Carboxypeptidase B protein monoclonal antibody, kit based on carboxypeptidase B protein monoclonal antibody and application of carboxypeptidase B protein monoclonal antibody

Abstract

The invention discloses a carboxypeptidase B protein monoclonal antibody, a kit based on the monoclonal antibody and application, and belongs to the technical field of antibody preparation and biological detection. Specifically, two monoclonal antibodies, namely, BE-1E12B8 and BE4-2B6A6, which have binding characteristics with carboxypeptidase B are obtained through screening, the BE-1E12B8 antibody has high titer, and the BE4-2B6A6 antibody has high titer. The BE-1E12B8 and the BE4-2B6A6 are matched for use and are used for sandwich ELISA (enzyme-linked immunosorbent assay), and the carboxypeptidase B can be efficiently detected. Therefore, a double-antibody sandwich ELISA method is established by taking the two monoclonal antibodies as a capture antibody and a detection antibody respectively, the monoclonal antibodies can be bound to different epitopes of carboxypeptidase B respectively, the binding positions are proper, so that the monoclonal antibodies can be jointly used for realizing the detection of the double-antibody sandwich method, and the monoclonal antibodies have better sensitivity and specificity compared with the existing detection method; the method has the advantages of high repeatability and high flux.

Description

Technical Field

[0001] This invention relates to the field of antibody preparation and biological detection technology, specifically to a carboxypeptidase B protein monoclonal antibody and a kit and application based thereon. Background Technology

[0002] Carboxypeptidase B (CPB, EC 3.4.17.2) is a zinc-containing extrapancreatic peptidase that specifically hydrolyzes the basic amino acids (arginine, lysine, or ornithine) at the C-terminus of peptide chains. CPB contains 306 amino acids and has a relative molecular mass of 35 kDa. Currently used CPB is extracted from animal pancreas, which is expensive and does not completely remove other proteases. CPB derived from the pancreas of humans, pigs, cattle, and rats has the same or substantially the same amino acid sequence and function. Recombinant carboxypeptidase B (rCPB) obtained by cloning and expressing the proenzyme B followed by protease digestion is cheaper and pollution-free compared to biological extraction. rCPB is used in the preparation of proinsulin C-peptide and has the same function as naturally extracted carboxypeptidase B.

[0003] The applications of carboxypeptidase B (CPB) in scientific research are primarily based on its cleavage properties. It can be used for protein and peptide sequencing and in medicine for diagnosing pancreatitis. Additionally, as a tool enzyme, CPB is widely used in protein sequencing, insulin production, cell and gene therapy, and calcitonin production. According to the requirements of various pharmacopoeias (such as the European Pharmacopoeia and the United States Pharmacopoeia) and drug regulatory agencies (FDA, NMPA), it is necessary to limit the detection of process-related impurities (including residual biological enzymes) in biopharmaceuticals. As a potential impurity, CPB requires the establishment of highly sensitive detection methods (typically requiring detection limits at ppm or ng / mg levels). Simultaneously, during the purification process development stage, monitoring CPB residues assesses the removal efficiency of different purification steps, thereby guiding the optimization of the purification process. Therefore, the quantitative detection and control of CPB protein is particularly important.

[0004] Currently, although enzyme-linked immunosorbent assay (ELISA) methods have been developed for the quantitative determination of recombinant carboxypeptidase B protein, commercially available kits suffer from drawbacks such as low sensitivity, poor specificity, and complex operation. Therefore, further improvements and the development of reagents with accurate results and high sensitivity are crucial for the quantitative (trace) detection of recombinant carboxypeptidase B protein. The core raw materials required for these reagents are specific antigens or antibodies.

[0005] Based on the above, the preparation of highly specific and sensitive CPB antibodies has become a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the deficiency of the lack of highly specific and sensitive CPB antibodies in the prior art, and to provide a highly potent and specific anti-CPB monoclonal antibody. Based on this antibody, antibody pairs are designed to construct a kit that can efficiently detect CPB.

[0007] The first objective of this invention is to provide a hybridoma cell line that secretes a monoclonal antibody against carboxypeptidase B (BE-1E12B8), the hybridoma cell line having the accession number CCTCC NO:C2025330.

[0008] A second objective of this invention is to provide a monoclonal antibody (BE-1E12B8) secreted from the above-mentioned hybridoma cell line.

[0009] Furthermore, the monoclonal antibody is an IgG antibody.

[0010] A third objective of this invention is to provide a monoclonal antibody pair comprising a first monoclonal antibody (BE-1E12B8) and a second monoclonal antibody (BE4-2B6A6), wherein the first monoclonal antibody is the aforementioned monoclonal antibody, and the second monoclonal antibody is secreted by a hybridoma cell line with accession number CCTCC NO:C2025347.

[0011] Furthermore, the second monoclonal antibody is an IgG antibody.

[0012] This invention also claims protection for: The polynucleotide encoding the above-mentioned monoclonal antibody or monoclonal antibody pair; Recombinant expression vectors containing the above-mentioned polynucleotides; expression vectors include, but are not limited to, DNA, RNA, adeno-associated virus vectors, plasmids, etc. Recombinant cells containing the above-mentioned recombinant expression vector; the host cell can be a prokaryotic cell or a eukaryotic cell, such as animal cells (CHO cells, 293T cells, etc.), microorganisms, etc. Monovalent antibodies, bivalent antibodies, multivalent antibodies, or recombinant proteins containing the above-mentioned monoclonal antibodies.

[0013] A fourth objective of this invention is to provide the application of the hybridoma cell line, monoclonal antibody, or monoclonal antibody pair in the preparation of carboxypeptidase B detection products.

[0014] The present invention provides a detection or diagnostic product containing the monoclonal antibody or monoclonal antibody pair, wherein the product is in the form of, but is not limited to, reagents, detection plates, kits, etc.

