An early warning kit for liver fibrosis and antibody preparation thereof

Abstract

The present invention relates to the technical field of fluorescent immunoassay, and more specifically, to an early warning kit for liver fibrosis and antibody preparation thereof. The present invention prepares a monoclonal antibody for specific recognition based on a shell enzyme protein marker, and uses it to construct a test strip based on fluorescent immunoassay. The test strip has excellent detection sensitivity, specificity and stability. Compared with the existing detection method, the detection method of the present invention is simpler and suitable for large-scale detection, significantly improves the detection efficiency, and has important scientific significance and application value for clinical early diagnosis of liver fibrosis.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent immunoassay, and more specifically, to an early warning kit for liver fibrosis and antibody preparation thereof. Background Art

[0002] Liver fibrosis is a pathophysiological process, which refers to the abnormal proliferation of connective tissue in the liver caused by various pathogenic factors. Any liver damage will cause liver fibrosis during the process of liver repair and healing. If the damaging factors cannot be removed for a long time, the fibrosis process will develop into cirrhosis. Therefore, it is not an independent disease. Therefore, early detection, early diagnosis, and early treatment are of great significance for patients with liver fibrosis.

[0003] At present, the common early detection methods of liver fibrosis include two categories: one is invasive examination, namely liver puncture biopsy; the other is non-invasive examination, including serum molecular markers and imaging examination. In recent years, with the further development of fluorescent immunoassay technology, the use of colloidal gold labeled monoclonal antibodies can also achieve early warning of the disease. However, the colloidal gold immunochromatography method has defects such as low sensitivity. In summary, it is urgent to establish a set of rapid, simple and accurate detection methods to provide strong value information for early screening, diagnosis, efficacy judgment, prognosis evaluation or recurrence monitoring of liver fibrosis. Summary of the invention

[0004] In view of the technical problems existing in the prior art, the present invention provides an early warning kit for liver fibrosis and its antibody preparation. The present invention prepares a monoclonal antibody for specific recognition based on the shell enzyme protein marker, and constructs a test strip based on fluorescent immunoassay. The test strip has excellent detection sensitivity, specificity and stability. Compared with the existing detection method, the detection method of the present invention is simpler and suitable for large-scale detection, which significantly improves the detection efficiency and has important scientific significance and application value for the clinical diagnosis of early liver fibrosis.

[0005] One of the purposes of the present invention is to provide an early warning test strip for liver fibrosis, the test strip comprising a PVC base plate, a sample pad, a conjugation pad, a chromatographic membrane and a water absorbent pad, wherein the chromatographic membrane, the water absorbent pad, the conjugation pad and the sample pad are sequentially overlapped on the PVC base plate; a detection line T and a quality control line C are provided on the chromatographic membrane, wherein the quality control line C is coated with a sheep anti-mouse IgG antibody, and the detection line T is coated with a monoclonal antibody 2 that specifically recognizes a chitinase protein; the detection line T is arranged at one end close to the conjugation pad, and the quality control line C is arranged at one end close to the water absorbent pad; the conjugation pad is coated with a fluorescently labeled monoclonal antibody 1 that specifically recognizes a chitinase protein.

[0006] Preferably, the monoclonal antibody 1 that specifically recognizes the shell enzyme protein comprises a heavy chain variable region and a light chain variable region, wherein CDR1-3 of the heavy chain variable region are respectively as shown in SEQ ID NO.1-3, and CDR4-6 of the light chain variable region are respectively as shown in SEQ ID NO.4-6.

[0007] Preferably, the heavy chain and light chain of the monoclonal antibody comprise the amino acids shown in SEQ ID NOs. 7 and 8;

[0008] Preferably, the monoclonal antibody 2 that specifically recognizes the shell enzyme protein comprises a heavy chain variable region and a light chain variable region, wherein CDR1-3 of the heavy chain variable region are respectively as shown in SEQ ID NOs.9-11, and CDR4-6 of the light chain variable region are respectively as shown in SEQ ID NOs.12-14.

