A MMP7 detection kit, preparation method and detection method thereof

Abstract

The present invention provides an MMP7 detection kit and use thereof in preparing a product for diagnosing MMP7-related diseases. The kit comprises a magnetic microsphere solution M, a biotinylated antibody solution R1 and a luminescent marker R2, and exhibits the advantages of stable performance, high sensitivity, wide linear range, convenient operation, strong clinical practicality, etc. in the detection of MMP7.

Description

Technical Field

[0001] The present invention belongs to the technical field of in vitro diagnosis, and specifically relates to a MMP7 detection kit, a preparation method and a detection method. Background Art

[0002] Matrix metalloproteinase 7 (MMP7) is a zinc-dependent endopeptidase. Its gene is located at 11q21-q22, with a total cDNA length of 1094bp and a molecular weight of 28KD. MMP7 is secreted in the form of zymogen. Under normal physiological conditions, MMP7 combines with its specific matrix metalloproteinase inhibitor (TIMP) to maintain the homeostasis of the extracellular matrix. MMP7 is a target and product of multiple profibrotic pathways, such as TGF-β / Smad, Rho / ROCK, Notch, MAPK and P13 signaling pathways. Abnormal expression of MMP7 is associated with liver fibrosis, and MMP7 can be used for non-invasive diagnosis of biliary atresia. Moreover, MMP7 may be involved in tumor metastasis and inflammatory processes, and upregulated MMP7 is associated with many malignant tumors.

[0003] At present, the detection of MMP7 mainly adopts enzyme-linked immunosorbent assay. In this detection method, specific anti-human MMP7 antibodies are pre-coated on high-affinity ELISA plates. Standards, samples to be tested and biotinylated detection antibodies are added to the wells of the ELISA plate. After incubation, the MMP7 present in the sample binds to the solid phase antibody and the detection antibody. After washing to remove unbound substances, horseradish peroxidase-labeled streptavidin is added. After washing, the color substrate TMB is added and the color is developed in the dark. The depth of the color reaction is proportional to the concentration of MMP7 in the sample. The stop solution is added to stop the reaction, and the absorbance value is measured at a wavelength of 450 nm (reference wavelength 570-630 nm). The concentration of human matrix metalloproteinase 7 in the sample is proportional to the OD value, and the concentration of human matrix metalloproteinase 7 in the sample is calculated by drawing a standard curve.

[0004] ELISA usually relies on manual operation, which is labor-intensive and time-consuming. Moreover, the operator's operating standards and proficiency greatly affect the accuracy and repeatability of the test results, leading to inaccurate results. At the same time, the detection sensitivity and linear range of ELISA are also low. It is mainly used in scientific research scenarios and has certain limitations in clinical testing, which limits the application and promotion of MMP7 detection in clinical practice.

[0005] Currently, there is still a lack of MMP7 detection kits and detection methods using the same that are fast, convenient, stable, sensitive, and can be automated on instruments on the market. Summary of the invention

[0006] The present invention provides a MMP7 detection kit, a preparation method thereof and a detection method. The kit can be used for automatic instrument detection, and can more quickly and accurately measure the MMP7 concentration in an in vitro sample. The present invention prepares a monoclonal MMP7 antibody and adjusts various actual reagent components, so that the kit has stable performance, high sensitivity, strong specificity, wide linear range, convenient operation, short detection time and strong clinical practicality.

[0007] In order to solve the above technical problems, the present invention provides a MMP7 detection kit, a preparation method thereof and a method for detecting the concentration of MMP7 in a sample using the kit.

[0008] Kit composition

[0009] In one aspect, the present invention provides a MMP7 detection kit, comprising:

[0010] A magnetic microsphere solution M, comprising streptavidin-coupled magnetic beads;

[0011] Biotinylated antibody solution R1, which contains biotinylated polyclonal MMP7 antibody;

[0012] Luminescent marker R2, which comprises an acridinium ester-labeled monoclonal MMP7 antibody.

[0013] Furthermore, the present invention uses a self-prepared monoclonal MMP7 antibody as a capture antibody, which has been proven to have excellent reactivity compared with the prior art and improves the detection sensitivity of the kit.

[0014] In some preferred embodiments, the monoclonal MMP7 antibody comprises:

[0015] (1) HCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 41;

[0016] (2) HCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 42;

[0017] (3) HCDR3, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 43;

[0018] (4) LCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 44;

[0019] (5) LCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 45; and

[0020] (6) LCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 46, or consists of the amino acid sequence.

[0021] In some preferred embodiments, the monoclonal MMP7 antibody comprises:

[0022] (a) HCDR1, which comprises or consists of the amino acid sequence shown in any one of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 7 or SEQ ID NO: 10;

[0023] (b) HCDR2, which comprises or consists of the amino acid sequence shown in any one of SEQ ID NO: 2 or SEQ ID NO: 5 or SEQ ID NO: 8 or SEQ ID NO: 11;

[0024] (c) HCDR3 comprising or consisting of the amino acid sequence shown in any one of SEQ ID NO: 3 or SEQ ID NO: 6 or SEQ ID NO: 9 or SEQ ID NO: 12;

[0025] (d) LCDR1, which comprises or consists of the amino acid sequence shown in any one of SEQ ID NO: 13 or SEQ ID NO: 16 or SEQ ID NO: 19 or SEQ ID NO: 22;

[0026] (e) LCDR2, which comprises or consists of the amino acid sequence shown in any one of SEQ ID NO: 14 or SEQ ID NO: 17 or SEQ ID NO: 20 or SEQ ID NO: 23; and

[0027] (f) LCDR3, which comprises or consists of the amino acid sequence shown in any one of SEQ ID NO: 15 or SEQ ID NO: 18 or SEQ ID NO: 21 or SEQ ID NO: 24.

[0028] In some preferred embodiments, the monoclonal MMP7 antibody comprises:

[0029] (a) HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:2, HCDR3 as shown in SEQ ID NO:3, LCDR1 as shown in SEQ ID NO:13, LCDR2 as shown in SEQ ID NO:14, LCDR3 as shown in SEQ ID NO:15;

[0030] (b) HCDR1 as shown in SEQ ID NO:4, HCDR2 as shown in SEQ ID NO:5, HCDR3 as shown in SEQ ID NO:6, LCDR1 as shown in SEQ ID NO:16, LCDR2 as shown in SEQ ID NO:17, LCDR3 as shown in SEQ ID NO:18;

[0031] (c) HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in SEQ ID NO:8, HCDR3 as shown in SEQ ID NO:9, LCDR1 as shown in SEQ ID NO:19, LCDR2 as shown in SEQ ID NO:20, LCDR3 as shown in SEQ ID NO:21; or

[0032] (d) HCDR1 as shown in SEQ ID NO:10, HCDR2 as shown in SEQ ID NO:11, HCDR3 as shown in SEQ ID NO:12, LCDR1 as shown in SEQ ID NO:22, LCDR2 as shown in SEQ ID NO:23, LCDR3 as shown in SEQ ID NO:24.

[0033] In some preferred embodiments, the monoclonal MMP7 antibody comprises:

[0034] A heavy chain variable region comprising an amino acid sequence as shown in any one of SEQ ID NOs: 25-28, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity compared to the amino acid sequence as shown in any one of SEQ ID NOs: 25-28, or an amino acid sequence having one or more (e.g., 2, 3 or more) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence as shown in any one of SEQ ID NOs: 25-28, or consisting of said amino acid sequence; and / or

[0035] A light chain variable region comprising an amino acid sequence as shown in any one of SEQ ID NOs: 29-32, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity compared to the amino acid sequence as shown in any one of SEQ ID NOs: 29-32, or an amino acid sequence having one or more (e.g., 2, 3 or more) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence as shown in any one of SEQ ID NOs: 29-32, or consisting of said amino acid sequence.

[0036] In some preferred embodiments, the monoclonal MMP7 antibody comprises:

[0037] (1) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 25, or an amino acid sequence having at least 80% sequence identity thereto, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 29, or an amino acid sequence having at least 80% sequence identity thereto;

[0038] (2) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:26, or an amino acid sequence having at least 80% sequence identity thereto, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:30, or an amino acid sequence having at least 80% sequence identity thereto;

[0039] (3) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:27, or an amino acid sequence having at least 80% sequence identity thereto, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:31, or an amino acid sequence having at least 80% sequence identity thereto; or

[0040] (4) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 28, or an amino acid sequence having at least 80% sequence identity thereto, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 32, or an amino acid sequence having at least 80% sequence identity thereto.

