A kit and a method for detecting pepsinogen II

Abstract

The present invention discloses a kit and a method for detecting pepsinogen II, and relates to the technical field of detection kits. The kit comprises a first reagent, a second reagent, a luminescent substrate solution and a pepsinogen II calibrator, wherein the first reagent comprises hydrophobic carboxyl magnetic beads coated with a pepsinogen II monoclonal antibody and a first buffer; the second reagent comprises an enzyme-labeled pepsinogen II monoclonal antibody and a second buffer. The present invention adopts enzymatic chemiluminescence technology and selects hydrophobic carboxyl magnetic beads, so that the obtained kit has good stability, high sensitivity, and a wide linear range, and does not require any light source irradiation, the analysis results are stable and reliable, the reaction speed is fast, the operation is simple, the automation program is high, and a large number of tests can be carried out at the same time, which is faster and more efficient in clinical applications.

Description

Technical Field

[0001] The present invention relates to the technical field of detection kits, and in particular to a kit and a method for detecting pepsinogen II. Background Art

[0002] Pepsinogen (PG) is the inactive precursor of pepsin, which is composed of a protein polypeptide chain of 375 amino acids and has a relative molecular mass of about 42×10 3 Samloff divided it into two subgroups based on its biochemical properties and immunogenicity. Groups 1 to 5 have similar immunogenicity and are called pepsinogen I (PGI), which is mainly secreted by gastric mucosal cells and neck mucous cells; Groups 6 to 7 are called pepsinogen II (PGII), which is mainly secreted in the entire gastric glands and duodenal Brunner glands, and can also be expressed in small amounts in the pancreas and prostate.

[0003] Helicobacter pylori (HP) infection and atrophic gastritis are two lesions closely related to gastric cancer. HP infection is one of the main causes of atrophic gastritis, and atrophic gastritis has been generally considered to be a precancerous lesion of gastric cancer. In the development chain of HP infection-atrophic gastritis-gastric cancer, pepsinogen (PG) changes are accompanied. Moreover, the latter has become a good diagnostic indicator for the previous three lesions, as well as a monitoring indicator in the process of treatment and preventive intervention. Differential function analysis shows that the best indicator for serological judgment of the histological state of the gastric fundus mucosa is the combination of the PGI / PGII ratio and the absolute value of PGII. Its total sensitivity and positive predictive value for judging the state of the gastric fundus mucosa is 70%, and it is known as the "serological biopsy" of the gastric fundus gland mucosa. In August 2009, the book "Technical Program for Cancer Screening, Early Diagnosis and Early Treatment in China" issued by the Disease Control Bureau of the Ministry of Health clearly stated that PGI and PGII were used as the judgment criteria for pre-endoscopic detection and gastric cancer screening.

[0004] At present, the methods for detecting pepsinogen II include enzyme-linked immunosorbent assay, immunoturbidimetry, fluorescent immunochromatography, chemiluminescent immunoreaction, etc. Among them, the sensitivity of enzyme-linked immunosorbent assay is relatively low in actual application, and it can only detect qualitatively or semi-quantitatively, and cannot dynamically observe the changes of PG II. In addition, the detection time is long and it cannot realize automated operation; immunoturbidimetry refers to the direct detection and recording of the concentration of the analyte. This method detects the physical concentration of the analyte by detecting the change of the optical density value. Its detection sensitivity is low and it is difficult to meet clinical needs; fluorescent immunochromatography includes direct method and indirect method. The sensitivity of direct method is low, and each time an antigen is checked, a fluorescent antibody needs to be prepared. If multiple antigens are detected, multiple corresponding fluorescent labeled antibodies need to be prepared. The sensitivity of indirect method is higher, about 10 times higher than that of direct method. The preparation of a fluorescent labeled antibody can be applied to multiple primary antibodies, but there are more factors participating in the reaction, which increases the chance of non-specific staining, and the cost of reagents and instruments is high; chemiluminescent immunoassay has poor stability and cannot be popularized in middle and grassroots hospitals and medical institutions. Summary of the invention

[0005] The main purpose of the present invention is to provide a kit and a method for detecting pepsinogen II, aiming to provide a kit for detecting pepsinogen II with good stability.

[0006] To achieve the above object, the present invention provides a kit, which comprises a first reagent, a second reagent, a luminescent substrate solution and a pepsinogen II calibrator.