[0015] Furthermore, the product can be used to detect carboxypeptidase B.

[0016] Furthermore, the products include, but are not limited to, reagents for detecting carboxypeptidase B using double-antibody sandwich ELISA, blocking ELISA, indirect ELISA, competitive ELISA, and colloidal gold test strips.

[0017] Furthermore, when using a double-antibody sandwich ELISA for detection: if the product contains the aforementioned monoclonal antibody pair, the capture antibody is the first monoclonal antibody, and the detection antibody is the second monoclonal antibody.

[0018] Furthermore, the product includes an ELISA plate coated with the monoclonal antibody, a reaction solution containing biotin or HRP-labeled monoclonal antibody, a washing solution, a diluent, a substrate chromogenic solution, a stop solution, a negative control, and a positive control.

[0019] More specifically: In one or more preferred embodiments, the kit includes: A solid-phase support, wherein the solid-phase support is coated with a first monoclonal antibody; and Container or package a, wherein the container or package a contains a second monoclonal antibody; The second monoclonal antibody carries a detectable marker; or the kit contains a detectable marker for linking to the second monoclonal antibody. In one or more preferred embodiments, the kit further comprises one or more components selected from the following: (a) a container or package b containing a standard (positive standard) of carboxypeptidase B protein; and / or (b) A container or package c containing a quality control of carboxypeptidase B protein; preferably, the quality control includes a positive quality control and a negative quality control. (c) Container or package d, wherein the container or package d contains a substrate corresponding to the detectable marker; (d) Container or package e containing a color developer; (e) A container or package f containing a coating liquid; (f) A container or package g containing a washing solution; (g) A container or package f containing a termination liquid.

[0020] In one or more preferred embodiments, the detectable markers include (but are not limited to): horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-D-galactosidase, urease, catalase, or glucosylamylase.

[0021] In one or more preferred embodiments, the solid support includes (but is not limited to): well plates (including microtiter plates), magnetic beads, microspheres, chips, glass slides, and test paper.

[0022] In one or more preferred embodiments, the linear range of the kit is 0.125-64 ng / ml.

[0023] In one or more preferred embodiments, the detection sensitivity of the kit is 0.141 ng / mL.

[0024] In one or more preferred embodiments, the kit is an enzyme-linked immunosorbent assay (ELISA) kit.

[0025] In one or more preferred embodiments, the carboxypeptidase B protein includes recombinant carboxypeptidase B protein.

[0026] In one or more preferred embodiments, the carboxypeptidase B protein comprises the carboxypeptidase B protein with the amino acid sequence shown in SEQ ID NO.1.

[0027] In one or more preferred embodiments, the detection includes quantitative detection, semi-quantitative detection, or qualitative detection. The detection method includes the following steps: S1. Construct a double-antibody sandwich ELISA detection system using the first monoclonal antibody as the capture antibody and the second monoclonal antibody as the detection antibody; wherein, the second monoclonal antibody is modified with a detectable marker; S2. Using the detection system of step S1, detect known concentrations of carboxypeptidase B protein and collect signals of detectable markers; S3. Based on step S2, establish a coordinate system between the signal (e.g., intensity) of the detectable marker and the content of carboxypeptidase B protein; S4. Take the sample to be tested and use the detection system of step S1 to detect it. Substitute the signal of the detectable marker obtained into the coordinate system of step S3 to calculate the content of carboxypeptidase B protein in the sample to be tested.

[0028] Furthermore, the detectable markers include, but are not limited to, horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-D-galactosidase, urease, catalase, and glucosylamylase.

[0029] Furthermore, the sample to be tested may or may not be diluted, and the dilution factor may be adjusted according to the actual sample.

[0030] Furthermore, the steps for detection using the detection system of step S1 specifically include: S11. Coat the first monoclonal antibody, and then wash and block after coating; S12. Take carboxypeptidase B protein standard or sample to be tested and incubate it with the system in step S11. Wash after incubation. S13. Mix the S12 system with the biotin-labeled second monoclonal antibody and continue incubation. Wash the mixture after incubation. S14. Mix the S13 system with streptavidin-labeled horseradish peroxidase and incubate again. Wash after incubation. S15. Develop the above system with a chromogenic substrate and perform signal detection.

[0031] In one or more preferred embodiments, the method for detecting carboxypeptidase B protein is an in vitro method.

[0032] In one or more preferred embodiments, the method for detecting carboxypeptidase B protein is a non-diagnostic method.

[0033] The beneficial effects of this invention are: (1) Because the kit of the present invention uses a monoclonal antibody with high affinity and high specificity for carboxypeptidase B protein to recognize different epitopes of carboxypeptidase B protein, the sensitivity and accuracy are very high. The ELISA kit can be used for the quantitative (trace) detection of recombinant carboxypeptidase B protein.

[0034] (2) Because the monoclonal antibody used has extremely excellent binding properties to carboxypeptidase B protein, the kit of the present invention can detect carboxypeptidase B protein in the sample extremely quickly, with a short time consumption, which is faster than ordinary ELISA kits of the same type.

[0035] (3) The kit of the present invention also has the characteristics of being simple and stable.

[0036] Preservation of biological materials The hybridoma cell line BE-1E12B8 was deposited at the China Center for Type Culture Collection (CCTCC) on November 12, 2025, with accession number CCTCC NO: C2025330, classified as Hybridoma cellline, and located at Wuhan University, China.

[0037] The hybridoma cell line BE4-2B6A6 was deposited at the China Center for Type Culture Collection (CCTCC) on November 12, 2025, with accession number CCTCC NO: C2025347, classified as Hybridoma cellline, and located at Wuhan University, China. Attached Figure Description

[0038] Figure 1The results show the specificity test results for the monoclonal antibodies BE-1E12B8 and BE4-2B6A6 in Example 2.