[0041] In some embodiments, compared with the amino acid sequence shown in any one of SEQ ID NOs: 25-28, the different amino acids in the amino acid sequence with at least 80% sequence identity are all located in the FR region. In some embodiments, compared with the amino acid sequence shown in SEQ ID NOs: 29-32, the different amino acids in the amino acid sequence with at least 80% sequence identity are all located in the FR region.

[0042] In some embodiments, the monoclonal MMP7 antibody further comprises a heavy chain constant region, a light chain constant region, or a combination thereof. In some embodiments, the heavy chain constant region is an IgG (including IgG1, IgG2, IgG3, or IgG4) heavy chain constant region or a variant thereof (e.g., LALA mutation, AAA mutation, DLE mutation, YTE mutation, and LS mutation known in the prior art), such as a mouse IgG, human IgG, or monkey IgG heavy chain constant region or a variant thereof. In some embodiments, the light chain constant region is a κ or λ chain constant region or a variant thereof. In some embodiments, the heavy chain constant region has an amino acid sequence as shown in SEQ ID NO: 47, and the light chain constant region has an amino acid sequence as shown in SEQ ID NO: 48.

[0043] In some embodiments, the monoclonal MMP7 antibody comprises:

[0044] A heavy chain comprising an amino acid sequence as shown in any one of SEQ ID NOs:33-36, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity compared to the amino acid sequence as shown in any one of SEQ ID NOs:33-36, or an amino acid sequence having one or more (e.g., 2, 3 or more) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence as shown in any one of SEQ ID NOs:33-36, or consisting of said amino acid sequence; and

[0045] A light chain comprising an amino acid sequence as shown in any one of SEQ ID NOs:37-40, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity compared to the amino acid sequence shown in any one of SEQ ID NOs:37-40, or an amino acid sequence having one or more (e.g., 2, 3 or more) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence shown in any one of SEQ ID NOs:37-40, or consisting of said amino acid sequence.

[0046] In some embodiments, the monoclonal MMP7 antibody comprises:

[0047] (1) a heavy chain having an amino acid sequence as shown in SEQ ID NO: 33, or an amino acid sequence having at least 80% sequence identity thereto, and a light chain having an amino acid sequence as shown in SEQ ID NO: 37, or an amino acid sequence having at least 80% sequence identity thereto;

[0048] (2) a heavy chain having an amino acid sequence as shown in SEQ ID NO:34, or an amino acid sequence having at least 80% sequence identity thereto, and a light chain having an amino acid sequence as shown in SEQ ID NO:38, or an amino acid sequence having at least 80% sequence identity thereto;

[0049] (3) a heavy chain having an amino acid sequence as shown in SEQ ID NO:35, or an amino acid sequence having at least 80% sequence identity thereto, and a light chain having an amino acid sequence as shown in SEQ ID NO:39, or an amino acid sequence having at least 80% sequence identity thereto; or

[0050] (4) a heavy chain having the amino acid sequence as shown in SEQ ID NO:36, or an amino acid sequence having at least 80% sequence identity thereto, and a light chain having the amino acid sequence as shown in SEQ ID NO:40, or an amino acid sequence having at least 80% sequence identity thereto.

[0051] In some embodiments, the different amino acids in the amino acid sequence having at least 80% sequence identity compared to the amino acid sequence shown in any one of SEQ ID NOs: 33-40 are located in the FR region, the heavy chain constant region, the light chain constant region, or a combination thereof.

[0052] In some embodiments, the magnetic microsphere solution M further comprises a magnetic bead storage solution, such as a magnetic bead storage solution having a pH of 6.5 to 7.8.

[0053] In some embodiments, the magnetic microsphere solution M comprises: streptavidin-coupled magnetic beads at a concentration of 0.2-2 mg / mL and a magnetic bead storage solution at a pH of 6.5-7.8.

[0054] In some embodiments, the magnetic bead storage solution comprises: a buffer, an inert protein, a surfactant, and a preservative. In some preferred embodiments, the magnetic bead storage solution comprises: a 1-20 mM buffer with a pH of 7 to 7.5, 0.5 wt% to 3 wt% inert protein, 0.1 wt% to 3 wt% surfactant, and 0.05 wt% to 0.3 wt% preservative.

[0055] In some embodiments, the buffer comprises one or more of a Tris buffer, a phosphate-buffered saline (PBS) buffer, and a 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer.

[0056] In some preferred embodiments, the inert protein is one or more selected from BSA (bovine serum albumin), sodium caseinate, fish skin gelatin, and the like.

[0057] In some preferred embodiments, the surfactant is one or more selected from Tween-20, Tween-80, TritonX-100, SDS, Brij-35 and the like.

[0058] In some preferred embodiments, the preservative is one or more selected from Proclin-300, Proclin-150, sodium azide, ProClean 150, ProClean 300, and the like.

[0059] In some preferred embodiments, the magnetic bead storage solution comprises: 10 mM PB buffer, 0.5wt%-3wt% BSA, 0.1wt%-3wt% Tween-20, and 0.05wt%-0.3wt% Proclin-300.

[0060] In some preferred embodiments, the magnetic bead storage solution comprises: 10 mM PB buffer, 1 wt% BSA, 0.4 wt% Tween-20, and 0.1 wt% Proclin-300.

[0061] In some preferred embodiments, the pH of the PB buffer is 7.4±0.05.

[0062] In some preferred embodiments, the particle size of the streptavidin-coupled magnetic beads is 1 μm-4 μm, and more preferably, the particle size of the magnetic beads is 1.5 μm-3 μm.

[0063] In some embodiments, the biotinylated antibody solution R1 further comprises a biotinylated antibody diluent, such as a biotinylated antibody diluent having a pH of 6.5 to 7.8.

[0064] In some embodiments, the biotinylated antibody solution R1 comprises: a biotinylated polyclonal MMP7 antibody and a biotinylated antibody diluent having a pH of 6.5 to 7.8.

[0065] In some embodiments, the biotinylated antibody diluent comprises: a buffer, an inert protein, a sugar, and a preservative. In some preferred embodiments, the biotinylated antibody diluent comprises: a 1-20 mM buffer with a pH of 7.0 to 7.5, 0.5 wt% to 3 wt% inert protein, 5 wt% to 15 wt% sugar, and 0.05 wt% to 0.3 wt% preservative.

[0066] In some embodiments, the buffer comprises one or more of a Tris buffer, a phosphate-buffered saline (PBS) buffer, and a 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer.

[0067] In some embodiments, the inert protein is one or more selected from BSA (bovine serum albumin), sodium caseinate (casein), fish skin gelatin, and the like.

[0068] In some preferred embodiments, the sugar is one or more selected from sucrose, trehalose, glucose and the like.

[0069] In some preferred embodiments, the preservative is one or more selected from Proclin-300, Proclin-150, sodium azide, ProClean 150, ProClean 300, and the like.

[0070] In some preferred embodiments, the biotinylated antibody diluent comprises: 10 mM PB buffer, 0.5wt%-3wt% casein, 5wt%-15wt% sucrose, and 0.05wt%-0.3wt% Proclin-300.

[0071] In some preferred embodiments, the biotinylated antibody diluent comprises: 10 mM PB buffer, 1 wt% casein, 10 wt% sucrose, and 0.1 wt% Proclin-300.

[0072] In some preferred embodiments, the pH of the PB buffer is 7.4±0.05.

[0073] In some preferred embodiments, in the biotinylated antibody solution R1, the volume ratio of the biotinylated antibody diluent to the biotinylated polyclonal MMP7 antibody is 1000-3000. In some preferred embodiments, the volume ratio of the biotinylated antibody diluent to the biotinylated polyclonal MMP7 antibody is 1000-2000.

[0074] In some preferred embodiments, the polyclonal MMP7 antibody is derived from mouse, rat, pig, monkey, alpaca, camel, or human. In some preferred embodiments, the polyclonal MMP7 antibody is of IgG isotype, such as IgG1 isotype, IgG2 isotype, IgG3 isotype, or IgG4 isotype.

[0075] Herein, the polyclonal MMP7 antibody may be any commercially available antibody known in the art, such as antibodies-online MMP 7, CUSABIO MMP 7, MyBioSource MMP 7, and the like.

[0076] In some embodiments, the luminescent marker R2 comprises: a labeling preservation solution and an acridinium ester-labeled monoclonal MMP7 antibody.

[0077] In some embodiments, the labeling preservation solution comprises: a buffer, an inert protein, a sugar, and a preservative. In some preferred embodiments, the labeling preservation solution comprises: a 1-20 mM buffer with a pH of 6.0 to 6.5, 0.5 wt% to 3 wt% inert protein, 1 wt% to 5 wt% sugar, and 0.05 wt% to 0.3 wt% preservative.