[0007] Wherein, the first reagent comprises hydrophobic carboxyl magnetic beads coated with pepsinogen II monoclonal antibody and a first buffer;

[0008] The second reagent includes an enzyme-labeled pepsinogen II monoclonal antibody and a second buffer.

[0009] Optionally, in the hydrophobic carboxyl magnetic beads coated with pepsinogen II monoclonal antibody, 5 to 40 μg of pepsinogen II monoclonal antibody is coated per 1 mg of hydrophobic carboxyl magnetic beads; and / or,

[0010] In the enzyme-labeled pepsinogen II monoclonal antibody, the mass ratio of the pepsinogen II monoclonal antibody to the enzyme is 5:6-10.

[0011] Optionally, the first buffer comprises MES, Tween-20, Proclin 300, NaCl and BSA, and has a pH value of 5.8-6.2.

[0012] Optionally, the first buffer comprises: 50 mM MES, 0.1% Tween-20 (v / v), 0.1% Proclin 300 (v / v), 0.9% NaCl (w / v) and 1.2% BSA (w / v).

[0013] Optionally, the concentration of the hydrophobic carboxyl magnetic beads coated with pepsinogen II monoclonal antibody is 0.05 mg / mL to 0.1 mg / mL.

[0014] Optionally, the second buffer comprises Tris, Tween-20, glycine, Proclin300, NaCl, BSA, MgCl2 and ZnCl2, and has a pH value of 7.1-7.3.

[0015] Optionally, the second buffer comprises: 50 mM Tris, 0.1% Tween-20 (v / v), 1% glycine (w / v), 0.1% Proclin300 (v / v), 0.9% NaCl (w / v), 1.2% BSA (w / v), 0.6 mol / L MgCl2 and 0.1 mol / L ZnCl2.

[0016] Optionally, the luminescent substrate solution includes AMPPD; and / or,

[0017] In the second reagent, the volume ratio of the enzyme-labeled pepsinogen II monoclonal antibody to the second buffer is 1:480-520.

[0018] Furthermore, the present invention also proposes a method for detecting pepsinogen II comprising the following steps:

[0019] Providing a kit as described above;

[0020] After incubating the first reagent, the second reagent and the sample to be tested, magnetic separation and cleaning are performed, and then a luminescent substrate solution is added to obtain a solution to be tested;

[0021] The luminescence value of the test solution is detected, and the content of pepsinogen II is calculated according to the luminescence value.

[0022] Optionally, the volume ratio of the first reagent, the second reagent and the sample to be tested is 5:10:2.

[0023] The kit provided by the present invention adopts the sandwich immunoassay method of enzymatic chemiluminescence technology, the PGII antigen in the sample to be tested reacts with the PGII antibody coated on the paramagnetic particles and the enzyme-labeled PGII antibody conjugate to form a sandwich (antibody-antigen-antibody) complex, under the action of the magnetic field, the sandwich complex is adsorbed to the wall of the reaction tube, the unbound substance is washed away by the cleaning solution, and the luminescent substrate liquid is added to the sandwich complex, and the content of PGII can be calculated by the luminescence value; with hydrophobic carboxyl magnetic bead superparamagnetic particles, the carboxyl group on the surface of the hydrophobic carboxyl magnetic bead can be directly chemically combined with the antibody containing amino group to form an amide bond, so that the stability of the hydrophobic carboxyl magnetic bead coated with the PGII monoclonal antibody is good. By adopting enzymatic chemiluminescence technology and selecting hydrophobic carboxyl magnetic beads, the kit provided by the present invention has good stability, high sensitivity, wide linear range, and does not require any light source irradiation, the analysis result is stable and reliable, the reaction speed is fast, the operation is simple, the automation program is high, and a large number of tests can be carried out at the same time, which is faster and more efficient in clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 This is a graph showing the linear range test results of the kit in Example 1 of the present invention;

[0026] Figure 2 This is a comparison chart of clinical sample tests of the kit in Example 1 of the present invention.

