Fluorescence chromatography test strip and kit for quantitatively detecting matrix metalloproteinase 7 (MMP-7) and application

Abstract

The invention relates to a fluorescence chromatography test strip capable of quantitatively detecting matrix metalloproteinase 7 (MMP-7) in human serum / plasma or whole blood, a kit and application of the fluorescence chromatography test strip. The anti-human MMP-7 rabbit monoclonal antibody I and the anti-human MMP-7 rabbit monoclonal antibody II are applied to the development of the double-antibody sandwich fluorescence immunochromatography kit, the fluorescence immunochromatography kit developed by utilizing the antibodies has the advantages of strong anti-interference capability, good specificity, wide linear range, high accuracy and the like, and the detection range is 2.0-150.0 ng / ml, so that the anti-human MMP-7 rabbit monoclonal antibody I and the anti-human MMP-7 rabbit monoclonal antibody II can be applied to the development of the double-antibody sandwich fluorescence immunochromatography kit. The linear correlation coefficient R2 of the kit is larger than 0.99, the whole detection process only needs 15 min, the kit can be used for bedside instant diagnosis (POCT), and great convenience is provided for clinical auxiliary diagnosis and postoperative follow-up visit of biliary atresia (BA) diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and specifically relates to a fluorescent chromatography test strip, a kit and an application thereof for quantitatively detecting matrix metalloproteinase 7 (MMP-7). Background Art

[0002] Matrix metalloproteinase 7 (MMP-7) is an important member of the matrix metalloproteinase family. It is secreted in the form of zymogen. Under normal physiological conditions, MMP-7 and its specific matrix metalloproteinase inhibitors jointly maintain the homeostasis of the extracellular matrix. MMP-7 is present in low levels in normal healthy liver tissue, but its expression level is greatly increased in liver tissue with biliary atresia (BA) and liver fibrosis, and is mainly distributed in the bile duct epithelial Kuffer cells and endothelial cells of children with BA. When the bile duct is obstructed or damaged by inflammation, the bile duct epithelial cells release MMP-7 into the lumen, and at this time, the increase in MMP-7 levels can be detected by serology. The latest studies have confirmed that using MMP-7 in the blood as a marker for the diagnosis of BA has good accuracy and clinical application prospects.

[0003] BA is a biliary obstructive disease that seriously threatens the life of infants. In September 2023, it was included in the "Notice on the Announcement of the Second Batch of Rare Diseases Catalogue" jointly issued by the National Health Commission, the National Medical Products Administration and other six departments. The Kasai operation is currently an effective means of treating biliary atresia, but the age of the child at the time of surgery plays a key role. Early and clear diagnosis and surgery can significantly improve the prognosis of children. Otherwise, the prognosis is mostly poor. Therefore, early diagnosis of biliary atresia is particularly important. The "Evidence-Based Practice Guidelines for the Diagnosis and Treatment of Biliary Atresia" issued by my country in 2022 recommends that children with cholestasis undergo matrix metalloproteinase 7 (MMP-7) to diagnose biliary atresia. For children with biliary atresia, regular follow-up after surgery is also of great significance. Problems in the rehabilitation process can be detected early and the survival rate of the autologous liver can be improved.

[0004] Since BA is a progressive intrahepatic and extrahepatic bile duct fibrosis disease, the disease is manifested by jaundice, hepatomegaly, and bilious stools. These clinical symptoms are very similar to other non-BA cholestatic liver diseases. There is no reliable non-invasive diagnostic method to distinguish BA from cholestasis caused by other reasons. At present, the gold standard diagnosis method for BA diagnosis in clinical practice is intraoperative cholangiography and liver biopsy, both of which are invasive tests. This detection method is easy to cause pain to patients and requires manual operation by expert physicians with rich clinical experience combined with auxiliary equipment such as medical imaging. For economically underdeveloped areas, due to the relatively backward medical facilities and resources and limited diagnosis and treatment level of medical units, it will be difficult to carry out the above medical diagnosis work, which is not conducive to the timely diagnosis of BA patients and is easy to miss the best window period for Kasai surgery. To this end, the development of a safe, effective, and rapid method for non-invasively diagnosing BA will enable patients with non-BA cholestatic liver disease to avoid excessive diagnosis and treatment such as invasive surgical examinations, alleviate patient suffering, and identify BA patients as early as possible, perform Kasai surgery in a timely manner, improve the prognosis of children, and increase the survival rate of their own liver.

[0005] Chinese application CN108267585A discloses a biliary atresia diagnostic kit based on serum marker MMP-7, which includes an anti-human monoclonal antibody coated ELISA plate, a negative control solution, a positive control solution, an ELISA reagent, an enzyme substrate solution, a blocking solution, a sample diluent, a washing solution and a stop solution. The kit can quantitatively determine the level of MMP-7 in human serum, which is helpful for early diagnosis of BA. However, the kit is only suitable for detecting serum sample types and cannot be used to detect plasma and whole blood samples. Secondly, the kit is based on an enzyme-linked immunosorbent assay (ELISA), which has cumbersome operating steps and involves multiple sample addition incubation reactions and washing operations. If the washing is not thorough, it is very easy to cause false positive results, and each test requires the calibration well to be set and the standard curve to be prepared at the same time, which increases the workload of the test operator and reduces the efficiency of the test kit. In addition, the operation is time-consuming, excluding the sample pretreatment time, and only the detection time of one sample is completed, which takes at least 3.5 hours, and requires professional test personnel to operate in the laboratory with the help of equipment such as an ELISA instrument, which is not suitable for the promotion and use of bedside instant diagnosis (POCT).

