Antibody, detection kit and application of type IV collagen α5

Abstract

The present invention discloses an antibody for detecting type IV collagen α5, a detection kit and an application thereof, and belongs to the field of immunoassay technology. The antibody for detecting type IV collagen α5 is COL4A5 monoclonal antibody, and the COL4A5 monoclonal antibody can specifically recognize the protein fragment of type IV collagen α5 as shown in SEQ ID NO:19. The present invention also discloses an antibody combination including the COL4A5 monoclonal antibody. The present invention also discloses the application of the COL4A5 monoclonal antibody or the antibody combination in the preparation of a type IV collagen detection kit and the kit prepared therefrom. The kit of the present invention is used to detect Alport syndrome, which has high specificity and strong sensitivity and has great clinical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunoassay, and in particular, relates to an antibody for type IV collagen α5, a detection kit and applications thereof. Background Art

[0002] Hereditary nephritis, namely Alport syndrome (AS), is a hereditary glomerular basement membrane disease with the main manifestations of hematuria, progressive renal function decline, sensorineural deafness and eye abnormalities. It is a disease caused by mutations in the gene encoding the main collagen component of the glomerular basement membrane - collagen IV. The incidence of gene mutation is about 1 / 10000 to 1 / 5000. According to the inheritance mode, it can be divided into: ① X-linked dominant inheritance (XL, accounting for about 80%), the pathogenic gene is on the X chromosome, and inheritance is related to gender. ② Autosomal recessive inheritance (AR; accounting for about 15%), the pathogenic gene is on the autosome. ③ Autosomal dominant inheritance (AD; very rare). The three inheritance modes are caused by mutations in the genes COL4A5, COL4A6, COL4A3 and / or COL4A4 that encode different α chains of type IV collagen.

[0003] The cause of hereditary nephritis is: the six alpha chains of type IV collagen polymerize into three triple helical molecular structures called monomers, which polymerize into dimers or tetramers, and then intertwine to form a collagen network structure. The gene mutation of X-linked dominant Alport syndrome mainly occurs in the gene encoding type IV collagen alpha 5 chain (COL4A5). The gene mutation of autosomal recessive Alport syndrome occurs in the gene encoding type IV collagen alpha 3 chain or alpha 4 chain on chromosome 2 (COL4A3 / COL4A4), while the genes of type IV collagen alpha 1 chain or type IV collagen alpha 2 chain are still expressed normally, that is, whether it is hereditary glomerulonephritis or normal glomeruli, type IV collagen alpha 1 chain or type IV collagen alpha 2 chain will be expressed normally.

[0004] The current detection methods for hereditary nephritis are: 1. Light microscopy. The pathological changes of hereditary nephritis are not characteristic. Before the application of electron microscopy, Alport syndrome was considered to be a chronic interstitial nephritis, and renal interstitial foam cells were mistakenly used as the basis for diagnosis. (2) Immunofluorescence detection of kidney and skin tissue using specific antibodies against different α chains of type IV collagen can diagnose X-linked dominant hereditary males and females and autosomal recessive hereditary Alport syndrome. (3) Electron microscopy observation: Alport syndrome characteristic pathological changes can be observed under electron microscopy, with extensive thickening or thinning of GBM and splitting of the dense layer as its typical lesions.

[0005] There are currently two types of kits in the prior art for differential diagnosis of hereditary nephritis: one is the Swedish company's wieslab Alprot's syndrome kit. The kit consists of monoclonal antibodies against mouse COL4A1 and COL4A3 and their corresponding FITC-coupled mouse secondary antibodies; monoclonal antibodies against rat COL4A5 and their corresponding FITC-coupled rat secondary antibodies, and the system 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 three type IV collagen antibodies, which is relatively complicated. The other is the fluorescent immunochemical staining kit of Japan's COSMO company, which uses COL4A2 and COL4A5 to mark red fluorescence and green fluorescence respectively, and then uses them as primary antibodies in a certain ratio to react with antigens on renal tissue. The results are observed using a fluorescence microscope. Theoretically, COL4A2 is located in the basement membrane of both the glomerulus and tubules, while the expression spectrum of COL4A5 is relatively narrow. Under normal circumstances, it is generally only located in the glomerulus, and the tubules are basically not stained. If hereditary nephritis (i.e., Alport syndrome) is present in the pathology, then COL4A5 is missing in the glomerulus.

