Immunochromatography reagent for detecting antiproteinase 3 antibody and application

Abstract

The invention relates to the technical field of immunodetection, and discloses an immunochromatography reagent for detecting an antiproteinase 3 antibody and application, which are used for detecting the antiproteinase 3 antibody in human serum, plasma or whole blood and assisting in diagnosing wegener granuloma. The sample pad is used for bearing a to-be-detected sample; the combination pad is coated with an anti-human immune globulin antibody or an anti-human IgG antibody marked by a marker; the detection line is coated with a recombinant protease 3 antigen, myeloperoxidase antigen fragments related to Wegner granuloma attack are added, and hydrophilic polysaccharides are adopted as spacer arms to be arranged at intervals; the quality control line is coated with a goat anti-mouse IgG antibody; the water absorption pad is used for absorbing liquid in the chromatography process and promoting the liquid to flow unidirectionally; the bottom plate is used for bearing all the components, and all the components are sequentially and mutually assembled on the bottom plate to form a detection test strip; the immunochromatography reagent is extremely simple and convenient to operate, professional laboratory equipment and complex operation skills are not needed, and a detection result can be obtained only by dropwise adding a sample onto the test strip.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and particularly relates to an immunochromatographic reagent for detecting anti-proteinase 3 antibody and its application. Background Art

[0002] Anti-proteinase 3 antibody (anti-proteinase 3 antibody, PR3-ANCA) is an autoantibody that is closely related to various autoimmune diseases, especially Wegener's granulomatosis (WG). Wegener's granulomatosis is a necrotizing granulomatous vasculitis that can affect multiple systems throughout the body, such as the respiratory tract, kidneys, eyes, etc. If not diagnosed and treated in a timely manner, the disease progresses rapidly and the prognosis is poor. Currently, the diagnosis of this disease depends on a comprehensive judgment of clinical symptoms, pathological biopsy, and various laboratory indicators, and the detection of anti-proteinase 3 antibody is of great significance for its early diagnosis, disease monitoring, and prognosis evaluation; traditional methods for detecting anti-proteinase 3 antibody, such as enzyme-linked immunosorbent assay (ELISA), although having high sensitivity and specificity, are cumbersome to operate, time-consuming, require professional laboratory equipment and technical personnel, and are difficult to meet the needs of rapid clinical diagnosis. Summary of the Invention

[0003] The purpose of the present invention is to solve the above problems and design an immunochromatographic reagent for detecting anti-proteinase 3 antibody and its application.

[0004] The first aspect of the present invention provides an immunochromatographic reagent for detecting anti-proteinase 3 antibody, and the immunochromatographic reagent for detecting anti-proteinase 3 antibody includes a sample pad, a conjugate pad, a test line, a control line, an absorbent pad, and a bottom plate. Among them,

[0005] The sample pad is used to carry the sample to be detected;

[0006] The conjugate pad is coated with an anti-human immunoglobulin antibody or anti-human IgG antibody labeled with a label;

[0007] The test line is coated with recombinant proteinase 3 antigen, and a myeloperoxidase antigen fragment related to the pathogenesis of Wegener's granulomatosis is added, and is arranged at intervals using a hydrophilic polysaccharide as a spacer arm;

[0008] The control line is coated with goat anti-mouse IgG antibody;

[0009] The absorbent pad is used to absorb the liquid during the chromatography process and promote the unidirectional flow of the liquid;

[0010] The bottom plate is used to carry the above components, and the components are sequentially assembled on the bottom plate to form a test strip.

[0011] Optionally, in the first implementation manner of the first aspect of the present invention, thermosensitive nanogels are embedded in the sample pad and the conjugate pad.

[0012] Optionally, in the second implementation manner of the first aspect of the present invention, the label is one of colloidal gold, quantum dots, latex microspheres or fluorescent microspheres.

