Antibody against complement protein C5 and its use

Abstract

The present application relates to the technical field related to the detection of complement protein C5 and soluble membrane attack complex SC5b-9, and specifically discloses an anti-complement protein C5 antibody and its use. An anti-complement protein C5 antibody includes a light chain and a heavy chain, and the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 in the variable region of the light chain are shown in SEQ ID NO.3, 4, and 5, respectively; the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 in the heavy chain variable region of the antibody are shown in SEQ ID NO.9, 10, and 11, respectively. The anti-complement protein C5 antibody based on the present application shows high affinity and high specificity for the soluble membrane attack complex SC5b-9, and can achieve quantitative detection of the soluble membrane attack complex SC5b-9.

Description

Technical Field

[0001] The present application relates to the technical field related to the detection of complement protein C5 and soluble membrane attack complex SC5b-9, and specifically to an anti-complement protein C5 antibody and its use. Background Art

[0002] The complement system is a crucial component of the body's innate immunity, composed of over 50 soluble and membrane-bound proteins. It plays a crucial role in a variety of life activities, including defense against foreign substances, cell lysis, inflammatory responses, dissolution of immune complexes, clearance of apoptotic cells, and enhancement of humoral immune responses. Complement activation occurs via three main pathways: the classical pathway, the alternative pathway, and the mannose-binding lectin (MBL) pathway.

[0003] Complement protein C5 is a major component of the complement system and a key part of the innate immune system. Complement protein C5 is a 190 kDa glycoprotein composed of two disulfide-linked polypeptide chains, α and β, with molecular masses of 115 kDa and 75 kDa. C5 convertase cleaves C5 at the arginine residue 74 amino acids downstream of the N-terminus of the α-chain, splitting C5 into C5a and C5b. Among them, C5a is an anaphylatoxin that can stimulate vasodilation through histamine released by basophils and giant cells; C5b binds to the cell surface and then forms a terminal complement complex (TCC) with complement proteins C6, C7, C8, and C9. TCC has two forms: (1) exists in plasma and binds to S protein to form a soluble membrane attack complex SC5b-9; (2) binds to the cell membrane and forms C5678(9)n with multiple C9 (C9n) and C5b-8, called membrane attack complex (MAC), which causes irreversible damage to the cell membrane and leads to cell lysis and destruction.

[0004] It can be seen from this that SC5b-9 is a hydrophilic, non-cytolytic macromolecular fragment formed by the common end product C5b-9 and S protein produced after activation of the complement system.

[0005] C5b-9's action on target cells can be categorized as fully lytic or sub-lytic. Different target cells produce different effects. When C5b-9 is assembled on the surface of anucleated cells (such as red blood cells), it can form transmembrane channels in the cell membrane, leading to cell lysis. When complement restriction factors (such as CD59) are present on the surface of nucleated cells, these complement regulatory proteins prevent C5b-9 from polymerizing on the cell membrane, interfering with C5b-9 formation. They also hinder C5b-9 insertion into the target cell's phospholipid bilayer, resulting in shallow insertion and preventing cell perforation and rupture. This type of C5b-9 is called a sub-lytic C5b-9 complex. C5b-9 can stimulate multiple biological activity changes in a variety of cells, including the production of inflammatory mediators and cytokines, and the induction of membrane protein expression. This cell-stimulating effect of C5b-9 plays a role in the pathogenesis and progression of various diseases, especially sublytic C5b-9, which has multiple pathogenic effects in diseases such as inflammation, neurological diseases, kidney disease and atherosclerosis.

[0006] Therefore, by detecting the content of SC5b-9 molecules in a sample, the activation of the complement system can be reflected. Summary of the Invention

[0007] In view of this, the present application provides an anti-complement protein C5 antibody and its use. The anti-complement protein C5 antibody based on the present application exhibits high affinity and high specificity for soluble membrane attack complex SC5b-9, and can achieve quantitative detection of soluble membrane attack complex SC5b-9.

[0008] In the first aspect of the present application, the present application provides an anti-complement protein C5 antibody or an antigen-binding fragment thereof, wherein the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 in the light chain variable region of the antibody or its antigen-binding fragment are shown as SEQ ID NOs. 3, 4, and 5, respectively; and the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 in the heavy chain variable region of the antibody or its antigen-binding fragment are shown as SEQ ID NOs. 9, 10, and 11, respectively.