[0027] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0029] In addition, the meaning of "and / or" appearing in the full text includes three parallel solutions. Taking "A and / or B" as an example, it includes solution A, solution B, or a solution that satisfies both A and B. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the ability of ordinary technicians in this field to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0030] The present invention provides a kit for detecting the content of pepsinogen II. In one embodiment, the kit comprises a first reagent, a second reagent, a luminescent substrate solution and a pepsinogen II calibrator, wherein the first reagent comprises hydrophobic carboxyl magnetic beads coated with a pepsinogen II monoclonal antibody and a first buffer; the second reagent comprises an enzyme-labeled pepsinogen II monoclonal antibody and a second buffer.

[0031] The kit provided by the present invention adopts the sandwich immunoassay method of enzymatic chemiluminescence technology, the PGII antigen in the sample to be tested reacts with the PGII antibody coated on the paramagnetic particles and the enzyme-labeled PGII antibody conjugate to form a sandwich (antibody-antigen-antibody) complex, under the action of the magnetic field, the sandwich complex is adsorbed to the wall of the reaction tube, the unbound substance is washed away by the cleaning solution, and the luminescent substrate liquid is added to the sandwich complex, and the content of PGII can be calculated by the luminescence value; with hydrophobic carboxyl magnetic bead superparamagnetic particles, the carboxyl group on the surface of the hydrophobic carboxyl magnetic bead can be directly chemically combined with the antibody containing amino group to form an amide bond, so that the stability of the hydrophobic carboxyl magnetic bead coated with the PGII monoclonal antibody is good. By adopting enzymatic chemiluminescence technology and selecting hydrophobic carboxyl magnetic beads, the kit provided by the present invention has good stability, high sensitivity, wide linear range, and does not require any light source irradiation, the analysis result is stable and reliable, the reaction speed is fast, the operation is simple, the automation program is high, and a large number of tests can be carried out at the same time, which is faster and more efficient in clinical applications.

[0032] Preferably, in the hydrophobic carboxyl magnetic beads coated with pepsinogen II monoclonal antibody, 5 to 40 μg of pepsinogen II monoclonal antibody is coated per 1 mg of hydrophobic carboxyl magnetic beads, so that the hydrophobic carboxyl magnetic beads and pepsinogen II monoclonal antibody can be fully utilized, and the binding of the hydrophobic carboxyl magnetic beads and the pepsinogen II monoclonal antibody is stable.

[0033] The present invention does not limit the specific components of the first buffer. In this embodiment, the first buffer includes MES (morpholineethanesulfonic acid), Tween-20, Proclin 300, NaCl and BSA (bovine serum albumin), and the pH value is 5.8 to 6.2, preferably, the pH value is 6.0. Proclin 300 is used as a preservative.

[0034] Furthermore, the first buffer comprises: 50 mM MES, 0.1% Tween-20 (v / v), 0.1% Proclin 300 (v / v), 0.9% NaCl (w / v) and 1.2% BSA (w / v). Under the above ratio, the stability and sensitivity of the first reagent are the best and the reaction efficiency is the fastest.

[0035] It should be noted that w / v in this article refers to the mass-to-volume ratio, with the unit of g / mL. For example, the concentration of BSA is 1.2%, which means that the mass of BSA in every 100 mL of the first buffer is 1.2 g; v / v refers to the volume ratio, for example, the concentration of Tween-20 is 0.1%, which means that 100 mL of the first dilution solution includes 0.1 mL of Tween-20. In addition, mM refers to mmol / L, for example, the concentration of MES is 50 mM, which means that the concentration of MES in the first buffer is 50 mmol / L.

[0036] In this embodiment, in the enzyme-labeled pepsinogen II monoclonal antibody, the mass ratio of the pepsinogen II monoclonal antibody to the enzyme is 5:6-10, that is, the mass ratio can be 5:6, 5:7, 5:7.6, 5:7.9, 5:9, 5:10, etc. Further, the enzyme includes alkaline phosphatase.

[0037] Preferably, in the first reagent, the concentration of the hydrophobic carboxyl magnetic beads coated with pepsinogen II monoclonal antibody is 0.05 mg / mL to 0.1 mg / mL. At this concentration, the pepsinogen II monoclonal antibody is conducive to reacting with the PG II antigen in the sample to be tested, and the reaction rate is fast.

[0038] In order to improve the stability of the second reagent, in this embodiment, the second buffer includes Tris, Tween-20, glycine, Proclin300, NaCl, BSA, MgCl2 and ZnCl2, and the pH value is 7.1-7.3, preferably, the pH value is 7.2.