[0013] Optionally, in the third implementation manner of the first aspect of the present invention, the method for preparing the immunochromatographic reagent for detecting anti-proteinase 3 antibody includes the following steps:

[0014] S1. Preparation of recombinant proteinase 3 antigen: By gene cloning technology, the gene fragment encoding proteinase 3 is introduced into Pichia pastoris for expression, and the expression product is purified by affinity chromatography and ion exchange chromatography methods to obtain high-purity recombinant proteinase 3 antigen;

[0015] S2. Preparation of the conjugate pad: Dilute the anti-human immunoglobulin antibody or anti-human IgG antibody labeled with the label with a buffer solution, and uniformly spray it on the nitrocellulose membrane by microfluidic spraying technology, and set aside after drying.

[0016] S3. Coating of the test line and the control line: Dilute the recombinant proteinase 3 antigen and goat anti-mouse IgG antibody with coating buffer respectively, and use a membrane scribing instrument to uniformly coat them on the nitrocellulose membrane as the test line and the control line at a certain line speed and spraying volume, and set aside after drying.

[0017] S4. Assembly of the test strip: Adopt a continuous microfluidic chip-based preparation process. In the microfluidic chip, regulate the flow rate, temperature and pressure of the microfluid, drive each component to complete immobilization and assembly in the chip area in sequence, and automatically construct a complete test strip structure integrating sample introduction, reaction, detection and absorption functions to obtain the test strip.

[0018] Optionally, in the fourth implementation manner of the first aspect of the present invention, the purification process in step S1 includes the following steps:

[0019] Collect the fermentation broth after induced expression, centrifuge to obtain the supernatant, filter it with a 0.45 μm filter membrane, load the filtered supernatant onto a nickel affinity chromatography column at a flow rate of 1 mL / min, wash the column with 5 column volumes of equilibration buffer to remove non-specifically bound impurities, elute the target protein with elution buffer, and collect the elution peak;

[0020] Dialyze the elution peak collected by affinity chromatography against the ion exchange starting buffer overnight, load the dialyzed sample onto an anion exchange chromatography column at a flow rate of 1 mL / min, wash the column with 5 column volumes of the starting buffer, and then elute with a linear gradient elution buffer to collect the target protein peak;

[0021] The purified recombinant protease 3 antigen was analyzed by SDS-PAGE electrophoresis, stained with Coomassie Brilliant Blue, and the purity of the protein band was observed. Through the analysis of the gel imaging system, the purity of the target protein should reach more than 95%.

[0022] Optionally, in the fifth implementation manner of the first aspect of the present invention, in step S2, the labeled antibody solution is diluted to 0.5 mg / mL with PBS buffer containing 2% sucrose and 0.1% Triton X-100. Using a microfluidic spraying device, the diluted labeled antibody solution is evenly sprayed on the nitrocellulose membrane. The spraying pressure is 20 psi, the spraying speed is 10 μL / cm, the spraying temperature is 25 °C, and the relative humidity is 40%.

[0023] Optionally, in the sixth implementation manner of the first aspect of the present invention, the specific process of step S3 includes: diluting the recombinant protease 3 antigen to 1 mg / mL with coating buffer, and diluting the goat anti-mouse IgG antibody to 0.5 mg / mL with coating buffer; using a membrane scribing instrument to coat the diluted recombinant protease 3 antigen and goat anti-mouse IgG antibody on the nitrocellulose membrane as the test line and the quality control line respectively. The membrane scribing line speed is 1 μL / cm, the spraying volume is 0.5 μL / cm, the membrane scribing temperature is 25 °C, and the relative humidity is 40%.

[0024] Optionally, in the seventh implementation manner of the first aspect of the present invention, the specific process of step S4 includes: installing the prepared sample pad, conjugate pad, coated nitrocellulose membrane and absorbent pad at the corresponding positions of the microfluidic chip respectively, regulating the microfluidic flow rate to 0.5 μL / s, the temperature to 30 °C, and the pressure to 200 mbar. Fix the sample pad at the entrance of the sample introduction channel, then overlap the conjugate pad with the sample pad and fix it at the front end of the reaction chamber. Then overlap the coated nitrocellulose membrane with the conjugate pad and fix it in the detection area. Finally, overlap the absorbent pad with the nitrocellulose membrane and fix it in the absorption area. After the assembly is completed, take out the microfluidic chip from the device and cut the chip into test strips with a width of 4 mm using a cutting machine to obtain the detection test strips.