[0009] In some specific embodiments of the present application, the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment is shown as SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is shown as SEQ ID NO.8.

[0010] In some specific embodiments of the present application, the amino acid sequence of the light chain constant region of the antibody is shown as SEQ ID NO. 6. In some specific embodiments of the present application, the amino acid sequence of the light chain of the antibody is shown as SEQ ID NO. 1.

[0011] In some specific embodiments of the present application, the amino acid sequence of the heavy chain constant region of the antibody is shown as SEQ ID NO. 12. In some specific embodiments of the present application, the amino acid sequence of the heavy chain of the antibody is shown as SEQ ID NO. 7.

[0012] In some specific embodiments of the present application, the antibody or antigen-binding fragment thereof is selected from Fab, Fab', F(ab')2, a monoclonal antibody, an animal-derived antibody, or a humanized antibody.

[0013] In some specific embodiments of the present application, the antibody is selected from a monoclonal antibody.

[0014] In some specific embodiments of the present application, the antibody is selected from IgG antibodies.

[0015] In the second aspect of the present application, the present application provides a polynucleotide encoding the antibody or antigen-binding fragment thereof described in the first aspect of the present application.

[0016] Based on the amino acid sequence of the antibody described in the first aspect of this application, those skilled in the art can obtain the nucleotide sequence of a polynucleotide encoding the antibody described in the first aspect of this application. Due to the degeneracy of codons, the nucleotide sequence of a polynucleotide is not unique. All polynucleotides capable of encoding the antibody described in the first aspect of this application are within the scope of protection of this application.

[0017] In the third aspect of the present application, the present application provides a biological material containing the polynucleic acid described in the second aspect of the present application.

[0018] The biological material includes, but is not limited to, an expression cassette, a vector, or a host cell. The expression cassette can be obtained by connecting a transcriptional or translational regulatory element, such as a promoter, upstream of the polynucleic acid and / or a transcriptional or translational regulatory element, such as a terminator, downstream thereof. The vector includes, but is not limited to, a plasmid vector, a phage vector, a viral vector, an artificial chromosome vector, and the like. The host cell includes a microbial cell, an insect cell, or other animal cell.

[0019] In the fourth aspect of the present application, the present application provides an antibody conjugate, which is obtained by conjugating the antibody or antigen-binding fragment thereof described in the first aspect of the present application with a label.

[0020] In some optional embodiments of the present application, the label is selected from at least one of an enzyme label, a biotin label, a fluorescent dye label, a chemiluminescent dye label, and a radioactive label.

[0021] In a fifth aspect of the present application, the present application provides use of any of the following items in the preparation of a product for detecting complement protein C5 or soluble membrane attack complex SC5b-9:

[0022] (I) the antibody or antigen-binding fragment thereof according to the first aspect of the present application; and / or

[0023] (II), the polynucleotide described in the second aspect of the present application; and / or

[0024] (III) the biomaterial described in the third aspect of the present application; and / or

[0025] (IV) The antibody conjugate described in the fourth aspect of the present application.

[0026] In the sixth aspect of the present application, the present application provides a detection reagent for detecting complement protein C5 or soluble membrane attack complex SC5b-9, wherein the detection reagent comprises the antibody or antigen-binding fragment thereof described in the first aspect of the present application, and / or the antibody conjugate described in the fourth aspect of the present application.

[0027] In the seventh aspect of the present application, the present application provides a detection kit for detecting complement protein C5 or soluble membrane attack complex SC5b-9, characterized in that the kit comprises the antibody or antigen-binding fragment thereof described in the first aspect of the present application, and / or the antibody conjugate described in the fourth aspect of the present application.

[0028] In some optional embodiments of the present application, the detection kit is selected from an enzyme-linked immunosorbent assay kit, a fluorescent immunoassay kit or a chemiluminescent immunoassay kit.

[0029] In some optional embodiments of the present application, the detection kit is selected from an enzyme-linked immunosorbent assay kit.

[0030] This application has the following beneficial effects:

[0031] First, the monoclonal antibody (anti-complement protein C5 antibody) produced by the present application using complement protein C5 as a target exhibits high affinity and high specificity for the soluble membrane attack complex SCb59. In addition, the anti-complement protein C5 antibody of the present application, combined with the anti-complement protein C9 antibody, can achieve quantitative detection of the soluble membrane attack complex SC5b-9 to predict the activation of the complement system in the sample.