[0078] In some embodiments, the buffer comprises one or more of a Tris buffer, a phosphate-buffered saline (PBS) buffer, and a 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer.

[0079] In some preferred embodiments, the inert protein is one or more selected from BSA (bovine serum albumin), sodium caseinate, fish skin gelatin, and the like.

[0080] In some preferred embodiments, the sugar is one or more selected from trehalose, trehalose, glucose and the like.

[0081] In some preferred embodiments, the preservative is one or more selected from Proclin-300, Proclin-150, sodium azide, ProClean 150, ProClean 300, and the like.

[0082] In some preferred embodiments, the labeling preservation solution comprises: 10 mM PB buffer, 0.5wt%-3wt% BSA, 1wt%-5wt% trehalose, and 0.05wt%-0.3wt% Proclin-300.

[0083] In some preferred embodiments, the labeling preservation solution includes: 10 mM PB buffer, 1 wt% BSA, 2 wt% trehalose, and 0.1 wt% Proclin-300.

[0084] In some preferred embodiments, the pH of the PB buffer is 6.3±0.05.

[0085] In some preferred embodiments, the volume ratio of the labeling preservation solution to the acridinium ester-labeled monoclonal MMP7 antibody is 50-800.

[0086] In some preferred embodiments, the monoclonal MMP7 antibody is produced by a hybridoma cell line. In some preferred embodiments, the monoclonal MMP7 antibody is of IgG isotype, such as IgG1 isotype, IgG2 isotype, IgG3 isotype, IgG4 isotype.

[0087] In some embodiments, the MMP7 detection kit further comprises an MMP7 quality control substance and an optional MMP7 calibrator.

[0088] In this article, calibrators are used to calibrate the test system. They are usually used when starting the test system. If the test system remains unchanged, it is not necessary to use it every time. Quality control products are mainly used for measuring precision and ensuring the uniformity and stability of the test.

[0089] In some embodiments, the MMP7 calibrator comprises an MMP7 protein and an antigen diluent. In some embodiments, the MMP7 protein is a human MMP7 protein, such as UniProt ID: P09237, or is isolated from a human ex vivo sample.

[0090] In some preferred embodiments, in the MMP7 calibrator, the concentration of MMP7 protein is one or more of 4.25-230 ng / mL, preferably one or more of 4.5-220 ng / mL, for example two.

[0091] In some preferred embodiments, in the MMP7 calibrator, the concentration of MMP7 protein is 4.25-5.75 ng / mL, and / or 170-230 ng / mL. In some preferred embodiments, the concentration of MMP7 protein is 4.5-5.5 ng / mL, and / or 180-220 ng / mL, such as 5±0.75 ng / mL, and 200±30.00 ng / mL.

[0092] In some preferred embodiments, the antigen diluent comprises: a buffer, an inert protein, and a preservative.

[0093] In some preferred embodiments, the antigen diluent comprises: 1-20 mM buffer, 0.5 wt%-3 wt% bovine serum albumin, and 0.05 wt%-0.3 wt% preservative.

[0094] In some embodiments, the buffer comprises one or more of a Tris buffer, a phosphate-buffered saline (PBS) buffer, and a 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer.

[0095] In some preferred embodiments, the inert protein is one or more selected from BSA (bovine serum albumin), sodium caseinate, fish skin gelatin, and the like.

[0096] In some preferred embodiments, the preservative is one or more selected from Proclin-300, Proclin-150, sodium azide, ProClean 150, ProClean 300, and the like.

[0097] In some preferred embodiments, the antigen diluent comprises: 10 mM PB buffer, 0.5%-3wt% BSA, 0.05wt%-0.3wt% Proclin-300.

[0098] In some preferred embodiments, the antigen diluent comprises: 10 mM PB buffer, 1 wt % BSA, 0.1 % Proclin-300.

[0099] In some preferred embodiments, the pH of the PB buffer is 7.4±0.05.

[0100] In some embodiments, the MMP7 quality control comprises an MMP7 protein and an antigen diluent. In some embodiments, the MMP7 protein is a human MMP7 protein, such as UniProt ID: P09237, or is isolated from a human ex vivo sample.

[0101] In some preferred embodiments, in the MMP7 quality control product, the concentration of MMP7 protein is one or more of 8.5-287.5 ng / mL, preferably one or more of 9-275 ng / mL, for example two.

[0102] In some preferred embodiments, in the MMP7 quality control, the concentration of MMP7 protein is 8.5-11.5 ng / mL, and / or 212.5-287.5 ng / mL. In some preferred embodiments, the concentration of MMP7 protein is 9-11 ng / mL, and / or 225-275 ng / mL, such as 10±1.50 ng / mL, and 250±37.50 ng / mL.

[0103] In some preferred embodiments, the antigen diluent comprises a buffer, an inert protein, and a preservative.

[0104] In some preferred embodiments, the antigen diluent comprises: 1-20 mM buffer with a pH of 7-7.5, 0.5 wt%-3 wt% inert protein, and 0.05%-0.3 wt% preservative.

[0105] In some embodiments, the buffer comprises one or more of a Tris buffer, a phosphate-buffered saline (PBS) buffer, and a 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer.

[0106] In some preferred embodiments, the inert protein is one or more selected from BSA (bovine serum albumin), sodium caseinate, fish skin gelatin, and the like.

[0107] In some preferred embodiments, the preservative is one or more selected from Proclin-300, Proclin-150, sodium azide, ProClean 150, and ProClean 300.

[0108] In some preferred embodiments, the antigen diluent comprises: 10 mM PB buffer, 0.5 wt%-3 wt% BSA, and 0.05 wt%-0.3 wt% Proclin-300.

[0109] In some preferred embodiments, the antigen diluent comprises: 10 mM PB buffer, 1 wt % BSA, 0.1 wt % Proclin-300.

[0110] In some preferred embodiments, the pH of the PB buffer is 7.4±0.05.

[0111] In some embodiments, the MMP7 detection kit further comprises a pre-stimulation solution and / or a stimulation solution.

[0112] Herein, the pre-initiating solution and the initiating solution may be any pre-initiating solution and initiating solution known in the art, including any commercially available solution that can cause acridinium ester to generate photons.

[0113] As an example, the pre-excitation solution may include, by volume percentage, 0.05%-2% of 60wt%-90wt% concentrated HNO3, 3%-6% of 30% H2O2 by g / mL, 0.1wt%-2% Triton-X100, and 0.1%-0.4% anhydrous ethanol.

[0114] As an example, the excitation solution may include, by volume percentage, 1%-2% NaOH, 1%-4% Triton-X100, 0.1%-0.4% anhydrous ethanol, and 0.1%-0.3% EDTA-Na.

[0115] Preparation of MMP7 detection kit

[0116] The present invention also provides a method for preparing an MMP7 detection kit, comprising: separately preparing a magnetic microsphere solution M, a biotinylated antibody solution R1 and a luminescent marker R2, and then packaging them separately and placing them in the same or different detection kits.

[0117] In some embodiments, the above-mentioned preparation method further comprises preparing MMP7 quality control materials and optional MMP7 calibrators.

[0118] In some embodiments, the preparation of magnetic microsphere solution M, the preparation of biotinylated antibody solution R1, the preparation of luminescent marker R2, and the preparation of MMP7 calibrator and MMP7 quality control are respectively shown below and can be used in combination or separately.

[0119] 1) Preparation of magnetic microsphere solution M

[0120] Place the streptavidin-coupled magnetic bead stock solution on a magnetic separator and let it stand, discard the supernatant, wash the magnetic beads 1-3 times with magnetic bead storage solution, add magnetic bead storage solution to the washed magnetic beads to dilute the magnetic beads to a predetermined concentration (e.g., 0.3 mg / mL) for later use.

[0121] In some embodiments, the magnetic bead storage solution comprises: a buffer, an inert protein, a surfactant, and a preservative. In some preferred embodiments, the magnetic bead storage solution comprises: a 1-20 mM buffer with a pH of 7 to 7.5, 0.5 wt% to 3 wt% inert protein, 0.1 wt% to 3 wt% surfactant, and 0.05 wt% to 0.3 wt% preservative.

[0122] In some embodiments, the buffer comprises one or more of a Tris buffer, a phosphate-buffered saline (PBS) buffer, and a 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer.

[0123] In some preferred embodiments, the inert protein is one or more selected from BSA (bovine serum albumin), sodium caseinate, fish skin gelatin, and the like.