[0039] Figure 2-3 A coordinate system was established for detecting carboxypeptidase B protein using the ELISA kit prepared in Example 5. Detailed Implementation

[0040] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0041] The technical solution involved in this invention is as follows: This invention, through in-depth research, isolates and obtains hybridoma cells that secrete monoclonal antibodies against carboxypeptidase B protein. The secreted monoclonal antibodies are highly specific, possess excellent antigen-binding properties, and target different epitopes of carboxypeptidase B protein. Based on the double-antibody sandwich method, a simple, accurate, rapid, and precise kit for analyzing the presence of carboxypeptidase B can be prepared, enabling accurate quantitative detection of recombinant carboxypeptidase B protein.

[0042] As those skilled in the art will understand, an antigen may contain multiple epitopes (antigenic determinants). Therefore, more than one antibody can be obtained against the same antigen, and these antibodies may have different binding properties (such as specificity). Therefore, those skilled in the art need to compare and screen for the same antigen to find a monoclonal antibody suitable for specific binding. Due to the spatial structure of antigens, many epitopes are contained within the spatial structure, making it difficult to find antibodies that can specifically bind to epitopes that are stably located outside the spatial structure. This invention has conducted in-depth research and isolated monoclonal antibodies with high specificity for carboxypeptidase B protein, particularly suitable for the double antibody sandwich method.

[0043] In this invention, the target of detection is carboxypeptidase B protein. In a preferred embodiment, the target of detection is recombinant carboxypeptidase B, and the antibody of this invention can achieve accurate detection of recombinant carboxypeptidase B. Single anti-carboxypeptidase B antibodies are easily interfered with during detection, resulting in poor specificity and large result deviations. Typically, if only one antibody is used to bind to carboxypeptidase B protein, non-specific binding may inevitably occur during the assay; however, by preparing the kit based on the double-antibody sandwich method, using two antibodies binding to different epitopes of the carboxypeptidase B protein antigen, the probability of non-specific binding is very low, thus resulting in high accuracy and precision.

[0044] Furthermore, the two highly specific antibodies of this invention adsorb and localize the target antigen carboxypeptidase B protein, resulting in better localization and amplification effects, thus achieving higher specificity and precision. Moreover, only a small sample volume is required for the assay.

[0045] Explanation of terms used in the following embodiments: In this invention, the terms "capture antibody," "coating antibody," "first monoclonal antibody," "first antibody," and "primary antibody" are used interchangeably and refer to antibodies specifically targeting carboxypeptidase B protein that are immobilized on a solid-phase support.

[0046] In this invention, the terms "detection antibody," "second monoclonal antibody," "second antibody," "enzyme-labeled antibody," and "secondary antibody" are used interchangeably and refer to antibodies that specifically target carboxypeptidase B protein and correspond to the corresponding first antibody in the kit. For the antigenic carboxypeptidase B protein, the corresponding first and second antibodies are different and can simultaneously bind to different epitopes (antigenic determinants) of the carboxypeptidase B protein.

[0047] In this invention, the term "antibody" refers to a protein composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain.

[0048] In this invention, "multivalent" refers to a fusion protein containing multiple variable regions of the antibodies of this invention or antibodies of this invention.

[0049] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0050] In this invention, "sample to be tested" encompasses a variety of sample types, including various objects requiring detection of carboxypeptidase B content. For example, the "sample to be tested" can be a biological polypeptide drug and gene product, such as the expression product of a recombinant fusion protein, a small molecule protein or polypeptide biological agent, or a biological reagent for a recombinant protein drug.

[0051] In this invention, the term "marker" or "detectable marker" refers to a marker located on / linkable to a second monoclonal antibody used to determine the presence and amount of carboxypeptidase B protein in a sample to be tested. Preferably, the marker may be selected from: horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, β-D-galactosidase, urease, catalase, or glucosylamylase.

[0052] In this invention, the "substrate corresponding to the detectable marker" refers to a marker that can be catalyzed by the second monoclonal antibody to produce a colorimetric reaction, displaying a recognition signal indicating the binding of the second antibody to carboxypeptidase B protein. Examples of such substrates include: o-phenylenediamine (OPD), tetramethylbenzidine (TMB), and ABTS for horseradish peroxidase; p-nitrophenyl phosphate (p-NPP) for alkaline phosphatase; and so on.

[0053] In this invention, the "double-antibody sandwich method" is a type of enzyme-linked immunosorbent assay (ELISA). The conventional procedure involves immobilizing a primary antibody on a carrier, reacting the primary antibody with the antigen, washing, reacting with a labeled secondary antibody, washing again, and finally performing a chemiluminescent or ELISA reaction to detect the signal. The double-antibody sandwich method is suitable for detecting antigens with two or more epitopes, but requires two antibodies that target different epitopes and have excellent binding properties. In this field, polyclonal antibodies are commonly used as the secondary antibody, but their specificity or sensitivity is often lacking.

[0054] In this invention: Carboxypeptidase B protein Carboxypeptidase B protein is a known protein, and its amino acid and nucleotide sequences are known in the art. The amino acid sequence of the recombinant carboxypeptidase B described in this invention is shown in SEQ ID NO.1.

[0055] Methods for producing carboxypeptidase B protein are also known. For example, recombinant carboxypeptidase B protein can be expressed or produced using the polynucleotide sequence of carboxypeptidase B protein through conventional recombinant DNA technology. Generally, the following steps are involved: (1) Transform or transduce suitable host cells with a polynucleotide (or variant) encoding carboxypeptidase B protein, or with a recombinant expression vector containing the polynucleotide. (2) Host cells cultured in a suitable culture medium; and (3) Isolate and purify proteins from culture media or cells.