[0032] Second, the anti-complement protein C5 antibody of the present application is also specific for binding to complement protein C5, and is suitable for testing plasma samples or samples from other tissue sources, and has clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1This is the standard curve for detecting soluble membrane attack complex SC5b-9 based on the double antibody sandwich method in Example 2 of the present application.

[0034] Figure 2 This is the binding activity curve of anti-complement protein C5 antibodies and complement protein C5 detected based on the direct ELISA method in Example 3 of the present application. DETAILED DESCRIPTION

[0035] The present application discloses an anti-complement protein C5 antibody and its use. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters for implementation. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art and are deemed to be included in this application. The methods and applications of this application have been described through preferred embodiments. Relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of this application to implement and apply the technology of this application.

[0036] Explanation of terms:

[0037] As used herein, "antibody" refers to an immunoglobulin (Ig) secreted by B lymphocytes after antigen stimulation, which possesses immune function (i.e., the ability to specifically bind to an antigen). Antibodies have a tetrameric structure, consisting of two identical heavy chains (H chains) and two identical light chains (L chains). The regions of the heavy and light chains near the N-terminus, where the amino acid sequence varies significantly, are called the variable region (V region). The amino acid sequences of the heavy and light chain variable regions exhibit significant variability primarily in three regions, known as the hypervariable regions (HVRs). These regions are designated as the complementarity determining regions (CDRs), as they are the sites where Ig binds to antigen epitopes. These regions are designated HVR1 (CDR1), HVR2 (CDR2), and HVR3 (CDR3), respectively, with CDR3 exhibiting the greatest variation. The regions of the heavy and light chains near the C-terminus, where the amino acid sequence remains relatively constant, are called the constant region (C region).

[0038] The "monoclonal antibody" used in this application is a specific antibody produced by B lymphocytes under the stimulation of specific antigens, which has an identical chemical structure and is complementary to the antigenic determinant.

[0039] As used herein, "amino acid difference" refers to amino acid modification (such as amino acid methylation modification) or amino acid replacement.

[0040] In order to make the purpose, technical solutions and advantages of this application clearer, the implementation scheme of this application will be further described in detail with reference to examples below.

[0041] Unless otherwise specified, the recombinant human complement protein C5 used in this application was purchased from Sanyou Biopharmaceuticals (Shanghai) Co., Ltd., catalog number #P217782.

[0042] Unless otherwise specified, the horseradish peroxidase (HRP)-labeled mouse anti-goat IgG antibody used in this application was purchased from Jackson Immunoresearch Inc., catalog number #205-035-108.

[0043] Unless otherwise specified, the recombinant human complement protein C5 used in this application was purchased from Sanyou Biopharmaceuticals (Shanghai) Co., Ltd., catalog number #P217782.

[0044] Example 1 Antibody against complement protein C5:

[0045] In this embodiment, the antibody against complement protein C5 is a monoclonal antibody, which is obtained by conventional mouse immunization and phage screening methods.

[0046] The method for preparing an antibody against complement protein C5 specifically comprises the following steps:

[0047] S101. Female Balb / c mice aged 6 to 8 weeks were selected and immunized with recombinant human complement protein C5 mixed with an equal volume of Freund's adjuvant and emulsified. The immunization cycle was two weeks. After three immunizations, blood was collected from the submandibular vein of the mice. The antibody titer of the mice's immune response to recombinant human complement protein C5 was detected by ELISA.

[0048] S102. After five immunizations, the mice were killed by cervical dislocation, and the spleens were removed aseptically. The total RNA from the mouse spleen tissue was extracted by squeezing and grinding in a plate, and then Trlzol reagent was added to extract the total RNA. The RNA was reverse transcribed into cDNA to obtain the gene sequence encoding the antibody after mouse immunization.

[0049] S103. Molecular cloning technology is used to construct the gene sequence encoding the antibody into a phage display vector. The constructed phage display vector is then electroporated into Escherichia coli to obtain a phage display library of mouse immune antibodies (anti-recombinant human complement protein C5).

[0050] S104. The phage library sequence is displayed on the phage surface by phage display technology. After that, the antibodies binding to the antigen are continuously enriched through multiple rounds of liquid / solid phase screening using biotinylated and non-biotinylated recombinant human complement protein C5, and the screening collection is detected by ELISA. The positive clones that specifically bind to the recombinant human complement protein C5 antigen are further screened by ELISA, and 34 positive clone antibodies are obtained by sequencing.