[0025] The second aspect of the present invention provides an application of an immunochromatographic reagent for detecting anti - protease 3 antibody, which is used to detect anti - protease 3 antibody in human serum, plasma or whole blood to assist in the diagnosis of Wegener's granulomatosis. The specific detection method is as follows: Drop the patient sample onto the sample pad of the test strip. Due to capillary action, the sample chromatographs forward along the test strip. If the sample contains anti - protease 3 antibody, this antibody first binds to the anti - human immunoglobulin antibody or anti - human IgG antibody labeled with a marker on the conjugate pad to form an antibody - marker complex. As the chromatographic process proceeds, this complex moves to the test line and specifically binds to the recombinant protease 3 antigen coated on the test line, causing the marker to aggregate at the test line to present a visible band to the naked eye, indicating that the sample is positive. If there is no anti - protease 3 antibody in the sample, no band appears at the test line.

[0026] As can be seen from the above solution, the immunochromatographic reagent of the present invention, with its unique multi - antigen composite test line design, significantly broadens the diagnostic scope, screens multiple key antibodies at one time, effectively avoids missed diagnosis caused by single - antigen detection, and greatly improves the diagnostic accuracy; the application of the self - calibrating marker system and intelligent response materials greatly enhances the accuracy and stability of detection, calibrates the interference of sample differences in real - time, and ensures reliable results; it accelerates the immune reaction, shortens the detection time, and meets the urgent need for rapid clinical diagnosis; the innovative preparation process, especially the continuous microfluidic chip - type preparation and biomimetic surface modification technology, realizes efficient and automated production, improves the product quality consistency, extends the shelf life of the reagent at the same time, reduces the production cost and the difficulty of storage and transportation; the extended multi - application mode deeply integrates mobile medical treatment, remote diagnosis and disease dynamic monitoring, breaks the time - space limitation of medical treatment, provides convenient and efficient whole - process medical services for patients, significantly improves the utilization efficiency of medical resources, and promotes the implementation of precision medicine. Brief Description of the Drawings

[0027] By reading the detailed description of the preferred embodiments below, various other advantages and benefits will become clear to those of ordinary skill in the art. The drawings are only for the purpose of showing the preferred embodiments and are not considered to be a limitation of the present invention.

[0028] Figure 1 It is a schematic diagram of the preparation method of the immunochromatographic reagent for detecting anti - protease 3 antibody provided by the embodiment of the present invention. Detailed Embodiments

[0029] In the description, claims and the above drawings of the present invention, the terms "first", "second", "third", "fourth", etc. (if any) are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged under appropriate circumstances so that the embodiments described herein can be implemented in an order other than that illustrated or described herein. In addition, the term "comprising" or "having" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, device, product or equipment comprising a series of steps or units need not be limited to those steps or units clearly listed, but may include other steps or units not clearly listed or inherent to these processes, methods, products or equipment.

[0030] For ease of understanding, the specific process of the embodiment of the present invention is described below. The immunochromatographic reagent for detecting anti-proteinase 3 antibody comprises a sample pad, a conjugate pad, a test line, a control line, an absorbent pad and a base plate. Among them, the sample pad is used to carry the sample to be detected; the conjugate pad is coated with an anti-human immunoglobulin antibody or an anti-human IgG antibody labeled with a label; the test line is coated with recombinant proteinase 3 antigen, and an antigen fragment related to the pathogenesis of Wegener's granulomatosis, myeloperoxidase antigen fragment, is added, and hydrophilic polysaccharide is used as a spacer arm for spaced arrangement; the control line is coated with goat anti-mouse IgG antibody; the absorbent pad is used to absorb the liquid during the chromatography process to promote the unidirectional flow of the liquid; the base plate is used to carry the above components, and the components are sequentially assembled on the base plate to form a test strip; a thermosensitive nanogel is embedded in the sample pad and the conjugate pad; the label is one of colloidal gold, quantum dots, latex microspheres or fluorescent microspheres.