[0006] Currently, there is a lack of COL4A3 and COL4A5 recombinant rabbit monoclonal antibodies in the prior art, and there is also a clear shortage of COL4A1 recombinant rabbit monoclonal antibodies for use in Alport syndrome. Summary of the invention

[0007] In order to solve at least one of the above technical problems, the present invention aims to provide three type IV collagens, namely type IV collagen α1 (COL4A1), type IV collagen α3 (COL4A3), and type IV collagen α5 (COL4A5) monoclonal antibodies and their variable region sequences, and thereby form a pathological diagnosis kit, and its application in the auxiliary diagnosis of Alport syndrome. In order to achieve this purpose, the technical solution adopted by the present invention is as follows:

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

[0009] The second aspect of the present invention provides a COL4A3 monoclonal antibody, which can specifically recognize the protein fragment of type IV collagen α3 as shown in SEQ ID NO: 9. In some embodiments of the present invention, the COL4A3 monoclonal antibody is obtained by cross-linking KLH with a human COL4A3 protein polypeptide fragment with an amino acid sequence as shown in SEQ ID NO: 9 and then immunizing a rabbit.

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

[0011] The third aspect of the present invention provides a COL4A5 monoclonal antibody, which can specifically recognize the protein fragment of type IV collagen α5 as shown in SEQ ID NO: 19. In some embodiments of the present invention, the COL4A5 monoclonal antibody is obtained by immunizing rabbits with a human COL4A5 protein polypeptide fragment with an amino acid sequence as shown in SEQ ID NO: 19, which is modified and cross-linked with KLH, preferably, the modification refers to adding a cysteine ​​to the amino terminus of the polypeptide amino acid sequence to facilitate the cross-linking reaction.

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

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

[0014] In some embodiments of the present invention, the antibody combination includes the COL4A1 monoclonal antibody described in the first aspect of the present invention, and also includes 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 other embodiments of the present invention, the antibody combination includes the COL4A3 monoclonal antibody described in the second aspect of the present invention, and also includes a COL4A1 antibody prepared or obtained by any method, preferably, also includes a COL4A5 antibody prepared or obtained by any method, preferably, the antibody is a monoclonal antibody.

[0016] In some other embodiments of the present invention, the antibody combination includes the COL4A5 monoclonal antibody described in the third aspect of the present invention, and also includes a COL4A1 antibody prepared or obtained by any method, preferably, also includes a COL4A3 antibody prepared or obtained by any method, preferably, the antibody is a monoclonal antibody.

[0017] The fifth aspect of the present invention provides use of the COL4A1 monoclonal antibody described in the first aspect of the present invention, the COL4A3 monoclonal antibody described in the second aspect of the present invention, the COL4A5 monoclonal antibody described in the third aspect of the present invention, or the antibody combination described in the fourth aspect of the present invention in the preparation of a type IV collagen immunoassay kit.

[0018] In some embodiments of the present invention, the immunodetection is selected from at least one of the group consisting of immunohistochemistry, immunofluorescence, enzyme-linked immunosorbent assay, and immunoblotting.

[0019] The sixth aspect of the present invention provides a type IV collagen immunoassay kit, wherein the kit comprises the COL4A1 monoclonal antibody described in the first aspect, the COL4A3 monoclonal antibody described in the second aspect of the present invention, the COL4A5 monoclonal antibody described in the third aspect of the present invention, or the antibody combination described in the fourth aspect of the present invention.

[0020] In some embodiments of the invention, the kit is used to diagnose Alport syndrome.

[0021] In some embodiments of the present invention, the kit comprises the COL4A3 monoclonal antibody described in the second aspect of the present invention and the COL4A1 monoclonal antibody prepared or obtained by any method, wherein the COL4A1 monoclonal antibody and the COL4A3 monoclonal antibody are respectively coupled to different fluoresceins, or the COL4A1 monoclonal antibody and the COL4A3 monoclonal antibody are respectively coupled to different enzyme markers. Preferably, the COL4A1 monoclonal antibody is the COL4A1 monoclonal antibody described in the first aspect of the present invention.

[0022] In some embodiments of the present invention, 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 invention, the COL4A1 monoclonal antibody and the COL4A3 monoclonal antibody are conjugated to Texas Red and FITC, respectively.

[0023] In some embodiments of the present invention, the enzyme markers are selected from two of horseradish peroxidase (HRP), alkaline phosphatase (AP), and β-galactosidase (β-Gal). In some embodiments of the present invention, the COL4A1 monoclonal antibody and the COL4A3 monoclonal antibody are coupled to HRP and AP, respectively.

[0024] In other embodiments of the present invention, the kit comprises the COL4A5 monoclonal antibody described in the third aspect of the present invention and the COL4A1 monoclonal antibody prepared or obtained by any method, wherein the COL4A1 monoclonal antibody and the COL4A5 monoclonal antibody are respectively coupled to different fluoresceins, or the COL4A1 monoclonal antibody and the COL4A5 monoclonal antibody are respectively coupled to different enzyme markers. Preferably, the COL4A1 monoclonal antibody is the COL4A1 monoclonal antibody described in the first aspect of the present invention.