[0006] Chinese application CN115561458A discloses a colloidal gold rapid test card for diagnosing biliary atresia, the test card comprising components such as a sample pad, a gold label binding pad, a nitrocellulose membrane and a water-absorbing pad which are sequentially overlapped on a bottom plate. The test card can be used for rapid detection of MMP-7 in human serum / plasma or whole blood samples, and is suitable for rapid screening and auxiliary diagnosis of patients with biliary atresia on site. However, the detection method in the application can only be used as a qualitative test for biliary atresia, and the double antibody sandwich method is used to detect the MMP-7 antigen in the sample. The detection antibody and capture antibody involved are both mouse monoclonal antibodies prepared by traditional mouse hybridoma cell fusion technology. The sandwich detection method for antigens established by using double mouse monoclonal antibodies may cause interference reactions of human anti-mouse antibodies (HAMA) in the sample to be tested, resulting in false positive or false negative test results. In addition, since the kit detection method in the present application is only used for qualitative detection, it is difficult to determine the diagnostic results for gray zone samples near the diagnostic critical value of BA, and false negative or false positive results are likely to occur, resulting in misdiagnosis.

[0007] Compared with the existing clinical gold standard methods for diagnosing BA, such as intraoperative cholangiography and liver biopsy, the fluorescent immunochromatography detection method overcomes the disadvantages of invasive detection, expensive instruments and equipment, and professional clinical physicians for detection operations. At the same time, compared with other detection methods based on immunological principles, such as enzyme-linked immunosorbent assay and colloidal gold method, it overcomes the shortcomings of cumbersome operation steps in the detection process, the need for professional detection personnel to perform inspection operations, and the low detection accuracy of critical gray zone samples and the difficulty in interpreting the test results. In addition, since MMP-7 belongs to the MMP family, it has similar structures and characteristics to other major family members and is prone to cross-reactivity. Therefore, the antibodies used to establish the sandwich method for detecting MMP-7 antigens are required to have high sensitivity and specificity, and strong anti-interference ability. Compared with other commonly used animal models such as mice, rabbits have a stronger immune system, and the antibodies produced have higher sensitivity, specificity, affinity and diversity.

[0008] At present, there are still technical difficulties in obtaining anti-human MMP-7 rabbit monoclonal antibodies with high sensitivity and good specificity, and applying them to the double antibody sandwich fluorescent immunochromatography method to prepare fluorescent immunochromatography test strips and kits with strong anti-interference ability, good specificity, wide linear range, and high accuracy, which can be used for auxiliary diagnosis of biliary atresia. Summary of the invention

[0009] In view of the problems existing in the prior art, the present invention provides a fluorescent immunochromatography kit and its application for quantitatively detecting MMP-7 in human serum / plasma or whole blood samples based on the double antibody sandwich method. The present invention successfully developed a pair of high-affinity anti-human MMP-7 rabbit monoclonal antibodies, and further developed a fluorescent immunochromatography detection kit for quantitatively detecting MMP-7 with a double antibody sandwich, which is used for rapid quantitative detection of MMP-7 in human serum / plasma or whole blood samples. The detection method based on the kit has the advantages of strong anti-interference ability, good specificity, wide linear range, small batch difference, high consistency of detection results with clinical gold standard diagnostic methods, etc., and the detection process is simple and rapid, which can be applied to the auxiliary diagnosis of clinical biliary atresia diseases, and has broad market application value.

[0010] The invention is realized in this way: a fluorescence chromatography test strip for quantitatively detecting MMP-7, the test strip comprising a sample pad, a binding pad, a coating membrane and a water-absorbing pad which are sequentially overlapped on a PVC bottom plate; the binding pad is coated with fluorescent microspheres labeled with anti-human MMP-7 rabbit monoclonal antibody I; the coating membrane is provided with a detection area (T line) and a quality control area (C line) with lines; the detection area (T line) is coated with anti-human MMP-7 rabbit monoclonal antibody II, and the quality control area (C line) is coated with goat anti-rabbit IgG; during detection, the sample to be detected is loaded through the sample pad, the MMP-7 in the sample is combined with the fluorescent microspheres labeled with anti-human MMP-7 rabbit monoclonal antibody I in the binding pad to form an immune complex, the complex migrates upward with the capillary action of the membrane to reach the detection area (T line), generates an immune reaction with the anti-human MMP-7 rabbit monoclonal antibody II on the membrane, generates a fluorescent signal after being excited by a specific wavelength excitation light source, and the strength of the signal is proportional to the MMP-7 content in the sample.

[0011] The present invention provides a monoclonal antibody that specifically binds to human MMP-7, which is any one of the following A)-C):

[0012] A) anti-human MMP-7 rabbit monoclonal antibody I, the antibody comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in the amino acid sequences of SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, and the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in the amino acid sequences of SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10;

[0013] B) anti-human MMP-7 rabbit monoclonal antibody II, the antibody comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in the amino acid sequences of SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, and the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in the amino acid sequences of SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20;

[0014] C) A combination of the above A) and B).