[0124] In some preferred embodiments, the surfactant is one or more selected from Tween-20, Tween-80, TritonX-100, SDS, Brij-35 and the like.

[0125] In some preferred embodiments, the preservative is one or more selected from Proclin-300, Proclin-150, sodium azide, ProClean 150, ProClean 300, and the like.

[0126] In some preferred embodiments, the magnetic bead storage solution comprises: 10 mM PB buffer, 0.5wt%-3wt% BSA, 0.1wt%-3wt% Tween-20, and 0.05wt%-0.3wt% Proclin-300.

[0127] In some preferred embodiments, the magnetic bead storage solution comprises: 10 mM PB buffer, 1 wt% BSA, 0.4 wt% Tween-20, and 0.1 wt% Proclin-300.

[0128] In some preferred embodiments, the pH of the PB buffer is 7.4±0.05.

[0129] In some preferred embodiments, the particle size of the streptavidin-coupled magnetic beads is 1 μm-4 μm, and more preferably, the particle size of the magnetic beads is 1.5 μm-3 μm.

[0130] 2) Preparation of biotinylated antibody solution R1

[0131] The purchased biotinylated antibody (stock solution) is thoroughly mixed according to the manufacturer's instructions, and the biotinylated antibody diluent is added to dilute the antibody (stock solution) to a predetermined concentration (eg, 0.2 μg / mL) for later use.

[0132] In some embodiments, the biotinylated antibody diluent comprises: a buffer, an inert protein, a sugar, and a preservative. In some preferred embodiments, the biotinylated antibody diluent comprises: a 1-20 mM buffer with a pH of 7.0 to 7.5, 0.5 wt% to 3 wt% inert protein, 5 wt% to 15 wt% sugar, and 0.05 wt% to 0.3 wt% preservative.

[0133] In some embodiments, the buffer comprises one or more of a Tris buffer, a phosphate-buffered saline (PBS) buffer, and a 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer.

[0134] In some embodiments, the inert protein is one or more selected from BSA (bovine serum albumin), sodium caseinate (casein), fish skin gelatin, and the like.

[0135] In some preferred embodiments, the sugar is one or more selected from sucrose, trehalose, glucose and the like.

[0136] In some preferred embodiments, the preservative is one or more selected from Proclin-300, Proclin-150, sodium azide, ProClean 150, ProClean 300, and the like.

[0137] In some preferred embodiments, the biotinylated antibody diluent comprises: 10 mM PB buffer, 0.5wt%-3wt% casein, 5wt%-15wt% sucrose, and 0.05wt%-0.3wt% Proclin-300.

[0138] In some preferred embodiments, the biotinylated antibody diluent comprises: 10 mM PB buffer, 1 wt% casein, 10 wt% sucrose, and 0.1 wt% Proclin-300.

[0139] In some preferred embodiments, the pH of the PB buffer is 7.4±0.05.

[0140] In some preferred embodiments, the biotinylated polyclonal MMP7 antibody is diluted with a biotinylated antibody diluent at a volume ratio of 1000-3000. In some preferred embodiments, the biotinylated polyclonal MMP7 antibody is diluted with a biotinylated antibody diluent at a volume ratio of 1000-2000.

[0141] 3) Preparation of luminescent marker R2

[0142] The monoclonal MMP7 antibody is incubated with an acridinium ester esterified with NHS in a buffer to obtain an acridinium ester-labeled monoclonal MMP7 antibody, and then the acridinium ester-labeled monoclonal MMP7 antibody is diluted with a labeling storage solution to obtain a luminescent marker R2. Specifically, for example, the luminescent marker R2 can be prepared as follows:

[0143] The monoclonal MMP7 antibody was thoroughly mixed with buffer 1 (e.g., 1× PBS buffer, pH 8.5), transferred to a 7 kD dialysis tube, and dialyzed in buffer 1 by rotation, with the buffer changed 6 times, each time for 1-2 hours;

[0144] In a centrifuge tube, NHS-esterified acridinium ester (NSP-SA-NHS) and the monoclonal MMP7 antibody reaction solution to be coated obtained above were mixed at room temperature at a molecular molar ratio of 20:1 and the labeling reaction was carried out for 0.5-1h;

[0145] Use buffer 1 to prepare a lysine buffer with a concentration of 0.01wt% to 0.1wt%, add 20μL of 0.01wt% to 0.1wt% lysine buffer to the above centrifuge tube, and continue to shake and mix for 0.5h; adding 0.01wt% to 0.1wt% lysine buffer to the solution containing acridinium ester effectively blocks the unoccupied binding sites and improves the sensitivity of detection.

[0146] After the reaction is completed, the mixed solution is transferred to a 7 kD dialysis tube and dialyzed in buffer 2 (e.g., 1× PBS buffer, pH 6.3) by rotation, and the solution is changed 6 times, each time for 1-2 hours, to obtain a labeled solution;

[0147] After dialysis overnight, remove the labeling solution, add labeling preservation solution to 1 mL, mix thoroughly and store at 2-8°C.

[0148] In some embodiments, the monoclonal MMP7 antibody is prepared by the following method, the method comprising culturing a host cell under conditions that allow the antibody to be expressed. For example, any appropriate conventional culture medium known in the art, appropriate temperature and culture time, etc., can be routinely determined by a person skilled in the art according to the specific type of host cell. The host cell includes a nucleic acid molecule or a recombinant expression vector comprising the nucleic acid molecule, wherein the nucleic acid molecule comprises a nucleotide sequence encoding the antibody or its antigen-binding fragment. Preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell, such as a CHO cell, a CHO-S cell, a 293 cell, or a monkey kidney cell.

[0149] In some embodiments, the labeling preservation solution comprises: a buffer, an inert protein, a sugar, and a preservative. In some preferred embodiments, the labeling preservation solution comprises: a 1-20 mM buffer with a pH of 6.0 to 6.5, 0.5 wt% to 3 wt% inert protein, 1 wt% to 5 wt% sugar, and 0.05 wt% to 0.3 wt% preservative.

[0150] In some embodiments, the buffer comprises one or more of a Tris buffer, a phosphate-buffered saline (PBS) buffer, and a 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer.

[0151] In some preferred embodiments, the inert protein is one or more selected from BSA (bovine serum albumin), sodium caseinate, fish skin gelatin, and the like.

[0152] In some preferred embodiments, the sugar is one or more selected from trehalose, trehalose, glucose and the like.

[0153] In some preferred embodiments, the preservative is one or more selected from Proclin-300, Proclin-150, sodium azide, ProClean 150, ProClean 300, and the like.

[0154] In some preferred embodiments, the labeling preservation solution comprises: 10 mM PB buffer, 0.5wt%-3wt% BSA, 1wt%-5wt% trehalose, and 0.05wt%-0.3wt% Proclin-300.

[0155] In some preferred embodiments, the labeling preservation solution includes: 10 mM PB buffer, 1 wt% BSA, 2 wt% trehalose, and 0.1 wt% Proclin-300.

[0156] In some preferred embodiments, the pH of the PB buffer is 6.3±0.05.

[0157] In some preferred embodiments, the acridinium ester-labeled monoclonal MMP7 antibody is diluted with the labeling preservation solution at a volume ratio of 50-800 to obtain the luminescent marker R2.

[0158] 4) Preparation of MMP7 Calibrators

[0159] The MMP7 calibrator was prepared by adding MMP7 protein to the antigen diluent.

[0160] In some embodiments, the MMP7 protein is a human MMP7 protein, such as UniProt ID: P09237, or is isolated from a human ex vivo sample.

[0161] In some preferred embodiments, in the MMP7 calibrator, the concentration of MMP7 protein is 4.25-230 ng / mL, preferably 4.5-220 ng / mL.

[0162] In some preferred embodiments, in the MMP7 calibrator, the concentration of MMP7 protein is 4.25-5.75 ng / mL, or 170-230 ng / mL. In some preferred embodiments, the concentration of MMP7 protein is 4.5-5.5 ng / mL, or 180-220 ng / mL.

[0163] In some preferred embodiments, the antigen diluent comprises: a buffer, an inert protein, and a preservative.

[0164] In some preferred embodiments, the antigen diluent comprises: 1-20 mM buffer, 0.5 wt%-3 wt% bovine serum albumin, and 0.05 wt%-0.3 wt% preservative.

[0165] In some embodiments, the buffer comprises one or more of a Tris buffer, a phosphate-buffered saline (PBS) buffer, and a 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer.

[0166] In some preferred embodiments, the inert protein is one or more selected from BSA (bovine serum albumin), sodium caseinate, fish skin gelatin, and the like.