[0025] In some embodiments of the present invention, 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 invention, the COL4A1 monoclonal antibody and the COL4A5 monoclonal antibody are conjugated with Texas Red and FITC, respectively.

[0026] In some embodiments of the present invention, the enzyme markers are selected from two of HRP, AP, and β-Gal. In some embodiments of the present invention, the COL4A1 monoclonal antibody and the COL4A3 monoclonal antibody are coupled to HRP and AP, respectively.

[0027] In some other embodiments of the present invention, the kit comprises: the antibody combination described in the fourth aspect of the present invention, and also comprises: a polyclonal antibody against the antibody combination, and a fluorescent dye combination equal to the sum of the number of monoclonal antibodies in the antibody combination and the number of the polyclonal antibodies, and the fluorescent colors of the fluorescent dyes in the fluorescent dye combination are all different.

[0028] In some specific embodiments of the present invention, the kit comprises: an antibody combination consisting of the COL4A1 monoclonal antibody described in the first aspect of the present invention and the COL4A3 monoclonal antibody described in the second aspect of the present invention, a polyclonal antibody against the COL4A1 monoclonal antibody and the COL4A3 monoclonal antibody, and a combination of three fluorescent dyes, wherein the fluorescent colors of the fluorescent dyes in the fluorescent dye combination are different.

[0029] In other specific embodiments of the present invention, the kit comprises: an antibody combination consisting of the COL4A1 monoclonal antibody described in the first aspect of the present invention and the COL4A5 monoclonal antibody described in the third aspect of the present invention, a polyclonal antibody against the COL4A1 monoclonal antibody and the COL4A5 monoclonal antibody, and a combination of three fluorescent dyes, wherein the fluorescent colors of the fluorescent dyes in the fluorescent dye combination are different.

[0030] In some further specific embodiments of the present invention, the kit comprises: an antibody combination consisting of the COL4A1 monoclonal antibody described in the first aspect of the present invention, the COL4A3 monoclonal antibody described in the second aspect of the present invention, and the COL4A5 monoclonal antibody described in the third aspect of the present invention, a polyclonal antibody against the COL4A1 monoclonal antibody, the COL4A3 monoclonal antibody, and the COL4A5 monoclonal antibody, and a combination of four fluorescent dyes, wherein the fluorescent colors of the fluorescent dyes in the fluorescent dye combination are all different.

[0031] In some embodiments of the present invention, the fluorescent dye can be any fluorescent dye known in the art, as long as the different fluorescent colors are different, the requirements of the present invention can be met. In some embodiments of the present invention, when 4 fluorescent dyes are required, the fluorescent dyes are, for example, 520, 570, 690 and DAPI.

[0032] Beneficial effects of the present invention

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] By selecting specific sequences of protein fragments and subsequently modifying and designing the immunogen, rabbit monoclonal antibodies with excellent sensitivity and specificity were prepared, which are suitable for various immunoassays, especially immunohistochemistry and immunofluorescence.

[0035] Immunohistochemistry (IHC) refers to an immunological detection technique in which specific antibodies labeled with colorants are used to qualitatively, locally or quantitatively determine the corresponding antigens in pathological tissues through antigen-antibody reactions and histochemical color reactions in situ in tissue cells. The principle of immunohistochemical detection is as follows: tissue sections are treated with antigen heat repair and then incubated with primary antibody reagents 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 then incubated with the enzyme-labeled polymer secondary antibody to further form an antigen-antibody-secondary antibody polymer complex in situ; finally, a colored deposit is formed at the antigen site by the enzyme-catalyzed substrate. The presence of the target antigen and its expression can be determined by observing the brown area under an optical microscope. Immunohistochemical staining can determine the type and morphology of pathological tissue cells, identify the source of tissue cell products, and determine the degree of differentiation of tissue cells; especially in clinical pathology, its application is most important, such as identifying the nature of lesions, discovering micro-lesions, exploring the origin or differentiation phenotype of tumors, determining tumor staging, guiding treatment and prognosis; assisting in disease diagnosis and classification, and finding the cause of infection. At present, the specific detection method for hereditary nephritis in clinical practice adopts the fluorescence method, which requires the labeling of fluorescein and the configuration of a fluorescence microscope for the result judgment. If an immunohistochemical detection kit can be developed, the result judgment will not require a fluorescence microscope, and an ordinary light microscope can be used.

