Monoclonal antibody specifically combined with enterokinase protein and application of monoclonal antibody in detection

Abstract

The invention provides a monoclonal antibody specifically combined with enterokinase and application of the monoclonal antibody in detection. The invention also provides a kit for detecting enterokinase, wherein the kit contains the monoclonal antibody. The enterokinase comprises recombinant enterokinase. The monoclonal antibody disclosed by the invention can be respectively combined to different epitopes of enterokinase, and the combination positions are proper, so that the monoclonal antibody can be jointly used for realizing detection of a double-antibody sandwich method, and has high specificity and high sensitivity.

Description

Technical Field

[0001] The invention belongs to the field of immune detection, and specifically relates to a hybridoma cell capable of secreting anti-enterokinase protein monoclonal antibody, a monoclonal antibody and application thereof. The enterokinase includes recombinant enterokinase. Background Art

[0002] Recombinant Bovine Enterokinase (REK, EC: 3.4.21.9) is a serine protease. The molecular weight is usually 26.3kD, with 3 glycosylation sites, and its glycosylation molecular weight is about 43kD. Enterokinase can specifically recognize the Asp-Asp-Asp-Asp-lys site and can efficiently hydrolyze the peptide bond after Lys without affecting the downstream purification process. It is reported that the recombinant enterokinase light chain has the enzymatic cleavage specificity of the whole enzyme in vitro, and the enzymatic cleavage activity of the genetically engineered fusion protein substrate is significantly enhanced compared with the purified bovine enterokinase.

[0003] Enterokinase is widely present in the duodenal mucosa of mammals. Due to the limited sources of natural enterokinase and the fact that enterokinase extracted from animal tissues is easily contaminated with other proteins, it brings difficulties to practical application. Recombinant enterokinase produced by genetic engineering has the characteristics of strong specificity and high hydrolysis efficiency. As an important tool enzyme for specific cutting of recombinant fusion protein drugs, it is widely used in the development and production of gene products such as low-molecular-weight bioactive polypeptide drugs. Therefore, the quantitative detection or control of recombinant enterokinase protein is particularly important.

[0004] At present, although the enzyme-linked immunosorbent assay method has been developed for the quantitative determination of recombinant enterokinase protein, the commercial kits currently on sale have the disadvantages of low sensitivity, poor specificity, and complex operation. Therefore, further improvement and development of reagents with accurate detection results and high detection sensitivity are of great significance for the quantitative (trace) detection of recombinant enterokinase protein, and the core raw materials required for the detection reagents are specific antigens or antibodies.

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

[0006] The purpose of the present invention is to provide a hybridoma cell capable of secreting anti-recombinant enterokinase protein (REK) monoclonal antibody, monoclonal antibody and application.

[0007] In a first aspect of the present invention, there is provided a kit for detecting enterokinase protein, the kit comprising: (i) a monoclonal antibody secreted by a hybridoma cell line (EK-10G9D1) with a deposit number of CCTCC NO: C2023161; and (ii) a monoclonal antibody secreted by a hybridoma cell line (EK-6-3A6) with a deposit number of CCTCC NO: C2023162.

[0008] In one or more preferred embodiments, the kit includes:

[0009] A solid-phase carrier, on which a first monoclonal antibody is coated, and the first monoclonal antibody is selected from the monoclonal antibodies of (i) or (ii); and

[0010] A container or package a, in which a second monoclonal antibody is contained, and the second monoclonal antibody is selected from the monoclonal antibodies of (ii) or (i);

[0011] wherein, the second monoclonal antibody carries a detectable label; or, the kit includes a detectable label for connecting with the second monoclonal antibody;

[0012] wherein, the first monoclonal antibody and the second monoclonal antibody are different and can bind to the protein simultaneously; preferably, the first monoclonal antibody is a monoclonal antibody secreted by a hybridoma cell line with a deposit number of CCTCC NO: C2023161, and the second monoclonal antibody is a monoclonal antibody secreted by a hybridoma cell line with a deposit number of CCTCC NO: C2023162.

[0013] In one or more preferred embodiments, the first monoclonal antibody is preferably the antibody of (i), and the second monoclonal antibody is preferably the antibody of (ii).

[0014] In one or more preferred embodiments, the kit further comprises one or more components selected from the following:

[0015] (a) A container or package b, in which a standard product (positive standard) of enterokinase protein is contained; and / or

[0016] (b) A container or package c, in which a quality control product of enterokinase protein is contained; preferably, the quality control product includes a positive quality control product and a negative quality control product;

[0017] (c) A container or package d, in which a substrate corresponding to the detectable label is contained;

[0018] (d) A container or package e, in which a chromogenic agent is contained;

[0019] (e) A container or package f containing a coating solution;

[0020] (f) A container or package g containing a washing solution;

[0021] (g) A container or package f containing a termination solution.

[0022] In one or more preferred embodiments, the detectable marker includes (but is not limited to): horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-D-galactosidase, urease, catalase, or glucoamylase.

[0023] In one or more preferred embodiments, the solid-phase carrier includes (but is not limited to): microtiter plates (including microtiter plates), magnetic beads, microspheres, chips, glass slides, test strips.

[0024] In one or more preferred embodiments, the linear range value of the kit is 0.0312 - 32 ng / ml.

[0025] In one or more preferred embodiments, the detection sensitivity of the kit is 0.043 ng / mL.