[0167] In some preferred embodiments, the preservative is one or more selected from Proclin-300, Proclin-150, sodium azide, ProClean 150, ProClean 300, and the like.

[0168] In some preferred embodiments, the antigen diluent comprises: 10 mM PB buffer, 0.5 wt%-3 wt% BSA, and 0.05 wt%-0.3 wt% Proclin-300.

[0169] In some preferred embodiments, the antigen diluent comprises: 10 mM PB buffer, 1 wt % BSA, 0.1 wt % Proclin-300.

[0170] In some preferred embodiments, the pH of the PB buffer is 7.4±0.05.

[0171] 5) Preparation of MMP7 quality control

[0172] Prepare MMP7 control by adding MMP7 protein to the antigen dilution.

[0173] In some embodiments, the MMP7 protein is a human MMP7 protein, such as UniProt ID: P09237, or is isolated from a human ex vivo sample.

[0174] In some preferred embodiments, in the MMP7 quality control product, the concentration of MMP7 protein is 8.5-287.5 ng / mL, preferably 9-275 ng / mL.

[0175] In some preferred embodiments, in the MMP7 quality control product, the concentration of MMP7 protein is 8.5-11.5 ng / mL, or 212.5-287.5 ng / mL. In some preferred embodiments, the concentration of MMP7 protein is 9-11 ng / mL, or 225-275 ng / mL.

[0176] In some preferred embodiments, the antigen diluent comprises a buffer, an inert protein, and a preservative.

[0177] In some preferred embodiments, the antigen diluent comprises: 1-20 mM buffer with a pH of 7-7.5, 0.5 wt%-3 wt% inert protein, and 0.05 wt%-0.3 wt% preservative.

[0178] In some embodiments, the buffer comprises one or more of a Tris buffer, a phosphate-buffered saline (PBS) buffer, and a 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer.

[0179] In some preferred embodiments, the inert protein is one or more selected from BSA (bovine serum albumin), sodium caseinate, fish skin gelatin, and the like.

[0180] In some preferred embodiments, the preservative is one or more selected from Proclin-300, Proclin-150, sodium azide, ProClean 150, and ProClean 300.

[0181] In some preferred embodiments, the antigen diluent comprises: 10 mM PB buffer, 0.5 wt%-3 wt% BSA, and 0.05 wt%-0.3 wt% Proclin-300.

[0182] In some preferred embodiments, the antigen diluent comprises: 10 mM PB buffer, 1 wt % BSA, 0.1 wt % Proclin-300.

[0183] In some preferred embodiments, the pH of the PB buffer is 7.4±0.05.

[0184] Detection Methods

[0185] In the method for detecting MMP7 using the kit of the present invention, after the magnetic microsphere solution M, the biotinylated antibody solution R1, and the luminescent marker R2 are placed in a fully automatic chemiluminescence immunoassay instrument, the fully automatic chemiluminescence immunoassay instrument automatically detects the MMP7 protein concentration in the sample.

[0186] In the method for detecting MMP7 by the kit of the present invention, the magnetic microsphere solution M, the luminescent marker R2, and the biotinylated antibody solution R1 are sequentially placed in a fully automatic chemiluminescence immunoassay instrument, and the fully automatic chemiluminescence immunoassay instrument automatically detects the MMP7 protein concentration in the sample. The order of adding the magnetic microsphere solution M, the luminescent marker R2, and the biotinylated antibody solution R1 is to first add the biotinylated antibody solution R1 and the luminescent marker R2 after adding the sample, and then add the magnetic microsphere solution M.

[0187] Preferably, the magnetic microsphere solution M is added 15-30 minutes after the biotinylated antibody solution R1 and the luminescent marker R2 are added.

[0188] Preferably, the pre-excitation solution and the excitation solution are added 5-10 minutes after the magnetic microsphere solution M is added.

[0189] In terms of volume percentage, the pre-excitation solution includes 0.05%-2% of concentrated HNO3 with a concentration of 60wt%-90wt%, 3%-6% of H2O2 with a concentration of 30% in terms of g / mL, 0.1%-2% Triton-X100, and 0.1%-0.4% anhydrous ethanol.

[0190] In terms of volume percentage, the above-mentioned excitation solution includes 1%-2% NaOH, 1%-4% Triton-X100, 0.1%-0.4% anhydrous ethanol, and 0.1%-0.3% EDTA-Na.

[0191] This kit adopts the double antibody sandwich method, with a solution containing biotinylated polyclonal MMP7 antibody as reagent R1, a solution containing acridinium ester-labeled monoclonal MMP7 antibody as reagent R2, and magnetic particles with streptavidin functional groups coupled to the surface as solid phase carriers.

[0192] The detection reaction includes the following steps: the first step is to incubate the sample with reagents R1 and R2. The MMP7 antibody can specifically recognize the MMP7 antigen in the sample to form a sandwich-like immune complex of antibody-antigen-antibody. The second step is to add the magnetic microsphere solution M, fix the immune complex on the surface of the solid phase carrier with the help of the strong affinity between biotin and streptavidin, and remove the remaining unreacted substances by washing. Finally, the pre-excitation solution and the excitation solution are added to make the acridinium ester generate photons, and the number of photons is linearly related to the MMP7 protein content in the sample. Substituting the number of photons into the calibration curve (previously constructed by measuring the luminescence value of the calibration product by the fully automatic chemiluminescence immunoassay) can calculate the concentration of MMP7 in the sample.

[0193] The magnetic microsphere solution M of the present invention is prepared by diluting the magnetic beads with the magnetic bead preservation solution mentioned above, has good dispersibility and stability, low background and good signal-to-noise ratio, and has good adaptability to the detection items.

[0194] As a luminescent substance, acridinium ester molecules generate extremely unstable N-methylacridone after adding the excitation liquid, release photons to return to the ground state, and the released photons are collected as reaction signals.

[0195] application

[0196] In one aspect, the present invention provides use of the MMP7 detection kit as described above in diagnosing MMP7-related diseases.

[0197] In one aspect, the present invention provides use of the MMP7 detection kit as described above in the preparation of a product for diagnosing MMP7-related diseases.

[0198] Herein, MMP7-related diseases refer to diseases or conditions that cause patients to show abnormal MMP7 expression during the onset and progression of the disease compared to healthy humans. In some embodiments, the MMP7-related diseases may be inflammatory diseases (e.g., chronic inflammation), autoimmune diseases, cancer, fibrotic diseases, cardiovascular diseases, neurological diseases, eye diseases, lung diseases, hepatobiliary diseases, and kidney diseases, but are not limited thereto.

[0199] In some embodiments, the MMP7-related disease can be selected from biliary atresia and liver fibrosis caused by biliary atresia, obstructive jaundice, cholangitis, cholestasis, liver fibrosis, renal fibrosis, chronic kidney disease, intrahepatic cholangiocarcinoma, necrotizing enterocolitis (NEC) or sepsis, ovarian cancer, pain, cystitis, arthritis, dry eyes, hepatitis, liver fibrosis, non-alcoholic fatty liver disease, megacolon, lupus erythematosus, acute respiratory distress syndrome (ARDS), pulmonary hypertension, idiopathic pulmonary fibrosis (IPF).

[0200] When the detection kit of the present invention is used to detect MMP7 protein, in particular, the monoclonal MMP7 antibody prepared by the present invention is used as the capture antibody, which has good specificity and high detection sensitivity.

[0201] When the kit of the present invention is used to measure samples, there is no need to manually dilute the samples, and the vacuum blood collection tubes of the sick children can be directly tested on the machine, further avoiding errors caused by manual dilution of samples.

[0202] The kit of the present invention has stable performance, high sensitivity, wide linear range, convenient operation, strong clinical practicality, and can help laboratory personnel quickly determine the concentration of MMP7 protein in in vitro serum samples, which effectively promotes the clinical detection application of this marker. The detection process only takes 28 minutes to obtain a test report, no human operation is required throughout the process, and the data does not need to be manually processed. BRIEF DESCRIPTION OF THE DRAWINGS

[0203] Figure 1 It is the calibration curve of the kit of the present invention on the fully automatic chemiluminescence immunoassay analyzer.

[0204] Figure 2 It is the performance evaluation curve (linear range) of the kit of the present invention.

[0205] Figure 3 This is the clinical compliance rate curve of the kit of the present invention.

[0206] Figure 4 The present invention is a correlation between the results of the kit of the present invention and the commercially available enzyme-linked immunosorbent assay kit for measuring the content of MMP7 in human serum. DETAILED DESCRIPTION

[0207] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0208] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. See, e.g., Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989); Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, NY (2009); Perbal, A Practical Guide to Molecular Cloning (1984).