[0036] The multi-label immunofluorescence staining kit is a multi-label counterstaining solution developed based on TSA, which allows the use of different primary antibodies from the same species in the same sample, and the covalent binding of various labeled tyramides to the target antigen in a sequential single staining manner (antigen direct labeling), followed by microwave heating to remove the antibody (the antibody is gone but the signal remains on the antigen). After removing the previous round of antibodies, you can continue staining with new antibodies without worrying about cross-reactions. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

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

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

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

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

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

[0046] Fig.10 The figure shows the purified peak shape of the COL4A5 rabbit monoclonal antibody coupled to FITC complex prepared in Example 4 of the present invention.

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

[0048] Fig.12 The figure shows the purified peak shape of the COL4A1 rabbit monoclonal antibody-Texas Red complex prepared in Example 4 of the present invention.

[0049] Fig.13 The staining results of the COL4A1 rabbit monoclonal antibody-coupled Texas Red complex prepared in Example 4 of the present invention and the unlabeled COL4A1 rabbit monoclonal antibody under a fluorescence microscope are shown.

[0050] Fig.14 The fluorescence staining results of three different concentration formulas of the COL4A1 and COL4A5 fluorescence dual staining kit prepared in Example 4 of the present invention are shown.

[0051] Fig.15 The staining results of Alport syndrome detected by the COL4A1-COL4A5 fluorescent dual staining kit prepared in Example 4 of the present invention are shown.

[0052] Fig.16 The results of Alport syndrome staining detected by the control one-step fluorescent double staining kit are shown.

[0053] Fig.17 The staining results of Alport syndrome detected by the 4-color multi-labeled immunofluorescence staining kit prepared in Example 5 of the present invention are shown.

[0054] Fig.18 The results of staining for Alport syndrome detected by using the anti-COL4A1-HRP and COL4A5-AP visible light detection kit prepared in Example 6 of the present invention are shown. DETAILED DESCRIPTION

[0055] Unless otherwise indicated, implied from the context, or customary in the prior art, all parts and percentages in this application are based on weight, and the test and characterization methods used are all current as of the filing date of this application. Where applicable, the contents of any patent, patent application or publication referred to in this application are fully incorporated herein by reference, and their equivalent patent families are also incorporated by reference, especially the definitions of synthesis techniques, product and processing designs, polymers, comonomers, initiators or catalysts disclosed in these documents in the art. If the definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition of the term provided in this application shall prevail.

[0056] Numerical ranges in this application are approximate, and therefore may include values ​​outside the range unless otherwise specified. Numerical ranges include all values ​​from the lower limit to the upper limit in increments of 1 unit, provided that there is a gap of at least 2 units between any lower value and any higher value. These are only specific examples of what is intended to be expressed, and all possible combinations of values ​​between the lowest and highest values ​​listed are considered to be clearly recorded in this application.

[0057] When used with respect to chemical compounds, unless expressly specified otherwise, the singular includes all isomeric forms and vice versa (e.g., "hexane" includes all isomers of hexane, individually or collectively). In addition, nouns using "a," "an," or "the" also include their plural forms unless expressly specified otherwise.

[0058] The terms "comprising", "including", "having" and their derivatives do not exclude the presence of any other components, steps or processes, and are irrelevant to whether these other components, steps or processes are disclosed in this application. To eliminate any doubt, unless explicitly stated, all compositions using the terms "comprising", "including", or "having" in this application may include any additional additives, adjuvants or compounds. On the contrary, except for those necessary for operating performance, the term "essentially consisting of..." excludes any other components, steps or processes from the scope of any description of the term below. The term "consisting of..." does not include any components, steps or processes that are not specifically described or listed. Unless explicitly stated, the term "or" refers to the listed individual members or any combination thereof.

[0059] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments.

[0060] Example

[0061] The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention.

[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 invention belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

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

[0064] The molecular biology experimental methods not specifically described in the following examples are all carried out in accordance with the specific methods listed in the book Molecular Cloning Experiment Guide (4th Edition) (J. Sambrook, MR Green, 2017), or in accordance with the kits and product instructions. Other experimental methods are conventional methods unless otherwise specified. The instruments and equipment used in the following examples are conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0065] Example 1 Preparation of COL4A1 Antibody

[0066] The preparation steps of 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, rabbit serum was collected and the antibody titer in the serum was detected using the ELISA method. The ELISA titer detection data is shown in Figure 1 Among them, rabbit number 3 had the highest serum titer.

[0068] (2) Whole blood of rabbit 3 was taken to separate PBMC (peripheral blood lymphocytes) for B cell cloning culture. After 9 days of culture, the B cell supernatant was sent for ELISA and IHC testing. The cells in the IHC positive wells were lysed with cell lysis buffer and stored at -80°C for later use.