[0026] In one or more preferred embodiments, the kit is an enzyme-linked immunosorbent assay (ELISA) detection kit.

[0027] In one or more preferred embodiments, the enterokinase protein includes a recombinant enterokinase protein.

[0028] In one or more preferred embodiments, the enterokinase protein includes an enterokinase protein having an amino acid sequence as shown in SEQ ID NO:1.

[0029] In another aspect of the present invention, there is provided a monoclonal antibody that specifically binds to an enterokinase protein, selected from: (i) a monoclonal antibody secreted by the hybridoma cell line (EK-10G9D1) with the deposit number CCTCC NO: C2023161; (ii) a monoclonal antibody secreted by the hybridoma cell line (EK-6-3A6) with the deposit number CCTCC NO: C2023162.

[0030] In one or more preferred embodiments, the monoclonal antibody is used for preparing a kit for detecting an enterokinase protein.

[0031] In one or more preferred embodiments, the detection includes quantitative detection, semi-quantitative detection, or qualitative detection.

[0032] In another aspect of the present invention, there is provided a hybridoma cell line that produces a monoclonal antibody specifically binding to enterokinase protein, selected from: the hybridoma cell line (EK-10G9D1) with the deposit number of CCTCC NO: C2023161; the hybridoma cell line (EK-6-3A6) with the deposit number of CCTCC NO: C2023162.

[0033] In another aspect of the present invention, there is provided a method for detecting enterokinase protein, the method comprising the following steps:

[0034] (a) Loading a sample to be tested onto a solid-phase carrier coated with a first monoclonal antibody, so that the enterokinase protein in the sample to be tested binds to the first monoclonal antibody on the solid-phase carrier, forming a solid-phase carrier with a "enterokinase protein - first monoclonal antibody" binary complex;

[0035] (b) Loading a second monoclonal antibody onto the solid-phase carrier obtained in (a), so as to form a solid-phase carrier with a "second monoclonal antibody - enterokinase protein - first monoclonal antibody" ternary complex; and the second monoclonal antibody carries a label;

[0036] (c) Detecting the label in the ternary complex, so as to determine the presence or absence and the amount of enterokinase protein in the sample to be detected;

[0037] The additional condition is that steps (a) and (b) can be carried out sequentially or simultaneously;

[0038] Wherein, the first monoclonal antibody and the second monoclonal antibody are selected from: monoclonal antibodies secreted by the hybridoma cell line with the deposit number of CCTCC NO: C2023161, monoclonal antibodies secreted by the hybridoma cell line with the deposit number of CCTCC NO: C2023162; preferably, the first monoclonal antibody is a monoclonal antibody secreted by the hybridoma cell line with the deposit number of CCTCC NO: C2023161, and the second monoclonal antibody is a monoclonal antibody secreted by the hybridoma cell line with the deposit number of CCTCC NO: C2023162.

[0039] In one or more preferred embodiments, the method for detecting enterokinase protein is an in vitro method.

[0040] In one or more preferred embodiments, the method for detecting enterokinase protein is a non-diagnostic method.

[0041] Other aspects of the present invention will be apparent to those skilled in the art from the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1Specificity test results of the monoclonal antibodies EK-10G9D1 and EK-6-3A6 in Example 2.

[0043] Figure 2 Determination results of the upper limit of detection of the kit in the implementation application.

[0044] Figure 3 Determination results of the lower limit of detection of the kit in the implementation application. Detailed implementation manners

[0045] Through in-depth research, the present inventors isolated hybridoma cells secreting monoclonal antibodies against enterokinase protein. The secreted monoclonal antibodies are highly specific, have excellent antigen-binding properties, and are directed against different epitopes of enterokinase protein. According to the principle of the sandwich immunoassay, a kit can be prepared that is simple to operate, highly accurate, rapid, and can accurately analyze the presence of enterokinase, enabling precise quantitative detection of recombinant enterokinase protein.

[0046] Those skilled in the art are aware that an antigen may contain multiple epitopes (antigenic determinants). Therefore, more than one antibody can be obtained against the same antigen, and the binding characteristics (such as specificity) of these antibodies to the antigen may all be different. Therefore, for the same antigen, those skilled in the art need to compare and screen to find a monoclonal antibody suitable for specific binding. Due to the spatial structure of the antigen, many epitopes are included inside the spatial structure, making it difficult to find antibodies that can specifically bind to epitopes stably present outside the spatial structure. The present inventors have conducted in-depth research work and isolated monoclonal antibodies with high specificity for enterokinase protein, especially suitable for the sandwich immunoassay.

[0047] In the present invention, the target object to be detected is enterokinase protein. In a preferred embodiment, the detection target object of the present invention is recombinant enterokinase, and the antibody of the present invention can achieve precise detection of recombinant enterokinase. A single anti-enterokinase protein antibody is prone to interference during detection, resulting in poor specificity and large result deviations. Usually, if one antibody is used to bind enterokinase protein, non-specific binding may inevitably occur during the determination process; while preparing a kit based on the principle of the sandwich immunoassay and using two antibodies that bind to different epitopes of enterokinase protein antigen, the probability of non-specific binding in this case is very low, so the accuracy and precision of the results are high.

[0048] Moreover, the two highly specific antibodies of the present invention adsorb and locate the target antigen enterokinase protein, and their localization and amplification effects are better, resulting in higher specificity and precision. And only a very small amount of sample is required for the determination.