[0015] Furthermore, the light chain variable region sequence of the anti-human MMP-7 rabbit monoclonal antibody I is the amino acid sequence shown in SEQ ID NO:3, and the heavy chain variable region sequence is the amino acid sequence shown in SEQ ID NO:4; the light chain variable region sequence of the anti-human MMP-7 rabbit monoclonal antibody II is the amino acid sequence shown in SEQ ID NO:13, and the heavy chain variable region sequence is the amino acid sequence shown in SEQ ID NO:14.

[0016] Furthermore, the light chain sequence of the anti-human MMP-7 rabbit monoclonal antibody I is the amino acid sequence shown in SEQ ID NO:1, and the heavy chain amino acid sequence is SEQ ID NO:2; the light chain sequence of the anti-human MMP-7 rabbit monoclonal antibody II is the amino acid sequence shown in SEQ ID NO:11, and the heavy chain amino acid sequence is the amino acid sequence shown in SEQ ID NO:12.

[0017] The present invention also provides a biomaterial, which is any one of the following D1) to D5):

[0018] D1) a nucleic acid molecule encoding the monoclonal antibody according to any one of claims 1 to 3;

[0019] D2) an expression cassette containing the nucleic acid molecule described in D1);

[0020] D3) a recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2);

[0021] D4) a recombinant microorganism containing the nucleic acid molecule described in D1), or a recombinant microorganism containing the expression cassette described in D2), or a recombinant microorganism containing the recombinant vector described in D3);

[0022] D5) A cell line containing the nucleic acid molecule described in D1), or a cell line containing the expression cassette described in D2), or a cell line containing the recombinant vector described in D3).

[0023] The present invention also provides the use of any of the above-mentioned monoclonal antibodies or compositions or kits or biological materials containing the same in preparing products for quantitatively detecting the content of MMP-7 in a sample or for diagnosing or assisting in the diagnosis of biliary atresia.

[0024] Furthermore, the product is a test kit, the test kit is a fluorescent chromatography test strip, and the sample of the product is taken from serum, plasma or whole blood.

[0025] The present invention also provides a kit for quantitatively detecting MMP-7, comprising the anti-human MMP-7 rabbit monoclonal antibody I and the anti-human MMP-7 rabbit monoclonal antibody II according to any one of claims 1 to 3 or the antigen binding portions thereof, and a matching detection reagent.

[0026] Furthermore, the kit is a fluorescent chromatography test strip, which includes a sample pad, a conjugation pad, a coating membrane and a water-absorbing pad sequentially overlapped on a PVC bottom plate; the conjugation pad is coated with fluorescent microspheres-labeled anti-human MMP-7 rabbit monoclonal antibody I; the coating membrane is provided with a detection area (T line) and a quality control area (C line) with lines; the detection area (T line) is coated with anti-human MMP-7 rabbit monoclonal antibody II, and the quality control area (C line) is coated with goat anti-rabbit IgG, wherein the detection antibody is anti-human MMP-7 rabbit monoclonal antibody I labeled with fluorescent microspheres, and the capture antibody is anti-human MMP-7 rabbit monoclonal antibody II.

[0027] The present invention also provides a method for preparing the above-mentioned fluorescence chromatography test strip for quantitatively detecting MMP-7, comprising the following steps:

[0028] 1) Preparation of sample pad: soak the sample pad in the treatment solution for 0.5 to 1 hour, place it in an oven with a humidity of less than 25% and a temperature of 45°C to 60°C, dry it for 12 to 24 hours, seal it and store it at room temperature;

[0029] 2) Preparation of the conjugate pad: dilute the fluorescent microsphere-labeled anti-human MMP-7 rabbit monoclonal antibody I conjugate with microsphere storage solution to a concentration of 0.05-0.1 mg / ml and spray it on the conjugate pad at a spray volume of 3-5 μl / cm. After spraying, place the conjugate pad in an oven at 45°C-60°C with a humidity of <25% for 12-24 hours and seal it for storage;

[0030] 3) Preparation of coating membrane: Anti-human MMP-7 rabbit monoclonal antibody II and goat anti-rabbit IgG were diluted with coating solution to a concentration of 0.5-1.0 mg / ml, and then sprayed on the detection area (T line) and quality control area (C line) of the coating membrane, respectively. The spraying amount was 1.0 μl / cm, and the interval between the T line and the C line was 5-7 mm. After spraying, the coating membrane was placed in an oven at 45°C to 60°C with a humidity of <25%, dried for 12-24 hours, and sealed for storage;

[0031] 4) Preparation of test strips: The prepared sample pad, conjugate pad, coating membrane, and absorbent pad are overlapped and pasted on a PVC base plate in sequence, and cut into test strips with a width of 3 to 4 mm to obtain the fluorescent chromatography test strips for quantitative detection of MMP-7.