[0031] In this embodiment, on the test line, in addition to coating the recombinant proteinase 3 antigen, other key antigen fragments related to the pathogenesis of Wegener's granulomatosis, such as myeloperoxidase (MPO) antigen fragments, are additionally added. Because there may be antibodies against multiple autoantigens in the bodies of some patients, this composite test line can screen multiple key antibodies at one time, improve the comprehensiveness of the diagnosis of complex conditions, and reduce the risk of missed diagnosis. By optimizing the ratio of antigens and the immobilization process, it is ensured that the activities of each antigen are not affected and can accurately identify the corresponding antibodies; a spacer molecule is used to separate different antigens to prevent interference between antigens. For example, hydrophilic polysaccharide is used as a spacer arm to make the antigens be orderly distributed on the test line, further improving the accuracy and specificity of the detection.

[0032] In this embodiment, nano-intelligent materials, such as thermosensitive nano-gels, are embedded in the sample pad or conjugate pad. When the ambient temperature is within the normal body temperature range (36 - 37 °C), the nano-gel will undergo slight volume changes or surface property alterations, prompting the anti-proteinase 3 antibody in the sample to bind more efficiently to the labeled antibody, accelerating the immune reaction process, shortening the detection time, and hopefully reducing the original detection duration of 10 - 15 minutes to 5 - 8 minutes. Meanwhile, this temperature-responsive property can also serve as an internal quality control indicator. If the detection ambient temperature deviates too much from the normal range, the credibility of the detection result will be automatically alerted and reduced.

[0033] In this embodiment, the internal reference marker and the anti-human immunoglobulin antibody or anti-human IgG antibody are co-labeled on the conjugate pad. The internal reference marker can specifically bind to a commonly present and relatively stable endogenous protein in the sample (such as albumin). During the detection process, by comparing the signal intensities of the test line, control line, and internal reference marker line, the detection deviation caused by sample differences (such as viscosity, protein content, etc.) can be calibrated in real time, making the detection result more accurate and reliable, especially suitable for some complex sample sources, such as blood samples from patients with hyperlipidemia.

[0034] Please refer to Figure 1 The schematic diagram of the preparation method of the immunochromatographic reagent for detecting anti-proteinase 3 antibody provided by the embodiment of the present invention, and this method specifically includes the following steps:

[0035] S1. Preparation of recombinant proteinase 3 antigen: Through gene cloning technology, the gene fragment encoding proteinase 3 is introduced into Pichia pastoris for expression, and the expression product is purified by affinity chromatography and ion exchange chromatography methods to obtain high-purity recombinant proteinase 3 antigen;

[0036] S2. Preparation of conjugate pad: The anti-human immunoglobulin antibody or anti-human IgG antibody labeled with a marker is diluted with a buffer solution and evenly sprayed on the nitrocellulose membrane by microfluidic spraying technology, and then dried for later use.

[0037] S3. Coating of test line and control line: The recombinant proteinase 3 antigen and goat anti-mouse IgG antibody are respectively diluted with a coating buffer solution, and a membrane scribing instrument is used to evenly coat them on the nitrocellulose membrane at a certain line speed and spraying volume as the test line and control line, and then dried for later use.

[0038] S4. Assembly of test strip: Adopting a continuous microfluidic chip-based preparation process, within the microfluidic chip, the flow rate, temperature, and pressure of the microfluid are regulated to drive each component to complete immobilization and assembly in the chip area in sequence, automatically constructing a complete test strip structure integrating functions of sample introduction, reaction, detection, and absorption to obtain the detection test strip.

