ is the amino acid sequence at the C-terminus of APOA2, wherein the anti-APOA2-ATQ terminus monoclonal antibody comprises the CDRs shown in the following (i) or (ii); (i) in the heavy chain, CDR1, CDR2, and CDR3 having the amino acid sequence represented by SEQ ID NOs: 3, 4, and 5, respectively, and in the light chain, CDR1, CDR2, and CDR3 having the amino acid sequence represented by SEQ ID NOS: 6, 7, and 8, respectively, and (ii) in the heavy chain, CDR1, CDR2, and CDR3 having the amino acid sequence represented by SEQ ID NOs: 9, 10, and 11, respectively, and in the light chain, CDR1, CDR2, and CDR3 having the amino acid sequence represented by SEQ ID NOS: 12, 13, and 14, respectively, and wherein the anti-APOA2 non-terminus monoclonal antibody comprises the CDRs shown in the following (iii) or (vi); (iii) in the heavy chain, CDR1, CDR2, and CDR3 having the amino acid sequence represented by SEQ ID NOs: 15, 16, and 17, respectively, and in the light chain, CDR1, CDR2, and CDR3 having the amino acid sequence represented by SEQ ID NOs: 18, 19, and 20, respectively, and (vi) in the heavy chain, CDR1, CDR2, and CDR3 having the amino acid sequence represented by SEQ ID NOS: 21, 22, and 23, respectively, and in the light chain, CDR1, CDR2, and CDR3 having the amino acid sequence represented by SEQ ID NOs: 24, 25, and 26, respectively.

Description

EXAMPLES

(1) The present invention will be described more specifically by the following Examples. However, the scope of the present invention is not limited by the Examples.

Example 1: Detection of Pancreatic Exocrine Dysfunction by Using the APOA2-ATQ Protein or the APOA2-AT Protein in Blood as a Biomarker (1)

(2) The relationship between the amounts of the indicated two APOA2 protein variants and the concentration of pancreatic amylase in serum obtained from patients with pancreatic ductal carcinoma was examined.

(3) Serum samples collected with informed consent from 6 patients with pancreatic ductal carcinoma (Samples A to F) and 10 healthy subjects were measured by ELISA for the concentrations of the APOA2-ATQ protein and the APOA2-AT protein to compare the result to the detection method for pancreatic exocrine function based on the concentration of pancreatic amylase.

(4) (Measurement of the Amount of the APOA2-ATQ Protein)

(5) The measurement of the amount of the APOA2-ATQ protein in serum was carried out by sandwich ELISA using a POD-labeled derivative of the anti-APOA2-ATQ terminus monoclonal antibody 7F2 and an anti-APOA2 non-terminus polyclonal antibody which recognizes the region of the APOA2 protein excluding the C-terminal region (Fitzgerald Industries International). The labeling of the antibody 7F2 with POD was carried out using the Peroxidase Labeling Kit-SH and the details of the labeling followed the appended protocol of the kit. An anti-APOA2 non-terminus polyclonal antibody solution in PBS was prepared at a concentration of 2 μg/mL and, then, 100 μL of the solution was dispensed into each well of an Immunoplate MaxiSorp plate (Nunc) for overnight immobilization. Next day, the above solution was discarded, 400 μL of PBS-T (0.05% Tween-20 in PBS) was added to each well for washing, and 400 μL of a blocking buffer solution (1% BSA and 0.05% Tween-20 in PBS) was added to each well and incubated at room temperature for one hour. Subsequently, the above solution was discarded to obtain an antibody-immobilized plate. Next, 100 μL of a plasma sample diluted with a dilution solution was added to each well to allow reaction at room temperature for one hour. In this case, the dilution factor was 10,000. After the antigen solution in each well was discarded, the well was washed with PBS-T and 100 μL of the POD-labeled derivative of the antibody 7F2 diluted with the dilution solution to a concentration of 0.2 μg/mL was added to each well to allow reaction at room temperature for one hour. After washing with PBS-T, 100 μL of TMB solution (manufactured by Pierce) was added to each well for an enzyme reaction, 100 μL of 0.5 N sulfuric acid was added to stop the reaction, and then the absorbance was measured at 450 nm. The concentration of the protein in blood was calculated based on the comparison of the obtained measured value to that from a recombinant human APOA2-ATQ protein antigen solution as a reference standard.

(6) (Measurement of the Amount of the APOA2-AT Protein)

(7) The measurement of the amount of the APOA2-AT protein in serum was carried out in the same serum samples as described above, similarly to the measurement of the amount of the APOA2-ATQ protein described above, by sandwich ELISA using an anti-APOA2-AT terminus polyclonal antibody and a POD-labeled derivative of the anti-APOA2 non-terminus polyclonal antibody. The labeling of the anti-APOA2 non-terminus polyclonal antibody with POD and the sandwich ELISA were performed similarly as described above. The concentration of the protein in blood was calculated based on the comparison of the obtained measured value to that from a recombinant human APOA2-AT protein antigen solution as a reference standard.