[0049] As used herein, the terms "capture antibody", "coated antibody", "first monoclonal antibody", "first antibody", and "primary antibody" are used interchangeably and refer to an antibody that is specific for enterokinase protein and is used for immobilization on a solid-phase carrier.

[0050] As used herein, the terms "detection antibody", "second monoclonal antibody", "second antibody", "enzyme-labeled antibody", and "secondary antibody" are used interchangeably and refer to an antibody that is specific for enterokinase protein and corresponds to the respective first antibody in the kit. For the antigen enterokinase protein, the corresponding first antibody and second antibody are different and can simultaneously bind to different epitopes (antigenic determinants) of the enterokinase protein.

[0051] As used herein, "sample to be tested" encompasses various sample types, including various objects for which enterokinase content detection is required. For example, the "sample to be tested" can be biopeptide drugs and gene products, such as expression products of recombinant fusion proteins, small molecule protein or polypeptide biologics, biological reagents of recombinant protein drugs, etc.

[0052] As used herein, the "label" or "detectable label" refers to a label that is located / attachable to the second monoclonal antibody and is used to determine the presence or absence and the amount of enterokinase protein in the sample to be tested. Preferably, the label can be selected from: horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, β-D-galactosidase, urease, catalase, or glucoamylase.

[0053] As used herein, the "substrate corresponding to the detectable label" refers to a substrate that can be catalytically colored by the label of the second monoclonal antibody and is used to display the recognition signal of the binding of the second antibody to enterokinase protein. Such substrates include, for example: o-phenylenediamine (OPD), 3,3',5,5'-tetramethylbenzidine (TMB), ABTS for horseradish peroxidase; p-nitrophenyl phosphate (p-NPP) for alkaline phosphatase; and so on.

[0054] As used herein, the "sandwich ELISA method" is a type of enzyme-linked immunosorbent assay. The conventional procedure is to immobilize the primary antibody on a carrier, then the primary antibody reacts with the antigen, after washing, it reacts with the labeled secondary antibody, and after washing again, finally, a chemiluminescence or enzyme-linked color reaction is performed to detect the signal. The sandwich ELISA method is applicable to the detection of antigens with two or more epitopes, but the prerequisite is that there are two antibodies that target different epitopes and have excellent binding performance. In the art, polyclonal antibodies are commonly used as the second antibody, but their specificity or sensitivity is often lacking.

[0055] Enterokinase protein

[0056] Enterokinase protein is a known protein, and both its amino acid sequence and nucleotide sequence are known in the art. The amino acid sequence of the recombinant enterokinase described in the present invention is shown in SEQ ID NO: 1.

[0057] Methods for producing enterokinase protein are also known. For example, through conventional recombinant DNA techniques, the polynucleotide sequence of enterokinase protein can be used to express or produce recombinant enterokinase protein. Generally, the following steps are involved:

[0058] (1). Transform or transduce a suitable host cell with a polynucleotide (or variant) encoding enterokinase protein, or with a recombinant expression vector containing the polynucleotide;

[0059] (2). Culture the host cell in a suitable medium; and

[0060] (3). Isolate and purify the protein from the medium or cells.

[0061] The recombinant enterokinase protein obtained above can be processed into a protein with a certain purity for formulating a standard product.

[0062] Monoclonal antibody against enterokinase protein

[0063] The antibody used in the present invention is a monoclonal antibody specific for enterokinase protein. Here, "specificity" means that the antibody can bind to enterokinase protein or its fragment. More particularly, it refers to those antibodies that can bind to enterokinase protein or fragment but do not recognize and bind to other non-related antigen molecules.

[0064] The present invention utilizes highly specific monoclonal antibodies against different epitopes of enterokinase protein, and according to the principle of the sandwich ELISA method, a kit for conveniently, rapidly and accurately analyzing enterokinase protein is prepared.

[0065] Hybridoma cell line EK-10G9D1, deposit number CCTCC NO: C2023161, IgG1 subtype;

[0066] Hybridoma cell line EK-6-3A6, deposit number CCTCC NO: C2023162, IgG1 subtype.

[0067] The monoclonal antibodies of the present 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, N.Y., 1981). The monoclonal antibodies of the present invention can be obtained by conventional immunization techniques using enterokinase protein or fragments or functional regions. In addition, they can be prepared by recombinant methods or synthesized using a polypeptide synthesizer.

[0068] In one example of the present invention, the monoclonal antibody can be prepared by the following preparation method, which includes the steps of: (1) providing adjuvant-pretreated mice; (2) inoculating the hybridoma cells into the peritoneal cavity of the mice and secreting monoclonal antibodies; (3) extracting ascites and isolating the monoclonal antibody. As a preferred method, the method for isolating monoclonal antibodies from ascites can be by collecting ascites, affinity purification, and dialysis. For example, it may include: collecting ascites, precipitating with ammonium sulfate and octanoic acid, and then purifying with a pre-packed Protein G chromatography column to obtain highly pure monoclonal antibodies.

[0069] In addition, according to the conventional animal cell culture method, the hybridoma cells can be cultured and amplified in vitro so that they secrete the monoclonal antibody.

[0070] After obtaining the antibodies of the present invention, those skilled in the art can obtain the sequence information of the monoclonal antibody through sequencing techniques known in the art, such as measuring the sequences of its heavy chain and light chain. Thus, it can be artificially prepared using bioengineering techniques.