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

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

[0071] The COL4A1 rabbit monoclonal antibody was analyzed using VBASE2 software. The results showed that the variable region sequence information of the antibody was as follows:

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

[0073] QEQLVESGGGLVQPEGSLTLTCTASGFSFSSIYYIYWVRQAPGKGLEWIACIYGGSSGTSYYASWAKGRFTVSKTSSTTVTLQMTSLTAADTATYFCARDAGYLGYDYVLWGPGTLVIVSS

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

[0075] DPVLTQTPASVSAAVGGTVTINCQASQNIANLLAWYQQKPGQPPKLLIYKASTLQSGVSSRFKGSGSGTEFTLTISDLECADAATYYCQSVAYGSTYVAAFGGGTEVVVKGDP

[0076] The CDR sequence information is as follows:

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

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

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

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

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

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

[0083] Example 2 Preparation of COL4A3 Antibody

[0084] Since human COL4A3 protein and rabbit COL4A3 protein have high homology, it is difficult to obtain good rabbit antibodies against human COL4A3 protein by immunizing with randomly designed antigens. In terms of antigen selection, the aa1557-1570 polypeptide of human COL4A3 protein was selected as the immunogen and detector. This segment of immunogen differs from the aa1381-1394 polypeptide of rabbit COL4A3 protein by 3 amino acids. The amino acid sequence of the former is: AIAVHSQTTDIPPC (SEQ ID NO: 9), and the amino acid sequence of the latter is: AIAIHSQSTDVPPC (SEQ ID NO: 10). The aa1557-1570 polypeptide of human COL4A3 protein can produce 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 polypeptide 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 is shown in Figure 4 Among them, rabbit number 2 had the highest serum titer.

[0087] (2) Whole blood was obtained 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, transfected into HEK293 cells, and 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 has specific positive staining only 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 includes: SSYWIC (SEQ ID NO: 13).

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

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

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

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

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

[0102] Example 3 Preparation of COL4A5 Antibody

[0103] Similar to COL4A3, since human COL4A5 protein and rabbit COL4A5 protein have high homology, it is difficult to obtain good rabbit antibodies against human COL4A5 protein by immunizing with randomly designed antigens. In terms of antigen selection, the aa1277-1294 polypeptide of human COL4A5 protein was selected as the immunogen and detector. This segment of immunogen differs from the aa1283-1300 polypeptide of rabbit COL4A5 protein by 4 amino acids. The amino acid sequence of the former is: NGGIKGEKGNPGQPGLPG (SEQ ID NO: 19), and the amino acid sequence of the latter is: NGAIKGERGNPGPPGQPG (SEQ ID NO: 20). As many antibodies as possible can be generated against the aa1277-1294 polypeptide of human COL4A5 protein.

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

[0105] (1) Using the aa1277-1294 polypeptide of the human COL4A5 protein, a cysteine ​​was added to the amino terminus for the convenience of subsequent cross-linking, i.e., the sequence was CNGGIKGEKGNPGQPGLPG, and four New Zealand white rabbits were immunized with cross-linked KLH. After four immunizations, rabbit serum was collected and the antibody titer in the serum was detected by ELISA. The ELISA titer detection data is 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 the 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 , Fig. 9 .Depend on Figure 8 It can be seen that the titer of the developed COL4A5 rabbit monoclonal antibody is 8.29 ng / mL. Fig. 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 analyzed 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 is as follows (SEQ ID NO: 21):

[0111] CQSVEESGGRLVTPGTPLTLTCTVSGFSLSSYGVSWVRQAPGKGLEYIGMVSTGGYIYYASWARGRSTITRNTNENTVTLKMTSPTTEDTATYFCSREYSNYGTGSFGLWGPGTLVTVSS

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

[0113] AQVLTQTPSSVSAAVGGTVTINCQASQSVYNNKNLAWYQQKPGQPPKLLIYKASTLASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCQGEFSCSSADCFGFGGGTEVVVKGDP

[0114] The CDR sequence information is as follows:

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

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

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

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

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

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

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

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

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

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

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

[0126] (3) Incubate at 37°C in the dark for 90 to 150 minutes.

[0127] (4) Use a desalting column (Sephadex G25) to remove free FITC. First, rinse the Sephadex G25 column with 25 mL PBS.

[0128] (5) Collect the first FITC-bound COL4A5 rabbit monoclonal antibody IgG protein peak eluted with PBS and determine the F / P ratio (i.e., the number of fluorescent molecules labeled on each protein molecule). The appropriate F / P ratio is 2 to 5. The second fluorescent peak is free fluorescent and is discarded.