[0209] Herein, the term "or" is intended to include "and" and vice versa unless the context clearly indicates otherwise. Herein, singular terms encompass plural referents and vice versa unless otherwise stated.

[0210] As used herein, unless otherwise specified, the terms “comprise, comprises and comprising” or their equivalents (e.g., contain, containing, include, including) are open-ended expressions and should be understood as “including but not limited to”, meaning that in addition to the listed elements, components and steps, other unspecified elements, components and steps may also be included.

[0211] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0212] The term "sequence identity" herein refers to the percentage of identical amino acid residues between the sequences to be compared, after aligning two or more sequences to be compared and, if necessary, introducing gaps to achieve the maximum sequence identity percentage, without considering any conservative substitutions as part of the sequence identity. The comparison of amino acid sequence identities can be performed in a variety of ways known in the art, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.

[0213] In this article, unless otherwise specified, the term "optional" or "optionally" means that the event or situation described later may or may not occur. For example, "the MMP7 detection kit includes an optional MMP7 calibrator" means that the MMP7 detection kit includes or does not include an MMP7 calibrator.

[0214] Unless otherwise specified, the CDR sequences in the present invention are defined according to the Kabat numbering scheme, but the CDR regions defined by known numbering schemes such as Kabat, AbM, Chothia, Contact and IMGT also fall within the scope of protection of the present invention.

[0215] Example

[0216] Next, the present invention is further described in detail by examples, but the protection scope of the present invention is not limited to these examples. Unless otherwise specified, each reagent, material and equipment used in the following examples can be prepared according to conventional means known in the art or are commercially available.

[0217] Unless otherwise specified, the samples mentioned in the following examples are serum samples collected from children with biliary atresia who underwent hepatobiliary surgery in the hepatobiliary surgery department of the hospital after the patients' guardians signed informed consent.

[0218] Example 1 Preparation and detection of kit

[0219] 1. Preparation steps of magnetic microsphere solution M

[0220] (1) Prepare magnetic bead storage solution, including 10 mM PBS buffer with a pH of 7.4, 1% BSA, 0.4% Tween-20, and 0.1% Proclin-300;

[0221] (2) Take 0.5 mL of streptavidin magnetic bead stock solution (particle size 1.5 μm, purchased from JSR) in a centrifuge tube, place it on a magnetic separator and let it stand for 3 minutes, then discard the supernatant, add 2 mL of magnetic bead storage solution to resuspend the magnetic beads, and repeatedly invert and mix 20 times. Let it stand and mix again, then discard the supernatant, and repeat the washing step once;

[0222] (3) Add magnetic bead storage solution and dilute to a working concentration of 0.3 mg / mL to obtain a magnetic microsphere solution.

[0223] 2. Preparation steps of biotinylated antibody solution R1

[0224] (1) Prepare biotinylated antibody diluent, including 10 mM PB buffer at pH 7.4, 1% casein, 10% sucrose, and 0.1% Proclin-300;

[0225] (2) The biotinylated polyclonal MMP7 antibody (mouse IgG1 antibody CUSABIOMMP 7, purchased from CUSABIO) was redissolved and diluted with a biotinylated antibody diluent to a polyclonal MMP7 antibody concentration of 0.2 μg / mL to obtain a biotinylated antibody solution.

[0226] 3. Preparation steps of luminescent marker R2:

[0227] 3.1 Synthesis of monoclonal MMP7 antibody

[0228] Female Balb / c mice aged 6-8 weeks were immunized with hMMP7, and re-immunized 2-3 weeks after the first immunization, and then boosted about 3 weeks later. After that, blood samples were taken from the tail vein of the mice for titer determination by ELISA, and the spleens of mice with high titers were removed to prepare spleen lymphocytes, and the spleen lymphocytes were fused with myeloma cells (Sp2 / 0 cells). The hybridoma cells were cultured with HAT selection medium to screen out hybridoma cells, and the culture supernatant was measured by ELISA to screen hybridomas by determining whether they effectively bind to the MMP7 protein. The positive hybridomas were further subcloned by limiting dilution to obtain stable monoclonal hybridoma cells, and finally 4 monoclonal hybridoma cell lines were screened.

[0229] According to the method mentioned in the literature (Bradbury, A. (2010). Cloning Hybridoma cDNA by RACE. Antibody Engineering. R. Kontermann and S. Dübel. Springer-Verlag Berlin Heidelberg: 15-20), specific primers were synthesized (synthesized by Sangon Biotech (Shanghai) Co., Ltd., the primer sequences are shown in Table 1 below), and RNA extraction kit and reverse transcription kit (both purchased from Beijing Quanshijin Biotechnology Co., Ltd.) were used to clone the hybridoma cell antibody target gene. The amplified heavy chain and light chain target fragments were cloned into the blunt-end cloning vector (purchased from Beijing Quanshijin Biotechnology Co., Ltd.), and then coated on LB agar plates (ampicillin resistance) respectively; after the clones grew out, clones were picked from each plate for Sanger sequencing.

[0230] Table 1 Primer sequences

[0231] Forward primer Reverse primer Heavy chain variable region GACAGTGGATARACMGATGG (SEQ ID NO:49) GAGGTSMARCTGCAGSAGTCWGG (M and R represent degenerate bases) (SEQ ID NO:50) Light chain variable region GGATACAGTTGGTGCAGCATC (SEQ ID NO:51) GAGGTSMARCTGCAGSAGTCWGG (SEQ ID NO:52) GATATTGTGATGACGCAGGCT (SEQ ID NO:53) GATATTGTGATAACCCAG (SEQID NO:54) GACATTGTGCTGACCCAATCT (SEQ ID NO:55) GACATTGTGATGACCCAGTCT (SEQ ID NO:56) GATATTGTGCTAACTCAGTCT (SEQ ID NO:57) GATATCCAGATGACACAGACT (SEQ ID NO:58) GACATCCAGCTGACTCAGTCT (SEQ ID NO:59) CAAATTGTTCTCACCCAGTCT (SEQ ID NO:60)

[0232] The genes of the heavy chain variable region sequence and the light chain variable region sequence of the antibody were obtained, codons were optimized using codon optimization software and commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd., and then the genes were cloned into the pcDNA3.1 expression vector using EcoRI / HinDIII restriction endonuclease to construct a monoclonal antibody expression plasmid, which was used to transfect 293F cells. After culturing at 37°C and 5% CO2, the cell supernatant was collected by centrifugation and purified using a protein A affinity column to obtain monoclonal MMP7 antibodies M1-M4.

[0233] Table 2 below shows the sequence information of each antibody involved in the present invention.

[0234] Table 2 Antibody sequence information

[0235]

[0236] Heavy chain variable region

[0237] M1 QVQLQQSGAELMKPGASVKLSCKASGYTFTDYYMNWVKQRPGQGLEWIGRINPKGGSTGSVPKFRSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGRIGTPLDYWGQGTLVTVSS (SEQ ID NO:25) M2 EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMYWVRQAPGQGLEWIGEIMPSWGDTIFNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCTRMPFLGAMDYWGQGTTVTVSS (SEQ ID NO:26) M3 EVQLVQSGAEVKKPGASVKVSCKASGYTFTYYDMYWVRQAPGQGLEWIGWINPSMGDTVFNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCAREPRYGAMDYWGQGTTVTVSS (SEQ ID NO:27) M4 QVQLQQSGAELMKPGASVKLSCKASGYTFTSYYMYWVKQRPGQGLEWIGEINPSNADTEFNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCTRWPLYGAMDYWGQGTSVTVSS (SEQ ID NO:28)

[0238] Light chain variable region

[0239] M1 DVQITQSPSYLAASPGETITINCRASRDISKRLAWYQEKPGKTNKLLIYKTSNLQSGIPSRFSGSGSGSDFTLTISSLEPEDFAMYYCQQLKNYPRTFGQGTNLEIK(SEQ ID NO:29) M2 DVQMTQSPSSLSASVGDRVTITCRASKSISKFLAWYQQKPGKAPKLLIYHGSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQENEFPETFGQGTKLEIK(SEQ ID NO:30) M3 DVQMTQSPSSLSASVGDRVTITCRASKSIFWRLAWYQQKPGKAPKLLIYWGSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQENEMPMTFGQGTKLEIK(SEQ ID NO:31) M4 DVQITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSGHWLQSGIPSRFSGSGSGSDFTLTISSLEPEDFAMYYCQQHFEYPWTFGAGTKLELK(SEQ ID NO:32)