[0009] Preferably, the heavy chain and light chain of the monoclonal antibody comprise the amino acids shown in SEQ ID NOs. 15 and 16;

[0010] Preferably, goat anti-mouse IgG antibody is purchased from Beijing Keyue Zhongkai Biotechnology Co., Ltd. with the product number MG23N303.

[0011] Preferably, the preparation of the fluorescently labeled monoclonal antibody 1 that specifically recognizes the shell enzyme protein is as follows: 150 μL of fluorescent microspheres (purchased from Xi'an Qiyue Biotechnology Co., Ltd., product number Q-0344571) are taken, activated with EDC.HCl and Sulfo-NHS, and then 300 μg of the monoclonal antibody 1 of the shell enzyme protein is added thereto, reacted at room temperature for 12 hours, and then centrifuged, placed in a suspension, and stored at 4°C to obtain the fluorescently labeled monoclonal antibody 1 that specifically recognizes the shell enzyme protein, which is prepared for use.

[0012] Another object of the present invention is to provide a method for preparing a test strip for detecting chitin protein, the method comprising the following steps:

[0013] (1) Preparation of the binding pad: soaking the binding pad in a binding pad blocking solution, and then drying it for later use; diluting a solution of a fluorescently labeled monoclonal antibody 1 that specifically recognizes the shell enzyme protein and spraying it evenly on the binding pad, and drying it to prepare a binding pad for later use;

[0014] (2) Preparation of sample pad: soak the sample pad in sample pad blocking solution and dry it for later use;

[0015] (3) Preparation of nitrocellulose membrane: Monoclonal antibody 2 that specifically recognizes the chitin protein is added to the coating solution and mixed, and then sprayed on the detection line T of the nitrocellulose membrane. Goat anti-mouse IgG antibody is sprayed on the nitrocellulose membrane at an interval of 10 mm from the detection line as the quality control line C. The nitrocellulose membrane is dried for later use;

[0016] (4) Assembly of the test strip: The sample pad, conjugate pad, chromatographic membrane, and absorbent pad are sequentially connected on a PVC base plate to obtain a test strip, wherein the detection line T is set at one end close to the conjugate pad, and the quality control line C is set at the other end close to the absorbent pad.

[0017] The conjugate pad blocking solution contains 5-10wt% trehalose, 5-10wt% BSA, 0.75-1.25wt% Tween-20, and 0.5-1.5wt% Triton X-100 in a boric acid solution.

[0018] Preferably, the sealing time is 2-6 hours and the temperature is 37°C.

[0019] Preferably, the sample pad blocking solution contains 0.75-1.5wt% Tween-20, 2-5wt% PEG20000, and 2-5wt% BSA in a boric acid solution.

[0020] Preferably, the boric acid solution has a concentration of 0.02-0.04 M and a pH of 8.0-8.5.

[0021] Preferably, the sealing time is 2-6 hours and the temperature is 37°C.

[0022] Preferably, the coating solution contains 0.9-2.5wt% NaCl, 0.2-1.0wt% NaH 2 PO 4 , a phosphate solution containing 3-6 wt% sucrose, wherein the concentration of the phosphate solution is 10 mM-50 mM and the pH is 7.0-7.4.

[0023] Another object of the present invention is to provide an early warning kit for liver fibrosis, characterized in that the kit comprises the following components: an early warning test strip for liver fibrosis, a conjugate pad blocking solution, a sample pad blocking solution and a coating solution.

[0024] Another object of the present invention is to provide a detection method for early warning of liver fibrosis, the method comprising the following steps:

[0025] 1) Dilute the sample and add it to the sample well of the test strip and let it stand at room temperature for 45-60 minutes;

[0026] 2) Observe the test strip results. If both the quality control line and the test line of the sample are colored, it is judged as positive; if only the quality control line is colored and the test line is not colored, it is judged as negative; if the quality control line is not colored, the test is invalid.

[0027] Another object of the present invention is to provide the use of the test strip for detecting chitin protein or the early warning kit for liver fibrosis in the preparation of an early warning product for liver fibrosis.