[0051] S105, using 34 positive cloned antibodies as templates, the antigen binding fragment (Fab) of the antibody molecule was obtained by PCR technology. The heavy chain Fab fragment was connected to pcDNA3.4-HC (goat-IgG), and the light chain Fab fragment was connected to pcDNA3.4-LC (goat-Kappa). The connected fragments were respectively transformed into Escherichia coli to obtain monoclonal colonies. Single colonies were selected for sequencing and identification. Single colonies with correct sequences were used for plasmid extraction to obtain heavy chain plasmids and light chain plasmids respectively. The heavy chain plasmid and light chain plasmid were co-transfected into 6×10 6 Expi CHO cells were transfected and cultured at 37°C, 7% CO₂ for 7 days. The supernatant was collected by centrifugation at 15,000 g and purified using MabSelect SuRe LX (Cytiva) resin to obtain anti-complement protein C5 antibodies.

[0052] The anti-complement protein C5 antibody comprises a light chain having an amino acid sequence as shown in SEQ ID NO.1 and a heavy chain having an amino acid sequence as shown in SEQ ID NO.7.

[0053] Among them, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2, the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 in the light chain variable region are shown in SEQ ID NO.3, 4, and 5, respectively; the amino acid sequence of the light chain constant region is shown in SEQ ID NO.6.

[0054] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.8, and the amino acid sequences of the complementarity determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NO.9, 10, and 11, respectively; the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.12.

[0055] Table 1 Sequence information of amino acid sequences SEQ ID NO. 1 to 10:

[0056]

[0057] Example 2 Enzyme-linked immunosorbent assay kit for detecting soluble membrane attack complex SC5b-9:

[0058] In this embodiment, the main components of the enzyme-linked immunosorbent assay reagent / enzyme-linked immunosorbent assay kit for detecting soluble membrane attack complex SC5b-9 include: capture antibody, detection antibody, enzyme-labeled antibody, diluent, washing solution, color development solution, and reaction termination solution.

[0059] Among them, the capture antibody: anti-human complement protein C9 antibody (purchased from Foshan Sichuang Biotechnology Co., Ltd., catalog number #SY-ab220505).

[0060] Detection antibody: the anti-complement protein C5 antibody of Example 1 of the present application.

[0061] Enzyme-labeled antibody: HRP-labeled mouse anti-goat IgG antibody.

[0062] Diluent: 0.1 M sodium bicarbonate buffer solution (pH = 9.6).

[0063] Washing solution: PBST buffer (0.05% by volume Tween-20, pH = 7.4).

[0064] Color developing solution: single-component 3,3',5,5'-tetramethylbenzidine (TMB) color developing solution.

[0065] Blocking solution: It is made by diluting bovine serum albumin (BSA) with PBS buffer (pH=7.4); the concentration of the blocking solution is 1% (mass volume ratio w / v).

[0066] Reaction termination solution: 1M HCl aqueous solution.

[0067] This kit uses a double-antibody sandwich assay to detect soluble membrane attack complex SC5b-9. First, an anti-human complement protein C9 antibody is coated onto an ELISA plate as the capture antibody. Then, a human SC5b-9 standard or sample is added to the wells of the plate. The human SC5b-9 protein in the standard or sample binds to the capture antibody immobilized on the plate. Next, an anti-complement protein C5 antibody is added to the wells of the plate. The anti-complement protein C5 antibody serves as the detection antibody, capturing the soluble membrane attack complex SC5b-9. Finally, an HRP-conjugated mouse anti-goat IgG antibody is added to the wells. Unbound HRP-conjugated mouse anti-goat IgG is washed away, and a chromogenic substrate (TMB) is added. TMB is converted to blue by horseradish peroxidase and to yellow by acid. The intensity of the color is positively correlated with the amount of human SC5b-9 protein in the sample. The absorbance (OD value) was measured at a wavelength of 450 nm using a microplate reader, and the concentration of human SC5b-9 protein in the sample was calculated by drawing a standard curve.

[0068] The detection kit is based on the double antibody sandwich method to detect soluble membrane attack complex SC5b-9, which specifically includes the following steps:

[0069] Step 201: Use a diluent to prepare an anti-complement protein C9 antibody working solution with a concentration of 2.5 μg / mL; add the anti-complement protein C9 antibody working solution to the ELISA plate at 50 μL / well, incubate at 4°C for 12 to 18 hours or at 37°C for 2 hours, and discard the solution in the microwells of the ELISA plate; then, add a washing solution to the microwells of the ELISA plate at 300 μL / well, and wash three times for 2 minutes each time.