[0240] Heavy chain

[0241] M1 QVQLQQSGAELMKPGASVKLSCKASGYTFTDYYMNWVKQRPGQGLEWIGRINPKGGSTGSVPKFRSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGRIGTPLDYWGQGTLVTVSSVLAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQID NO:33) M2 EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMYWVRQAPGQGLEWIGEIMPSWGDTIFNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCTRMPFLGAMDYWGQGTTVTVSSAKTTPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQ ID NO:34) M3 EVQLVQSGAEVKKPGASVKVSCKASGYTFTYYDMYWVRQAPGQGLEWIGWINPSMGDTVFNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCAREPRYGAMDYWGQGTTVTVSSAKTTPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQ ID NO:35) M4 QVQLQQSGAELMKPGASVKLSCKASGYTFTSYYMYWVKQRPGQGLEWIGEINPSNADTEFNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCTRWPLYGAMDYWGQGTSVTVSSAKTTPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQ ID NO:36)

[0242] Light chain

[0243] M1 DVQITQSPSYLAASPGETITINCRASRDISKRLAWYQEKPGKTNKLLIYKTSNLQSGIPSRFSGSGSDSDFTLTISSLEPEDFAMYYCQQLKNYPRTFGQGTNLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID NO:37) M2 DVQMTQSPSSLSASVGDRVTITCRASKSISKFLAWYQQKPGKAPKLLIYHGSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQENEFPETFGQGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID NO:38) M3 DVQMTQSPSSLSASVGDRVTITCRASKSIFWRLAWYQQKPGKAPKLLIYWGSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQENEMPMTFGQGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO:39) M4 DVQITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSGHWLQSGIPSRFSGSGSGSDFTLTISSLEPEDFAMYYCQQHFEYPWTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO:40)

[0244]

[0245] 3.2 Preparation of luminescent marker R2

[0246] (1) Prepare acridinium ester labeling buffer 1 (0.01 M phosphate buffer, pH 8.5);

[0247] (2) Prepare acridinium ester labeling buffer 2 (0.01 M phosphate buffer, pH 6.3);

[0248] (3) Prepare the labeling preservation solution, including 10 mM PB buffer at pH 6.3, 1% BSA, 2% trehalose, and 0.1% Proclin-300;

[0249] (4) Labeling with acridinium ester: dilute the anti-MMP7 monoclonal antibody M1 with buffer 1 to a monoclonal MMP7 antibody solution concentration of 0.1 mg / mL, transfer to a 7 kD dialysis tube, and perform rotary dialyzing in buffer 1. Change the solution 6 times, each time for 1-2 hours;

[0250] (5) The monoclonal MMP7 antibody reaction solution was then transferred to a centrifuge tube, and the NHS-esterified acridinium ester (NSP-SA-NHS) and the antibody reaction solution were mixed at room temperature at a molecular molar ratio of 20:1 and the labeling reaction was carried out for 0.5-1h;

[0251] (6) Prepare 0.05% lysine buffer using acridinium ester labeling buffer 1, add 20 μl of 0.05% lysine buffer to the centrifuge tube in step (5), and continue to shake and mix for 0.5 h;

[0252] (7) After the reaction is completed, the resulting mixture is transferred to a 7 kD dialysis tube and dialyzed against acridinium ester labeled buffer 2 by rotation, with the solution changed 6 times, each time for 1-2 hours;

[0253] (8) The obtained acridinium ester-labeled monoclonal MMP7 antibody is diluted with a labeling storage buffer solution at a dilution ratio of 100 times to obtain a luminescent labeling solution R2.

[0254] 4. Preparation steps of MMP7 calibrator:

[0255] The calibrator was prepared by adding MMP7 protein (recombinantly prepared according to UniProt ID: P09237) to the antigen diluent. The antigen diluent (including 10mM PB buffer at pH 7.4, 1% BSA, 0.1% Proclin-300) was prepared, and the MMP7 protein was dissolved in the antigen diluent and prepared into solutions of different concentrations with little matrix effect.

[0256] MMP7 protein was diluted with antigen diluent to a concentration of 5ng / mL and 200ng / mL. The prepared calibrator was assigned values ​​using the company's working calibrator (obtained by diluting MMP7 protein with antigen diluent to a concentration of 0.1ng / mL, 0.6ng / mL, 3ng / mL, 15ng / mL, 75ng / mL, and 300ng / mL) to obtain the MMP7 calibrator, which was used to calibrate the user's measurement program and assign values ​​to clinical samples.

[0257] 5. Preparation steps of MMP7 quality control:

[0258] The quality control was prepared by adding MMP7 protein (recombinantly prepared according to UniProt ID: P09237) to the antigen diluent. The antigen diluent (including 10mM PB buffer at pH 7.4, 1% BSA, 0.1% Proclin-300) was prepared, and the MMP7 protein was dissolved in the antigen diluent and prepared into solutions of different concentrations with little matrix effect.

[0259] The MMP7 protein was diluted with antigen diluent to a concentration of 10 ng / mL and 250 ng / mL.

[0260] 6. Test process of the kit

[0261] The kit prepared above was loaded into the SMART 500S fully automatic chemiluminescence immunoassay analyzer produced by Chongqing Cosma Biotechnology Co., Ltd. The master curve information of the kit was read by scanning, and the calibration master curve of the project was calibrated by calibrator 1 (5 ng / mL) and calibrator 2 (200 ng / mL). Figure 1 as shown).

[0262] The detection steps are:

[0263] 1) Place the calibrator and quality control product in the reagent rack for calibration and quality control (if the detection system is not started for the first time on the same day, it is not necessary to calibrate each time if the detection system remains unchanged). After the calibration is passed and the quality control is qualified, place the serum sample to be tested in the reagent rack, wherein the sample volume of each sample (including the quality control product, calibrator and sample to be tested) is set to 10 μL, and the order of sample addition is sample → biotinylated antibody solution R1 → luminescent marker R2 (the order of R1 and R2 can be reversed). After incubation for 15 minutes, add the magnetic microsphere solution M. After incubation for 5 minutes, remove the remaining unreacted substances by washing, and add the excitation solution and pre-excitation solution;

[0264] 2) After editing the detection instructions, click OK to start the detection;

[0265] 3) Choose to export data or directly print the test report to analyze the results.

[0266] Example 2 Methodological indicators of the kit

[0267] According to the detection process of the kit in Example 1, various methodological indicators of the kit in Example 1 were measured.

[0268] 1. Linearity experiment

[0269] The high-value samples close to the upper limit of the linear range were diluted to at least 5 concentrations according to a certain ratio, among which the low-value concentration samples were close to the lower limit of the linear range. Each concentration of the sample was tested 3 times, and the average value was calculated. The average value of the result and the dilution ratio were fitted with a linear fit using the least squares method, and the linear correlation coefficient (r) was calculated. Table 3 below, Figure 2 is the linear range of the kit of Example 1. From the results, it can be seen that in the range of 0.1-300 ng / mL, the linear correlation coefficient r is 1.0000.

[0270] Table 3 Linear range of the kit

[0271]

[0272] 2. Repeatability Experiment

[0273] The serum samples at three concentration levels were tested, each repeated 10 times, and the coefficient of variation (CV) was calculated. The mean value M, standard deviation SD, and coefficient of variation (CV) of the measurement results of each sample were calculated respectively. The following Table 4 shows the repeatability measurement results of the kit of Example 1, and the results show that the coefficient of variation CV of the kit is ≤5%.

[0274] Table 4 Repeatability test results of the kit

[0275] Detection Serial Number Sample 1 (ng / mL) Sample 2 (ng / mL) Sample 3 (ng / mL) 1 1.94 59.52 248.85 2 1.93 62.44 246.39 3 1.98 58.15 251.78 4 2.08 57.00 248.33 5 2.11 62.44 244.54 6 1.97 60.34 253.09 7 2.04 61.89 252.13 8 2.06 59.19 244.17 9 1.88 54.88 252.57 10 1.95 59.08 246.91 M 2.00 59.49 248.88 SD 0.08 2.43 3.36 CV 3.8% 4.1% 1.35%

[0276] 3. Interference Experiment

[0277] For serum samples with a concentration of 0 ng / mL (i.e., samples that have been inactivated), the interference of 33 g / L fat emulsion, 200 mg / L bilirubin, 2 g / L hemoglobin, 100 U / mL rheumatoid factor, and 68 ng / mL anti-mouse heterophilic antibody (HAMA) on the determination results was detected, and each test was repeated 3 times. The reaction values ​​of these reagents were all lower than 0.1 ng / mL. Table 5 below is the anti-interference evaluation of the kit of Example 1. From the results, it can be seen that the kit of the present invention has no effect on the determination results in the presence of the above-mentioned interfering substances.