[0056] The recombinant carboxypeptidase B protein obtained above can be processed into a protein with a certain purity for use in the preparation of standards.

[0057] Monoclonal antibodies against carboxypeptidase B protein The antibodies used in this invention are monoclonal antibodies specific to carboxypeptidase B protein. Here, "specific" means that the antibody can bind to carboxypeptidase B protein or fragments thereof. More specifically, it refers to antibodies that can bind to carboxypeptidase B protein or fragments but do not recognize or bind to other unrelated antigen molecules.

[0058] This invention utilizes highly specific monoclonal antibodies targeting different epitopes of carboxypeptidase B protein and, based on the double-antibody sandwich method, prepares a kit for convenient, rapid, and accurate analysis of carboxypeptidase B protein.

[0059] Hybridoma cell line BE-1E12B8, preservation number CCTCC NO:C2025330, IgG1 subtype; Hybridoma cell line BE4-2B6A6, preservation number CCTCC NO:C2025347, IgG1 subtype.

[0060] The monoclonal antibodies of this invention can be prepared using hybridoma technology (see Kohler et al., Nature 256;495, 1975; Kohler et al., Eur. J. Immunol. 6:511, 1976; Kohler et al., Eur. J. Immunol. 6:292, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, NY, 1981). The monoclonal antibodies of this invention can be obtained using carboxypeptidase B protein, fragments, or functional regions via conventional immunoassay techniques. Alternatively, they can be prepared using recombinant methods or synthesized using a peptide synthesizer.

[0061] In one embodiment of the present invention, the monoclonal antibody can be prepared by the following method, the method comprising the steps of: (1) providing mice pretreated with adjuvants; (2) inoculating the hybridoma cells into the peritoneal cavity of the mice and secreting monoclonal antibodies; and (3) aspirating ascites fluid and separating the monoclonal antibody. As a preferred embodiment, the method for separating the monoclonal antibody from the ascites fluid can employ ascites fluid collection, affinity purification, and dialysis. For example, it may include: collecting the ascites fluid, precipitating it with ammonium sulfate and caprylic acid, and then purifying it with a Protein G pre-packed chromatography column to obtain a high-purity monoclonal antibody.

[0062] Alternatively, the hybridoma cells can be cultured and expanded in vitro using conventional animal cell culture methods, thereby causing them to secrete the monoclonal antibodies.

[0063] After obtaining the antibody of the present invention, those skilled in the art can obtain the sequence information of the monoclonal antibody using sequencing techniques known in the art, such as determining the sequences of its heavy and light chains. This allows for artificial preparation using bioengineering techniques.

[0064] The monoclonal antibody of the present invention can be prepared using recombinant methods or synthesized using a peptide synthesizer. Those skilled in the art will understand that, after obtaining the hybridoma cell line of the monoclonal antibody or identifying the monoclonal antibody through sequencing or other means, they can easily obtain the antibody.

[0065] The monoclonal antibodies of this invention are derived from specific hybridoma cell lines. It should be understood that after obtaining the antibodies of this invention from these cell lines, various modifications can be made to the antibodies, and they can also be sequenced. Optimization and modification can then be performed based on the sequenced antibody structure; all these antibody modifications are included within the scope of this invention. Therefore, the antibodies of this invention can be complete immunoglobulin molecules or antigen-binding fragments, including but not limited to Fab, F(ab'), F(ab')2, Fv, dAb, Fd, complementarity-determining region (CDR) fragments, single-chain antibodies (scFv), bivalent single-chain antibodies, single-chain antibodies, bispecific double-chain antibodies, triple-chain antibodies, and quadruple-chain antibodies.

[0066] This invention also provides an immunoconjugate comprising the antibody described herein and further comprising at least one other type of functional molecule. The functional molecule includes, but is not limited to, a detectable marker. The antibody and the functional molecule can form the conjugate through covalent linkage, coupling, attachment, cross-linking, or other means.

[0067] After determining the coating antibody and / or detection antibody used in the kit of the present invention, various detectable markers conventionally used in the art for binding with the detection antibody for detection can be employed. The present invention does not impose any particular limitation on the markers used; any marker capable of binding to the monoclonal antibody of the present invention and, after appropriate processing, accurately indicating the presence and amount of the target protein in the sample to be tested is acceptable. The detectable markers may include, but are not limited to: colloidal gold, fluorescent markers, chromogenic markers; such as: enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals, and non-radioactive paramagnetic metal ions. More than one marker may also be included. The labeling used for antibody labeling for detection and / or analytical purposes depends on the specific detection / analysis technique and / or method used, such as immunohistochemical staining of (tissue) samples, flow cytometry, etc. Suitable markers for detection / analysis techniques and / or methods known in the art are well known to those skilled in the art.

[0068] The marker can be directly applied to the detection antibody; alternatively, it can be applied to an anti-antibody specific to the second antibody. Those skilled in the art can select a suitable marker based on the type and characteristics of the antibody used. For example, the marker can be selected from: horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, β-D-galactosidase, urease, catalase, glucosylamylase, or biotin. When using some of the enzyme markers mentioned above, a substrate that binds to the corresponding enzyme is also required, so that the presence or amount of the marker can be reported by colorimetric methods. In this invention, the "substrate corresponding to the marker" refers to a substrate that can be catalyzed by the marker to produce a colorimetric reaction, used to display a recognition signal indicating the binding of the second antibody to the target protein. Examples of substrates include: o-phenylenediamine (OPD), tetramethylbenzidine (MB), and ABTS for horseradish peroxidase; p-nitrophenyl phosphate (p-NPP) for alkaline phosphatase; phycoerythrin (streptavidin-phycoerythrin, also known as PE-labeled streptavidin, SA-R-PE) for biotin; and so on. Those skilled in the art can select suitable substrates based on the type and characteristics of the labeled material used.