[0071] The monoclonal antibodies of the present invention can be prepared by recombinant methods or synthesized using a polypeptide synthesizer. Those skilled in the art understand that after obtaining the hybridoma cell line of the monoclonal antibody or knowing the monoclonal antibody by means such as sequencing, those skilled in the art can conveniently obtain the antibody.

[0072] The monoclonal antibodies of the present invention are derived from a specific hybridoma cell line. It should be understood that after obtaining the antibodies of the present invention from this cell line, various modifications can be made to the antibodies, and they can also be sequenced, and optimized modifications can be made based on the antibody structure after sequencing. All these antibody variation methods are included within the scope of the present invention. Therefore, the antibodies of the present invention can be intact 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-stranded antibodies, trispecific antibodies, and tetravalent antibodies.

[0073] The present invention also provides an immunoconjugate, which comprises the antibody described herein and further comprises at least one other type of functional molecule. The functional molecules include but are not limited to: detectable labels. The antibody and the functional molecule can form a conjugate by means of covalent linkage, coupling, attachment, crosslinking, etc.

[0074] After determining the coating antibody and / or detection antibody used in the kit of the present invention, various detectable labels conventionally used in the art for binding to the detection antibody for detection can be used. The present invention places no particular limitation on the labels used, as long as they can bind to the monoclonal antibodies of the present invention and can accurately indicate the presence or absence and the amount of the target protein in the sample to be detected after appropriate treatment. The detectable labels can include but are not limited to: colloidal gold, fluorescent labels, chromogenic labels; such as: enzymes, cofactors, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals, and non-radioactive paramagnetic metal ions. It can also contain more than one label. The labels used for labeling antibodies for detection and / or analysis purposes depend on the specific detection / analysis techniques and / or methods used, such as immunohistochemical staining (tissue) samples, flow cytometry, etc. Labels suitable for the detection / analysis techniques and / or methods known in the art are well known to those skilled in the art.

[0075] The marker described above can be directly set on the detection antibody; alternatively, the marker can also be set on the anti-antibody specific to the second antibody. Those skilled in the art can select a suitable marker according to 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, glucoamylase or biotin. When using some of the enzyme markers as described above, some substrates that bind to the corresponding enzymes are also required, so that the presence or amount of the marker can be reported by means of color development or the like. As used herein, the "substrate corresponding to the marker" refers to a substance that can be catalyzed and colored by the marker and is used to display the recognition signal of the binding of the second antibody to the target protein. Examples of the substrate include: o-phenylenediamine (OPD), 3,3',5,5'-tetramethylbenzidine (MB), 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 a suitable substrate according to the type and characteristics of the marker used.

[0076] As a specific example, the detectable marker is HRP. As a display marker, when combined with OPD, MB or ABTS, etc., a signal is generated.

[0077] Kit

[0078] After obtaining the monoclonal antibody of the present invention, those skilled in the art can sensitively detect whether enterokinase protein exists or its concentration in a sample through various means, and the techniques used can be those commonly used in the field of immunology. It includes qualitative detection and quantitative detection methods. Preferably, the qualitative detection includes: identifying the presence of enterokinase protein by immunoblotting or immunofluorescence methods; for example, determining enterokinase protein by methods such as ELISA, immunogold test strip, immunofluorescence test strip, homogeneous enzyme immunoassay, etc. The present invention provides a kit for detecting enterokinase protein, and the kit can be used to detect enterokinase protein and the like.

[0079] The kit contains the monoclonal antibody of the present invention. In a preferred manner, one of the monoclonal antibodies is immobilized on a solid-phase carrier, and the other is used as a detection antibody.

[0080] As an optimal embodiment of the present invention, the first monoclonal antibody is a monoclonal antibody secreted by the hybridoma cell line (EK-10G9D1) with the preservation number of CCTCC NO: C2023161; and the second monoclonal antibody is a monoclonal antibody secreted by the hybridoma cell line (EK-6-3A6) with the preservation number of CCTCC NO: C2023162. Surprisingly, using EK-10G9D1 as the coating antibody and EK-6-3A6 as the detection antibody, such a combination can produce the best detection effect for enterokinase protein, and its detection sensitivity reaches about 0.043 ng / mL.

[0081] The first monoclonal antibody described above is coated on a solid-phase carrier. The present invention has no particular limitation on the solid-phase carrier used, as long as it can be coupled (connected) with the first monoclonal antibody. For example, the solid-phase carrier is selected from: microtiter plates, microspheres, etc.

[0082] As an operation example, the solid-phase carrier used can be a microtiter plate (ELISA plate).

[0083] In order to eliminate false positives and false negatives, it is advisable to set up quality control (control) during the detection process. In an example of the present invention, the quality control product uses an enterokinase protein standard.

[0084] In addition, in order to obtain quantitative results, standards containing multiple enterokinase proteins with known concentrations can be set up during the detection process. The method for setting up the standards can adopt conventional methods.

[0085] Using the above standards, the standard curve is set as follows: using the OD value detection results of the standards as the ordinate (Y-axis) and the standard concentration as the abscissa (X-axis), a quantitative standard curve of the enterokinase protein kit is plotted. Thus, according to the OD value obtained from the detection of the sample to be tested, the concentration of enterokinase protein in the sample to be tested can be calculated using the standard curve.