[0039] In this embodiment, the purification process in step S1 includes the following steps: collecting the fermentation broth after induced expression, centrifuging to obtain the supernatant, filtering with a 0.45 μm filter membrane, loading the filtered supernatant onto a nickel affinity chromatography column at a flow rate of 1 mL / min, washing the column with 5 column volumes of equilibration buffer to remove impurities with non-specific binding, eluting the target protein with elution buffer, and collecting the elution peak; dialyzing the elution peak collected by affinity chromatography against the starting buffer for ion exchange using a dialysis bag overnight, loading the dialyzed sample onto an anion exchange chromatography column at a flow rate of 1 mL / min, washing the column with 5 column volumes of the starting buffer, and then eluting with a linear gradient elution buffer to collect the target protein peak; performing SDS-PAGE electrophoresis analysis on the purified recombinant protease 3 antigen, staining with Coomassie Brilliant Blue, observing the purity of the protein band, and analyzing through a gel imaging system. The purity of the target protein should reach over 95%.

[0040] In this embodiment, in step S2, the labeled antibody solution is diluted to 0.5 mg / mL with PBS buffer containing 2% sucrose and 0.1% Triton X-100. Using a microfluidic spraying device, the diluted labeled antibody solution is evenly sprayed onto a nitrocellulose membrane. The spraying pressure is 20 psi, the spraying speed is 10 μL / cm, the spraying temperature is 25 °C, and the relative humidity is 40%.

[0041] In this embodiment, the specific process of step S3 includes: diluting the recombinant protease 3 antigen to 1 mg / mL with coating buffer, and diluting the goat anti-mouse IgG antibody to 0.5 mg / mL with coating buffer; using a membrane scribing instrument to coat the diluted recombinant protease 3 antigen and goat anti-mouse IgG antibody onto the nitrocellulose membrane as the test line and the quality control line respectively. The membrane scribing line speed is 1 μL / cm, the spraying volume is 0.5 μL / cm, the membrane scribing temperature is 25 °C, and the relative humidity is 40%.

[0042] In this embodiment, the specific process of step S4 includes: installing the prepared sample pad, conjugate pad, coated nitrocellulose membrane, and absorbent pad at the corresponding positions of the microfluidic chip, regulating the microfluidic flow rate to 0.5 μL / s, the temperature to 30 °C, and the pressure to 200 mbar. Fix the sample pad at the entrance of the sample introduction channel, then overlap the conjugate pad with the sample pad and fix it at the front end of the reaction chamber. Then overlap the coated nitrocellulose membrane with the conjugate pad and fix it in the detection area. Finally, overlap the absorbent pad with the nitrocellulose membrane and fix it in the absorption area. After assembly, take out the microfluidic chip from the device and cut the chip into test strips with a width of 4 mm using a cutting machine to obtain the detection test strips.

[0043] The present invention is further illustrated by the following detailed embodiments, but the scope of the present invention is not limited thereto.

[0044] Example 1: Antigen Preparation and Optimization

[0045] Expression of recombinant proteinase 3 antigen and MPO antigen: The optimized codon proteinase 3 and MPO genes were cloned into the Pichia pastoris expression vector pPIC9K respectively to construct recombinant plasmids pPIC9K-PR3 and pPIC9K-MPO, which were introduced into Pichia pastoris GS115 competent cells by electrotransformation. Positive transformants were screened using MD and MM plates. Single colonies were picked and inoculated into BMGY medium, and cultured with shaking at 30 °C and 250 rpm until the OD600 was about 2 - 3. The cells were collected by centrifugation, resuspended with BMMY medium, and induced to express for 72 h by adding 0.5% methanol. Methanol was replenished every 24 h during the period;

[0046] Protein purification: The supernatant was taken by centrifuging the fermentation broth, and two-step purification was carried out successively through nickel affinity chromatography (eluting the impurity proteins with PBS buffer containing 20 mM imidazole and the target protein with 250 mM imidazole) and size exclusion chromatography (selecting a gel column with an appropriate cut-off molecular weight and eluting with PBS buffer). The target peak was collected, and small molecule impurities such as imidazole were removed by dialysis. The purity of proteinase 3 antigen and MPO antigen was identified by SDS-PAGE electrophoresis to be over 95%, and their immunogenicity was verified by immunoblotting.

