An antibody against collagen type iv α3, a detection kit and application thereof

Abstract

The application discloses a kind of antibody for detecting collagen type IV alpha 3, detection kit and its application, belong to immunodetection technical field.Wherein, the antibody for detecting collagen type IV alpha 3 is COL4A3 monoclonal antibody, the COL4A3 monoclonal antibody can specifically recognize collagen type IV alpha 3, such as the protein fragment shown in SEQ ID NO:9.The application further discloses an antibody combination comprising the COL4A3 monoclonal antibody.The application further discloses the application of the COL4A3 monoclonal antibody or antibody combination in the preparation of collagen type IV detection kit and the kit prepared by the same.The kit of the application has very great clinical application value for detecting Alport syndrome with high specificity and strong sensitivity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of immunodetection technology, and particularly relates to an antibody of collagen type IV α3, a detection kit and application thereof. BACKGROUND

[0002] Hereditary nephritis, i.e. Alport syndrome (AS), is a hereditary glomerular basement membrane disease mainly manifested as hematuria, progressive reduction of renal function, sensorineural hearing loss and ocular abnormalities, and is a disease caused by mutation of a gene encoding a main collagen component of glomerular basement membrane, i.e. collagen type IV. The incidence of gene mutation is about 1 / 10000-1 / 5000. According to the genetic mode, it can be divided into: ① X-linked dominant (XL, about 80%), the pathogenic gene is on the X chromosome, and the inheritance is related to gender. ② autosomal recessive (AR; about 15%), the pathogenic gene is on an autosome. ③ autosomal dominant (AD; a very small number). The three genetic modes are caused by mutations of genes COL4A5, COL4A6, COL4A3 and / or COL4A4 encoding different α chains of collagen type IV, respectively.

[0003] The pathogenesis of hereditary nephritis is that six α chains of collagen type IV are polymerized into three triple helix molecular structures called monomers, the monomers are polymerized into dimers or tetramers, and then the dimers or tetramers are intertwined to form a collagen network structure. The gene mutation of X-linked dominant Alport syndrome mainly occurs in the gene (COL4A5) encoding the α5 chain of collagen type IV. The gene mutation of autosomal recessive Alport syndrome occurs in the gene (COL4A3 / COL4A4) encoding the α3 chain or the α4 chain of collagen type IV on chromosome 2, while the genes of the α1 chain of collagen type IV or the α2 chain of collagen type IV are normally expressed, i.e. the α1 chain of collagen type IV or the α2 chain of collagen type IV is normally expressed whether it is hereditary glomerulonephritis or normal glomerulus.

[0004] At present, the detection methods for hereditary nephritis are: 1. Light microscopic detection. The pathological changes of hereditary nephritis are not characteristic, and before the application of electron microscopy technology, Alport syndrome was considered to be chronic interstitial nephritis, and the foam cells in the renal interstitium were misused as a diagnostic basis. (2) Immunofluorescence detection in kidney and skin tissues using specific antibodies against different α chains of collagen type IV can diagnose X-linked dominant male, female and autosomal recessive Alport syndrome. (3) Electron microscopic observation: under electron microscopy, the characteristic pathological changes of Alport syndrome can be observed, such as extensive thickening or thinning of GBM and splitting of the dense layer as the typical pathological changes.

[0005] Currently, there are two types of kits in the prior art for the differential diagnosis of genetic nephritis: one is the wieslab Alprot's syndrome kit of a Swedish company. The kit composition includes anti-mouse COL4A1, COL4A3 monoclonal antibodies and their corresponding FITC-conjugated mouse secondary antibodies; anti-rat COL4A5 monoclonal antibody and its corresponding FITC-conjugated rat secondary antibody, which is suitable for immunofluorescence cytochemical staining. In addition, the kit can also be used for paraffin-embedded tissues for immunohistochemical staining, but this method requires separate IHC staining of the three type IV collagen antibodies, which is a relatively complex method. The other is the fluorescence immunochemical staining kit of the Japanese COSMO company. This method uses COL4A2 and COL4A5 to label red fluorescence and green fluorescence, respectively, and then uses a certain ratio as a primary antibody to bind to the antigens on the kidney tissue. The results are observed using a fluorescence microscope. In theory, COL4A2 is localized on the basement membrane of the glomerulus and the renal tubule, and the expression profile of COL4A5 is relatively narrow, which is generally only localized on the glomerulus, and the renal tubule is basically not stained. If there is genetic nephritis (i.e., Alport syndrome) in the pathology, then COL4A5 is missing on the glomerulus.

[0006] Currently, there is a lack of COL4A3 and COL4A5 recombinant rabbit monoclonal antibodies in the prior art, and the application of COL4A1 recombinant rabbit monoclonal antibodies in Alport syndrome is also significantly insufficient. SUMMARY

[0007] In order to solve at least one of the above technical problems, the present application aims to provide three type IV collagen monoclonal antibodies, i.e., type IV collagen alpha 1 (COL4A1), type IV collagen alpha 3 (COL4A3), and type IV collagen alpha 5 (COL4A5), and their variable region sequences, and to provide a pathological diagnosis kit composed of the same, and the application thereof in the auxiliary diagnosis of Alport syndrome. In order to achieve this purpose, the technical solutions adopted by the present application are as follows:

[0008] The present application provides a COL4A1 monoclonal antibody, the heavy chain variable region of which is shown in SEQ ID NO: 1, and the light chain variable region of which is shown in SEQ ID NO: 2; the amino acid sequences of the CDR-H1, CDR-H2, CDR-H3 of the heavy chain variable region and the CDR-L1, CDR-L2, CDR-L3 of the light chain variable region of the COL4A1 monoclonal antibody are shown in SEQ ID NO: 3-8, respectively.

[0009] The second aspect of the present application provides a COL4A3 monoclonal antibody, which can specifically recognize a protein fragment of collagen type IV alpha 3 as shown in SEQ ID NO: 9. In some embodiments of the present application, the COL4A3 monoclonal antibody is obtained by immunizing a rabbit with a polypeptide fragment of human COL4A3 protein as shown in SEQ ID NO: 9 after cross-linking with KLH.

[0010] In some embodiments of the present application, the heavy chain variable region of the COL4A3 monoclonal antibody is as shown in SEQ ID NO: 11, and the light chain variable region is as shown in SEQ ID NO: 12; the amino acid sequences of CDR-H1, CDR-H2, CDR-H3 of the heavy chain variable region and CDR-L1, CDR-L2, CDR-L3 of the light chain variable region of the COL4A3 monoclonal antibody are as shown in SEQ ID NO: 13-18, respectively.