[0086] In addition, in order to make the kit of the present invention more convenient during detection, the kit preferably further contains some other auxiliary reagents. The auxiliary reagents are some reagents commonly used in the double antibody sandwich method, and the characteristics of these reagents and their preparation methods are well known to those skilled in the art. The reagents are, for example (but not limited to): chromogenic agents, washing solutions, termination solutions, and sensitizing diluents.

[0087] The beneficial effects of the present invention mainly lie in:

[0088] (1) Since the kit of the present invention uses monoclonal antibodies that have a high affinity for enterokinase protein and highly specifically recognize different epitopes of enterokinase protein, the sensitivity and accuracy are very high. Its linear range value is 0.0312 - 32 ng / ml. The ELISA kit can be used for quantitative (trace) detection of recombinant enterokinase protein.

[0089] (2) Since the monoclonal antibodies used have extremely excellent binding properties for enterokinase protein, the kit of the present invention can detect enterokinase protein in blood extremely quickly, with a short time-consuming, and is faster than ordinary similar ELISA kits.

[0090] (3) The kit of the present invention also has the characteristics of simplicity and stability.

[0091] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions such as those described in Molecular Cloning: A Laboratory Manual, Third Edition, edited by J. Sambrook et al., published by Science Press, or according to the conditions recommended by the manufacturer.

[0092] Example 1: Obtaining of Hybridoma Cell Lines Capable of Secreting Monoclonal Antibodies Against Recombinant Enterokinase Protein

[0093] The recombinant enterokinase protein (REK) used as an immunogen has the following amino acid sequence (SEQ ID NO:1):

[0094] IVGGSDSREGAWPWVVALYFDDQQVCGASLVSRDWLVSAAHCVYGRNMEPSKWKAVLGLHMASNLTSPQIETRLIDQIVINPHYNKRRKDNDIAMMHLEMKVNYTDYIQPICLPEENQVFSPGRICSIAGWGTLIYQGSTADVLQEADVPLLSNEKCQQQMPEYNITENMVCAGYEAGGVDSCQGDSGGPLMCQENNRWLLAGVTSFGYQCALPNRPGVYARVPRFTEWIQSFLH

[0095] Analysis was carried out based on the full-length REK sequence. Restriction enzyme sites EcoRⅠ and SalⅠ and protective bases were added to both ends of the coding sequence for total gene synthesis. After double digestion of the vector PET28A(+), it was ligated with the REK gene fragment digested with the same enzymes and then transformed. The recombinant plasmid was constructed and sequenced. The correctly sequenced REK recombinant plasmid was transformed into BL21 cells. After collecting and lysing the cells, the recombinant REK protein was purified. The purified recombinant enterokinase protein (REK) was formulated with physiological saline at a concentration of 500 μg / mL and mixed with an equal volume of sigma-Freund's complete adjuvant, and 100 μL was injected into the dorsal muscles of 2 6-week-old Balb / c mice for immunization; then every 14 days, the recombinant enterokinase protein (REK) was formulated with physiological saline at a concentration of 500 μg / mL and mixed with an equal volume of sigma-Freund's incomplete adjuvant, and the immunization was boosted 2 times in the same way. After the third injection, blood was taken from the tails of the mice, and the serum was separated. The serum titer was detected by the indirect ELISA method, and the titer reached the fusion requirement. Three days before cell fusion, the spleen was boosted with 100 μg of protein.

[0096] Balb / c mouse spleen cells and SP2 / 0 myeloma cells in the logarithmic growth phase were mixed at a ratio of 9:1 and then fused under the action of 50% PEG1500. The fused cells were cultured in 1640-HAT complete medium containing 10% FBS (fetal bovine serum) for 6 - 7 days. After picking cell clones and screening and culturing them in 1640-HT complete medium for 5 days, the antibody specificity in the cell culture supernatant was detected by the indirect ELISA method. Monoclonal cell lines that could stably secrete antibodies were obtained. After expansion culture, they were cryopreserved with FBS containing 10% DMSO, and the cell density was 10 6 cells / mL. Ascites was prepared by the in vivo induction method. The ascites was purified by Protein G affinity chromatography to obtain the purified antibody. After dialysis in 0.01 mol / L phosphate buffer, the protein concentration was measured at OD280nm, and multiple monoclonal antibodies were selected.

[0097] Antibody pairing analysis was carried out: A variety of antibodies secreted by the obtained multiple monoclonal cell lines were respectively coated on microtiter plates and labeled with biotin, and then paired with each other in a complete combination. The screening method was the double antibody sandwich ELISA method. The screening principle was: After obtaining the monoclonal antibodies, two monoclonal antibodies were taken out, one as the capture antibody and the other as the labeled antibody. The capture antibody was coated on the antigen plate. First, the antigen was added, incubated, and then the unbound antigen was washed away. Then the labeled antibody was added, incubated, and the unbound labeled antibody was washed away. Finally, the chromogenic solution was added for color development. If color development could occur, it indicated that the labeled antibody specifically bound to the antigen, and the capture antibody and the labeled antibody were a pair of paired antibodies. If color development could not occur, it indicated that the labeled antibody could not bind to the antigen and was thus eluted, and the capture antibody and the labeled antibody were not paired antibodies.

[0098] A large number of monoclonal cell lines were analyzed (the comparison results of some antibodies are shown in Table 1), and the two best cell lines that could stably secrete monoclonal antibodies and could be paired for sandwich ELISA were screened out, and were named hybridoma cell line EK-10G9D1 and EK-6-3A6 that could secrete monoclonal antibodies against recombinant enterokinase protein (REK), respectively.