[0069] As a specific example, the detectable marker is an HRP, which acts as a display marker and generates a signal when combined with OPD, MB, or ABTS.

[0070] Reagent test kit After obtaining the monoclonal antibody of this invention, those skilled in the art can sensitively detect the presence or concentration of carboxypeptidase B protein in a sample through various methods, employing techniques commonly used in the field of immunology. These include qualitative and quantitative detection methods. Preferably, the qualitative detection includes: identifying the presence of carboxypeptidase B protein using immunoblotting or immunofluorescence methods; for example, measuring carboxypeptidase B protein using ELISA, immunogold test strips, immunofluorescence test strips, homogeneous enzyme immunoassay, etc. This invention provides a kit for detecting carboxypeptidase B protein, which can be used to detect carboxypeptidase B protein, etc.

[0071] The kit contains the monoclonal antibody described in this invention. In a preferred embodiment, one of the monoclonal antibodies is immobilized on a solid-phase support, and the other serves as a detection antibody.

[0072] As a preferred embodiment of the present invention, the first monoclonal antibody is a monoclonal antibody secreted by the hybridoma cell line (BE-1E12B8) with accession number CCTCC NO: C2025330; and the second monoclonal antibody is a monoclonal antibody secreted by the hybridoma cell line (BE4-2B6A6) with accession number CCTCC NO: C2025347. Surprisingly, using BE-1E12B8 as the coating antibody and BE4-2B6A6 as the detection antibody produces the best detection effect for carboxypeptidase B protein, with a detection sensitivity of approximately 0.141 ng / mL.

[0073] The first monoclonal antibody is coated on a solid-phase support. This invention does not impose any particular limitation on the solid-phase support used, as long as it can be coupled (linked) to the first monoclonal antibody. For example, the solid-phase support can be selected from: microtiter plates, microspheres, etc.

[0074] As an example of operation, the solid support used can be a microtiter plate (ELISA plate).

[0075] To eliminate false positives and false negatives, it is advisable to include quality controls (checks) during the testing process. In one embodiment of the present invention, the quality control material is a carboxypeptidase B protein standard.

[0076] In addition, to obtain quantitative results, multiple carboxypeptidase B protein standards containing known concentrations can be set up during the detection process. Conventional methods can be used for setting up these standards.

[0077] Using the aforementioned standards, the standard curve is set as follows: the OD values ​​of the standards are plotted on the ordinate (Y-axis), and the standard concentrations are plotted on the x-axis (X-axis) to create a quantitative standard curve for the carboxypeptidase B protein kit. Therefore, based on the OD values ​​obtained from the test sample, the concentration of carboxypeptidase B protein in the test sample can be calculated using the standard curve.

[0078] Furthermore, to facilitate detection using the kit of the present invention, the kit preferably also includes other auxiliary reagents. These auxiliary reagents are commonly used in the double-antibody sandwich method, and their properties and preparation methods are well known to those skilled in the art. Examples of these reagents include (but are not limited to): chromogenic agents, washing solutions, stop solutions, and sensitizing diluents.

[0079] Example 1: Obtaining a hybridoma cell line capable of secreting monoclonal antibodies against recombinant carboxypeptidase B protein The recombinant carboxypeptidase B protein (rCPB), which serves as an immunogen, has the following amino acid sequence (SEQ ID NO.1): ASGHSYTKYNNWETIEAWIQQVATDNPDLVTQSVIGTFEGRNMYVLKIGKTRPNKPAIFIDCGFHAREWISPAFCQWFVREAVRTYNQEIHMKQLLDELDFYVLPVVNIDGYVYTWTKDRMWRKTRSTMAGSSCLGVDPNRNFNAGWCEVGA SRSPCSETYCGPAPESEKETKALADFIRNNLSTIKAYLTIHSYSQMMLYPYSYDYKLPENYEELNALVKGAAKELATLHGTKYTYGPGATTIYPAAGGSDDWSYDQGIKYSFTFELRDTGFFGFLLPESQIRQTCEETMLAVKYIANYVREHPY Based on the full-length rCPB sequence analysis, EcoRI and SalRI restriction enzyme sites and protective bases were added to both ends of the coding sequence to synthesize the whole gene. The PET28A(+) vector was double-digested and ligated with the similarly digested rCPB gene fragment for transformation, constructing a recombinant plasmid and sequencing it. The rCPB recombinant plasmid with correct sequencing results was transformed into BL21 cells, and the cells were collected, lysed, and purified to obtain the recombinant rCPB protein. The purified recombinant carboxypeptidase B protein (rCPB) was diluted with physiological saline to a concentration of 400 μg / mL and mixed with an equal volume of Sigma-French complete adjuvant. 100 μL of this mixture was injected intramuscularly into the back of four 8-week-old Balb / c mice. Subsequently, every 10-14 days, the recombinant carboxypeptidase B protein (rCPB) was diluted with physiological saline to a concentration of 400 μg / mL and mixed with an equal volume of Sigma-French incomplete adjuvant, and two booster immunizations were administered in the same manner. Blood was collected from the tail of mice after the third injection. Serum was separated and serum titer was detected using indirect ELISA. The titer met the fusion requirements. Three days before cell fusion, the spleen was boosted with 100 μg of protein.

[0080] Balb / c mouse spleen cells and logarithmically growing SP2 / 0 myeloma cells were fused at a ratio of 9:1 using 50% PEG1500. After fusion, the cells were cultured in 1640-HAT complete medium containing 10% FBS (fetal bovine serum) for 6-7 days. Cell clones were then selected and cultured in 1640-HT complete medium for 5 days. The antibody specificity in the cell culture supernatant was detected using an indirect ELISA method. A stable antibody-secreting monoclonal cell line was obtained. After expansion culture, the cells were cryopreserved in FBS containing 20% ​​DMSO at a cell density of 10T. 6Ascites fluid was prepared using an in vivo induction method. The ascites fluid was purified by ammonium octanoate-sulfate precipitation and Protein G affinity chromatography to obtain purified antibodies. After dialyzing in 0.01 mol / L phosphate buffer, the protein concentration was measured at OD280 nm, and multiple monoclonal antibodies were selected.