[0028] The advantages of the present invention are as follows: the present invention prepares a monoclonal antibody for the first time for the early detection of protein markers for liver fibrosis, and uses it to construct a test strip based on fluorescent immunoassay. The test strip has excellent detection sensitivity, specificity and stability. Compared with existing detection methods, the detection method of the present invention is simpler and suitable for large-scale detection, which significantly improves the detection efficiency and has important scientific significance and application value for the clinical diagnosis of early liver fibrosis. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0030] The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the protection scope of the present invention.

[0031] In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0032] Example 1

[0033] The present invention provides a monoclonal antibody 1 that specifically recognizes a chitinase protein, the monoclonal antibody comprising a heavy chain variable region and a light chain variable region, the CDR1-3 of the heavy chain variable region are respectively as shown in SEQ ID NO.1-3, and the CDR4-6 of the light chain variable region are respectively as shown in SEQ ID NO.4-6. Preferably, the heavy chain and light chain of the monoclonal antibody comprise the amino acids shown in SEQ ID NO.7 and 8;

[0034] The present invention provides a monoclonal antibody 2 that specifically recognizes a chitinase protein, wherein the monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein CDR1-3 of the heavy chain variable region are respectively as shown in SEQ ID NOs.9-11, and CDR4-6 of the light chain variable region are respectively as shown in SEQ ID NOs.12-14. Preferably, the heavy chain and light chain of the monoclonal antibody comprise amino acids as shown in SEQ ID NOs.15 and 16;

[0035] The sheep anti-mouse IgG antibody provided by the present invention was purchased from Beijing Keyue Zhongkai Biotechnology Co., Ltd. with the product number of MG23N303.

[0036] Preparation of fluorescently labeled monoclonal antibody 1 that specifically recognizes the enzyme protein: Take 150 μL of fluorescent microspheres (purchased from Xi'an Qiyue Biotechnology Co., Ltd., product number Q-0344571), activate them with EDC.HCl and Sulfo-NHS, and then add 300 μg of monoclonal antibody 1 to the enzyme protein. React at room temperature for 12 hours and then centrifuge. Place in a suspension and store at 4°C to obtain the fluorescently labeled monoclonal antibody 1 that specifically recognizes the enzyme protein, which is ready for use.

[0037] Example 2

[0038] An early warning test strip for liver fibrosis, the test strip comprising a PVC base plate, a sample pad, a conjugation pad, a chromatographic membrane and a water absorbent pad, wherein the chromatographic membrane, the water absorbent pad, the conjugation pad and the sample pad are sequentially overlapped on the PVC base plate; a detection line T and a quality control line C are provided on the chromatographic membrane, wherein the quality control line C is coated with a sheep anti-mouse IgG antibody, and the detection line T is coated with a monoclonal antibody 2 that specifically recognizes a chitinase protein; the detection line T is arranged at one end close to the conjugation pad, and the quality control line C is arranged at one end close to the water absorbent pad; the conjugation pad is coated with a fluorescently labeled monoclonal antibody 1 that specifically recognizes a chitinase protein.

[0039] A method for preparing a test strip for detecting chitin protein, the method comprising the following steps:

[0040] (1) Preparation of the binding pad: soaking the binding pad in a binding pad blocking solution, and then drying it for later use; diluting a solution of a fluorescently labeled monoclonal antibody 1 that specifically recognizes the shell enzyme protein and spraying it evenly on the binding pad, and drying it to prepare a binding pad for later use;

[0041] (2) Preparation of sample pad: soak the sample pad in sample pad blocking solution and dry it for later use;

[0042] (3) Preparation of nitrocellulose membrane: Monoclonal antibody 2 that specifically recognizes the chitin protein is added to the coating solution and mixed, and then sprayed on the detection line T of the nitrocellulose membrane. Goat anti-mouse IgG antibody is sprayed on the nitrocellulose membrane at an interval of 10 mm from the detection line as the quality control line C. The nitrocellulose membrane is dried for later use;

[0043] (4) Assembly of the test strip: The sample pad, conjugate pad, chromatographic membrane, and absorbent pad are sequentially connected on a PVC base plate to obtain a test strip, wherein the detection line T is set at one end close to the conjugate pad, and the quality control line C is set at the other end close to the absorbent pad.