[0032] Furthermore, the treatment solution includes: 0.2-1% bovine serum albumin, 1%-5% sucrose, 0.2-1% trehalose, 0.03-0.1% Tween-20, 0.02-0.05% Proclin300, and 0.02M PBS buffer at pH7.4; the microsphere preservation solution includes: 0.8-1.2% bovine serum albumin, 1-5% trehalose, 0.05-0.1% Tween-20, 0.02-0.05% Proclin300, and 0.25M Tris buffer at pH8.5; the coating solution includes: 1%-2% sucrose, 0.02-0.05% Proclin300, and 0.1M PBS buffer at pH7.4.

[0033] The present invention also provides a method for quantitatively detecting the content of MMP-7 in a sample for purposes other than disease diagnosis or treatment. The above-mentioned kit is used for detection. During the detection, the sample to be detected is loaded through the sample pad, and the MMP-7 in the sample combines with the fluorescent microsphere-labeled anti-human MMP-7 rabbit monoclonal antibody I in the binding pad to form an immune complex. The complex migrates upward to the detection area (T line) with the capillary action of the membrane, and reacts immune-wise with the anti-human MMP-7 rabbit monoclonal antibody II on the membrane. After being excited by an excitation light source of a specific wavelength, a fluorescent signal is generated, and the strength of the signal is proportional to the content of MMP-7 in the sample.

[0034] Furthermore, the sample is taken from serum, plasma or whole blood.

[0035] Further, the following steps are included:

[0036] (1) Return the test kit and the sample to be tested to room temperature;

[0037] (2) Use the fluorescence immunochromatography analyzer (model: IGreat-IF-F10) produced by Igreat (Wuhan) Technology Co., Ltd. for testing: turn on the fluorescence analyzer, insert the ID card with the same batch number as the reagent, and correctly select the corresponding "sample type" and "test item" on the instrument;

[0038] (3) Pipette 20 μl of the sample to be tested and add it to the matching sample diluent and mix thoroughly. Take 100 μl of the mixed sample and vertically drop it into the sample addition area of ​​the test card and start timing;

[0039] (4) 15 minutes after adding the sample, immediately insert the test card into the card slot of the fluorescence analyzer (produced by Igreat (Wuhan) Technology Co., Ltd., model: IGreat-IF-F10) and press the "Quick Test" button. The instrument will automatically test and display the results.

[0040] The advantages and positive effects of the present invention are:

[0041] (1) The present invention uses matrix metalloproteinase 7 (MMP-7) as a biomarker for diagnosing biliary atresia, successfully develops a pair of high-affinity anti-human MMP-7 rabbit monoclonal antibodies, and further develops a double antibody sandwich fluorescent immunochromatography detection kit that can quantitatively detect MMP-7. The present invention uses eukaryotic mammalian cells to express MMP-7 recombinant protein and uses it as an immunogen to immunize New Zealand white rabbits, separate single antigen-specific B lymphocytes from rabbit spleen cells, construct the heavy chain and light chain genes corresponding to the positive clone B lymphocytes into expression vectors, respectively, obtain recombinant expression plasmids and transfect 293F cells, and screen and purify anti-human MMP-7 rabbit monoclonal antibodies I and II that can be used in the double antibody sandwich fluorescent immunochromatography method from the cell culture supernatant.

[0042] (2) The fluorescent immunochromatographic detection method for detecting MMP-7 based on the anti-human MMP-7 rabbit monoclonal antibody provided by the present invention has high specificity and anti-interference ability, wherein the detection antibody is the anti-human MMP-7 rabbit monoclonal antibody I of the present invention labeled with fluorescent microspheres, and the capture antibody is the anti-human MMP-7 rabbit monoclonal antibody II of the present invention. No cross-reaction occurred when detecting other member proteins of the MMP family. When the abnormal samples with obvious hemolysis, lipemia, and jaundice were tested and verified, there was no interference and significant impact on the test results compared with the normal control.

[0043] (3) The fluorescent immunochromatographic test strip and kit of the present invention are suitable for the rapid quantitative detection of MMP-7 in human serum / plasma or whole blood samples, and can be used for the auxiliary diagnosis of clinical biliary atresia. The detection process is simple and rapid, and the kit has strong anti-interference ability, good specificity, wide linear range, small batch difference, and high consistency with the detection results of the clinical gold standard diagnostic method. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0045] Figure 1It is the standard curve of the fluorescent immunochromatographic detection method established based on anti-human MMP-7 rabbit monoclonal antibody I and anti-human MMP-7 rabbit monoclonal antibody II.

[0046] Figure 2 It is the linear interval validation fitting curve of the MMP-7 fluorescence immunochromatography detection method. DETAILED DESCRIPTION

[0047] In order to make the objectives, technical solutions and advantages of the present invention clearer and easier to understand, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments; it should be understood that the specific embodiments described herein are only used to explain the present invention and should not be construed as limiting the present invention.

[0048] The reagents or instruments used in the examples of the present invention without indicating the manufacturer are all conventional reagent products that can be obtained through commercial purchase.