[0039] Glycine is a zwitterion containing an amino group and a carboxyl group, has a strong buffering property, and can form a buffer system with Tris-HCl to maintain the second buffer in a relatively stable pH range, so that the enzyme-labeled pepsinogen II monoclonal antibody is always in a stable pH range, thereby maintaining its biological activity. The present invention greatly improves the stability of the second reagent by adding glycine to the second buffer.

[0040] Preferably, the second buffer comprises: 50 mM Tris, 0.1% Tween-20 (v / v), 1% glycine (w / v), 0.1% Proclin300 (v / v), 0.9% NaCl (w / v), 1.2% BSA (w / v), 0.6 mol / L MgCl2 and 0.1 mol / L ZnCl2. Under the above ratio, the stability of the second reagent is optimal. Wherein, mol / L can be abbreviated as M.

[0041] Furthermore, in the second reagent, the volume ratio of the enzyme-labeled pepsinogen II monoclonal antibody to the second buffer is 1:480-520, that is, it can be 1:480, 1:485, 1:490, 1:500, 1:505, 1:520, etc., preferably 1:500.

[0042] Wherein, the pepsinogen II calibrator comprises a diluent and pepsinogen II. In this embodiment, the concentrations of the pepsinogen II calibrator provided are 15 ng / mL and 50 ng / mL, respectively.

[0043] Preferably, the luminescent substrate solution includes AMPPD, which is a 1,2-dioxetane derivative and is the latest ultra-sensitive alkaline phosphatase substrate in the field of biochemistry. It has the following characteristics: fast reaction speed and can provide accurate and reliable results in a very short time.

[0044] The present invention does not limit the specific preparation method of the kit, as long as the prepared kit meets the above conditions. In a specific embodiment, the preparation method of the kit comprises the following steps:

[0045] (1) Preparation of the first reagent: 10 mg of hydrophobic carboxyl magnetic beads were washed three times, each washing step being: adding 1 mL of 50 mM MES buffer with a pH of 6.2, mixing well, and then standing on a magnetic rack for about 1 minute, and then removing the supernatant; then adding 1 mL of 50 mM MES buffer with a pH of 6.2, mixing well, and then adding 100 ug of pepsinogen II monoclonal antibody, and mixing well at room temperature for 1 to 3 hours; after the reaction is completed, adding 1% BSA buffer with a pH of 6.5 for blocking, and standing on a magnetic rack for about 1 minute, and then removing the supernatant to obtain hydrophobic carboxyl magnetic beads coated with pepsinogen II monoclonal antibody; the hydrophobic carboxyl magnetic beads coated with pepsinogen II monoclonal antibody were washed three times, and each washing step was: adding 1 mL of 0.1M PBS buffer with a pH value of 7.0 was mixed and allowed to stand on a magnetic rack for about 1 minute, and the supernatant was removed; after washing, it was resuspended in the first buffer to obtain the first reagent.

[0046] (2) Preparation of the second reagent: Take 100uL of pepsinogen II monoclonal antibody, add 250uL of 0.1M PBS buffer with a pH value of 7.0, mix well, add 20ul of a newly prepared 10.0mg / ml EDC aqueous solution, activate for 30 minutes, then add 200ul of a 5mg / ml alkaline phosphatase solution and mix well, store at room temperature in the dark for 2 hours, take out, block the reaction with 0.1M Tris buffer with a pH value of 7.4 containing 1% BSA at room temperature for 30min, then add 0.5% diethyl phosphate and continue the reaction at room temperature for 30min, desalt and purify with a 30KD ultrafiltration column to obtain enzyme-labeled pepsinogen II monoclonal antibody, add half of glycerol to the remaining volume of the collected solution, and store at -20°C for future use; resuspend the obtained enzyme-labeled pepsinogen II monoclonal antibody in the second buffer to obtain the second reagent.

[0047] The present invention also provides a method for detecting pepsinogen II. In one embodiment, the detection method comprises the following steps:

[0048] Step S10, providing the kit as described above;

[0049] Step S20, after incubating the first reagent, the second reagent and the sample to be tested, magnetic separation and cleaning are performed, and then a luminescent substrate solution is added to obtain a solution to be tested;

[0050] Among them, the volume ratio of the first reagent, the second reagent and the sample to be tested is 5:10:2.