[0044] The conjugate pad blocking solution is a boric acid solution containing 10wt% trehalose, 5wt% BSA, 0.75wt% Tween-20, and 0.75wt% Triton X-100. The blocking time is 4 hours at a temperature of 37°C.

[0045] The sample pad blocking solution is a boric acid solution containing 0.75 wt% Tween-20, 2.5 wt% PEG20000, and 2.5 wt% BSA. The blocking time is 4 hours at a temperature of 37°C.

[0046] Wherein, the concentration of the boric acid solution is 0.02M and the pH is 8.0.

[0047] The coating solution contains 0.9 wt% NaCl, 0.2 wt% NaH 2 PO 4 , a phosphate solution containing 5 wt% sucrose, wherein the concentration of the phosphate buffer is 10 mM and the pH is 7.0.

[0048] Example 3

[0049] An early warning kit for liver fibrosis comprises the following components: an early warning test strip for liver fibrosis, a conjugate pad blocking solution, a sample pad blocking solution and a coating solution.

[0050] A detection method for early warning of liver fibrosis, the method comprising the following steps:

[0051] 1) Dilute the sample and add it to the sample well of the test strip and let it stand at room temperature for 45 minutes;

[0052] 2) Observe the test strip results. If both the quality control line and the test line of the sample are colored, it is judged as positive; if only the quality control line is colored and the test line is not colored, it is judged as negative; if the quality control line is not colored, the test is invalid.

[0053] Example 4

[0054] Fluorescence immunoassay to evaluate the sensitivity, specificity and repeatability of test strips

[0055] (1) Sensitivity testing of early warning test strips for liver fibrosis

[0056] Dilute the 100 ng / mL shell enzyme protein stock solution 10 times in sequence (100 ng / mL, 10 ng / mL, 1 ng / mL, 100 pg / mL, 10 pg / mL and 1 pg / mL), and drop it onto the sample pad of the test strip. Use the cell culture supernatant as a negative control and the shell enzyme protein dilution as a blank control to observe whether a T line appears and establish the sensitivity of the test strip.

[0057] The test results showed that when the shell enzyme protein was diluted to 1pg / mL, only the C line appeared, no T line appeared, and the test was negative; when the shell enzyme protein was diluted to 10pg / mL, both the C line and the T line appeared at the same time, and the test was positive. It can be seen that the detection line can reach 10pg / mL, indicating that the prepared early warning test strip for liver fibrosis has a high sensitivity.

[0058] (2) Specificity of early warning test strips for liver fibrosis

[0059] 100 pg / mL of chitin, laminin, interleukin-4 and β-actin protein were dripped onto the sample pad of the test strip in sequence.

[0060] The test results showed that only the test strip with the addition of shell enzyme protein had both T line and C line appear, indicating a positive result, while the rest of the strips had only C line appearing, indicating a negative result. This test result indicated that the prepared test strip had good specificity.

[0061] (3) Stability testing of early warning test strips for liver fibrosis

[0062] The test strips were placed in an aluminum foil bag with desiccant, evacuated and stored at 4°C. The test strips were taken out for testing at 1, 30 and 60 days to compare the color intensity.

[0063] The results showed that after 60 days of low-temperature storage at 4°C, the color intensity of the T line and C line of the test strip remained basically unchanged, indicating that the test strip has strong resistance to low temperature and can be used for low-temperature storage.