[0011] The third aspect of the present application provides a COL4A5 monoclonal antibody, which can specifically recognize a protein fragment of collagen type IV alpha 5 as shown in SEQ ID NO: 19. In some embodiments of the present application, the COL4A5 monoclonal antibody is obtained by immunizing a rabbit with a polypeptide fragment of human COL4A5 protein as shown in SEQ ID NO: 19 after cross-linking with KLH, preferably, the modification refers to adding a cysteine at the amino terminal of the polypeptide amino acid sequence to facilitate the cross-linking reaction.

[0012] In some embodiments of the present application, the heavy chain variable region of the COL4A4 monoclonal antibody is as shown in SEQ ID NO: 21, and the light chain variable region is as shown in SEQ ID NO: 22; the amino acid sequences of CDR-H1, CDR-H2, CDR-H3 of the heavy chain variable region and CDR-L1, CDR-L2, CDR-L3 of the light chain variable region of the COL4A5 monoclonal antibody are as shown in SEQ ID NO: 23-28, respectively.

[0013] The fourth aspect of the present application provides an antibody combination for detecting collagen type IV, which comprises the COL4A1 monoclonal antibody of the first aspect of the present application, and further comprises the COL4A3 monoclonal antibody of the second aspect of the present application and / or the COL4A5 monoclonal antibody of the third aspect of the present application.

[0014] In some embodiments of the present application, the antibody combination comprises the COL4A1 monoclonal antibody of the first aspect of the present application, and further comprises a COL4A3 antibody prepared or obtained by any method and / or a COL4A5 antibody prepared or obtained by any method, preferably, the antibodies are monoclonal antibodies.

[0015] In some embodiments of the application, the antibody combination comprises the COL4A3 mAb of the second aspect of the application, and further comprises any COL4A1 antibody prepared or obtained by any method, preferably further comprises any COL4A5 antibody prepared or obtained by any method, preferably the antibodies are mAbs.

[0016] In some embodiments of the application, the antibody combination comprises the COL4A5 mAb of the third aspect of the application, and further comprises any COL4A1 antibody prepared or obtained by any method, preferably further comprises any COL4A3 antibody prepared or obtained by any method, preferably the antibodies are mAbs.

[0017] The fifth aspect of the application provides use of the COL4A1 mAb of the first aspect of the application, the COL4A3 mAb of the second aspect of the application, the COL4A5 mAb of the third aspect of the application or the antibody combination of the fourth aspect of the application in the preparation of a collagen type IV immunodetection kit.

[0018] In some embodiments of the application, the immunodetection is at least one selected from the group consisting of immunohistochemistry, immunofluorescence, enzyme-linked immunoassay, immunoblotting.

[0019] The sixth aspect of the application provides a collagen type IV immunodetection kit, which comprises the COL4A1 mAb of the first aspect of the application, the COL4A3 mAb of the second aspect of the application, the COL4A5 mAb of the third aspect of the application or the antibody combination of the fourth aspect of the application.

[0020] In some embodiments of the application, the kit is used for diagnosing Alport syndrome.

[0021] In some embodiments of the application, the kit comprises the COL4A3 mAb of the second aspect of the application and any COL4A1 antibody prepared or obtained by any method, wherein the COL4A1 mAb and the COL4A3 mAb are respectively conjugated with different fluoresceins, or the COL4A1 mAb and the COL4A3 mAb are respectively conjugated with different enzyme labels. Preferably, the COL4A1 mAb is the COL4A1 mAb of the first aspect of the application.

[0022] In some embodiments of the application, the fluorophores are selected from two of FITC, Alexa Fluor 488, GFP, Fluo-3, PE, PI, PE-Cy5, PE-Cy5.5, PerCP, PerCP-Cy5.5, Texas Red, 7-AAD, PE-Cy7, PE-AlexaFluor 750, APC, Cy5, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 700, APC-Cy7, APC-Alexa Fluor 750, Hoechst 33342-Blue, DAPI, Hoechst 33342-Red, Alexa Fluor 405, Pacific Blue and Pacific Orange. In some embodiments of the application, the COL4A1 mAb and the COL4A3 mAb are conjugated to Texas Red and FITC, respectively.

[0023] In some embodiments of the application, the enzyme labels are selected from two of horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase (β-Gal), and in some embodiments of the application, the COL4A1 mAb and the COL4A3 mAb are conjugated to HRP and AP, respectively.

[0024] In some embodiments of the application, the kit comprises the COL4A5 mAb of the third aspect of the application and the COL4A1 mAb prepared or obtained by any of the methods, wherein the COL4A1 mAb and the COL4A5 mAb are conjugated to different fluorophores, or the COL4A1 mAb and the COL4A5 mAb are conjugated to different enzyme labels, respectively. Preferably, the COL4A1 mAb is the COL4A1 mAb of the first aspect of the application.

[0025] In some embodiments of the present application, the fluorescein is selected from two of FITC, Alexa Fluor 488, GFP, Fluo-3, PE, PI, PE-Cy5, PE-Cy5.5, PerCP, PerCP-Cy5.5, Texas Red, 7-AAD, PE-Cy7, PE-AlexaFluor 750, APC, Cy5, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 700, APC-Cy7, APC-Alexa Fluor 750, Hoechst33342-Blue, DAPI, Hoechst33342-Red, Alexa Fluor 405, Pacific Blue and Pacific Orange. In some embodiments of the present application, the COL4A1 mAb and the COL4A5 mAb are coupled with Texas Red and FITC, respectively.

[0026] In some embodiments of the present application, the enzyme label is selected from two of HRP, AP, β-Gal, and in some embodiments of the present application, the COL4A1 mAb and the COL4A3 mAb are coupled with HRP and AP, respectively.

[0027] In yet some embodiments of the present application, the kit comprises the antibody combination of the fourth aspect of the present application, further comprises a polyclonal antibody against the antibody combination, and a fluorescent dye combination, the fluorescent dyes in the fluorescent dye combination are all different in fluorescent color, and the number of the fluorescent dyes in the fluorescent dye combination is equal to the sum of the number of the mAbs in the antibody combination and the number of the polyclonal antibody.

[0028] In some specific embodiments of the present application, the kit comprises the antibody combination of the COL4A1 mAb of the first aspect of the present application and the COL4A3 mAb of the second aspect of the present application, further comprises a polyclonal antibody against the COL4A1 mAb and the COL4A3 mAb, and a fluorescent dye combination, the fluorescent dyes in the fluorescent dye combination are all different in fluorescent color, and the number of the fluorescent dyes in the fluorescent dye combination is three.

[0029] In some specific embodiments of the present application, the kit comprises the antibody combination of the COL4A1 mAb of the first aspect of the present application and the COL4A5 mAb of the third aspect of the present application, further comprises a polyclonal antibody against the COL4A1 mAb and the COL4A5 mAb, and a fluorescent dye combination, the fluorescent dyes in the fluorescent dye combination are all different in fluorescent color, and the number of the fluorescent dyes in the fluorescent dye combination is three.