[0051] Step S30, detecting the luminescence value of the test solution, and calculating the content of pepsinogen II according to the luminescence value.

[0052] In a specific embodiment, the detection method includes the following steps: using a fully automatic chemiluminescence analyzer (CL-2000) as a detection tool, adding 100 μL of R2 reagent and 20 μL of the sample to be tested and incubating for 5 minutes, then adding 50 μL of R1 reagent and incubating for 5 minutes to obtain a mixed solution, subjecting the mixed solution to magnetic separation and cleaning, and after cleaning, the instrument automatically adds 300 μL of substrate solution to obtain a solution to be tested, and automatically moves the solution to be tested to a photoelectric module to detect the luminescence value, and then the instrument automatically converts it to the concentration of pepsinogen II in the solution to be tested and displays the test results.

[0053] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0054] Example 1 Kit

[0055] The kit comprises:

[0056] The first reagent includes hydrophobic carboxyl magnetic beads coated with pepsinogen II monoclonal antibody and a first buffer, wherein each 1 mg of hydrophobic carboxyl magnetic beads is coated with 10 μg of pepsinogen II monoclonal antibody, the concentration of the hydrophobic carboxyl magnetic beads coated with pepsinogen II monoclonal antibody is 0.07 mg / mL, and the first buffer comprises: 50 mM MES, 0.1% Tween-20 (v / v), 0.1% Proclin 300 (v / v), 0.9% NaCl (w / v) and 1.2% BSA (w / v), and the pH value is 6.0.

[0057] The second reagent: an alkaline phosphatase-labeled pepsinogen II monoclonal antibody and a second buffer, wherein the mass ratio of the pepsinogen II monoclonal antibody to the alkaline phosphatase is 5:8, the volume ratio of the enzyme-labeled pepsinogen II monoclonal antibody to the second buffer is 1:500, and the second buffer comprises: 50 mM Tris, 0.1% Tween-20 (v / v), 1% glycine (w / v), 0.1% Proclin300 (v / v), 0.9% NaCl (w / v), 1.2% BSA (w / v), 0.6 mol / L MgCl2 and 0.1 mol / L ZnCl2, and the pH value is 7.2;

[0058] Luminescent substrate solution (AMPPD);

[0059] Pepsinogen II calibrator (the concentrations of the pepsinogen II calibrator are 15 ng / mL and 50 ng / mL respectively).

[0060] Example 2 Kit

[0061] The kit comprises:

[0062] The first reagent includes hydrophobic carboxyl magnetic beads coated with pepsinogen II monoclonal antibody and a first buffer, wherein each 1 mg of hydrophobic carboxyl magnetic beads is coated with 5 μg of pepsinogen II monoclonal antibody, the concentration of the hydrophobic carboxyl magnetic beads coated with pepsinogen II monoclonal antibody is 0.05 mg / mL, and the first buffer comprises: 50 mM MES, 0.1% Tween-20 (v / v), 0.1% Proclin300 (v / v), 0.9% NaCl (w / v) and 1.2% BSA (w / v), and the pH value is 6.2.

[0063] The second reagent: an alkaline phosphatase-labeled pepsinogen II monoclonal antibody and a second buffer, wherein the mass ratio of the pepsinogen II monoclonal antibody to the alkaline phosphatase is 5:7, the volume ratio of the enzyme-labeled pepsinogen II monoclonal antibody to the second buffer is 1:480, and the second buffer comprises: 50 mM Tris, 0.1% Tween-20 (v / v), 1% glycine (w / v), 0.1% Proclin300 (v / v), 0.9% NaCl (w / v), 1.2% BSA (w / v), 0.6 mol / L MgCl2 and 0.1 mol / L ZnCl2, and the pH value is 7.3;

[0064] Luminescent substrate solution (AMPPD);

[0065] Pepsinogen II calibrator (the concentrations of the pepsinogen II calibrator are 15 ng / mL and 50 ng / mL respectively).

[0066] Example 3 Kit

[0067] The kit comprises:

[0068] The first reagent includes hydrophobic carboxyl magnetic beads coated with pepsinogen II monoclonal antibody and a first buffer, wherein each 1 mg of hydrophobic carboxyl magnetic beads is coated with 20 μg of pepsinogen II monoclonal antibody, the concentration of the hydrophobic carboxyl magnetic beads coated with pepsinogen II monoclonal antibody is 0.1 mg / mL, and the first buffer comprises: 50 mM MES, 0.1% Tween-20 (v / v), 0.1% Proclin 300 (v / v), 0.9% NaCl (w / v) and 1.2% BSA (w / v), and the pH value is 5.8.