[0049] Example 1 Preparation of anti-human MMP-7 rabbit monoclonal antibody

[0050] 1) Animal immunization: In order to obtain rabbit monoclonal antibodies that recognize human MMP-7, self-produced recombinant MMP-7 protein was used as an immunogen to immunize New Zealand white rabbits; each white rabbit was immunized with 200ug. For the first immunization, the immunogen was mixed with an equal amount of complete Freund's adjuvant to make an emulsion, which was injected subcutaneously at multiple points in the abdomen and back. At intervals of 3 weeks, 100ug of the immunogen was mixed with an equal amount of incomplete Freund's adjuvant to make an emulsion, which was injected subcutaneously at multiple points in the abdomen and back. The immunization was boosted twice. After three immunizations, the serum titer was determined by ELISA. Rabbits with high serum titers were selected and boosted once with 200ug of the immunogen by subcutaneous injection at multiple points. The spleen was obtained three days later.

[0051] 2) Spleen cell separation: This is a conventional method known in the art.

[0052] 3) B lymphocyte sorting: This is a conventional method known in the art, for example, see patent 201910125091.4: A method for efficiently separating single antigen-specific B lymphocytes from spleen cells.

[0053] 4) Cloning of the gene encoding rabbit monoclonal antibody

[0054] The supernatant of cultured B cells was used to identify positive clones by antigen-coated ELISA. The cells of the positive clones were collected and lysed, and RNA was extracted and reverse transcribed into cDNA. Using the PCR method, the naturally paired rabbit monoclonal antibody light and heavy chain variable region genes (VH and VL) were amplified from the cDNA of the corresponding positive clones, and the sequences were determined by sequencing.

[0055] 5) Production and purification of monoclonal antibodies

[0056] In order to obtain multiple rabbit monoclonal antibodies that recognize human MMP-7, the heavy chain and light chain genes of rabbit monoclonal antibodies were loaded on expression vectors, and the plasmids were transfected into 293F cells; the culture supernatant containing recombinant rabbit monoclonal antibodies that recognize human MMP-7 was obtained after 72-96 hours of transfection. The recombinant rabbit monoclonal antibodies that recognize human MMP-7 were purified from the culture supernatant after transfection using protein A affinity gel resin, and the antibodies were packaged after identification and stored at -20°C for future use.

[0057] The present invention relates to anti-human MMP-7 rabbit monoclonal antibody I and anti-human MMP-7 rabbit monoclonal antibody II.

[0058] in:

[0059] The light chain amino acid sequence of anti-human MMP-7 rabbit monoclonal antibody I is SEQ ID NO: 1; the heavy chain amino acid sequence is SEQ ID NO: 2; the amino acid sequence of the light chain variable region is SEQ ID NO: 3; the amino acid sequence of the heavy chain variable region is SEQ ID NO: 4; the amino acid sequences of the light chain complementary determining regions CDR1, CDR2 and CDR3 are SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, respectively; the amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2 and CDR3 are SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

[0060] The light chain amino acid sequence of the anti-human MMP-7 rabbit monoclonal antibody II is SEQ ID NO: 11; the heavy chain amino acid sequence is SEQ ID NO: 12; the amino acid sequence of the light chain variable region is SEQ ID NO: 13; the amino acid sequence of the heavy chain variable region is SEQ ID NO: 14; the amino acid sequences of the light chain complementary determining regions CDR1, CDR2 and CDR3 are SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively; the amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2 and CDR3 are SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively.

[0061] Example 2 Preparation of Fluorescence Chromatography Test Strips for Quantitative Detection of MMP-7

[0062] A fluorescent chromatography test strip for quantitatively detecting MMP-7, the test strip comprising a sample pad, a binding pad, a coating membrane and a water-absorbing pad which are sequentially overlapped on a PVC bottom plate; the binding pad is coated with fluorescent microspheres-labeled anti-human MMP-7 rabbit monoclonal antibody I; the coating membrane is provided with a detection area (T line) and a quality control area (C line) with lines; the detection area (T line) is coated with anti-human MMP-7 rabbit monoclonal antibody II, and the quality control area (C line) is coated with goat anti-rabbit IgG; during detection, a sample to be detected is loaded through the sample pad, and the MMP-7 in the sample is combined with the fluorescent microsphere-labeled anti-human MMP-7 rabbit monoclonal antibody I in the binding pad to form an immune complex, the complex migrates upward to the detection area (T line) with the capillary action of the membrane, reacts with the anti-human MMP-7 rabbit monoclonal antibody II on the membrane, and generates a fluorescent signal after being excited by a specific wavelength excitation light source, and the strength of the signal is proportional to the MMP-7 content in the sample.

[0063] The method for preparing the above-mentioned fluorescence chromatography test strip for quantitative detection of MMP-7 comprises the following steps:

[0064] 1) Preparation of sample pad: soak the sample pad in the treatment solution for 0.5-1 h, place it in an oven with a humidity of less than 25% and a temperature of 45°C-60°C, dry it for 12-24 h, seal it and store it at room temperature.

[0065] 2) Preparation of the conjugate pad: dilute the fluorescent microsphere-labeled anti-human MMP-7 rabbit monoclonal antibody I conjugate with microsphere preservation solution to a concentration of 0.05-0.1 mg / ml and spray it on the conjugate pad at a spray volume of 3-5 μl / cm. After spraying, place the conjugate pad in an oven at 45°C-60°C with a humidity of <25% for 12-24 hours, seal it and store it at room temperature.