[0099] Table 1. Pairing situation of different monoclonal antibodies *

[0100]

[0101]

[0102] *Note: - means unable to pair; + means able to pair with weak positive result; ++ means able to pair with strong positive result; +++ means able to pair with the strongest positive result.

[0103] Hybridoma cell lines EK-10G9D1 and EK-6-3A6 were deposited in the China Center for Type Culture Collection, and the deposit numbers were CCTCC NO:C2023161 and CCTCC NO:C2023162, respectively. Hybridoma cell lines EK-10G9D1 and EK-6-3A6 can secrete monoclonal antibodies against recombinant enterokinase protein (REK). The monoclonal antibodies secreted by hybridoma cell lines EK-10G9D1 and EK-6-3A6 were named monoclonal antibody EK-10G9D1 and EK-6-3A6, respectively.

[0104] Example 2. Preparation, purification and titer of monoclonal antibodies EK-10G9D1 and EK-6-3A6

[0105] I. Preparation of monoclonal antibodies EK-10G9D1 and EK-6-3A6

[0106] 1. Incremental culture method

[0107] Preparation method of cell culture medium: Fetal bovine serum was added to RPMI-1640 medium, and the final concentration of fetal bovine serum was 10% (mass percentage).

[0108] Hybridoma cell lines EK-10G9D1 and EK-6-3A6 were respectively 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 EK-10G9D1 and EK-6-3A6 with a purity of more than 95% (stored at -20 °C).

[0109] 2. Ascites preparation

[0110] Balb / c mice were intraperitoneally injected with sterilized paraffin oil (0.5 mL / mouse). After 15 days, they were intraperitoneally injected with hybridoma cell lines EK-10G9D1 and EK-6-3A6 (1×10 6 cells / mouse). After 7-10 days, ascites were collected, purified by Protein G affinity chromatography, then transferred to a dialysis bag and dialyzed in PBS buffer at pH 7.4 and 0.01 M. Then the liquid in the dialysis bag was collected to obtain monoclonal antibodies EK-10G9D1 and EK-6-3A6 with a purity of over 95% respectively. After filtration through a 0.22 μm sterile filter membrane, they were stored at -20°C.

[0111] The monoclonal antibodies EK-10G9D1 and EK-6-3A6 used in the subsequent steps and subsequent examples were all prepared from ascites.

[0112] II. Titer Detection (Indirect ELISA Method)

[0113] 1. Take an ELISA plate, add the coating solution (100 μL / well), and coat overnight at 4°C. Coating solution: Take recombinant enterokinase protein (REK), dilute it with carbonate buffer at pH 9.6 and 0.05 M to a protein concentration of 5 μg / mL, which is the coating solution.

[0114] 2. After completing step 1, take the ELISA plate, wash it 3 times with PBST solution, then add the blocking solution (200 μL / well), and incubate at 37°C for 2 hours. Blocking solution: PBST solution containing 0.5 g / 100 mL BSA.

[0115] 3. After completing step 2, take the ELISA plate, wash it 3 times with PBST solution, and dilute the two monoclonal antibodies EK-10G9D1 and EK-6-3A6 to a basic concentration of 1 mg / ml with PBS buffer at pH 7.4 and 0.01 M respectively. Antibody diluent: Take monoclonal antibodies EK-10G9D1 and EK-6-3A6, and on the basis of an antibody concentration of 1 mg / ml (regarded as 1-fold or the original concentration), perform serial dilution with PBS buffer at pH 7.4 and 0.01 M respectively to obtain antibody diluents with different concentrations. Then add the antibody diluent (100 μL / well) and incubate at 37°C for 1 h.

[0116] 4. After completing step 3, take the ELISA plate, wash it 3 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. Working solution of goat anti-mouse HRP-labeled secondary antibody: 1:5000 dilution of HRP-labeled secondary antibody. HRP-labeled secondary antibody: Kangwei Century brand, product number CW0102S.

[0117] 5. After completing Step 4, take the ELISA plate, wash it 3 times with PBST solution, then add TMB substrate reaction solution for color development for 10 minutes, then add 2M sulfuric acid solution to terminate the color development, and measure the absorbance at 450 nm with an ELISA reader (see Table 2).

[0118] Table 2. Results of monoclonal antibody titer determination at different concentrations

[0119]

[0120] As shown in the results of Table 2, the titer of monoclonal antibody EK-10G9D1 ≥ 640,000, and the titer of EK-6-3A6 ≥ 320,000.

[0121] Example 3. Antibody specificity test of monoclonal antibodies EK-10G9D1 and EK-6-3A6

[0122] The test method is the same as Step 2 in Example 2, but the coating solution and antibody dilution solution are replaced.

[0123] Select other enzyme protein preparations of the same protein expression system as recombinant enterokinase protein (REK) respectively: recombinant carboxypeptidase B protein (RCPB), recombinant trypsin protein (RPT), sperm-specific protein (SP10), bovine serum albumin (BSA) expressed by other E. coli systems, negative serum, and recombinant REK protein (prepared in Example 1) for coating the plate, and add purified REK antibodies EK-10G9D1 and EK-6-3A6 at a concentration of 0.1 μg / mL respectively to test the reaction of the antibodies with these proteins.