[0069] The second reagent: an alkaline phosphatase-labeled pepsinogen II monoclonal antibody and a second buffer, wherein the mass ratio of the pepsinogen II monoclonal antibody to the alkaline phosphatase is 5:6, the volume ratio of the enzyme-labeled pepsinogen II monoclonal antibody to the second buffer is 1:520, and the second buffer comprises: 50 mM Tris, 0.1% Tween-20 (v / v), 1% glycine (w / v), 0.1% Proclin300 (v / v), 0.9% NaCl (w / v), 1.2% BSA (w / v), 0.6 mol / L MgCl2 and 0.1 mol / L ZnCl2, and the pH value is 7.2;

[0070] Luminescent substrate solution (AMPPD);

[0071] Pepsinogen II calibrator (the concentrations of the pepsinogen II calibrator are 15 ng / mL and 50 ng / mL respectively).

[0072] Example 4 Kit

[0073] The kit comprises:

[0074] The first reagent includes hydrophobic carboxyl magnetic beads coated with pepsinogen II monoclonal antibody and a first buffer, wherein each 1 mg of hydrophobic carboxyl magnetic beads is coated with 40 μg of pepsinogen II monoclonal antibody, the concentration of the hydrophobic carboxyl magnetic beads coated with pepsinogen II monoclonal antibody is 0.1% (w / v), and the first buffer comprises: 50 mM MES, 0.1% Tween-20 (v / v), 0.1% Proclin 300 (v / v), 0.9% NaCl (w / v) and 1.2% BSA (w / v), and the pH value is 6.1.

[0075] The second reagent: an alkaline phosphatase-labeled pepsinogen II monoclonal antibody and a second buffer, wherein the mass ratio of the pepsinogen II monoclonal antibody to the alkaline phosphatase is 5:10, the volume ratio of the enzyme-labeled pepsinogen II monoclonal antibody to the second buffer is 1:520, and the second buffer comprises: 50 mM Tris, 0.1% Tween-20 (v / v), 1% glycine (w / v), 0.1% Proclin300 (v / v), 0.9% NaCl (w / v), 1.2% BSA (w / v), 0.6 mol / L MgCl2 and 0.1 mol / L ZnCl2, and the pH value is 7.1;

[0076] Luminescent substrate solution (AMPPD);

[0077] Pepsinogen II calibrator (the concentrations of pepsinogen II are 15 ng / mL and 50 ng / mL respectively).

[0078] Example 5 Detection Method of Pepsinogen II

[0079] Using a fully automatic chemiluminescence analyzer (CL-2000) as the detection tool, add 100 μL of R2 reagent and 20 μL of the sample to be tested and incubate for 5 minutes, then add 50 μL of R1 reagent and incubate for 5 minutes to obtain a mixed solution, which is magnetically separated and cleaned. After cleaning, the instrument automatically adds 300 μL of substrate solution to obtain a test solution, which is automatically moved to the photoelectric module to detect the luminescence value, and then the instrument automatically converts it to the pepsinogen I concentration in the test solution and displays the test results.

[0080] Performance test of the kit:

[0081] 1. Sensitivity detection

[0082] Referring to the experimental scheme recommended by CLSI EP17-A document, the sensitivity of the kit in Example 1 was calculated, and it was found that the sensitivity of the kit for detecting pepsinogen II was <0.5 ng / mL.

[0083] 2. Linear range

[0084] The kit in Example 1 was used to perform linear analysis on pepsinogen II reference products with concentrations of 1 ng / mL, 6 ng / mL, 30 ng / mL, 75 ng / mL, and 100 ng / mL. The results are shown in Table 1 below.

[0085] Table 1 Linearity test results

[0086] Concentration (ng / mL) Luminescence value (RLU value) 0 2981 1 229993 6 1584619 30 6010613 75 13607390 100 17942399

[0087] According to the test results shown in Table 1, we can get Figure 1 The linear curve shown, Figure 1 In the figure, the horizontal axis is the concentration (ng / mL) and the vertical axis is the luminescence value (RLU value). Figure 1 It can be seen that the linear correlation coefficient r≥0.99, indicating that the linear range of the kit is 1-100 ng / mL with good linearity, and can accurately measure the test samples with a pepsinogen II concentration of 1-100 ng / mL.