[0030] In still some embodiments of the present application, the kit comprises: the antibody combination of the COL4A1 monoclonal antibody according to the first aspect of the present application, the COL4A3 monoclonal antibody according to the second aspect of the present application and the COL4A5 monoclonal antibody according to the third aspect of the present application, and further comprises the polyclonal antibody against the COL4A1 monoclonal antibody, the COL4A3 monoclonal antibody and the COL4A5 monoclonal antibody, and the combination of four fluorescent dyes, wherein the fluorescent colors of the fluorescent dyes in the combination are all different.

[0031] In some embodiments of the present application, the fluorescent dyes can be any fluorescent dyes well known in the art, as long as the fluorescent colors are all different, which meets the requirements of the present application. In some embodiments of the present application, when four fluorescent dyes are needed, the fluorescent dyes are, for example, 520, 570, 690 and DAPI.

[0032] Advantages of the present application

[0033] Compared with the prior art, the present application has the following advantages:

[0034] Through specific sequence selection of protein fragments and subsequent modification design of immunogens, rabbit monoclonal antibodies with excellent sensitivity and specificity are prepared, which are suitable for various immunological detection, especially immunohistochemical method and immunofluorescence method.

[0035] Immunohistochemistry (IHC) refers to a specific antibody labeled with a color developing agent to qualitatively, quantitatively or locally determine the corresponding antigen in pathological tissues through antigen-antibody reaction and histochemical color reaction in situ. The principle of immunohistochemical detection is as follows: after the tissue section is treated by antigen heat repair, the primary antibody reagent is incubated to form an antigen-antibody complex of the primary antibody and the target antigen in situ; the primary antibody molecules in the antigen-antibody complex are further combined with the enzyme-labeled polymer secondary antibody through incubation to form an antigen-antibody-secondary antibody polymer complex in situ; finally, the colored deposit is formed at the antigen site through the enzyme-catalyzed substrate. The presence or absence of the target antigen and its expression are determined by observing the brown site under an optical microscope. The cell type and morphology of pathological tissues, the source of tissue cell products, and the differentiation degree of tissue cells can be determined by immunohistochemical staining; especially in clinical pathology, the most important applications are to identify the nature of the lesion, find the micro lesion, explore the origin or differentiation phenotype of the tumor, determine the tumor stage, guide treatment and prognosis; assist disease diagnosis and classification, and find the cause of infection. The specific detection method for genetic nephritis in the clinic currently adopts fluorescence method, which needs to be labeled with fluorescein, and a fluorescence microscope is needed for result judgment. If an immunohistochemical detection kit can be developed, the result can be judged by an ordinary light microscope instead of a fluorescence microscope.

[0036] Multi-label immunofluorescence staining kit is a multi-label re-staining protocol developed on the basis of TSA, which allows the use of different primary antibodies of the same species in the same sample to covalently bind each color-labeled tyramide to the target antigen in a sequential single staining manner (antigen direct labeling), and then remove the antibody by microwave heating (antibody is gone but the signal remains on the antigen). After removing the previous round of antibody, a new antibody can be used for continuous staining without worrying about cross-reaction. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 The COL4A1 polyclonal serum prepared in Example 1 of the present application is shown in the indirect ELISA detection titer.

[0038] Figure 2 The ELISA binding curve of the COL4A1 rabbit monoclonal antibody prepared in Example 1 of the present application is shown.

[0039] Figure 3 The IHC detection results of the COL4A1 rabbit monoclonal antibody prepared in Example 1 of the present application in normal kidney and brain tissues are shown.

[0040] Figure 4 The COL4A3 polyclonal serum prepared in Example 2 of the present application is shown in the indirect ELISA detection titer.

[0041] Figure 5 The ELISA binding curve of the COL4A3 rabbit monoclonal antibody prepared in Example 2 of the present application is shown.

[0042] Figure 6 The IHC detection results of the COL4A3 rabbit monoclonal antibody prepared in Example 2 of the present application in normal kidney and brain tissues are shown.

[0043] Figure 7 The COL4A5 polyclonal serum prepared in Example 3 of the present application is shown in the indirect ELISA detection titer.

[0044] Figure 8 The ELISA binding curve of the COL4A5 rabbit monoclonal antibody prepared in Example 3 of the present application is shown.

[0045] Figure 9 The IHC detection results of the COL4A5 rabbit monoclonal antibody prepared in Example 3 of the present application in normal kidney and brain tissues are shown.

[0046] Figure 10 The purification peak shape diagram of the COL4A5 rabbit monoclonal antibody prepared in Example 4 of the present application is shown.

[0047] Figure 11The fluorescence staining results of COL4A5 rabbit monoclonal antibody conjugated FITC complex prepared in Example 4 of the present application and unlabeled COL4A5 rabbit monoclonal antibody in kidney tissue are shown.

[0048] Figure 12 The purification peak profile of COL4A1 rabbit monoclonal antibody conjugated Texas Red complex prepared in Example 4 of the present application is shown.

[0049] Figure 13 The staining results of COL4A1 rabbit monoclonal antibody conjugated Texas Red complex prepared in Example 4 of the present application and unlabeled COL4A1 rabbit monoclonal antibody under fluorescence microscope are shown.

[0050] Figure 14 The fluorescence staining results of three different concentration formulations of COL4A1 and COL4A5 fluorescence double staining kit prepared in Example 4 of the present application are shown.

[0051] Figure 15 The staining results of COL4A1 and COL4A5 fluorescence double staining kit prepared in Example 4 of the present application in detecting Alport syndrome are shown.

[0052] Figure 16 The staining results of control one-step fluorescence double staining kit in detecting Alport syndrome are shown.

[0053] Figure 17 The staining results of 4-color multi-label immunofluorescence staining kit prepared in Example 5 of the present application in detecting Alport syndrome are shown.

[0054] Figure 18 The staining results of anti-COL4A1-HRP and COL4A5-AP visible light detection kit prepared in Example 6 of the present application in detecting Alport syndrome are shown. DETAILED DESCRIPTION

[0055] Unless otherwise indicated, all parts and percentages in the present application are based on weight and all test and characterization methods are synchronous with the filing date of the present application. Where applicable, the contents of any patent, patent application or publication in the present application are incorporated by reference in their entirety, and equivalent homologous patents in other jurisdictions are also incorporated by reference, particularly the definitions of the synthetic techniques, products and processing designs, polymers, comonomers, initiators or catalysts, etc. disclosed in these documents. If the definition of a specific term disclosed in the prior art is inconsistent with any definition provided in the present application, the definition provided in the present application shall prevail.

[0056] Numerical ranges are approximations, and thus the endpoints should be considered to be approximations also, unless otherwise indicated. A numerical range includes all values from and including the lower and to and including the upper value of the range. These are only approximations, and the numerical values designated are approximate values, which can vary depending on the specific embodiments. Any numerical value, however, inherently contains certain errors necessarily resulting from the round-off or measurement of its constituents. It is also possible, however, that the end points of the ranges are themselves intended to be literally included in the range, unless it is clearly indicated otherwise.