[0070] Step 202: Add blocking solution to the ELISA plate at 300 μL / well, incubate at 37°C for 1-2 hours, and discard the solution in the microwells of the ELISA plate; then, add washing solution to the microwells of the ELISA plate at 300 μL / well, and wash three times for 2 minutes each time.

[0071] Step 203: Add gradient concentration standard solution or sample solution to be tested to the microwells of the ELISA plate at 50 μL / well, incubate at 37°C for 1 hour, and discard the solution in the microwells of the ELISA plate. Then, add washing solution to the microwells of the ELISA plate at 300 μL / well, and wash three times for 2 minutes each time.

[0072] Wherein, the gradient concentration standard solution: the terminal complement complex SC5b-9 is prepared into a gradient concentration standard solution of 0 ng / mL to 500 ng / mL using a diluent.

[0073] Sample solution to be tested: Dilute the blood sample 10 times with diluent; specifically: 20 μL of blood sample, 180 μL of diluent.

[0074] Step 204: Use a diluent to prepare an anti-complement protein C5 antibody working solution with a concentration of 0.062 μg / mL to 0.25 μg / mL; add the anti-complement protein C5 antibody working solution to the microwells of the ELISA plate at 50 μL / well, incubate at 37°C for 1 hour, and discard the solution in the microwells of the ELISA plate; then, add a washing solution to the microwells of the ELISA plate at 300 μL / well, and wash three times for 2 minutes each time.

[0075] Step 205: HRP-labeled mouse anti-goat IgG antibody is diluted with diluent to form HRP-labeled mouse anti-goat IgG antibody working solution at a dilution ratio of 1:10,000 to 1:20,000 (mass to volume ratio w / v); 50 μL / well of the HRP-labeled mouse anti-goat IgG antibody working solution is added to the microwells of the ELISA plate, and the plate is incubated at 37°C for 1 hour. The solution in the microwells of the ELISA plate is discarded; then, 300 μL / well of washing solution is added to the microwells of the ELISA plate, and the plate is washed three times for 2 minutes each time.

[0076] Step 206: Add chromogenic solution to the microwells of the ELISA plate, 90 μL / well, and react at 37° C. in the dark for 5 to 10 minutes.

[0077] Step 207: Add the reaction stop solution to the microwells of the ELISA plate, 50 μL / well, and gently shake the ELISA plate until the color is evenly developed.

[0078] Step 208: Use a microplate reader to read the OD value of each well in the microplate at a test wavelength of 450 nm, and calculate the concentration of human SC5b-9 protein in the sample by drawing a standard curve.

[0079] From this we get, Figure 2 The standard curve for detecting soluble membrane attack complex SC5b-9 protein is shown.

[0080] from Figure 1 It can be seen that the anti-complement protein C5 antibody and soluble membrane attack complex SC5b-9 of the present application have binding specificity and high affinity, are suitable for plasma sample detection or sample detection from other tissue sources, and have clinical application prospects.

[0081] The kit of this example detects the concentration of human SC5b-9 in blood samples numbered 1# to 32# based on the double antibody sandwich method. The detection results are shown in Table 2.

[0082] Table 2 Detection results of SC5b-9 concentrations in blood samples numbered 1# to 32#:

[0083]

[0084] Example 3 Enzyme-linked immunosorbent assay reagent / enzyme-linked immunosorbent assay kit for detecting complement protein C5:

[0085] In this embodiment, the main components of the enzyme-linked immunosorbent assay reagent / enzyme-linked immunosorbent assay kit for detecting complement protein C5 are: primary antibody, enzyme-labeled secondary antibody, diluent, washing solution, color development solution, and reaction stop solution.

[0086] Among them, the primary antibody is the anti-complement protein C5 antibody of Example 1 of the present application.

[0087] Enzyme-labeled secondary antibody: HRP-labeled mouse anti-goat IgG antibody.

[0088] Diluent: 0.1 M sodium bicarbonate buffer solution (pH = 9.6).

[0089] Washing solution: PBST buffer (0.05% by volume Tween-20, pH = 7.4).

[0090] Color developing solution: single-component 3,3',5,5'-tetramethylbenzidine (TMB) color developing solution.