[0088] 3. Clinical sample testing and comparison

[0089] The kit in Example 1 and the Abbott kit were used to simultaneously detect the pepsinogen II content in clinical serum, with the result measured by the Abbott kit as the horizontal coordinate X, the concentration in ng / mL, and the result measured by the present invention as the vertical coordinate y, the concentration unit in ng / mL. The results are shown in FIG. Figure 2 shown.

[0090] Depend on Figure 2 It can be seen that the regression equation is: y=1.0008x+0.0181, the correlation coefficient is 0.9959, and the K value is 1.0008, indicating that the correlation is very good.

[0091] 4. Accuracy

[0092] The kit in Example 1 was used to measure the pepsinogen II calibrator (concentrations of 15 ng / mL and 50 ng / mL) therein, and the parallel determination was repeated 3 times to obtain the average value. The recovery rate was in the range of 85.0%-115.0%. The test results are shown in Table 2 below.

[0093] Table 2 Accuracy test results

[0094] Determine the concentration of the calibrator (ng / mL) Mean value of test results (ng / mL) Recovery rate (%) 15 15.1 99.3％ 50 49.8 100.4％

[0095] It can be seen from Table 2 that the kit for detecting pepsinogen II provided in Example 1 has high accuracy and the recovery rate meets the requirements.

[0096] 5. Batch Difference

[0097] Three batches of the kit in Example 1 were used to test the pepsinogen II calibrator (with a concentration of 15 ng / mL and 50 ng / mL), respectively, with each batch repeated 10 times. The coefficient of variation CV was calculated, and the results are shown in Table 3 below.

[0098] Table 3 Batch difference test results

[0099]

[0100] It can be seen from Table 3 that the coefficient of variation CV is less than 8%, which meets the requirement that the coefficient of variation CV of the kit is less than 15%.

[0101] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. A kit for detecting the content of pepsinogen II, characterized in that: The kit comprises a first reagent, a second reagent, a luminescent substrate solution and a pepsinogen II calibrator. The first reagent comprises hydrophobic carboxyl magnetic beads coated with pepsinogen II monoclonal antibody and a first buffer; in the hydrophobic carboxyl magnetic beads coated with pepsinogen II monoclonal antibody, 10 μg of pepsinogen II monoclonal antibody is coated per 1 mg of hydrophobic carboxyl magnetic beads; the concentration of the hydrophobic carboxyl magnetic beads coated with pepsinogen II monoclonal antibody is 0.07 mg / mL; the first buffer comprises: 50 mM MES, 0.1% Tween-20 (v / v), 0.1% Proclin300 (v / v), 0.9% NaCl (w / v) and 1.2% BSA (w / v), with a pH value of 6.0; The second reagent includes an alkaline phosphatase-labeled pepsinogen II monoclonal antibody and a second buffer; in the alkaline phosphatase-labeled pepsinogen II monoclonal antibody, the mass ratio of the pepsinogen II monoclonal antibody to the alkaline phosphatase is 5:8; the volume ratio of the alkaline phosphatase-labeled pepsinogen II monoclonal antibody to the second buffer is 1:500; the second buffer includes: 50mM Tris, 0.1% Tween-20 (v / v), 1% glycine (w / v), 0.1% Proclin300 (v / v), 0.9% NaCl (w / v), 1.2% BSA (w / v), 0.6mol / L MgCl2 and 0.1mol / L ZnCl2, and the pH value is 7.2; The luminescent substrate solution includes AMPPD.

2. A method for detecting pepsinogen II, characterized in that: The following steps are involved: Providing a kit as claimed in claim 1; After incubating the first reagent, the second reagent and the sample to be tested, magnetic separation and cleaning are performed, and then a luminescent substrate solution is added to obtain a solution to be tested; The luminescence value of the test solution is detected, and the content of pepsinogen II is calculated according to the luminescence value.

3. The method for detecting pepsinogen II according to claim 2, characterized in that: The volume ratio of the first reagent, the second reagent and the sample to be tested is 5:10:2.

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