[0047] Example 2: Preparation of Immunochromatographic Test Strip

[0048] Preparation of conjugate pad: Quantum dots with a particle size of 30 nm were prepared, modified with mercaptoethylamine, and then conjugated with anti-human immunoglobulin antibody, anti-human IgG antibody and internal reference quantum dot label (specifically binding to albumin), and mixed in a ratio of 2:2:1. It was diluted to 0.5 mg / mL with PBS buffer containing 5% trehalose derivative, and uniformly sprayed on the glass cellulose membrane at a rate of 8 μL / cm 2 by a microfluidic spraying system and dried in an environment of 30 °C and 50% humidity for 3 h;

[0049] Coating of test line and control line: The nitrocellulose membrane was modified by biomimetic method to construct a sugar-containing polymer brush. The composite antigen obtained in Example 1 was diluted to 1.2 mg / mL with 0.01 M PBS (pH 7.4) buffer containing 0.1% polyelectrolyte (which can fine-tune antigen immobilization in response to humidity), and the goat anti-mouse IgG antibody was diluted to 0.6 mg / mL. The test line and control line were coated with an automated membrane scribing instrument at a line speed and spraying volume of 1.5 μL / cm, and dried at 40 °C for 2 h;

[0050] Test strip assembly: Using a continuous microfluidic chip-based preparation process, raw materials such as sample pads, conjugate pads, coated nitrocellulose membranes, and absorbent pads are injected into the microfluidic chip according to a preset program. Driven by a microelectric field, the liquid flows through each functional area in sequence to complete in-situ immobilization and assembly, resulting in an integrated test strip. The temperature inside the chip is controlled at 30 °C, the flow rate is 0.5 μL / s, and the pressure is 200 mbar. The test strip is cut into 5 mm wide strips and loaded into a special plastic cartridge, which contains a micro sample premixing pool and a detection slot.

[0051] Example 3: Comprehensive evaluation of reagent performance

[0052] Sensitivity and specificity detection: Dilute the known concentration anti-proteinase 3 antibody standard and positive serum samples containing other autoantibodies (ANA, dsDNA, etc.) in a serial dilution with PBS buffer. Starting from 5 U / mL anti-proteinase 3 antibody, take 100 μL of each and drop it onto the test strip. Repeat 15 times for each concentration and observe the signal changes of the test line and the internal reference line. The results show that the sensitivity of the reagent of the present invention is as low as 5 U / mL, there is no cross-reaction with other autoantibodies, and the specificity reaches 98%;

[0053] Stability evaluation: Place the test strips in environments of 4 °C, 25 °C, and 37 °C respectively, and take out 100 U / mL anti-proteinase 3 antibody standard for detection every month for 9 months. Under the condition of 4 °C, the detection performance is stable for 9 months; at 25 °C, the results are reliable within 7 months; at 37 °C high temperature, good detection results are maintained within 4 months, far exceeding traditional reagents.

[0054] Adaptability test of complex samples: Collect blood samples from patients with hyperlipidemia and hyperbilirubinemia and control samples from healthy people, and detect anti-proteinase 3 antibody respectively. The results show that even in the face of complex samples, the reagent of the present invention is accurately calibrated by the self-calibrating marker system, and the detection results are accurate compared with healthy samples, without false positives or false negatives.

[0055] Example 4: Clinical multi-application practice

[0056] Point-of-care testing: Collect 50 samples from suspected autoimmune disease patients, and medical staff use the test strips of the present invention for on-site detection and compare with the ELISA method. The results show that the coincidence rate of point-of-care diagnosis reaches 94%, the positive coincidence rate is 92%, the negative coincidence rate is 95%, and the detection is completed within 10 minutes, effectively assisting clinical rapid decision-making;

[0057] Mobile healthcare and remote diagnosis: Recruit 30 volunteers in remote areas to operate the test strips at home by themselves and transmit the results to the remote medical center using the supporting intelligent terminal. Compare the remote diagnosis of doctors with the subsequent local confirmed results, and the coincidence rate is 90%, proving the feasibility of remote diagnosis and facilitating medical care in remote areas;

[0058] Disease Dynamic Monitoring and Early Warning: Twenty patients diagnosed with Wegener's granulomatosis were selected and wore wearable micro blood sample collection devices for 2 weeks. During this period, the detection terminal captured abnormal fluctuations in anti-proteinase 3 antibodies in 3 patients, giving an early warning. Medical staff intervened and adjusted the treatment in a timely manner, and the patients' conditions were stable, demonstrating the advantages of dynamic monitoring.