[0081] Antibody pairing analysis was performed: Multiple antibodies secreted by various monoclonal cell lines were coated onto microplates and labeled with biotin, then paired with each other completely. The screening method was a double-antibody sandwich ELISA. The screening principle was as follows: After obtaining the monoclonal antibodies, two types were selected: one as a capture antibody and the other as a labeling antibody. The capture antibody was coated onto an antigen plate. Antigen was added first, incubated, and then unbound antigen was washed away. Then, the labeling antibody was added, incubated, and then unbound labeling antibody was washed away. Finally, chromogenic buffer was added for color development. If color development occurred, it indicates that the labeling antibody specifically binds to the antigen, and the capture antibody and labeling antibody are a paired antibody pair. If color development did not occur, it indicates that the labeling antibody could not bind to the antigen and was eluted, and the capture antibody and labeling antibody are not paired antibodies.

[0082] A large number of monoclonal cell lines were analyzed (the comparison results of some antibodies are shown in Table 1). The two best cell lines that could stably secrete monoclonal antibodies and could be paired for double antibody sandwich assay were selected and named hybridoma cells BE-1E12B8 and BE4-2B6A6, respectively, which can secrete monoclonal antibodies against recombinant carboxypeptidase B protein (rCPB).

[0083] Table 1. Pairing of different monoclonal antibodies Note: - indicates no pairing; + indicates pairing, weak positive result; ++ indicates pairing, strong positive result; +++ indicates pairing, strongest positive result.

[0084] Hybridoma cells BE-1E12B8 and BE4-2B6A6 are deposited at the China Center for Type Culture Collection (CCTCC), with accession numbers CCTCC NO: C2025330 and CCTCC NO: C2025347, respectively. Hybridoma cells BE-1E12B8 and BE4-2B6A6 can secrete monoclonal antibodies against recombinant carboxypeptidase B protein (rCPB). The monoclonal antibodies secreted by hybridoma cell lines BE-1E12B8 and BE4-2B6A6 are named monoclonal antibodies BE-1E12B8 and BE4-2B6A6, respectively.

[0085] Example 2: Preparation, purification, and titer of monoclonal antibodies BE-1E12B8 and BE4-2B6A6 I. Preparation of monoclonal antibodies BE-1E12B8 and BE4-2B6A6 1. Incremental cultivation method Preparation method of cell culture medium: Add fetal bovine serum to RPMI-1640 medium to a final concentration of 10% (mass percentage).

[0086] Hybridoma cell lines BE-1E12B8 and BE4-2B6A6 were placed in cell culture medium and cultured at 37°C for 3-4 days. The cell culture supernatant was purified by Protein G affinity chromatography to obtain monoclonal antibodies BE-1E12B8 and BE4-2B6A6 with a purity of over 95% (stored at -20±5°C).

[0087] 2. Preparation of ascites Balb / c mice were intraperitoneally injected with Freund's incomplete adjuvant (0.5 mL / mouse), and 10 days later, they were intraperitoneally injected with hybridoma cell lines BE-1E12B8 and BE4-2B6A6 (1×10⁻⁶). 6 (1 cell / animal) Ascites fluid was collected after 7-10 days, purified by Protein G affinity chromatography, and then transferred to a dialysis bag for dialysis in 0.01M PBS buffer at pH 7.4. The fluid in the dialysis bag was then collected to obtain monoclonal antibodies BE-1E12B8 and BE4-2B6A6 with a purity of over 95%. After filtration through a 0.22µm sterile filter membrane, the antibodies were stored at -20℃.

[0088] The monoclonal antibodies BE-1E12B8 and BE4-2B6A6 used in subsequent steps and examples were prepared from ascites fluid.

[0089] II. Potency Testing (Indirect ELISA Method) 1. Take an ELISA plate, add coating buffer (100 μL / well), and coat overnight at 2-8℃. Coating buffer: Take recombinant carboxypeptidase B protein (rCPB), dilute with pH 9.6, 0.05M carbonate buffer to a protein concentration of 5 µg / mL, which is the coating buffer.

[0090] 2. After completing step 1, take the ELISA plate, wash it three times with PBST solution, then add blocking buffer (200 μL / well) and incubate at 37°C for 2 hours. Blocking buffer: PBST solution containing 0.5 g / 100 mL fish gelatin.

[0091] 3. After completing step 2, take the enzyme-labeled plate, wash it three times with PBST solution, and dilute the two monoclonal antibodies BE-1E12B8 and BE4-2B6A6 to a baseline concentration of 1 mg / ml using 0.01 M PBS buffer at pH 7.4. Antibody dilution solution: Take monoclonal antibodies BE-1E12B8 and BE4-2B6A6, and perform serial dilutions with 0.01 M PBS buffer at pH 7.4 to obtain antibody dilution solutions of different concentrations, based on the antibody concentration of 1 mg / ml (considered as 1-fold or original-fold). Then add antibody dilution solution (100 μL / well) and incubate at 37°C for 1 h.

[0092] 4. After completing step 3, take the enzyme-labeled plate, wash it three times with PBST solution, then add the working solution of goat anti-mouse HRP-labeled secondary antibody, and incubate at 37°C for 0.5 h. HRP-labeled secondary antibody working solution: 1:5000 dilution of HRP-labeled secondary antibody. HRP-labeled secondary antibody: Kangwei Century brand, catalog number CW0102S.