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

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

[0131] Fig.10 This is the peak shape of the purification of COL4A5 rabbit monoclonal antibody coupled with FITC (monitoring OD 495 ), where the first peak from the left is the FITC-bound COL4A5 monoclonal antibody IgG protein peak, and the second peak is the free fluorescein.

[0132] Labeling concentration: The concentration of labeled fluorescein in this example was calculated by the formula to be 2.18 mg / mL, and the coupling 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 conjugate complex and the unlabeled COL4A5 rabbit monoclonal antibody. The specific steps are as follows.

[0134] ① The paraffin sections of renal tissue were baked at 60℃ for 1h, dewaxed and hydrated in the usual way, repaired with high temperature and normal pressure Tris-EDTA (pH9.0) repair solution, and then 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 coupling complex to one slide, and add 100 μL of 50 μg / mL unlabeled COL4A5 rabbit monoclonal antibody to the other slide, and incubate at room temperature for 30 min;

[0138] ⑤After 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 at room temperature away from light, and observe the staining results under a fluorescence microscope.

[0139] Fig.11 The staining results of COL4A5-FITC coupled complex and COL4A5 monoclonal antibody without FITC labeling under fluorescence microscope. The results showed that COL4A5-FITC coupled complex had green fluorescence staining on the basement membrane of Bowman's capsule of glomerulus, and the cell nucleus was blue ( Fig.11 Left); Unlabeled COL4A5 monoclonal antibody has no green fluorescence staining under fluorescence microscope, only blue staining of nuclei ( Fig.11 Right). The results showed that FITC was successfully coupled to 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 with Texas Red fluorescent dye

[0141] The COL4A1 rabbit monoclonal antibody prepared in Example 1 was coupled with Texas Red, and the specific steps were as follows:

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

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

[0144] (3) Incubate the reaction at room temperature in the dark for 60 to 120 minutes.

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

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

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

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

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

[0150] Fig.12 This is the peak shape of the purification of COL4A1 rabbit monoclonal antibody coupled to Texas Red (monitoring OD 595 ), where the first peak is the Texas Red-bound COL4A1 rabbit monoclonal antibody IgG protein peak, and the second peak is the free fluorescein.

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

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

[0153] Fig.13The staining results of COL4A1-Texas Red coupled complex and COL4A1 monoclonal antibody without Texas Red under fluorescence microscope. COL4A1-Texas Red coupled complex has red fluorescence staining on the basement membrane of Bowman's capsule of glomerulus, and the cell nucleus is blue ( Fig.13 Left); unlabeled COL4A1 monoclonal antibody showed no staining under fluorescence microscopy ( Fig.13 Right). The results showed that Texas Red was successfully coupled to COL4A1 rabbit monoclonal antibody and can be used to form a reagent combination or packaged into a kit for detection.

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

[0155] The COL4A5 rabbit monoclonal antibody-FITC complex prepared in step 1 and the COL4A1 rabbit monoclonal antibody-TexasRed complex prepared in step 2 were mixed according to the following table, and then immunofluorescence verification was performed using kidney tissue.

[0156] Recipe Number Final concentration of COL4A5 rabbit monoclonal antibody-FITC complex Final concentration of COL4A1 rabbit monoclonal antibody-Texas Red complex 1 2 μg / mL 2 μg / mL 2 5 μg / mL 5 μg / mL 3 10 μg / mL 10 μg / mL

[0157] Fig.14 These are the fluorescence staining pictures of three different concentration formulas of the COL4A1 and COL4A5 fluorescent double staining kit. From the staining results of formula 1, we can see that the fluorescence staining of the two antibodies is weak; from the staining results of formula 2, we can see that the fluorescence staining of the two antibodies is moderate, and no fluorescence background is generated; from the staining results of formula 3, we can see that the fluorescence staining of the two antibodies is strong, but there is a high fluorescence background.

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

[0159] Therefore, this embodiment provides a COL4A1 and COL4A5 fluorescent dual staining kit, comprising:

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

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

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

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

[0164] Sample type: paraffin section or frozen section

[0165] (1) Paraffin sections of normal renal tissue and Alport syndrome-positive renal tissue, 2 sections for each tissue, baked, dewaxed, hydrated, and then normal pressure / high pressure antigen repaired using Tris-EDTA (pH 9.0), cooled to room temperature. After washing with water, circle with an immunohistochemistry pen and soak in PBST. If the tissue type is frozen section, baking, dewaxing, and hydration are not required.

[0166] (2) Add 2-3 drops of component 1 to block endogenous peroxidase and incubate at room temperature for 10 min.