[0057] As used in connection with chemical compounds, the singular includes all isomeric forms, unless specifically indicated otherwise (e.g., "hexane" alone or in combination includes all isomers of hexane). Additionally, the use of "a", "an", or "the" to describe a noun also includes the plural of that noun, unless specifically indicated otherwise.

[0058] The terms "comprising", "including", "containing", and variations thereof, do not exclude the presence of other components, steps or processes, and are used synonymously with the term "including". For the avoidance of doubt, the term "consisting essentially of to expressly exclude any component, step or process not specifically recited. The term "consisting of expressly excludes the presence of anything other than the specific components, steps or processes recited. Unless specifically stated otherwise, the term "or" as used herein refers to all possible combinations of the individual members of the enumeration.

[0059] In order to make the technical problems solved by the present application, the technical solutions and beneficial effects clearer, the present application will be further explained in detail below in combination with embodiments.

[0060] Embodiments

[0061] The following examples are presented to demonstrate preferred embodiments of the present application. Those skilled in the art will appreciate that the technology disclosed in the following examples represents the best attempts of the inventors to use the technology discovered by the inventors to practice the present application, and therefore can be considered preferred modes of practicing the present application. However, those skilled in the art will appreciate from the present disclosure that the particular examples disclosed herein can be modified in many ways without departing from the spirit or scope of the present application.

[0062] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. References to materials, articles, or tools are intended to include all possible forms thereof which are reasonably likely to be used by those in the art, unless otherwise indicated.

[0063] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the application described herein. Such equivalents are intended to be encompassed by the following claims.

[0064] The molecular biology experimental methods not specifically described in the following examples were performed according to the specific methods listed in the book of Molecular Cloning: A Laboratory Manual (Fourth Edition) (J. Sambrook, M. R. Green, 2017) or according to the kit and product instructions. Other experimental methods, if not specifically described, are conventional methods. The instruments and equipment used in the following examples, if not specifically described, are conventional laboratory instruments and equipment; the experimental materials used in the following examples, if not specifically described, are purchased from conventional biochemical reagent stores.

[0065] Example 1 Preparation of COL4A1 antibody

[0066] The preparation steps of the anti-human COL4A1 rabbit monoclonal antibody are as follows:

[0067] (1) Four New Zealand white rabbits were immunized with human COL4A1 natural protein. After four immunizations, the rabbit serum was taken, and the antibody titer in the serum was detected by ELISA method. The ELISA titer detection data are shown in Figure 1 Among them, the serum titer of rabbit No. 3 was the highest.

[0068] (2) The whole blood of rabbit No. 3 was taken to separate PBMC (peripheral blood lymphocytes), and B cell cloning culture was performed. After 9 days of culture, the B cell supernatant was detected by ELISA and IHC. The cells in the IHC positive well were lysed using cell lysis solution, and were stored at -80°C for standby.

[0069] (3) The heavy chain plasmid and light chain plasmid of the antibody were obtained by recombinant technology, and HEK293 cells were transfected. After 5-8 days, the cell culture supernatant was harvested. The obtained COL4A1 rabbit monoclonal antibody was purified using Protein A.

[0070] (4) The COL4A1 rabbit monoclonal antibody was identified by iELISA and IHC, and the identification results are shown in Figure 2 , Figure 3 It can be seen from Figure 2 that the titer of the developed COL4A1 rabbit monoclonal antibody is 2.09 ng / mL. It can be seen from Figure 3 that the obtained anti-human COL4A1 rabbit monoclonal antibody has positive brown staining in the glomerulus, renal tubule and vascular basement membrane of human kidney tissue; and has positive brown staining in the vascular basement membrane of brain tissue. It shows that the antibody has accurate positive localization, good specificity, and no non-specific staining.

[0071] The COL4A1 rabbit monoclonal antibody was analyzed by using VBASE2 software, and the variable region sequence information of the antibody was as follows:

[0072] The heavy chain variable region sequence was as follows (SEQ ID NO: 1):

[0073] QEQLVESGGGLVQPEGSLTLTCTASGFSFSSIYYIYWVRQAPGKGLEWIACIYGGSSGTSYYASWAKGRFTVSKTSSTTVTLQMTSLTAADTATYFCARDAGYLGYDYVLWGPGTLVIVSS

[0074] The light chain variable region sequence was as follows (SEQ ID NO: 2):

[0075] DPVLTQTPASVSAAVGGTVTINCQASQNIANLLAWYQQKPGQPPKLLIYKASTLQSGVSSRFKGSGSGTEFTLTISDLECADAATYYCQSVAYGSTYVAAFGGGTEVVVKGDP

[0076] The CDR sequence information was as follows:

[0077] (1) The CDR-H1 amino acid sequence included SIYYIY (SEQ ID NO: 3).

[0078] (2) The CDR-H2 amino acid sequence included CIYGGSSGTSYYASWAKG (SEQ ID NO: 4).

[0079] (3) The CDR-H3 amino acid sequence included DAGYLGYDYVL (SEQ ID NO: 5).

[0080] (4) The CDR-L1 amino acid sequence included QASQNIANLLA (SEQ ID NO: 6).

[0081] (5) The CDR-L2 amino acid sequence included KASTLQS (SEQ ID NO: 7).

[0082] (6) The CDR-L3 amino acid sequence included QSVAYGSTYVAA (SEQ ID NO: 8).

[0083] Example 2 Preparation of COL4A3 antibody

[0084] Because human and rabbit COL4A3 proteins share a high degree of homology, it is difficult to obtain high-quality rabbit antibodies against human COL4A3 by arbitrarily designing antigens for immunization. In this study, we selected the human COL4A3 peptide aa1557-1570 as both the immunogen and the detector. This peptide differs from the rabbit COL4A3 peptide aa1381-1394 by three amino acids: the amino acid sequence of the former is AIAVHSQTTDIPPC (SEQ ID NO:9), while the amino acid sequence of the latter is AIAIHSQSTDVPPC (SEQ ID NO:10). This peptide aa1557-1570 allows for the generation of as many antibodies as possible that recognize human COL4A3.

[0085] Specifically, the preparation steps of anti-human COL4A3 rabbit monoclonal antibody are as follows:

[0086] (1) Four New Zealand white rabbits were immunized with KLH using the aa1557-1570 peptide of the human COL4A3 protein. After four immunizations, rabbit serum was collected and the antibody titer in the serum was detected using the ELISA method. The ELISA titer detection data are shown in Figure 4 Among them, rabbit number 2 had the highest serum titer.

[0087] (2) Whole blood was collected from rabbit 2 to separate PBMCs for B cell cloning. The supernatant was cultured for about 9 days and sent for ELISA and IHC testing. The cells in the IHC-positive wells were lysed using cell lysis buffer and stored at -80°C for later use.