[0093] 5. After completing step 4, take the enzyme-labeled plate, wash it 3 times with PBST solution, then add TMB substrate reaction solution for color development for 10 minutes, then add 2N sulfuric acid solution to stop the color development, and measure the absorbance at 450nm using an enzyme-labeled plate reader (see Table 2).

[0094] Table 2. Results of monoclonal antibody titer assay at different concentrations As shown in Table 2, the monoclonal antibody BE-1E12B8 had a titer ≥1.28 million, and the monoclonal antibody BE4-2B6A6 had a titer ≥320,000.

[0095] Example 3: Antibody specificity test of monoclonal antibodies BE-1E12B8 and BE4-2B6A6 The testing method is the same as step two in Example 2, but the coating solution and antibody diluent are replaced.

[0096] Other enzyme protein preparations with the same protein expression system as recombinant carboxypeptidase B protein (rCPB) were selected and plated: recombinant enterokinase protein (REK), recombinant trypsin protein (RPT), sperm-specific protein (SP10) expressed by other Escherichia coli systems, bovine serum albumin (BSA), negative serum, and recombinant rCPB protein (prepared in Example 1). 0.1 μg / mL of purified rCPB antibody BE-1E12B8 and BE4-2B6A6 were added to each plate, and the reaction between the antibody and these proteins was tested.

[0097] The results are as follows Figure 1It can be seen that the monoclonal antibodies BE-1E12B8 and BE4-2B6A6 only interact (bind) with rCPB and do not cross-react with other proteins, exhibiting very high specificity.

[0098] Example 4: Identification of antibody subtypes for monoclonal antibodies BE-1E12B8 and BE4-2B6A6 Antibody subtypes were identified using a commercially available antibody Ig class / subclass / subtype identification kit (Luoyang Baitong Experimental Materials Center, catalog number C060101-L). The results are shown in Table 3.

[0099] Table 3. Identification results of BE-1E12B8 and BE4-2B6A6 antibody subtypes Note: The values ​​in the table represent the absorbance at OD450nm.

[0100] The results showed that antibody BE-1E12B8 and antibody BE4-2B6A6 were both IgG1 subtypes, and the light chain of both antibodies was Kappa.

[0101] Example 5: Preparation of ELISA Detection Kit I. Preparation of HRP-labeled antibodies Weigh 1 mg HRP and dissolve it in deionized water to a concentration of 10 mg / ml (0.1 ml volume). Add 0.1 ml of freshly prepared 0.1 mol / L NaIO4 solution to the prepared HRP solution, and add an equal volume of NaIO4 solution to the enzyme solution dropwise. Incubate at 2-8°C in the dark for 30 minutes until the reaction solution turns dark green. Add 0.1 ml of 0.5% ethylene glycol dropwise, mix well, and incubate at 2-8°C in the dark for 30 minutes until the reaction solution turns brown. Add 1 mg of monoclonal antibody BE4-2B6A6, mix well, and place the antibody and activating enzyme into a dialysis bag. Dialyze in 50 mM CBS buffer (pH 9.6) overnight at 2-8°C. Collect the dialysis fraction and add 20 μL of freshly prepared 5 mg / ml NaIO4 solution. Incubate in NaBH4 solution for 1 hour in the dark; then add an equal volume of saturated (NH4)2SO4 solution to precipitate the protein, centrifuge at 5000 rpm for 30 min; remove the supernatant, resuspend the precipitate in 50% (NH4)2SO4 solution, centrifuge at 5000 rpm for 30 min; dissolve the precipitate in PBS, then desalt using a desalting column, add an equal volume of glycerol as a protective agent, and obtain the enzyme-labeled antibody BE4-2B6A6, aliquot and store at -20℃.

[0102] II. Preparation of the Coating Plate Dilute the monoclonal antibody BE-1E12B8 to a final concentration of 4 μg / ml with carbonate buffer, and pipette 100 μL / well of the diluted antibody into a PVC microplate. Cover the plate with sealing film and incubate overnight at 2–8°C. Discard the coating solution, wash the plate three times with PBST, and block the plate with PBST buffer containing 0.5% BSA, adding 200 μL to each well. Cover the plate with sealing film and incubate at 37°C for 2 hours, then wash three times with PBST. After drying, seal the plate in an aluminum foil bag to obtain the coated plate, and store at 2–8°C.

[0103] III. Instructions for using the ELISA test kit (double antibody sandwich method) Remove the coated plate (coated with BE-1E12B8) and allow it to return to room temperature. Using a pipette, pipette 100 μL of positive standard (32 ng / mL recombinant rCPB; dilute the positive standard with phosphate buffer as needed to obtain different concentrations of positive standard solution), blank control (0.01 M pH 7.4 phosphate buffer), and the sample to be tested, and add them to the corresponding reaction wells. Then, add 100 μL of enzyme-labeled antibody BE4-2B6A6 (250 ng / mL) to each reaction well, gently vortex for 10-15 seconds, cover the plate with the cap or sealing film, and incubate at 37°C for 0.5 h. After incubation, remove the cap or sealing film, quickly invert the plate, discard the liquid in the wells into the waste container, and pat dry on absorbent paper. Wash the plate 3-5 times with PBST, then add 100 μL of... After developing the TMB substrate solution for 10 minutes, add 50 μL of stop solution (2N sulfuric acid); measure the optical density of each well at 450 nm using a microplate reader. Plot a standard curve with the concentration of the standard on the x-axis (X) and the OD value on the y-axis (Y). Substitute the OD values ​​of the sample wells into the standard curve equation to obtain the corresponding protein quantification values ​​for the samples.

[0104] Example 6: Determination of the sensitivity and specificity of the kit I. Sensitivity Testing of ELISA Detection Kits The kit from Example 5 was analyzed to test its sensitivity. 0.01M PBS dilution was used as the test sample (30 samples of 0 ng / ml each), as shown in Table 4.