[0059] Through the implementation of the above solution, the immunochromatographic reagent of the present invention is extremely easy to operate, without the need for professional laboratory equipment and complex operation skills. Just drop the sample onto the test strip, and the test result can be obtained by naked eye observation or a supporting reader within a few minutes. It is applicable to various scenarios such as primary medical institutions, emergency rooms, bedside testing, etc., achieving rapid point-of-care testing; using recombinant proteinase 3 antigen as the detection line coating, it has strong specificity and can effectively avoid cross-reaction with other antibodies, ensuring high specificity of the detection; combined with highly sensitive antibodies labeled with markers, the overall detection sensitivity can meet the requirements of clinical early diagnosis, contributing to the early detection and early treatment of diseases such as Wegener's granulomatosis; the reagent has good stability and can be stored at room temperature for a long time, facilitating storage and transportation, reducing the use cost and dependence on cold chain logistics, and being easy to promote and apply.

[0060] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and descriptions in the specification are only preferred examples of the present invention and are not used to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. An immunochromatographic reagent for detecting anti - protease 3 antibody and its application, characterized in that, The immunochromatographic reagent for detecting anti-proteinase 3 antibody comprises a sample pad, a conjugate pad, a test line, a control line, an absorbent pad and a base plate. Among them, The sample pad is used to carry the sample to be detected; The conjugate pad is coated with an anti-human immunoglobulin antibody or an anti-human IgG antibody labeled with a label; The test line is coated with recombinant proteinase 3 antigen, and a myeloperoxidase antigen fragment related to the pathogenesis of Wegener's granulomatosis is added, and is arranged at intervals with a hydrophilic polysaccharide as a spacer arm; The control line is coated with goat anti-mouse IgG antibody; The absorbent pad is used to absorb the liquid during the chromatography process and promote the unidirectional flow of the liquid; The base plate is used to carry the above components, and the components are sequentially assembled on the base plate to form a test strip.

2. The immunochromatographic reagent for detecting anti - protease 3 antibody according to claim 1, characterized in that, A thermosensitive nanogel is embedded in the sample pad and the conjugate pad.

3. An immunochromatographic reagent for detecting anti-proteinase 3 antibody according to claim 1, characterized in that, The label is one of colloidal gold, quantum dots, latex microspheres or fluorescent microspheres.

4. The immunochromatographic reagent for detecting anti-proteinase 3 antibody according to claim 1, characterized in that, The preparation method of the immunochromatographic reagent for detecting anti-proteinase 3 antibody comprises the following steps: S1. Preparation of recombinant proteinase 3 antigen: By gene cloning technology, the gene fragment encoding proteinase 3 is introduced into Pichia pastoris for expression, and the expression product is purified by affinity chromatography and ion exchange chromatography methods to obtain a high-purity recombinant proteinase 3 antigen; S2. Preparation of the conjugate pad: Dilute the anti-human immunoglobulin antibody or anti-human IgG antibody labeled with a label with a buffer solution, and uniformly spray it on a nitrocellulose membrane by microfluidic spraying technology, and reserve it after drying treatment. S3. Coating of the test line and the control line: Dilute the recombinant proteinase 3 antigen and goat anti-mouse IgG antibody with a coating buffer solution respectively, and use a membrane scribing instrument to uniformly coat them on the nitrocellulose membrane at a certain line speed and spraying volume as the test line and the control line, and reserve them after drying. S4. Assembly of the test strip: Adopt a continuous microfluidic chip-based preparation process. In the microfluidic chip, regulate the flow rate, temperature and pressure of the microfluid, drive each component to complete immobilization and assembly in the chip area in turn, and automatically construct a complete test strip structure integrating sample introduction, reaction, detection and absorption functions to obtain a test strip.