[0088] (3) Using recombinant technology, the heavy chain plasmid and light chain plasmid of the antibody were obtained, and HEK293 cells were transfected. The cell culture supernatant was harvested after 5-8 days; the COL4A3 rabbit monoclonal antibody was obtained by purification using Protein A.

[0089] (4) The monoclonal antibody was identified by iELISA and IHC using conventional indirect ELISA and IHC methods. The identification results are shown in Figure 5 、 Figure 6 .Depend on Figure 5 It can be seen that the titer of the developed COL4A3 rabbit monoclonal antibody is 20.04 ng / mL. Figure 6 It can be seen that the antibody only has specific positive staining in the glomerular basement membrane of normal kidney tissue and the basement membrane of cerebral blood vessels, and the antibody has no brown staining in the basement membrane of the renal tubules, which is consistent with the immunohistochemical expression spectrum of the antibody.

[0090] (5) The obtained COL4A3 rabbit monoclonal antibody was sequenced and analyzed using VBASE2 software. The results showed that the variable region sequence information of the antibody was as follows:

[0091] The heavy chain variable region sequence is as follows (SEQ ID NO: 11):

[0092] CQQLEQSGGGAEGGLVKPGGSLELYCKASGFSLSSSYWICWVRQAPGKGLEWIGCIYVGSASSTYYASWVNGRFTLSRDIDQSTGCLQLNSLTAADTAMYYCAGYASRSGDFWYFNLWGQGTLVTVSS

[0093] The light chain variable region sequence is as follows (SEQ ID NO: 12):

[0094] AIKMTQTPASVSEPVGGTVTINCQASEDIESNLAWYQQKPGQPPKLLIYYTSTLASGVPSRFKGSGSGTEYTLTISGVQCDDAATYYCQSTYYSGSVDTMNTFGGGTEVVVKGDP

[0095] The CDR sequence information is as follows:

[0096] (1) The CDR-H1 amino acid sequence comprises: SSIYWIC (SEQ ID NO: 13).

[0097] (2) The CDR-H2 amino acid sequence comprises: CIYVGSASSTYYASWVNG (SEQ ID NO: 14).

[0098] (3) The CDR-H3 amino acid sequence comprises; YASRSGDFWYFNL (SEQ ID NO: 15).

[0099] (4) The CDR-L1 amino acid sequence comprises: QASEDIESNLA (SEQ ID NO: 16).

[0100] (5) The CDR-L2 amino acid sequence comprises: YTSTLAS (SEQ ID NO: 17).

[0101] (6) The CDR-L3 amino acid sequence comprises: QSTYYSGSVDTMNT (SEQ ID NO: 18).

[0102] Example 3 COL4A5 antibody preparation

[0103] Similar to COL4A3, human COL4A5 shares a high degree of homology with rabbit COL4A5, making it difficult to generate high-quality rabbit antibodies against it through arbitrarily designed antigens. Regarding antigen selection, the human COL4A5 peptide aa1277-1294 was selected as both the immunogen and the detector. This immunogen differs from the rabbit COL4A5 peptide aa1283-1300 by four amino acids: the amino acid sequence of the former is NGGIKGEKGNPGQPGLPG (SEQ ID NO:19), while the amino acid sequence of the latter is NGAIKGERGNPGPPGQPG (SEQ ID NO:20). This approach maximizes the production of antibodies against the human COL4A5 peptide aa1277-1294.

[0104] The specific steps for preparing anti-human COL4A5 rabbit monoclonal antibody are as follows:

[0105] (1) Using the aa1277-1294 peptide of the human COL4A5 protein, a cysteine ​​was added to the amino terminus to facilitate subsequent cross-linking, i.e., the sequence was CNGGIKGEKGNPGQPGLPG. Four New Zealand white rabbits were immunized with KLH. After four immunizations, rabbit serum was collected and the antibody titer in the serum was detected by ELISA. ELISA titer detection data are shown in Figure 7 Among them, rabbit number 3 had the highest serum titer.

[0106] (2) Whole blood was collected from rabbit 3 to separate PBMCs, and B cell cloning was performed using the Bailing rabbit monoclonal antibody development platform technology. The supernatant was cultured for about 9 days and sent for ELISA testing. The cells in the ELISA-positive wells were lysed using cell lysis buffer and stored at -80°C for later use.

[0107] (3) Using Bailing rabbit monoclonal antibody recombination platform technology, the heavy chain plasmid and light chain plasmid of the antibody were prepared, transfected into HEK293 cells, and the cell culture supernatant was harvested after 5-8 days; the COL4A5 rabbit monoclonal antibody was obtained by purification using Protein A.

[0108] (4) The monoclonal antibody was identified by iELISA and IHC using conventional indirect ELISA and IHC methods. The identification results are shown in Figure 8 、 Figure 9 .Depend on Figure 8 It can be seen that the titer of the developed COL4A5 rabbit monoclonal antibody is 8.29 ng / mL. Figure 9 It can be seen that this antibody only has positive staining in the glomerular basement membrane of normal kidney tissue, while the basement membrane of renal tubules, renal blood vessels and cerebral vascular cells is negative.

[0109] (5) The obtained COL4A5 rabbit monoclonal antibody was sequenced, and the COL4A5 rabbit monoclonal antibody was analyzed by using VBASE2 software. The results showed that the variable region sequence information of the antibody was as follows:

[0110] The heavy chain variable region sequence was as follows (SEQ ID NO: 21):

[0111] CQSVEESGGRLVTPGTPLTLTCTVSGFSLSSYGVSWVRQAPGKGLEYIGMVSTGGYIYYASWARGRSTITRNTNENTVTLKMTSPTTEDTATYFCSREYSNYGTGSFGLWGPGTLVTVSS

[0112] The light chain variable region sequence was as follows (SEQ ID NO: 22):

[0113] AQVLTQTPSSVSAAVGGTVTINCQASQSVYNNKNLAWYQQKPGQPPKLLIYKASTLASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCQGEFSCSSADCFGFGGGTEVVVKGDP

[0114] The CDR sequence information was as follows:

[0115] (1) The CDR-H1 amino acid sequence comprises: SYGVS (SEQ ID NO: 23).

[0116] (2) The CDR-H2 amino acid sequence comprises: MVSTGYIYYASWARG (SEQ ID NO: 24).

[0117] (3) The CDR-H3 amino acid sequence comprises: EYSNYGTGSFG (SEQ ID NO: 25).

[0118] (4) The CDR-L1 amino acid sequence comprises: QASQSVYNNKNLA (SEQ ID NO: 26).

[0119] (5) The CDR-L2 amino acid sequence comprises: KASTLAS (SEQ ID NO: 27).

[0120] (6) The CDR-L3 amino acid sequence comprises: QGEFSCSSADCFG (SEQ ID NO: 28).