[0066] 3) Preparation of coating membrane: Anti-human MMP-7 rabbit monoclonal antibody II and goat anti-rabbit IgG were diluted with coating solution to a concentration of 0.5-1.0 mg / ml, and then sprayed on the detection area (T line) and quality control area (C line) of the coating membrane, respectively. The spraying amount was 1.0 μl / cm, and the interval between the T line and the C line was 5-7 mm. After spraying, the coating membrane was placed in an oven at 45°C to 60°C with a humidity of <25%, dried for 12-24 hours, and sealed and stored at room temperature.

[0067] 4) Preparation of test strips: The prepared sample pad, conjugate pad, coating membrane, and absorbent pad are overlapped and pasted on a PVC base plate in sequence, and cut into test strips with a width of 3 to 4 mm to obtain the fluorescent chromatography test strips for quantitative detection of MMP-7.

[0068] The treatment solution comprises: 0.2-1% bovine serum albumin, 1%-5% sucrose, 0.2-1% trehalose, 0.03-0.1% Tween-20, 0.02-0.05% Proclin 300, and 0.02M PBS buffer at pH 7.4;

[0069] The microsphere preservation solution comprises: 0.8-1.2% bovine serum albumin, 1-5% trehalose, 0.05-0.1% Tween-20, 0.02-0.05% Proclin300, and 0.25M Tris buffer at pH 8.5;

[0070] The coating solution comprises: 1% to 2% sucrose, 0.02 to 0.05% Proclin 300, and 0.1 M PBS buffer solution with a pH of 7.4.

[0071] Example 3 Preparation of standard curve and blank limit analysis of MMP-7 fluorescence immunochromatography detection method

[0072] The MMP-7 recombinant protein was gradiently diluted with phosphate buffer containing 0.5% casein, 0.4% Tween-20, and 0.02% Triton-X100 to different concentrations, namely 200ng / ml, 100ng / ml, 50ng / ml, 25ng / ml, 10ng / ml, 5ng / ml, 2ng / ml, and 0.5ng / ml, and tested as samples. Each concentration was tested 3 times and the average value was taken. The logarithmic fitting was performed using the logarithmic value of the theoretical concentration of the MMP-7 recombinant protein as the abscissa and the T / C light intensity ratio of the test card read by the IGreat-IF-F10 fluorescent immunochromatography analyzer produced by Agilite (Wuhan) Technology Co., Ltd. as the ordinate, and the equation was established and the standard curve was fitted. The fitted standard curve is shown in FIG. Figure 1 As shown in the figure, the R 2 The linearity is good, and the concentration of MMP-7 in the sample can be quantitatively analyzed by this standard curve.

[0073] Using 0 ng / ml blank control as the sample, repeat the measurement 20 times, calculate the mean (M) and standard deviation (SD) of the 20 T / C light intensity ratios, calculate the T / C light intensity ratio of M+2SD, substitute it into the calibration curve, and calculate the corresponding concentration value, which is the blank limit sensitivity. The test results are shown in Table 1.

[0074] Table 1 Blank limit sensitivity test results

[0075]

[0076]

[0077] The experimental results showed that the blank limit sensitivity of the established MMP-7 fluorescent immunochromatography method reached 0.115 ng / ml.

[0078] Example 4 Verification of the measurable linear range of the MMP-7 fluorescence immunochromatography detection method

[0079] One serum sample with a high clinical value of MMP-7 close to the upper limit of the expected detection range and one serum sample with a low clinical value close to 0 were selected and mixed in different proportions to obtain 7 linear samples with different concentrations of 160 ng / ml, 80 ng / ml, 40 ng / ml, 20 ng / ml, 10 ng / ml, 2.5 ng / ml, and 1.25 ng / ml. Each concentration of the sample was tested three times, and the average concentration was calculated. The average value of the measured concentration was fitted with the calibrated theoretical concentration value by the least squares method to obtain the linear regression equation, and the linear correlation coefficient R was calculated. 2 The results were in the range of 1.25 to 160.0 ng / mL, and the correlation coefficient R 2 >0.99, indicating that the measurement linearity is good within the selected measurement range of 2.0 to 150.0 ng / ml. The test results and curves are shown in Table 2 and Figure 2 shown.

[0080] Table 2 Measurable linear interval verification test results

[0081]

[0082]

[0083] Example 5 Cross-reactivity analysis of MMP-7 fluorescence immunochromatography detection method

[0084] The seven major analogs of MMP-7 belonging to the same MMP family are MMP-3, MMP-4, MMP-5, MMP-6, MMP-8, MMP-9, and MMP-10, which are used to verify the cross-reactivity of the MMP-7 fluorescence immunochromatography detection method. The seven MMP-7 family analog recombinant proteins were diluted to 200 ng / ml with MMP-7 serum samples near the critical value (26.21 ng / ml) as samples for detection, and each sample was tested three times. The mean and relative deviation of the sample test results, i.e., the interference rate, were calculated to evaluate whether there was cross-reactivity when detecting samples containing MMP-7 family analog recombinant protein samples. The test results are shown in Table 3.

[0085] Table 3 Cross-reactivity test results

[0086]

[0087]

[0088] The results showed that the MMP-7 fluorescent immunochromatographic detection method did not produce cross-reactions when detecting these seven major analog recombinant proteins belonging to the same MMP family.