5. An immunochromatographic reagent for detecting anti-proteinase 3 antibody according to claim 4, characterized in that, The purification process in the step S1 includes the following steps: Collect the fermentation broth after induced expression, centrifuge to take the supernatant, filter it with a 0.45μm filter membrane, load the filtered supernatant onto a nickel affinity chromatography column at a flow rate of 1mL / min, wash the column with 5 column volumes of equilibration buffer to remove non-specifically bound impurities, elute the target protein with elution buffer, and collect the elution peak; Dialyze the elution peak collected by affinity chromatography against the ion exchange starting buffer overnight in a dialysis bag, load the dialyzed sample onto an anion exchange chromatography column at a flow rate of 1mL / min, wash the column with 5 column volumes of the starting buffer, and then elute with a linear gradient elution buffer to collect the target protein peak; Perform SDS-PAGE electrophoresis analysis on the purified recombinant proteinase 3 antigen, stain it with Coomassie brilliant blue, observe the purity of the protein band, and analyze it by a gel imaging system. The purity of the target protein should reach more than 95%.

6. The immunochromatographic reagent for detecting anti-proteinase 3 antibody according to claim 4, wherein In step S2, the labeled antibody solution is diluted to 0.5 mg / mL with PBS buffer containing 2% sucrose and 0.1% Triton X-100. Using a microfluidic spraying device, the diluted labeled antibody solution is evenly sprayed on the nitrocellulose membrane. The spraying pressure is 20 psi, the spraying speed is 10 μL / cm, the spraying temperature is 25°C, and the relative humidity is 40%.

7. The immunochromatographic reagent for detecting anti - protease 3 antibody according to claim 4, wherein The specific process of step S3 includes: diluting the recombinant protease 3 antigen to 1 mg / mL with coating buffer and diluting the goat anti-mouse IgG antibody to 0.5 mg / mL with coating buffer; using a membrane scribing instrument to coat the diluted recombinant protease 3 antigen and goat anti-mouse IgG antibody on the nitrocellulose membrane as the test line and the quality control line respectively. The membrane scribing line speed is 1 μL / cm, the spraying volume is 0.5 μL / cm, the membrane scribing temperature is 25°C, and the relative humidity is 40%.

8. The immunochromatographic reagent for detecting anti-proteinase 3 antibody according to claim 4, wherein The specific process of step S4 includes: installing the prepared sample pad, conjugate pad, coated nitrocellulose membrane and absorbent pad at the corresponding positions of the microfluidic chip respectively, regulating the microfluidic flow rate to 0.5 μL / s, the temperature to 30°C, and the pressure to 200 mbar. Fix the sample pad at the entrance of the sample introduction channel, then overlap the conjugate pad with the sample pad and fix it at the front end of the reaction chamber. Then overlap the coated nitrocellulose membrane with the conjugate pad and fix it in the detection area. Finally, overlap the absorbent pad with the nitrocellulose membrane and fix it in the absorption area. After assembly, take out the microfluidic chip from the device and cut the chip into test strips with a width of 4 mm using a cutting machine to obtain the test strip for detection.

9. Use of an immunochromatographic reagent for detecting anti-proteinase 3 antibody, characterized in that, It is used to detect anti-protease 3 antibodies in human serum, plasma or whole blood to assist in the diagnosis of Wegener's granulomatosis. The specific detection method is as follows: Drop the patient sample onto the sample pad of the test strip. Due to capillary action, the sample chromatographs forward along the test strip. If the sample contains anti-protease 3 antibodies, the antibody first binds to the anti-human immunoglobulin antibody or anti-human IgG antibody labeled with a marker on the conjugate pad to form an antibody-marker complex. As the chromatographic process proceeds, the complex moves to the test line and specifically binds to the recombinant protease 3 antigen coated on the test line, causing the marker to aggregate at the test line to present a visible band to the naked eye, indicating that the sample is positive. If there is no anti-protease 3 antibody in the sample, no band will appear at the test line.

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