[0121] Example 4 One-step double staining kit and application thereof

[0122] 1. COL4A5 rabbit monoclonal antibody conjugated with fluorescein isothiocyanate (FITC)

[0123] The COL4A5 rabbit monoclonal antibody prepared in Example 3 was conjugated with FITC, and the specific steps were as follows:

[0124] (1) The IgG of the purified COL4A5 rabbit monoclonal antibody was dialyzed overnight using a carbonate buffer with a pH of 8.5-9.5, and the antibody after dialysis was moved into a centrifuge tube for standby.

[0125] (2) 0.5 mL (2 mg / mL) of the COL4A5 rabbit monoclonal antibody to be conjugated was taken into a clean centrifuge tube, and 150 μL of 1 mg / mL FITC solution (Uelandy, Cat# F5027) was added, and mixed quickly.

[0126] (3) Reaction at 37°C in the dark for 90-150 min.

[0127] (4) The free FITC was removed using a desalting column (Sephadex G25), and the Sephadex G25 column was first rinsed with 25 mL of PBS.

[0128] (5) The first FITC-conjugated COL4A5 rabbit monoclonal antibody IgG protein peak was collected, and the F / P ratio (i.e., the number of labeled fluorescein molecules per protein molecule) was determined, and the appropriate F / P ratio was 2-5. The second fluorescein peak was free fluorescein, which was discarded.

[0129] Calculation: F / P = (2.87 x OD 495 ) / (OD 280 - 0.35 x OD 495 )

[0130] IgG amount (mg / mL) = (OD 280 - OD 495 x 0.35) / 1.4

[0131] Figure 10 The purified peak shape diagram of the COL4A5 rabbit monoclonal antibody conjugated with FITC (monitoring OD 495 ), in which the first peak from the left was the FITC-conjugated COL4A5 monoclonal antibody IgG protein peak, and the second peak was free fluorescein.

[0132] The labeled concentration was calculated by the formula to be 2.18 mg / mL, and the conjugation ratio (F / P) was 3.87, which met the requirements.

[0133] (6) Labeled complex identification method: Normal human kidney tissue was used to perform immunofluorescence staining of the labeled COL4A5-FITC coupled complex and unlabeled COL4A5 rabbit monoclonal antibody. The specific steps are as follows.

[0134] ① Renal tissue paraffin sections were baked at 60°C for 1 hour, dewaxed and hydrated, and then repaired using high-temperature and normal-pressure Tris-EDTA (pH 9.0) repair solution and cooled to room temperature;

[0135] ② Use 3% hydrogen peroxide to block endogenous peroxidase on the tissue at room temperature for 10 minutes.

[0136] ③ Use an immunohistochemistry pen to circle the tissue on the slide, and then use 10% goat serum to block the non-specific binding sites on the tissue at room temperature for 30 minutes.

[0137] ④ After washing the tissue slides three times with PBST, add 100 μL of 50 μg / mL COL4A5-FITC conjugate complex to one slide and 100 μL of 50 μg / mL unlabeled COL4A5 rabbit monoclonal antibody to the other slide and incubate at room temperature for 30 min.

[0138] ⑤ After washing the tissue slides 3 times with PBST, add 1 drop of DAPI (containing anti-fluorescence quencher) on the slides, seal the slides with coverslips, place them in the dark at room temperature, and observe the staining results under a fluorescence microscope.

[0139] Figure 11 The staining results of COL4A5-FITC coupled complex and unlabeled FITC COL4A5 monoclonal antibody under fluorescence microscope. The results show that COL4A5-FITC coupled complex has green fluorescence staining on the basement membrane of Bowman's capsule of glomerulus, and the cell nucleus is blue ( Figure 11 Left); Unlabeled COL4A5 monoclonal antibody showed no green fluorescence staining under a fluorescence microscope, only blue staining of the nucleus ( Figure 11 The results showed that FITC was successfully conjugated to the COL4A5 rabbit monoclonal antibody and can be used to form a reagent combination or packaged into a kit for detection.

[0140] 2. COL4A1 rabbit monoclonal antibody conjugated to Texas Red fluorescent dye

[0141] The COL4A1 rabbit monoclonal antibody prepared in Example 1 was conjugated to Texas Red in the following steps:

[0142] (1) The purified anti-COL4A1 rabbit monoclonal antibody IgG was dialyzed overnight using a carbonate buffer solution of pH 8.5-pH 9.5. The dialyzed antibody was transferred to a centrifuge tube for later use.

[0143] (2) Take 0.5 mL (2 mg / mL) COL4A1 rabbit monoclonal antibody to be conjugated into a clean centrifuge tube, add 200 μL of 1 mg / mL Texas Red solution (Uelandy, Cat# T5082), and mix quickly.

[0144] (3) React at room temperature for 60-120 min in the dark.

[0145] (4) Use a desalting column (Sephadex G25) to remove free fluorescein, first rinse the Sephadex G25 column with 25 mL of PBS.

[0146] (5) Collect the first Texas Red-bound COL4A1 monoclonal IgG protein peak eluted by PBS, and determine the F / P ratio (i.e., the number of labeled fluorescein molecules per protein molecule). The appropriate F / P ratio is 2-4. The second fluorescein peak is free fluorescein, which is discarded.

[0147] Calculation: IgG concentration (M) = (OD 280 - OD 595 x 0.18) / 203000

[0148] Where 203000 is the molar extinction coefficient (ε) of a typical IgG in cm -1 M -1 at 280 nm, and 0.18 is the correction factor for the contribution of the fluorescein group to absorbance at 280 nm.

[0149] F / P = (OD595 x dilution factor) / (80000 x protein molar concentration)

[0150] Figure 12 Purification peak profile of COL4A1 rabbit monoclonal antibody conjugated with Texas Red (monitoring OD 595 ), where the first peak is the Texas Red-bound COL4A1 rabbit monoclonal IgG protein peak, and the second peak is free fluorescein.

[0151] Labeling concentration: The labeling concentration of this example is calculated to be 1.95 mg / mL by the formula, and the conjugation ratio (F / P) is 2.85, which meets the requirements.

[0152] (6) Labeling compound identification method: same as the immunofluorescence staining method of COL4A5 rabbit monoclonal antibody-FITC complex described above.

[0153] Figure 13Staining results of COL4A1-Texas Red conjugate complex and COL4A1 monoclonal antibody without Texas Red under fluorescence microscope. COL4A1-Texas Red conjugate complex has red fluorescence staining on the basement membrane of the podocyte of the glomerulus, and the nucleus is blue (left); COL4A1 monoclonal antibody without Texas Red has no staining under fluorescence microscope (right). The results show that Texas Red is successfully conjugated to COL4A1 rabbit monoclonal antibody, which can be used for the next group of reagent combinations or packaged into a kit for detection. Figure 13 Figure 13 The results show that Texas Red is successfully conjugated to COL4A1 rabbit monoclonal antibody, which can be used for the next group of reagent combinations or packaged into a kit for detection.