[0089] Example 6 Anti-interference verification analysis of MMP-7 fluorescence immunochromatography detection method

[0090] Two fresh serum samples with different concentration levels of MMP-7 were selected (no hemolysis, no lipemia, non-icteric), the MMP-7 concentration of sample 1# was 26.21ng / ml, and the concentration of sample 2# was 75.36ng / ml. The interfering substances bilirubin, triglycerides, and hemoglobin were added to the two serum samples with different concentrations, respectively, so that the final concentrations of the interfering substances were 400μg / ml for bilirubin, 32mg / ml for triglycerides, and 20mg / ml for hemoglobin. The MMP-7 concentrations in the two samples before and after the addition of the three interfering substances were tested with the fluorescent immunochromatographic detection card prepared in Example 1. Each sample was tested 3 times, and the mean and relative deviation of the sample test results, i.e., the interference rate, were calculated to evaluate whether the conventional clinical interfering substances had a significant effect on the MMP-7 determination results. The test results are shown in Table 4.

[0091] Table 4 Interfering substance test results

[0092]

[0093]

[0094] As shown in Table 4, the relative deviations of the detection results of MMP-7 in each sample before and after the addition of interfering substances were all within the range of ±10%, which is within the acceptable range. That is, the MMP-7 fluorescence chromatography test strip prepared by the present invention has excellent anti-interference performance.

[0095] Example 7: Consistency test between MMP-7 fluorescence immunochromatography detection method and gold standard detection method

[0096] The MMP-7 fluorescence immunochromatography method and the MMP-7 colloidal gold method (refer to patent CN115561458A, a colloidal gold rapid test card for diagnosing biliary atresia) were used to simultaneously test serum samples from 30 patients with cholestatic liver disease, including 15 clinically confirmed BA samples (P1-P15) and 15 non-BA samples (N1-N15). The test results of the two methods were compared with the clinical gold standard diagnosis results to examine their consistency with the gold standard test method. The test results are shown in Table 5.

[0097] Table 5 Comparison test results with the gold standard method

[0098]

[0099]

[0100]

[0101] According to the above test of clinical samples, it can be seen from Table 5 that when the positive judgment value of the MMP-7 fluorescence immunochromatography detection method is set to 26.0ng / ml, the concentration values ​​of MMP-7 measured by the fluorescence immunochromatography method for 15 BA samples (P1-P15) are all greater than 26ng / ml, and the results are judged as BA, while the colloidal gold method test results have 2 false negative results (samples P5 and P14); among the 15 non-BA samples (N1-N15), the results measured by the fluorescence immunochromatography method, the MMP-7 concentration value measured by sample N7 is greater than 26.0ng / ml, and the result is judged as BA, while the colloidal gold method test results have 2 false positive results (samples N7 and N8). The above comparative test results show that the fluorescence immunochromatography method has better consistency with the clinical gold standard diagnostic method, with a sensitivity and specificity of 100.0% and 93.3% respectively, and a total compliance rate of 96.7%. However, the colloidal gold method is prone to false negative or false positive results when testing samples near the critical value. The sensitivity and specificity are 86.7% and 86.7% respectively, and the overall compliance rate is 86.7%.

[0102] It can be seen from the above embodiments that the fluorescent chromatographic test strip and kit provide a fluorescent immunochromatographic method for quantitatively detecting the content of MMP-7 in blood samples, which can be used for auxiliary diagnosis of biliary atresia. The blank detection limit is as low as 0.115ng / ml, and the linear relationship is good in the detection range of 2.0-150.0ng / ml, the anti-interference ability is strong, there is no cross-reactivity with other proteins of the same family, and the compliance rate with the clinical gold standard detection method is high. The entire detection process only takes 15 minutes, which provides a novel non-invasive and rapid detection method for auxiliary diagnosis of biliary atresia, and has broad market application value.

[0103] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A monoclonal antibody that specifically binds to human MMP-7, characterized in that: Any one of the following A)-C): A) anti-human MMP-7 rabbit monoclonal antibody I, the antibody comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in the amino acid sequences of SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, and the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in the amino acid sequences of SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10; B) anti-human MMP-7 rabbit monoclonal antibody II, the antibody comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in the amino acid sequences of SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, and the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in the amino acid sequences of SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20; C) A combination of the above A) and B).

2. The monoclonal antibody according to claim 1, characterized in that The light chain variable region sequence of the anti-human MMP-7 rabbit monoclonal antibody I is the amino acid sequence shown in SEQ ID NO:3, and the heavy chain variable region sequence is the amino acid sequence shown in SEQ ID NO:4; the light chain variable region sequence of the anti-human MMP-7 rabbit monoclonal antibody II is the amino acid sequence shown in SEQ ID NO:13, and the heavy chain variable region sequence is the amino acid sequence shown in SEQ ID NO:

14.

3. The monoclonal antibody according to any one of claims 1-2, characterized in that: The light chain sequence of the anti-human MMP-7 rabbit monoclonal antibody I is the amino acid sequence shown in SEQ ID NO: 1, and the heavy chain amino acid sequence is SEQ ID NO: 2; the light chain sequence of the anti-human MMP-7 rabbit monoclonal antibody II is the amino acid sequence shown in SEQ ID NO: 11, and the heavy chain amino acid sequence is the amino acid sequence shown in SEQ ID NO:

12.