[0091] Blocking solution: It is made by diluting bovine serum albumin (BSA) with PBS buffer (pH=7.4); the concentration of the blocking solution is 1% (mass volume ratio w / v).

[0092] Reaction termination solution: 1M HCl aqueous solution.

[0093] This assay kit uses a double-antibody sandwich assay to detect complement protein C5. First, an anti-complement protein C5 antibody is coated onto an ELISA plate. Then, a complement protein C5 standard or sample is added to the microwells of the plate, allowing the complement protein C5 in the standard or sample to bind to the primary antibody immobilized on the plate. Next, an HRP-conjugated mouse anti-goat IgG antibody is added. Unbound HRP-conjugated mouse anti-goat IgG is washed away, and a chromogenic substrate (TMB) is added. TMB is converted to blue by HRP catalysis and to yellow by acid. The intensity of the color is positively correlated with the amount of complement protein C5 in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader, and the concentration of complement protein C5 in the sample is calculated by plotting a standard curve.

[0094] The detection kit is based on the double antibody sandwich method to detect complement protein C5, which specifically includes the following steps:

[0095] Step S301: Use a diluent to prepare an anti-complement protein C5 antibody working solution with a concentration of 2.5 μg / mL; add the anti-complement protein C5 antibody working solution to the ELISA plate at 50 μL / well, incubate at 4°C for 12 to 18 hours or at 37°C for 2 hours, and discard the solution in the microwells of the ELISA plate; then, add a washing solution to the microwells of the ELISA plate at 300 μL / well, and wash three times for 2 minutes each time.

[0096] Step S302: Add blocking solution to the ELISA plate at 300 μL / well, incubate at 37°C for 1-2 hours, and discard the solution in the microwells of the ELISA plate; then, add washing solution to the microwells of the ELISA plate at 300 μL / well, and wash three times for 2 minutes each time.

[0097] Step S303: Add gradient concentration standard solution or sample solution to be tested to the microwells of the ELISA plate at 50 μL / well, incubate at 37°C for 1 hour, and discard the solution in the microwells of the ELISA plate; then, add washing solution to the microwells of the ELISA plate at 300 μL / well, and wash three times for 2 minutes each time.

[0098] Gradient concentration standard solution: Prepare a gradient concentration standard solution of complement protein C5 using diluent. Sample solution to be tested: Dilute the blood sample 10-fold using diluent; specifically: 20 μL of blood sample and 180 μL of diluent.

[0099] Step S304: HRP-labeled mouse anti-goat IgG antibody is diluted with diluent to form HRP-labeled mouse anti-goat IgG antibody working solution at a dilution ratio of 1:10,000 to 1:20,000 (mass to volume ratio w / v); 50 μL / well of the HRP-labeled mouse anti-goat IgG antibody working solution is added to the microwells of the ELISA plate, and the plate is incubated at 37°C for 1 hour. The solution in the microwells of the ELISA plate is discarded; then, 300 μL / well of washing solution is added to the microwells of the ELISA plate, and the plate is washed three times for 2 minutes each time.

[0100] Step S305: Add color developing solution to the microwells of the ELISA plate, 90 μL / well, and react at 37° C. in the dark for 5 to 10 minutes.

[0101] Step S306: Add the reaction stop solution to the microwells of the ELISA plate, 50 μL / well, and gently shake the ELISA plate until the color is evenly developed.

[0102] Step S307: Using a microplate reader to read the OD value of each well in the microplate at a test wavelength of 450 nm, and calculating the concentration of complement protein C5 in the sample by drawing a standard curve.

[0103] The enzyme-linked immunosorbent assay kit for detecting complement protein C5 in this embodiment uses a direct enzyme-linked immunosorbent assay (ELISA) method to detect the binding activity of anti-complement protein C5 antibodies and complement protein C5.

[0104] The method for detecting the binding activity of anti-complement protein C5 antibodies and complement protein C5 specifically comprises the following steps:

[0105] Step S401: Use diluent to prepare an antigen working solution with a concentration of 8 μg / mL of recombinant human complement protein C5; add the antigen working solution to the microwells of the ELISA plate at 50 μL / well, coat at 4°C for 12 to 18 hours or at 37°C for 2 hours, and discard the solution in the microwells of the ELISA plate; then, add washing solution to the microwells of the ELISA plate at 300 μL / well, and wash three times for 2 minutes each time.