[0154] 3. COL4A1 and COL4A5 fluorescent double staining kit

[0155] The COL4A5 rabbit monoclonal antibody-FITC complex prepared in step 1 and the COL4A1 rabbit monoclonal antibody-Texas Red complex prepared in step 2 are mixed as follows, and then verified by immunofluorescence using kidney tissue.

[0156] Formulation No. COL4A5 Rabbit Monoclonal-FITC Complex Final Concentration COL4A1 Rabbit Monoclonal-Texas Red Complex Final Concentration 1 2 μg / mL 2 μg / mL 2 5 μg / mL 5 μg / mL 3 10 μg / mL 10 μg / mL

[0157] Figure 14 The results of the three different concentration formulations of the COL4A1 and COL4A5 fluorescent double staining kit show that the fluorescence staining of the two antibodies is weak from the staining results of formulation 1; the fluorescence staining of the two antibodies is moderate from the staining results of formulation 2, and no fluorescence background is generated; the fluorescence staining of the two antibodies is strong, but there is a high fluorescence background.

[0158] Therefore, the inventors select the final concentration of the two fluorescently labeled antibodies to be 5 μg / mL for the assembly of the kit and subsequent detection.

[0159] Therefore, the inventors select the final concentration of the two fluorescently labeled antibodies to be 5 μg / mL for the assembly of the kit and subsequent detection.

[0160] ① Component 1: peroxidase blocking agent (3% H2O2);

[0161] ② Component 2: COL4A5 rabbit monoclonal antibody-FITC complex and COL4A1 monoclonal antibody-Texas Red complex (same concentration);

[0162] ③ Component 3: anti-fluorescence quenching mounting agent (containing DAPI).

[0163] 4. Application of COL4A1 and COL4A5 fluorescent double staining kit in detection of Alport syndrome (hereditary glomerulonephritis)

[0164] Sample type: paraffin section or frozen section​

[0165] (1) Two slices of each of normal kidney tissue and Alport syndrome positive kidney tissue paraffin sections were baked, deparaffinated, hydrated, and then subjected to Tris-EDTA (pH 9.0) antigen retrieval under normal pressure / high pressure, and then cooled to room temperature. After washing with water, the immunohistochemical pen was used to circle, and then the slices were soaked in PBST. If the tissue type is a frozen section, baking, deparaffination, and hydration are not required.

[0166] (2) 2-3 drops of component 1 were added to block endogenous peroxidase, and the reaction was allowed to proceed at room temperature for 10 min.

[0167] (3) 2-3 drops of component 2 (COL4A1 and COL4A5 fluorescent double staining kit) were added to one slice of normal kidney tissue and one slice of Alport syndrome positive kidney tissue, respectively, and the reaction was allowed to proceed at room temperature for 30 min.

[0168] (4) After washing with PBST for 3 times, 1 drop (about 20 μL) of component 3 was added for sealing, and the reaction was allowed to proceed at room temperature for 5-10 min.

[0169] The tissue slices were placed under a fluorescence microscope, and green and red fluorescence channels were used for photographing, respectively, and finally merged.

[0170] The results of the fluorescent staining are shown in Figure 15 , all normal renal tubules and glomerular basement membranes were red fluorescently stained, and green fluorescent staining was also observed in the glomerular basement membranes. In the fluorescent staining of the Alport syndrome positive kidney tissue, the renal tubules and glomerular basement membranes were red fluorescently stained, but no green fluorescent signal was observed. Figure 16 The results of the staining of the control in the normal kidney tissue and the Alport syndrome positive kidney tissue can be seen from Figure 16 , the staining results of the kit formula in the normal kidney tissue were weaker than those of the COL4A1 and COL4A5 fluorescent double staining kit prepared in the present embodiment. The above results show that the kit prepared in the present embodiment has higher sensitivity than the control kit.

[0171] Example 5 4-color multi-label immunofluorescent staining kit and application thereof

[0172] 1. 4-color multi-label immunofluorescent staining kit

[0173] The present example utilizes COL4A1 rabbit monoclonal antibody, COL4A3 rabbit monoclonal antibody and COL4A5 rabbit monoclonal antibody prepared in Examples 1-3 as primary antibodies to prepare a 4-color multi-label immunofluorescence staining kit, which comprises the following components:

[0174] ① Component 1: Peroxidase blocking agent (3% H2O2);

[0175] ② Component 2: COL4A1 rabbit monoclonal antibody (concentration: 5 μg / mL);

[0176] ③ Component 3: COL4A3 rabbit monoclonal antibody (concentration: 10 μg / mL);

[0177] ④ Component 4: COL4A5 rabbit monoclonal antibody (concentration: 10 μg / mL);

[0178] ⑤ Component 5: HRP-labeled goat anti-rabbit IgG (Jackson, cat#111-035-144; concentration: 1 μg / mL, or technical index is the same as that of the same type of reagent);

[0179] ⑥ Component 6: Four-color multi-label fluorescent dye combination (520, 570, 690, DAPI);

[0180] ⑦ Component 7: Tyramide signal amplification reaction solution.

[0181] 2. Application of 4-color multi-label immunofluorescence staining kit in detection of Alport syndrome

[0182] The specific experimental steps are as follows:

[0183] Sample type: paraffin section or frozen section

[0184] (1) After the paraffin section or frozen section of normal kidney tissue or kidney tissue positive for Alport syndrome is baked, dewaxed and hydrated, Tris-EDTA (pH 9.0) is used for high-temperature normal-pressure / high-pressure antigen retrieval, and then cooled to room temperature. After washing with water, the immunohistochemical pen is used to draw a circle and then the section is soaked in PBST. If the tissue type is a frozen section, baking, dewaxing and hydration are not required.

[0185] (2) Add 2-3 drops of component 1 to block endogenous peroxidase, and incubate at room temperature for 10 min.

[0186] (3) Add 2-3 drops of component 2, incubate at room temperature for 30 min, and then wash the section with PBST for 3 times.

[0187] (4) Add 2-3 drops of component 5, incubate at room temperature for 30 min, and then wash the section with PBST for 3 times.

[0188] (5) Dilute the fluorescent dye 520 in component 6 with component 7 (100:1), 100 μL / slide is added to the tissue area on the slide, incubate at room temperature for 10 min; wash the slide with PBST for 3 times.

[0189] (6) Put the slide into the boiled antigen repair buffer working solution (Fulinyang (Hangzhou) Biotechnology Co., Ltd., Cat#I30082B) and boil for 10-15 min, repeat step (2).

[0190] (7) Add 2-3 drops of component 3, incubate at room temperature for 30 min, then wash the slide with PBST for 3 times, repeat step (4).

[0191] (8) Dilute the fluorescent dye 570 in component 6 with component 7 (100:1), incubate at room temperature for 10 min; wash the slide with PBST for 3 times.