[0167] (3) Add 2-3 drops of component 2 in the COL4A1 and COL4A5 fluorescent double staining kit (the final concentrations of COL4A5 rabbit monoclonal antibody-FITC complex and COL4A1 monoclonal antibody-Texas Red complex were both 5 μg / mL) to one normal renal tissue section and one Alport syndrome-positive renal tissue section respectively; add 2-3 drops of control substance (COSMO, Cat#SGE-CFT45325) to the other two sections and incubate at room temperature in the dark for 30 min.

[0168] (4) After washing three times with PBST, add 1 drop (about 20 μL) of component 3 to seal the slide and place it at room temperature away from light for 5-10 min.

[0169] The tissue sections were placed under a fluorescence microscope and photographed using the green and red fluorescence channels respectively, and then merged.

[0170] Fluorescence staining results are shown in Fig.15 All normal renal tubules and glomerular basement membranes are stained with red fluorescence, and the glomerular basement membrane also has green fluorescence staining. In the fluorescent staining of renal tissue positive for Alport syndrome, the renal tubules and glomerular basement membranes are stained with red fluorescence, but no green fluorescence signal is seen. Fig.16 The staining results of the control substance in normal renal tissue and Alport syndrome positive renal tissue are shown in Figure 2. Fig.16 It can be seen that the staining results of the kit formula in normal kidney tissue are weaker than the staining results of the COL4A1 and COL4A5 fluorescent dual staining kit prepared in this embodiment. The above results show that the kit prepared in this embodiment has higher sensitivity than the control kit.

[0171] Example 5 4-color multi-labeled immunofluorescence staining kit and its application

[0172] 1.4-color multi-label immunofluorescence staining kit

[0173] In this example, the COL4A1 rabbit monoclonal antibody, COL4A3 rabbit monoclonal antibody and COL4A5 rabbit monoclonal antibody prepared in Examples 1 to 3 were used as primary antibodies to prepare a 4-color multi-labeled immunofluorescence staining kit, which includes the following components:

[0174] ① Component 1: peroxidase blocker (3% H2O2);

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

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

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

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

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

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

[0181] Application of 2.4-color multi-label immunofluorescence staining kit in the detection of Alport syndrome

[0182] The specific experimental steps are as follows:

[0183] Sample type: paraffin section or frozen section

[0184] (1) After baking, dewaxing, and hydrating, paraffin sections of normal renal tissue and Alport syndrome-positive renal tissue are subjected to high-temperature, normal-pressure, and high-pressure antigen retrieval using Tris-EDTA (pH 9.0) and then cooled to room temperature. After washing with water, circles are drawn with an immunohistochemistry pen and then soaked in PBST. If the tissue type is frozen section, baking, dewaxing, and hydration are not required.

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

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

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

[0188] (5) Use component 7 to dilute the fluorescent dye 520 in component 6 (100:1), add 100 μL / slide to the tissue area on the slide, and incubate at room temperature for 10 minutes; wash the slide three times with PBST.

[0189] (6) Place the slide in boiled antigen retrieval buffer (Tuling (Hangzhou) Biotechnology Co., Ltd., Cat#I30082B) and boil for 10 to 15 minutes, and repeat step (2).

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

[0191] (8) Use component 7 to dilute the fluorescent dye 570 in component 6 (100:1) and incubate at room temperature for 10 min; wash the slides three times with PBST.

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

[0193] (10) Use component 7 to dilute the fluorescent dye 690 in component 6 (100:1) and incubate at room temperature for 10 min; wash the slides three times with PBST.

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

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

[0196] Fig.17 It is a 4-color multi-label immunofluorescence staining kit for detecting normal renal tissue and renal tissue of patients with Alport syndrome. Fig.17 The middle left shows that COL4A1 is positively localized in the basement membrane of normal glomeruli and renal tubules, and COL4A3 and COL4A5 are positively localized in the basement membrane of Bowman's capsule of normal glomerulus. Fig.17 The right side shows that COL4A1 is positively localized in the basement membrane of the glomeruli and tubules of patients with Alport syndrome, while COL4A3 and COL4A5 are not positively localized in the basement membrane of the Bowman capsule of the glomerulus of patients with Alport syndrome.

[0197] The above results show that the 4-color multi-labeled immunofluorescence staining reagent prepared in this example has very high specificity and sensitivity in detecting Alport syndrome, and this is the first time in the art that this method has been used to detect hereditary glomerulonephritis.