[0105] Table 4. Results of reagent kit sensitivity assay Calculate the mean and variance of the above 30 samples, and substitute them into the equation Y = (OD mean + 2 × OD variance). Substitute the Y value into the equation of the standard curve to obtain the X concentration value, which is the sensitivity.

[0106] The detection sensitivity of the kit in Example 5 was determined to be 0.141 ng / mL.

[0107] II. Specificity Detection of the Reagent Kit The kit from Example 5 was analyzed to test its specificity.

[0108] Other enzyme protein preparations from the same protein expression system as recombinant carboxypeptidase B protein (rCPB) were selected: 1-recombinant enterokinase protein (100 ng / ml), 2-recombinant trypsin protein (100 ng / ml), 3-sperm-specific protein SP10 expressed in other E. coli systems (100 ng / ml), 4-1% BSA, 5-LB blank medium for E. coli system, 6-YPD blank medium for yeast system; 7-DMEM cell blank medium, 8-MEM cell blank medium, 9-1640 cell blank medium; 10-mouse negative serum as a negative control sample. 11-pH 7.4, 0.01M PBS buffer as a blank control sample; 12-recombinant carboxypeptidase B protein (rCPB) (100 ng / ml) as a positive control sample. The absorbance at 450 nm was measured using a microplate reader (see Table 5). Specific detection methods were the same as described above. The results are shown in Table 5.

[0109] Table 5 Results of kit specificity assay The results showed that the monoclonal antibody in the kit reacted only with rCPB and did not cross-react with other proteins, demonstrating ideal specificity.

[0110] III. Detection Scope of the Reagent Kit The kit from Example 5 was analyzed to test its detection limit.

[0111] Recombinant carboxypeptidase B protein (rCPB) was serially diluted using 0.01M pH 7.4 phosphate buffer as the diluent, and these diluted samples were used as test samples. 0.01M pH 7.4 phosphate buffer was used as a blank sample. The test kit's lower limit of detection is specified.

[0112] The results are as follows Figure 2 and Figure 3 As shown, the detection limit of the recombinant carboxypeptidase B assay kit is as low as 0.125 ng / ml, and detection can also be achieved at a carboxypeptidase B concentration of 64 ng / ml.

[0113] Example 7: Determination of residual carboxypeptidase B protein in biological enzyme preparations The kit from Example 5 was analyzed to test the residual amount of recombinant carboxypeptidase B protein in the bio-enzyme preparation.

[0114] Three batches of V8 protease were serially diluted using 0.01M pH 7.4 phosphate buffer as the diluent and used as test samples. Recombinant carboxypeptidase B protein (rCPB) was also serially diluted and used as spiked test samples. 0.01M pH 7.4 phosphate buffer was used as a blank sample. All three batches of V8 protease were samples taken from non-specific production stages, and the production processes of both protein preparations involved the use of recombinant carboxypeptidase B protein (rCPB). See Table 6.

[0115] The results are shown in Table 6. The recombinant carboxypeptidase B detection kit can effectively detect residual recombinant carboxypeptidase B protein in different batches of V8 protease samples. The sample spike recovery rate is in the range of 80%-120%, and it has good repeatability.

[0116] Table 6. Results of recombinant carboxypeptidase B protein (rCPB) assay The embodiments described above are merely preferred embodiments for fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention.

Claims

1. A hybridoma cell line, characterized in that, The hybridoma cell line has the accession number CCTCC NO:C2025330.

2. A monoclonal antibody secreted by the hybridoma cell line of claim 1.

3. A monoclonal antibody pair, characterized in that, It includes a first monoclonal antibody and a second monoclonal antibody, wherein the first monoclonal antibody is the monoclonal antibody as described in claim 2, and the second monoclonal antibody is secreted by a hybridoma cell line with accession number CCTCC NO:C2025347.

4. A hybridoma cell pair, characterized in that, It includes a first hybridoma cell and a second hybridoma cell, wherein the first hybridoma cell is the hybridoma cell described in claim 1, and the second hybridoma cell has the accession number CCTCC NO:C2025347.

5. A test kit, characterized in that, Contains the monoclonal antibody of claim 2 or the monoclonal antibody pair of claim 3.

6. The detection kit as described in claim 5, characterized in that, This includes reagents for detecting carboxypeptidase B using double-antibody sandwich ELISA, blocking ELISA, indirect ELISA, competitive ELISA, or colloidal gold.

7. The detection kit as described in claim 5 or 6, characterized in that, When using a double-antibody sandwich ELISA for detection, if the product contains the aforementioned monoclonal antibody pair, the capture antibody is the first monoclonal antibody, and the detection antibody is the second monoclonal antibody.

8. The use of the hybridoma cell line of claim 1, the monoclonal antibody of claim 2, the monoclonal antibody pair of claim 3, the hybridoma cell pair of claim 4, or the detection kit of any one of claims 5-7 in the preparation of a carboxypeptidase B detection product.

9. The application as described in claim 8, characterized in that, The detection method includes the following steps: S1. A double-antibody sandwich ELISA detection system was constructed using the first monoclonal antibody as the capture antibody and the second monoclonal antibody as the detection antibody; wherein, the second monoclonal antibody was modified with a detectable marker. S2. Using the detection system of step S1, detect known concentrations of carboxypeptidase B protein and collect signals of detectable markers; S3. Based on step S2, establish a coordinate system between the signal of the detectable marker and the content of carboxypeptidase B protein; S4. Take the sample to be tested and use the detection system of step S1 to detect it. Substitute the signal of the detectable marker obtained into the coordinate system of step S3 to calculate the content of carboxypeptidase B protein in the sample to be tested.

10. The application as described in claim 9, characterized in that, The detectable markers include horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-D-galactosidase, urease, catalase, or glucosylamylase.

Images