4. Biomaterial, characterized in that The biological material is any one of the following D1) to D5): D1) a nucleic acid molecule encoding the monoclonal antibody according to any one of claims 1 to 3; D2) an expression cassette containing the nucleic acid molecule described in D1); D3) a recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2); D4) a recombinant microorganism containing the nucleic acid molecule described in D1), or a recombinant microorganism containing the expression cassette described in D2), or a recombinant microorganism containing the recombinant vector described in D3); D5) A cell line containing the nucleic acid molecule described in D1), or a cell line containing the expression cassette described in D2), or a cell line containing the recombinant vector described in D3).

5. Use of the monoclonal antibody according to any one of claims 1 to 3, or a composition or kit containing the same, or the biomaterial according to claim 4 in preparing a product for quantitatively detecting the content of MMP-7 in a sample or for diagnosing or assisting in the diagnosis of biliary atresia.

6. The use according to claim 5, characterized in that The product is a test kit, which is a fluorescent chromatography test strip, and the sample of the product is taken from serum, plasma or whole blood.

7. A kit for quantitative detection of MMP-7, characterized in that: The invention comprises the anti-human MMP-7 rabbit monoclonal antibody I and the anti-human MMP-7 rabbit monoclonal antibody II as described in any one of claims 1 to 3, or the antigen binding parts of the two, and a matching detection reagent.

8. The kit according to claim 7, characterized in that The kit is a fluorescent chromatography test strip, which includes a sample pad, a conjugation pad, a coating membrane and a water-absorbing pad which are sequentially overlapped on a PVC bottom plate; the conjugation pad is coated with fluorescent microsphere-labeled anti-human MMP-7 rabbit monoclonal antibody I; the coating membrane is provided with a detection area and a quality control area with lines; the detection area is coated with anti-human MMP-7 rabbit monoclonal antibody II, and the quality control area is coated with goat anti-rabbit IgG, wherein the detection antibody is anti-human MMP-7 rabbit monoclonal antibody I labeled with fluorescent microspheres, and the capture antibody is anti-human MMP-7 rabbit monoclonal antibody II.

9. The method for preparing the fluorescent chromatography test strip for quantitative detection of MMP-7 according to claim 8, comprising the following steps: 1) Preparation of sample pad: soak the sample pad in the treatment solution for 0.5 to 1 hour, place it in an oven with a humidity of less than 25% and a temperature of 45°C to 60°C, dry it for 12 to 24 hours, seal it and store it at room temperature; 2) Preparation of the conjugate pad: dilute the fluorescent microsphere-labeled anti-human MMP-7 rabbit monoclonal antibody I conjugate with microsphere storage solution to a concentration of 0.05-0.1 mg / ml and spray it on the conjugate pad at a spray volume of 3-5 μl / cm. After spraying, place the conjugate pad in an oven at 45°C-60°C with a humidity of <25% for 12-24 hours and seal it for storage; 3) Preparation of coating membrane: Anti-human MMP-7 rabbit monoclonal antibody II and goat anti-rabbit IgG were diluted with coating solution to a concentration of 0.5-1.0 mg / ml, and then sprayed on the detection area (T line) and quality control area (C line) of the coating membrane, respectively. The spraying amount was 1.0 μl / cm, and the interval between the T line and the C line was 5-7 mm. After spraying, the coating membrane was placed in an oven at 45°C to 60°C with a humidity of <25%, dried for 12-24 hours, and sealed for storage; 4) Preparation of test strips: The prepared sample pad, conjugate pad, coating membrane, and absorbent pad are overlapped and pasted on a PVC base plate in sequence, and cut into test strips with a width of 3 to 4 mm to obtain the fluorescent chromatography test strips for quantitative detection of MMP-7.

10. The method according to claim 9, characterized in that The treatment solution comprises: 0.2-1% bovine serum albumin, 1%-5% sucrose, 0.2-1% trehalose, 0.03-0.1% Tween-20, 0.02-0.05% Proclin 300, and 0.02M PBS buffer at pH 7.4; The microsphere preservation solution comprises: 0.8-1.2% bovine serum albumin, 1-5% trehalose, 0.05-0.1% Tween-20, 0.02-0.05% Proclin300, and 0.25M Tris buffer at pH 8.5; The coating solution comprises: 1% to 2% sucrose, 0.02 to 0.05% Proclin 300, and 0.1M PBS buffer solution with a pH of 7.

4.

11. A method for quantitatively detecting the content of MMP-7 in a sample for purposes other than disease diagnosis or treatment, characterized in that: The kit described in claim 8 is used for detection. During the detection, the sample to be tested is loaded through the sample pad, and the MMP-7 in the sample combines with the fluorescent microsphere-labeled anti-human MMP-7 rabbit monoclonal antibody I in the binding pad to form an immune complex. The complex migrates upward to the detection area with the capillary action of the membrane, and reacts immune-wise with the anti-human MMP-7 rabbit monoclonal antibody II on the membrane. After being excited by an excitation light source of a specific wavelength, a fluorescent signal is generated, and the strength of the signal is proportional to the MMP-7 content in the sample.

12. The method according to claim 11, characterized in that The sample is obtained from serum, plasma or whole blood.

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