[0064] It is necessary to point out that the above embodiments are limited to further elaboration and explanation of the technical solution of the present invention, and are not further limitations of the technical solution of the present invention. The method of the present invention is only a preferred implementation scheme, and is not used to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An early warning test strip for liver fibrosis, characterized in that: The test strip comprises a PVC bottom plate, a sample pad, a conjugation pad, a chromatographic membrane and a water-absorbing pad, wherein the chromatographic membrane, the water-absorbing pad, the conjugation pad and the sample pad are sequentially overlapped on the PVC bottom plate; a detection line T and a quality control line C are arranged on the chromatographic membrane, wherein the quality control line C is coated with a sheep anti-mouse IgG antibody, and the detection line T is coated with a monoclonal antibody 2 that specifically recognizes the shell enzyme protein; the detection line T is arranged at one end close to the conjugation pad, and the quality control line C is arranged at one end close to the water-absorbing pad; the conjugation pad is coated with a fluorescently labeled monoclonal antibody 1 that specifically recognizes the shell enzyme protein, wherein the monoclonal antibody 1 comprises a heavy chain variable region and a light chain variable region, and CDR1-3 of the heavy chain variable region are respectively as shown in: SEQ ID NO.1-3, and CDR4-6 of the light chain variable region are respectively as shown in: SEQ ID NO.4-6; the monoclonal antibody 2 comprises a heavy chain variable region and a light chain variable region, and CDR1-3 of the heavy chain variable region are respectively as shown in: SEQ ID As shown in SEQ ID NO.9-11, the monoclonal antibody 2 includes a heavy chain variable region and a light chain variable region, and CDR4-6 of the light chain variable region are shown in SEQ ID NO.12-14 respectively; Among them, the preparation of fluorescently labeled monoclonal antibody 1 that specifically recognizes shell enzyme protein: take 150 μL of fluorescent microspheres, activate them with EDC.HCl and Sulfo-NHS, and then add 300 μg of monoclonal antibody 1 of shell enzyme protein thereto, react at room temperature for 12 hours, then centrifuge, place in a suspension, and store at 4°C to obtain the preparation of fluorescently labeled monoclonal antibody 1 that specifically recognizes shell enzyme protein, which is set aside.

2. The method for preparing the early warning test strip for liver fibrosis according to claim 1, characterized in that: The method comprises the following steps: (1) Preparation of the binding pad: soaking the binding pad in a binding pad blocking solution, and then drying it for later use; diluting a solution of a fluorescently labeled monoclonal antibody 1 that specifically recognizes the shell enzyme protein and spraying it evenly on the binding pad, and drying it to prepare a binding pad for later use; (2) Preparation of sample pad: soak the sample pad in sample pad blocking solution and dry it for later use; (3) Preparation of nitrocellulose membrane: Monoclonal antibody 2 that specifically recognizes the chitin protein is added to the coating solution and mixed, and then sprayed on the detection line T of the nitrocellulose membrane. Goat anti-mouse IgG antibody is sprayed on the nitrocellulose membrane at an interval of 10 mm from the detection line as the quality control line C. The nitrocellulose membrane is dried for later use; (4) Assembly of the test strip: The sample pad, conjugate pad, chromatographic membrane, and absorbent pad are sequentially connected on a PVC base plate to obtain a test strip, wherein the detection line T is set at one end close to the conjugate pad, and the quality control line C is set at the other end close to the absorbent pad.

3. The preparation method according to claim 2, characterized in that: The conjugate pad blocking solution contains 5-10wt% trehalose, 5-10wt% BSA, 0.75-1.25wt% Tween-20, and 0.5-1.5wt% Triton X-100 in boric acid solution.

4. The preparation method according to claim 2, characterized in that: The sample pad blocking solution contains 0.75-1.5wt% Tween-20, 2-5wt% PEG20000, and 2-5wt% BSA in boric acid solution.

5. The preparation method according to claim 2, characterized in that: The coating solution contains 0.9-2.5wt% NaCl, 0.2-1.0wt% NaH2PO4, and 3-6wt% sucrose in a phosphate solution.

6. An early warning kit for liver fibrosis, characterized in that: The kit comprises the following components: an early warning test strip for liver fibrosis according to any one of claims 1 to 2, a conjugated pad blocking solution, a sample pad blocking solution and a coating solution; wherein the conjugated pad blocking solution contains a boric acid solution of 5-10wt% trehalose, 5-10wt% BSA, 0.75-1.25wt% Tween-20 and 0.5-1.5wt% Triton X-100; the sample pad blocking solution contains a boric acid solution of 0.75-1.5wt% Tween-20, 2-5wt% PEG20000 and 2-5wt% BSA; and the coating solution contains a phosphate solution of 0.9-2.5wt% NaCl, 0.2-1.0wt% NaH2PO4 and 3-6wt% sucrose.