[0278] Table 5 Anti-interference evaluation results

[0279]

[0280] 4. Stability test

[0281] The kit of Example 1 was placed under 37°C for accelerated aging for 7 days, and after being taken out, it was tested on the machine together with the reagent stored at 4°C to determine the calibration product, and the signal retention rate was calculated. Table 6 below shows the stability evaluation results of the kit of Example 1. From the results, it can be seen that the kit of the present invention has excellent stability, and the signal value does not show a significant decrease after 7 days of acceleration at 37°C, and the average signal retention rate reaches 95.34%.

[0282] Table 6 Stability evaluation results

[0283]

[0284] 5. Specificity and sensitivity testing:

[0285] Depend on Figure 3It can be seen that when the kit of Example 1 was used to detect clinical samples and compared with the clinical diagnosis results, a total of 200 clinical samples (all with clear clinical diagnosis results) were detected. When the CUTOFF value was 15-25 ng / mL, the sensitivity reached 95.7%, the specificity reached 97.4%, and the AUC was 0.9830.

[0286] Example 3 Performance of the kit in clinical assays

[0287] The performance of the kit of Example 1 was compared with an ELISA kit purchased from R&D Company in the United States. The results are shown in Table 7 below. The following performances of the kit of the present invention are better than those of the ELISA kit.

[0288] Table 7 Performance comparison between the kit of the present invention and the enzyme-linked immunosorbent assay kit

[0289]

[0290] The MMP7 chemiluminescent detection kit of the present invention was compared with the enzyme-linked immunosorbent assay kit purchased from R&D Company in the United States to determine the content of MMP7 in serum samples. The data results are as follows: Figure 4 As shown. Correlation coefficient R 2 The value is 0.9992, indicating that the kit of the present invention has a good correlation with the enzyme-linked immunosorbent assay kit in the measurement of MMP7 in serum.

Claims

1. A MMP7 detection kit, comprising: A magnetic microsphere solution M, which comprises streptavidin-coupled magnetic beads; Biotinylated antibody solution R1, which contains biotinylated polyclonal MMP7 antibody; Luminescent marker R2, which comprises an acridinium ester-labeled monoclonal MMP7 antibody; Wherein, the monoclonal MMP7 antibody comprises: HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:2, HCDR3 as shown in SEQ ID NO:3, LCDR1 as shown in SEQ ID NO:13, LCDR2 as shown in SEQ ID NO:14, LCDR3 as shown in SEQ ID NO:

15.

2. The detection kit according to claim 1, wherein The monoclonal MMP7 antibody comprises: A heavy chain variable region having the amino acid sequence shown in SEQ ID NO:25, and a light chain variable region having the amino acid sequence shown in SEQ ID NO:

29.

3. The detection kit according to claim 2, wherein The monoclonal MMP7 antibody further comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is an IgG heavy chain constant region, and the light chain constant region is a κ or λ chain constant region.

4. The detection kit according to claim 1, wherein The monoclonal MMP7 antibody comprises: A heavy chain having the amino acid sequence shown in SEQ ID NO:33, and a light chain having the amino acid sequence shown in SEQ ID NO:

37.

5. The detection kit according to any one of claims 1 to 4, wherein: The luminescent marker R2 comprises: a labeling storage solution and the acridinium ester-labeled monoclonal MMP7 antibody.

6. The detection kit according to claim 5, wherein The volume ratio of the labeling preservation solution to the acridinium ester-labeled monoclonal MMP7 antibody is 50-100.

7. The detection kit according to claim 6, wherein The labeling preservation solution includes: 10mM PB buffer, 0.5wt%-3wt% BSA, 1wt%-5wt% trehalose, and 0.05wt%-0.3wt% Proclin-300.

8. The detection kit according to claim 1, wherein The magnetic microsphere solution M also contains a magnetic bead storage solution.

9. The detection kit according to claim 8, wherein The magnetic microsphere solution M comprises: the streptavidin-coupled magnetic beads at a concentration of 0.2-2 mg / mL and the magnetic bead storage solution at a pH of 6.5-7.

8.

10. The detection kit according to claim 8, wherein The magnetic bead storage solution comprises: 10mM PB buffer, 0.5wt%-3wt% BSA, 0.1wt%-3wt% Tween-20, and 0.05wt%-0.3wt% Proclin-300.

11. The detection kit according to any one of claims 1 and 8-10, wherein The particle size of the streptavidin-coupled magnetic beads is 1 μm-4 μm.

12. The detection kit according to claim 1, wherein The biotinylated antibody solution R1 further comprises a biotinylated antibody diluent.

13. The detection kit according to claim 12, wherein The biotinylated antibody solution R1 comprises: the biotinylated polyclonal MMP7 antibody and the biotinylated antibody diluent with a pH of 6.5-7.

8.

14. The detection kit according to claim 12, wherein The biotinylated antibody diluent comprises: 10 mM PB buffer, 0.5 wt%-3 wt% casein, 5 wt%-15 wt% sucrose, and 0.05 wt%-0.3 wt% Proclin-300.

15. The detection kit according to any one of claims 12 to 14, wherein The volume ratio of the biotinylated antibody diluent to the biotinylated polyclonal MMP7 antibody is 1000-3000.

16. The detection kit according to any one of claims 1 and 12-14, wherein The polyclonal MMP7 antibody is derived from mice, rats, pigs, monkeys, alpacas, camels and humans.

17. The detection kit according to claim 16, wherein The polyclonal MMP7 antibody was of IgG isotype.

18. The detection kit according to claim 17, wherein The polyclonal MMP7 antibody is antibodies-online MMP 7, CUSABIO MMP 7 or MyBioSource MMP 7.

19. The detection kit according to claim 1, wherein The MMP7 detection kit also includes an MMP7 quality control product.

20. The detection kit according to claim 19, wherein The MMP7 detection kit also includes an MMP7 calibrator.

21. The detection kit according to claim 20, wherein The MMP7 calibrator includes MMP7 protein and antigen diluent, wherein the antigen diluent contains: 10mM PB buffer, 0.5wt%-3wt% BSA, 0.05wt%-0.3wt% Proclin-300, and the pH of the PB buffer is 7.0-7.

5.

22. The detection kit according to claim 21, wherein The concentration of the MMP7 protein is one or more of 4.25-230 ng / mL.

23. The detection kit according to claim 19, wherein The MMP7 quality control product includes MMP7 protein and antigen diluent, wherein the antigen diluent contains: 10mM PB buffer, 0.5wt%-3wt% BSA, 0.05wt%-0.3wt% Proclin-300, and the pH of the PB buffer is 7.0-7.

5.

24. The detection kit according to claim 23, wherein The concentration of the MMP7 protein is one or more of 8.5-287.5 ng / mL.

25. The detection kit according to claim 21 or 23, wherein The MMP7 protein is a human MMP7 protein.

26. The detection kit according to any one of claims 1 to 3, wherein: The MMP7 detection kit also includes a pre-stimulation solution and / or a stimulation solution.

27. The detection kit according to claim 26, wherein In terms of volume percentage, the pre-excitation solution includes 0.05%-2% of concentrated HNO3 with a concentration of 60wt%-90wt%, 3%-6% of H2O2 with a concentration of 30% in terms of g / mL, 0.1wt%-2% Triton-X100, and 0.1%-0.4% anhydrous ethanol.

28. The detection kit according to claim 26, wherein In terms of volume percentage, the excitation solution includes 1%-2% NaOH, 1%-4% Triton-X 100, 0.1%-0.4% anhydrous ethanol, and 0.1%-0.3% EDTA-Na.

29. Use of the MMP7 detection kit according to any one of claims 1 to 28 in preparing a product for diagnosing MMP7-related diseases, wherein: The MMP7-related disease is selected from kidney disease, intrahepatic cholangiocarcinoma, ovarian cancer, pulmonary hypertension or idiopathic pulmonary fibrosis, biliary atresia, obstructive jaundice, liver fibrosis, hepatitis, non-alcoholic fatty liver disease, cholangitis, cholestasis, necrotizing enterocolitis or sepsis, cystitis, arthritis, and lupus erythematosus.

30. The use according to claim 29, wherein The MMP7-related disease is selected from liver fibrosis, renal fibrosis, or chronic kidney disease caused by biliary atresia.

31. A method for preparing the MMP7 detection kit according to any one of claims 1 to 28, comprising: The magnetic microsphere solution M, the biotinylated antibody solution R1 and the luminescent marker R2 are prepared separately, and then packaged and placed in the same or different detection kits.

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