[0198] Example 6 Visible light detection kit for type IV collagen and its application in Alport syndrome

[0199] In this example, the COL4A1 rabbit monoclonal antibody prepared in Example 1 and the COL4A5 rabbit monoclonal antibody prepared in Example 3 were used 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. Visible light detection kit for type IV collagen

[0201] The COL4A1 rabbit monoclonal antibody-HRP complex and the COL4A5 rabbit monoclonal antibody-AP coupling complex were used to prepare a type IV collagen visible light detection kit, which contains:

[0202] ① Component 1: peroxidase blocker (3% H2O2);

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

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

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

[0206] ⑤ Component 5: Fast-Red chromogen (150×), Fast-RED Buffer (Abcam, Cat#ab103741 or reagents with the same technical specifications);

[0207] ⑥Hematoxylin staining solution.

[0208] 2. Application of Type IV Collagen Visible Light Detection Kit in Hereditary Glomerulonephritis

[0209] The specific experimental steps are as follows:

[0210] Sample type: paraffin section or frozen section

[0211] (1) After baking, dewaxing, and hydrating, paraffin sections of normal renal tissue and Alport syndrome-positive renal tissue are subjected to high-temperature, normal-pressure, and high-pressure antigen retrieval using Tris-EDTA (pH 9.0) and then cooled to room temperature. After washing with water, circles are drawn with an immunohistochemistry pen and then soaked in PBST. If the tissue type is frozen section, baking, dewaxing, and hydration are not required.

[0212] (2) Add 2 to 3 drops of component 1 to block endogenous peroxidase and incubate at room temperature for 10 min.

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

[0214] (4) Add 2 to 3 drops of component 3 to the two sections that have been incubated at room temperature. After incubation at room temperature for 30 minutes, wash the slides three times with PBST.

[0215] (5) Add 100 μL of the working solution of component 4 onto the tissue sections. The working solution formula is DAB chromogen (20×): DAB Buffer = 1:19. Incubate at room temperature for 2 to 10 min. Wash the slides three times with PBST.

[0216] (6) Add 100 μL of the working solution of component 4 onto the tissue section. The working solution formula is Fast-Red chromogen: Fast-RED Buffer = 1:150. Incubate at room temperature for 10-15 min. Wash the slides three times with tap water.

[0217] (7) Use component 6 for counterstaining, followed by dehydration, transparency, and sealing.

[0218] Take pictures under visible light, and see the results Fig.18 .

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

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

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

Claims

1. A COL4A5 monoclonal antibody against COL4A5, characterized in that: The COL4A5 monoclonal antibody specifically recognizes type IV collagen α5, and the amino acid sequence of its heavy chain variable region CDR-H1 is shown in SEQ ID NO: 23, the amino acid sequence of CDR-H2 is shown in SEQ ID NO: 24, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO: 25, the amino acid sequence of the light chain variable region CDR-L1 is shown in SEQ ID NO: 26, the amino acid sequence of CDR-L2 is shown in SEQ ID NO: 27, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO:

28.

2. An antibody combination for detecting type IV collagen, characterized in that: The antibody combination includes anti-COL4A1 COL4A1 monoclonal antibody and anti-COL4A5 COL4A5 monoclonal antibody according to claim 1, and the anti-COL4A1 COL4A1 monoclonal antibody specifically recognizes type IV collagen α1, and the amino acid sequence of its heavy chain variable region CDR-H1 is shown in SEQ ID NO: 3, the amino acid sequence of CDR-H2 is shown in SEQ ID NO: 4, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO: 5, the amino acid sequence of the light chain variable region CDR-L1 is shown in SEQ ID NO: 6, the amino acid sequence of CDR-L2 is shown in SEQ ID NO: 7, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO:

8.

3. The antibody combination according to claim 2, characterized in that The antibody combination also includes an anti-COL4A3 COL4A3 monoclonal antibody, which specifically recognizes type IV collagen α3, and the amino acid sequence of its heavy chain variable region CDR-H1 is shown in SEQ ID NO: 13, the amino acid sequence of CDR-H2 is shown in SEQ ID NO: 14, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO: 15, the amino acid sequence of the light chain variable region CDR-L1 is shown in SEQ ID NO: 16, the amino acid sequence of CDR-L2 is shown in SEQ ID NO: 17, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO:

18.

4. Use of the anti-COL4A5 COL4A5 monoclonal antibody according to claim 1 or the antibody combination according to any one of claims 2 to 3 in the preparation of a type IV collagen immunoassay kit.

5. The use according to claim 4, characterized in that: The immunoassay is selected from at least one of the group consisting of immunohistochemistry, immunofluorescence, enzyme-linked immunosorbent assay, and immunoblotting.

6. The use according to claim 5, characterized in that: The type IV collagen immunoassay kit is used for diagnosing Alport syndrome.

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