[0124] The results are as Figure 1 , it can be seen that monoclonal antibodies EK-10G9D1 and EK-6-3A6 only interact (bind) with REK and do not cross-react with other proteins, showing very high specificity.

[0125] Example 4. Antibody subtype identification of monoclonal antibodies EK-10G9D1 and EK-6-3A6

[0126] Use a commercial antibody Ig class / subclass / subtype identification kit (Luoyang BioTong Experimental Materials Center, product number C060101-L) to identify the antibody subtype. The results are shown in Table 3.

[0127] Table 3. Identification results of EK-10G9D1 and EK-6-3A6 antibody subtypes *

[0128] Subtype EK-10G9D1 EK-6-3A6 Positive control Negative control IgG1 2.365 2.353 2.912 0.059 IgG2a 0.061 0.069 2.966 0.065 IgG2b 0.061 0.054 2.892 0.065 IgG3 0.087 0.068 2.920 0.063 IgM 0.064 0.061 2.997 0.066 IgA 0.058 0.067 2.944 0.058 Kappa 2.012 1.955 2.835 0.064 Lambda 0.071 0.062 2.893 0.065

[0129] * Note: The values in the table represent the OD450nm absorbance value.

[0130] The results showed that the antibody EK-10G9D1 was of IgG1 subtype, the antibody EK-6-3A6 was of IgG1 subtype, and the light chains of both antibodies were Kappa.

[0131] Example 5. Preparation of ELISA Detection Kit

[0132] I. Preparation of HRP-labeled Antibody

[0133] Weigh 1 mg of HRP and dissolve it in deionized water to a concentration of 10 mg / ml and a volume of 0.1 ml. To the prepared HRP solution, add 0.1 ml of freshly prepared 0.1 mol / L NaIO4 solution, which is of the same volume as the enzyme solution, and add it drop by drop. React in the dark at 4°C for 30 min until the reaction solution turns dark green. Then add 0.1 ml of 0.5% ethylene glycol drop by drop, mix well, and react in the dark at 4°C for 30 min until the reaction solution turns brown. Add 1 mg of monoclonal antibody EK-6-3A6, mix well, put the antibody and the activated enzyme into a dialysis bag, and dialyze in CBS buffer at 50 mM, pH 9.6 overnight at 4°C. Take out the dialyzed component, add 20 μL of freshly prepared 5 mg / ml NaBH4 solution, and let it stand in the dark for 1 h. Then add an equal volume of saturated (NH4)2SO4 solution to precipitate the protein, and centrifuge at 5000 rpm for 30 min. Remove the supernatant, resuspend the precipitate in 50% (NH4)2SO4 solution, and centrifuge at 5000 rpm for 30 min. Dissolve the precipitate in PBS, then desalt it using a desalting column, and add an equal volume of glycerol as a protective agent to obtain the enzyme-labeled antibody EK-6-3A6. After aliquoting, store it at -20°C.

[0134] II. Preparation of Coated Plate

[0135] Dilute the monoclonal antibody EK-10G9D1 with carbonate buffer to a final concentration of 4 μg / ml, and pipette 100 μL / well of the diluted antibody into a PVC microtiter plate. Cover the microtiter plate with a sealing film and incubate overnight at 4°C. Discard the coating solution, wash the plate 3 times with PBST, and block the microtiter plate with PBST buffer containing 0.5% BSA, adding 200 μL per well. Cover the microtiter plate with a sealing film and incubate in a 37°C incubator for 2 hours, then wash the plate 3 times with PBST. After drying, seal it with an aluminum foil bag to obtain the coated plate, and store it at 4°C.

[0136] III. Usage Method of ELISA Detection Kit (Double Antibody Sandwich Method)

[0137] Take out the coated plate (coated with EK-10G9D1), restore it to room temperature, and use a pipette to aspirate 100 μL of positive standard control (16 ng / mL recombinant REK, dilute the positive standard with phosphate buffer according to requirements to obtain positive control solutions with different series of concentrations), blank control (0.01 M phosphate buffer at pH 7.4), and the sample to be tested, and add them to the corresponding reaction wells; then add 100 μL of enzyme-labeled antibody EK-6-3A6 (250 ng / mL) to each reaction well, gently shake for 10 - 15 s, cover the plate with a plate cover or cover it with a sealing film, and incubate at 37 °C for 0.5 h; after incubation, remove the plate cover or sealing film, quickly invert the enzyme-linked immunosorbent assay (ELISA) plate, discard the liquid in the plate wells into the waste liquid tank, and pat it dry on absorbent paper. After washing the plate 3 - 5 times with PBST, add 100 μL of TMB substrate solution for color development for 10 minutes, and then add 50 μL of stop solution (2 M sulfuric acid); measure the optical density of each well at 450 nm with an ELISA reader. Take the concentration of the standard product as the abscissa (X) and the OD value as the ordinate (Y) to draw a standard curve. Substitute the OD value of the sample well into the standard curve equation to obtain the protein quantification value of the corresponding sample.

[0138] Example 6. Determination of the sensitivity and specificity of the kit

[0139] I. Sensitivity test of the ELISA detection kit

[0140] Analyze the kit in Example 3 to test its sensitivity. Use 0.01 M PBS dilution as the sample to be tested (the number of samples selected at 0 ng / ml ≥ 30), as shown in Table 4.