(8) (Measurement of the Amount of Pancreatic Amylase)

(9) The measurement of the amount of pancreatic amylase in serum was carried out in the same serum samples as described above by using the Pancreatic Amylase Human ELISA Kit (Abcam PLC). The antibody-immobilized 96-well plate, the washing solution, the biotinylated anti-pancreatic amylase antibody and the POD-streptavidin conjugate, and the pancreatic amylase standard for generating a standard curve which were all included in the kit were used. The serum samples were diluted 20 times with the dilution solution included in the kit and then dispensed in 50 μL per well into the antibody-immobilized plate, followed by reaction for one hour. Subsequently, the diluted samples were discarded, the plate was washed with the washing solution, and 50 μL of the biotinylated anti-pancreatic amylase antibody diluted with the dilution solution was dispensed into each well of the plate for another one hour of reaction. Furthermore, the biotinylated anti-pancreatic amylase antibody solution was discarded and the plate was washed with the washing solution. The POD-streptavidin conjugate diluted with the dilution solution was dispensed in 50 μL per well for 30 minutes of reaction. Subsequently, the POD-streptavidin conjugate solution was discarded and the plate was washed with the washing solution and, then, 50 μL of chromogen-substrate solution was added to each well. After 15 minutes of reaction, a stop solution was dispensed in 50 μL per well to stop the reaction and the absorbance at 450 nm was measured. A standard curve was generated based on the measured values of the pancreatic amylase standard for generating a standard curve, based on which the concentration of pancreatic amylase in serum was calculated for each sample from the obtained measured value.

(10) The concentrations of the APOA2-ATQ protein, the APOA2-AT protein and pancreatic amylase in serum from each of the pancreatic ductal carcinoma patients represented by Samples A to F and the ranges of concentrations of those items in 10 samples from the healthy subjects are shown in Table 1. The concentrations of the APOA2-ATQ protein, the APOA2-AT protein and pancreatic amylase were measured in 10 samples from the healthy subjects and the means±1 SD of those measured values are presented as the ranges of concentrations in the healthy subjects.

(11) The concentration of pancreatic amylase in the samples from the pancreatic exocrine dysfunction patients tends to deviate from the range of concentration derived from the healthy subjects. When the concentration of pancreatic amylase is below the range of concentration derived from the healthy subjects, the pancreatic exocrine dysfunction can be evaluated as insufficient production of pancreatic juice. When the concentration of pancreatic amylase is above the range of concentration, the pancreatic exocrine dysfunction can be evaluated as disturbance of pancreatic juice flow.

(12) From the measurements on the healthy subjects, the concentration of pancreatic amylase in the healthy subjects was found to be in the range of 65.7 to 177.4 (U/L). On the other hand, it was found in the sample patients with pancreatic ductal carcinoma that a concentration below the range of concentration in the healthy subjects was observed in Samples A, B, and C while a concentration above the range of concentration was observed in Sample D. Consequently, it was found that the concentration of pancreatic amylase allowed the detection of reduced production of pancreatic juice in Samples A, B, and C and disturbance of pancreatic juice flow in Sample D. However, the measured values from Samples E and F stayed in the reference interval for the healthy subjects, suggesting that pancreatic exocrine dysfunction was not detectable based on the measured values of pancreatic amylase in those samples.

(13) On the other hand, the concentrations of the APOA2-ATQ protein and the APOA2-AT protein in the healthy subjects were found to be in the range of 26.8 to 76.4 μg/mL and in the range of 22.7 to 158.3 μg/mL, respectively.

(14) In the measurements using the samples from the pancreatic ductal carcinoma patients, the concentration of the APOA2-ATQ protein in Sample D from a pancreatic ductal carcinoma patient was below the range of concentration in the healthy subjects. Thus, we successfully determined that the subject represented by this sample had pancreatic exocrine dysfunction caused by disturbance of pancreatic juice flow. Furthermore, in Samples A to C, E and F from pancreatic ductal carcinoma patients, the concentration of the APOA2-ATQ protein was equal to or more than that in the healthy subjects while the concentration of the APOA2-AT protein was below the range of concentration in the healthy subjects. Thus, we successfully determined that the subjects represented by these samples had pancreatic exocrine dysfunction caused by insufficient production of pancreatic juice.

(15) According to the above results, the use of the APOA2-AT protein and the APOA2-ATQ protein allowed the possibility of pancreatic exocrine dysfunction to be indicated even in the samples represented by Samples E and F, in which pancreatic exocrine dysfunction was not detectable by the method using pancreatic amylase. This indicates that test subjects who can be medically determined to have pancreatic exocrine dysfunction by a tube test may easily be selected by the detection method according to the present invention.