[0106] Step S402: Add blocking solution to the ELISA plate at 300 μL / well, incubate at 37°C for 1-2 hours, and discard the solution in the microwells of the ELISA plate; then, add washing solution to the microwells of the ELISA plate at 300 μL / well, and wash three times for 2 minutes each time.

[0107] Step S403: Using a diluent, prepare an anti-complement protein C5 antibody working solution with seven concentration gradients (starting at a concentration of 10 μg / mL, and serially diluted by a factor of 3 into seven concentration gradients); add 50 μL / well of the anti-complement protein C5 antibody working solution to the microwells of the ELISA plate, incubate at 37°C for 1 hour, and discard the solution in the microwells of the ELISA plate; then, add 300 μL / well of a washing solution to the microwells of the ELISA plate, and wash three times for 2 minutes each time.

[0108] Step S304: HRP-labeled mouse anti-goat IgG antibody is diluted with diluent to form HRP-labeled mouse anti-goat IgG antibody working solution at a dilution ratio of 1:10,000 to 1:20,000 (mass to volume ratio w / v); 50 μL / well of the HRP-labeled mouse anti-goat IgG antibody working solution is added to the microwells of the ELISA plate, and the plate is incubated at 37°C for 1 hour. The solution in the microwells of the ELISA plate is discarded; then, 300 μL / well of washing solution is added to the microwells of the ELISA plate, and the plate is washed three times for 2 minutes each time.

[0109] Step S405: Add color developing solution to the microwells of the ELISA plate, 90 μL / well, and react at 37° C. in the dark for 5 to 10 minutes.

[0110] Step S406: Add the reaction stop solution to the microwells of the ELISA plate, 50 μL / well, and gently shake the ELISA plate until the color is evenly developed.

[0111] Step S407: using a microplate reader to read the OD value of each well in the microplate at a test wavelength of 450 nm.

[0112] From this we get, Figure 1 The binding activity curve of anti-complement protein C5 antibody and complement protein C5 is shown.

[0113] from Figure 2 It can be seen that the binding between the anti-complement protein C5 antibody of the present application and complement protein C5 is specific, suitable for the detection of plasma samples or samples from other tissue sources, and has clinical application prospects.

[0114] The above is a detailed introduction to an anti-complement protein C5 antibody and its use provided by the present disclosure. This article uses specific examples to illustrate the principles and implementation methods of the present disclosure. The description of the above examples is only used to help understand the method of the present disclosure and its core idea. It should be pointed out that for those skilled in the art, without departing from the principles of the present disclosure, several improvements and modifications can be made to the present disclosure, and these improvements and modifications also fall within the scope of protection of the claims of the present disclosure.

Claims

1. An anti-complement protein C5 antibody or an antigen-binding fragment thereof, characterized in that: The amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 in the light chain variable region of the antibody or its antigen-binding fragment are shown in SEQ ID NOs. 3, 4, and 5, respectively; the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 in the heavy chain variable region of the antibody or its antigen-binding fragment are shown in SEQ ID NOs. 9, 10, and 11, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO.

8.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that The antibody or antigen-binding fragment thereof is selected from Fab, Fab', F(ab')2, monoclonal antibody, animal-derived antibody or humanized antibody. 4 . A polynucleotide encoding the antibody or antigen-binding fragment thereof according to claim 1 .

5. A biological material containing the polynucleotide according to claim 4.

6. An antibody conjugate, characterized in that The antibody conjugate is obtained by coupling the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 with a label, and the label is selected from at least one of an enzyme label, a biotin label, a fluorescent dye label, a chemiluminescent dye label, and a radioactive label.

7. Use of any of the following in the preparation of a detection reagent or a detection kit for detecting complement protein C5 or soluble membrane attack complex SC5b-9: (I), the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; and / or (II), the polynucleotide according to claim 4; and / or (III) the biomaterial according to claim 5; and / or (IV) The antibody conjugate according to claim 6.

8. A detection reagent for detecting complement protein C5 or soluble membrane attack complex SC5b-9, characterized in that: The detection reagent comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, and / or the antibody conjugate according to claim 6.

9. A detection kit for detecting complement protein C5 or soluble membrane attack complex SC5b-9, characterized in that: The kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, and / or the antibody conjugate according to claim 6.

10. The detection kit according to claim 9, characterized in that The detection kit is selected from an enzyme-linked immunosorbent assay kit, a fluorescent immunoassay kit or a chemiluminescent immunoassay kit.

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