[0192] (9) After repeating step (6), add 2-3 drops of component 4, incubate at room temperature for 30 min, then wash the slide with PBST for 3 times, repeat step (4).

[0193] (10) Dilute the fluorescent dye 690 in component 6 with component 7 (100:1), incubate at room temperature for 10 min; wash the slide with PBST for 3 times.

[0194] (11) Add 1 drop (about 20 μL) of DAPI in component 6 for mounting.

[0195] (12) Observe the stained tissue under a fluorescence microscope and take a photo.

[0196] Figure 17 The 4-color multi-label immunofluorescence staining kit is used for detecting normal kidney tissues and kidney tissues of patients with Alport syndrome, Figure 17 The left side of the figure shows that COL4A1 has positive localization in the basement membrane of normal glomerulus and renal tubule, and COL4A3 and COL4A5 have positive localization in the basement membrane of normal glomerulus. Figure 17 The right side of the figure shows that COL4A1 has positive localization in the basement membrane of glomerulus and renal tubule of patients with Alport syndrome, and COL4A3 and COL4A5 have no positive localization in the basement membrane of glomerulus of patients with Alport syndrome.

[0197] The above results show that the 4-color multi-label immunofluorescence staining kit prepared in the embodiment has very high specificity and sensitivity for detecting Alport syndrome, which is the first time to detect genetic glomerulonephritis by using the method in the technical field.

[0198] Example 6: Collagen type IV visible light detection kit and its application in Alport syndrome

[0199] This embodiment utilizes the COL4A1 rabbit monoclonal antibody prepared in Example 1 and the COL4A5 rabbit monoclonal antibody prepared in Example 3 to prepare a COL4A1 rabbit monoclonal antibody-horseradish peroxidase (HRP) complex (NaIO4) and a COL4A5 rabbit monoclonal antibody-alkaline phosphatase (AP) complex, respectively.

[0200] 1. A visible light detection kit for type IV collagen

[0201] A visible light detection kit for type IV collagen is prepared using the COL4A1 rabbit monoclonal antibody-HRP complex and the COL4A5 rabbit monoclonal antibody-AP complex described above, and the kit comprises:

[0202] ① Component 1: Peroxidase blocking agent (3% H2O2);

[0203] ② Component 2: COL4A1 rabbit monoclonal antibody-HRP complex (concentration: 3-5 μg / mL);

[0204] ③ Component 3: COL4A5 rabbit monoclonal antibody-AP complex (concentration: 5-10 μg / mL);

[0205] ④ Component 4: DAB chromogen (20x) (Hangzhou Bailing Biotechnology Co., Ltd., Cat# BX10002), DAB Buffer (Hangzhou Bailing Biotechnology Co., Ltd., Cat# BX10003);

[0206] ⑤ Component 5: Fast-Red chromogen (150x), Fast-RED Buffer (Abeam, Cat#ab103741 or technical specifications of the same type of reagent);

[0207] ⑥ Hematoxylin staining solution.

[0208] 2. Application of the visible light detection kit for type IV collagen in genetic glomerulonephritis

[0209] The specific experimental steps are as follows:

[0210] Sample type: paraffin section or frozen section

[0211] (1) After the paraffin section or frozen section of normal kidney tissue or kidney tissue positive for Alport syndrome is baked, deparaffinized, and hydrated, Tris-EDTA (pH 9.0) is used for high-temperature normal-pressure / high-pressure antigen retrieval, and then cooled to room temperature. After washing with water, the immunohistochemical pen is used to draw a circle and then the PBST is used for soaking. If the tissue type is a frozen section, baking, deparaffinization, and hydration are not required.

[0212] (2) 2-3 drops of component 1 are added to block endogenous peroxidase, and the reaction is allowed to proceed at room temperature for 10 min.

[0213] (3) Add 2-3 drops of component 2 on normal kidney tissue and kidney tissue positive for Alport syndrome, incubate at room temperature for 30 min, then wash the slide with PBST for 3 times.

[0214] (4) Add 2-3 drops of component 3 on the two slices incubated at room temperature, incubate at room temperature for 30 min, then wash the slide with PBST for 3 times.

[0215] (5) Add 100 μL of working solution of component 4 on the tissue slice, the working solution is prepared as DAB colorant (20x) : DAB Buffer = 1:19, incubate at room temperature for 2-10 min, then wash the slide with PBST for 3 times.

[0216] (6) Add 100 μL of working solution of component 4 on the tissue slice, the working solution is prepared as Fast-Red colorant: Fast-RED Buffer = 1:150, incubate at room temperature for 10-15 min, then wash the slide with tap water for 3 times.

[0217] (7) Use component 6 for re-staining, then dehydrate, transparentize and mount the slide.

[0218] Take photos under a microscope, and the results are shown in Figure 18 .

[0219] Figure 18 The staining results on the left side of the middle show that COL4A1 using the collagen type IV visible light detection kit has brown color staining on the glomerular basement membrane and the tubular basement membrane of normal kidney tissue, and COL4A5 has positive red staining on the glomerular basement membrane of normal kidney tissue. Figure 18 The staining results on the right side of the middle show that COL4A1 using the collagen type IV visible light detection kit has brown color staining on the glomerular basement membrane and the tubular basement membrane of kidney tissue of Alport syndrome patients, and COL4A5 has no any positive staining on the glomerular basement membrane of kidney tissue of Alport syndrome.

[0220] The above results show that the detection kit can realize the pathological auxiliary diagnosis of genetic glomerulonephritis under the visible light of a microscope, has a broad application prospect, and can assist pathologists in judging the lesions.

[0221] All the documents mentioned in the present application are cited as references in the present application, as if each document is cited as a reference individually. In addition, it should be understood that those skilled in the art can make various modifications or amendments to the present application after reading the above teaching of the present application, and these equivalent forms also fall within the scope defined by the claims attached to the present application.

Claims

1. An anti-COL4A3 monoclonal antibody, characterized in that: The anti-COL4A3 monoclonal antibody can specifically recognize the protein fragment of type IV collagen α3 as shown in SEQ ID NO:

9. The amino acid sequences of the heavy chain variable regions CDR-H1, CDR-H2, CDR-H3 and the light chain variable regions CDR-L1, CDR-L2, CDR-L3 of the anti-COL4A3 monoclonal antibody are shown in SEQ ID NOs: 13 to 18, respectively.

2. Use of the anti-COL4A3 monoclonal antibody according to claim 1 in the preparation of a type IV collagen immunoassay kit.

3. The use according to claim 2, characterized in that The immunoassay is selected from at least one of the group consisting of immunohistochemistry, immunofluorescence, enzyme-linked immunosorbent assay, and immunoblotting.

4. The use according to claim 3, characterized in that The type IV collagen immunoassay kit is used for diagnosing Alport syndrome.

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