(16) TABLE-US-00001 TABLE 1 Concentration of Concentration of Concentration of APOA2-ATQ APOA2-AT amylase in blood Samples (μg/mL) (μg/mL) (U/L) Sample A 114.5  4.3 33.3  Sample B 111.0  0.6 18.2  Sample C 122.4  0.3 8.0 Sample D 0.6 92.7  303.4  Sample E 91.6  8.7 97.4  Sample F 101.3  6.3 70.1  The average 51.6  90.5  121.55  for 10 samples from healthy subjects The range of 26.8 to 76.4 22.7 to 158.3 65.7 to 177.4 concentration for 10 samples from healthy subjects

Example 2: Detection of Pancreatic Exocrine Dysfunction by Using the APOA2-ATQ Protein or the APOA2-AT Protein in Blood as a Biomarker (2)

(17) The relationship between the amounts of the indicated two APOA2 protein variants and the concentration of pancreatic amylase in plasma obtained from patients with chronic pancreatitis was examined.

(18) Plasma samples collected with informed consent from 8 patients who had been diagnosed with chronic pancreatitis by an imaging examination (Samples G to N) and 60 healthy subjects were measured by ELISA for the concentrations of the APOA2-ATQ protein and the APOA2-AT protein to compare the result to the detection method for pancreatic exocrine function based on the concentration of pancreatic amylase. The measurement of the amount of each APOA2 protein or enzyme was performed according to a method similar to that in Example 1.

(19) The concentrations of the APOA2-ATQ protein, the APOA2-AT protein and pancreatic amylase in plasma from each of the chronic pancreatitis patients represented by Samples G to N, are shown in Table 2. The concentrations of the APOA2-ATQ protein, the APOA2-AT protein and pancreatic amylase were measured in 60 samples from the healthy subjects and the means±1 SD of those measured values are presented as the ranges of concentrations in the healthy subjects.

(20) The concentration of pancreatic amylase in the healthy subjects was found to be in the range of 38.7 to 85.9 (U/L). On the other hand, it was found in the sample patients with chronic pancreatitis that a concentration below the range of concentration in the healthy subjects was observed in Sample L and, moreover, a concentration above the range of concentration was observed in Samples H, J, and M. Consequently, it was found that the concentration of pancreatic amylase allowed the detection of reduced production of pancreatic juice in Sample L and disturbance of pancreatic juice flow in Samples H, J, and M. However, the measured values from Samples G, I, K, and N stayed in the reference interval for the healthy subjects, suggesting that pancreatic exocrine dysfunction was not detectable based on the measured values of pancreatic amylase in those samples.

(21) On the other hand, the concentrations of the APOA2-ATQ protein and the APOA2-AT protein in the healthy subjects were found to be in the range of 32.9 to 85.8 μg/mL and in the range of 28.9 to 151.5 μg/mL, respectively.

(22) In the measurements using the samples from the chronic pancreatitis patients, the concentration of the APOA2-ATQ protein in Samples H, J, and M from chronic pancreatitis patients was below the range of concentration in the healthy subjects. Thus, we successfully determined that the subjects represented by these samples had pancreatic exocrine dysfunction caused by disturbance of pancreatic juice flow. Furthermore, in Samples G, I, K, L, and N from chronic pancreatitis patients, the concentration of the APOA2-ATQ protein was equal to or more than that in the healthy subjects while the concentration of the APOA2-AT protein was below the range of concentration in the healthy subjects. Thus, we successfully determined that the subjects represented by these samples had pancreatic exocrine dysfunction caused by insufficient production of pancreatic juice.

(23) According to the above results, the use of the APOA2-AT protein and the APOA2-ATQ protein allowed the possibility of pancreatic exocrine dysfunction to be indicated even in the samples represented by Samples G, I, K, and N, in which pancreatic exocrine dysfunction was not detectable by the method using pancreatic amylase. This indicates that test subjects who can be medically determined to have pancreatic exocrine dysfunction by a tube test may easily be selected by the detection method according to the present invention.

(24) TABLE-US-00002 TABLE 2 Concentration of APOA2-ATQ APOA2-AT amylase in blood Samples (μg/mL) (μg/mL) (U/L) Sample G 151.2  2.2 41.1  Sample H 1.7 23.3  412.2  Sample I 112.9  1.2 46.1  Sample J 27.6  89.3  151.8  Sample K 193.1  0.8 39.7  Sample L 121.7  4.4 27.8  Sample M 1.7 489.6  321.3  Sample N 67.3  12.2  51.3  The average 59.4  90.2  62.3  for 60 samples from healthy subjects The range of 32.9 to 85.8 28.9 to 151.5 38.7 to 85.9 concentration for 60 samples from healthy subjects

(25) According to the present invention, pancreatic exocrine dysfunction can effectively be detected by a simple test method imposing a lesser burden on a subject, which consequently allows weak abnormalities in pancreatic exocrine function to be detected, diagnosed and treated. Moreover, according to the method of the present invention, pancreatic exocrine dysfunction can be detected non-invasively by using blood, which consequently allows pancreatic exocrine dysfunction to be simply and quickly detected.

(26) All publications, patents and patent applications cited in this specification shall be directly incorporated in this specification by reference.