[0141] Table 4. Results of the determination of the sensitivity of the kit

[0142]

[0143]

[0144] Calculate the average value and variance value of the above 30 samples, and substitute them into the equation Y = average OD value + 2 × variance value of OD. Then substitute the Y value into the drawn standard curve equation, and the obtained X concentration value is the sensitivity.

[0145] After determination, the detection sensitivity of the kit in Example 3 is 0.043 ng / mL.

[0146] II. Specificity detection of the kit

[0147] Analyze the kit in Example 3 to test its specificity.

[0148] Select other enzyme protein preparations with the same protein expression system as recombinant enterokinase protein (REK): 1 - recombinant carboxypeptidase protein (100 ng / ml), 2 - recombinant trypsin protein (100 ng / ml), 3 - sperm-specific protein SP10 expressed by other E. coli systems (100 ng / ml), 4 - 1% BSA, 5 - blank medium of E. coli system, 6 - blank medium of yeast system; 7 - blank medium of DMEM cells, 8 - blank medium of MEM cells, 9 - blank medium of 1640 cells; 10 - mouse negative serum as a negative control sample. 11 - PBS buffer at pH 7.4 and 0.01 M as a blank control sample; 12 - recombinant enterokinase protein (REK) (100 ng / ml) as a positive control sample. Measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay reader (see Table 5). The specific detection method is the same as described above. The results are shown in Table 5.

[0149] Table 5. Specificity determination results of the kit

[0150]

[0151] The results show that the monoclonal antibody in the kit only reacts with REK and does not cross-react with other proteins, and the specificity is ideal.

[0152] III. Determination of the detection limit range of the kit

[0153] Analyze the kit in Example 3 and test its detection limit.

[0154] Using 0.01 M phosphate buffer at pH 7.4 as a diluent, gradient-dilute recombinant enterokinase protein (REK) and use it as a sample to be tested. 0.01 M phosphate buffer at pH 7.4 is used as a blank sample. Test the upper detection value and lower detection value of the kit. As Figure 2 and Figure 3 shown.

[0155] The results are as Figure 2 and Figure 3 shown. The upper detection value of the recombinant enterokinase detection kit is 32 ng / ml, and the lower detection value is as low as 0.0312 ng / ml.

[0156] Example 7. Determination of the residual amount of enterokinase protein in biological enzyme preparations

[0157] Analyze the kit in Example 3 and test the residual amount of recombinant enterokinase protein in biological enzyme preparations.

[0158] Using 0.01M phosphate buffer at pH 7.4 as the diluent, V8 protease, T7 enzyme, and methyltransferase were serially diluted and used as test samples. At the same time, recombinant enterokinase protein (REK) was serially diluted and used as the spiked test sample. 0.01M phosphate buffer at pH 7.4 was used as the blank sample. Among them, the samples of V8 protease, T7 enzyme, and methyltransferase were all taken from non-specific production processes, and the production processes of the above three protein preparations all involved the use of recombinant enterokinase protein (REK). See Table 6.

[0159] As shown in Table 6, the recombinant enterokinase detection kit can effectively detect the residual recombinant enterokinase protein in the samples of V8 protease, T7 enzyme, and methyltransferase, and the sample spiked recovery rate reaches 90%-110%.

[0160] Table 6. Determination results of recombinant enterokinase protein (REK)

[0161]

[0162]

[0163] Biological material preservation

[0164] The hybridoma cell line EK-10G9D1 isolated in the present invention, Hybridoma cell line EK-10G9D1, has been deposited at the China Center for Type Culture Collection (abbreviation: CCTCC; Wuhan, China, Wuhan University), with the deposit date: June 9, 2023, and the deposit number: CCTCC NO: C2023161. It was detected as viable by the deposit center.

[0165] The hybridoma cell line EK-6-3A6 isolated in the present invention, Hybridoma cell line EK-6-3A6, has been deposited at the China Center for Type Culture Collection (abbreviation: CCTCC; Wuhan, China, Wuhan University), with the deposit date: June 9, 2023, and the deposit number: CCTCC NO: C2023162. It was detected as viable by the deposit center.

[0166] The above embodiments only represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent should be subject to the appended claims. At the same time, all the documents mentioned in the present invention are cited as references in this application, just as each document is cited separately as a reference.

Claims

1. A kit for detecting enterokinase protein, the kit comprising: (i) a monoclonal antibody secreted by a hybridoma cell line with the preservation number CCTCC NO: C2023161; and (ii) a monoclonal antibody secreted by a hybridoma cell line with the preservation number CCTCC NO: C2023162.

2. The kit according to claim 1, wherein It includes: A solid-phase carrier, on which a first monoclonal antibody is coated, and the first monoclonal antibody is selected from the monoclonal antibodies of (i) or (ii); and A container or package a, in which a second monoclonal antibody is contained, and the second monoclonal antibody is selected from the monoclonal antibodies of (ii) or (i); Wherein, the second monoclonal antibody carries a detectable label; or, the kit contains a detectable label for connecting with the second monoclonal antibody; Wherein, the first monoclonal antibody and the second monoclonal antibody are different and can bind to enterokinase protein simultaneously; preferably, the first monoclonal antibody is a monoclonal antibody secreted by a hybridoma cell line with the preservation number CCTCC NO: C2023161, and the second monoclonal antibody is a monoclonal antibody secreted by a hybridoma cell line with the preservation number CCTCC NO: C2023162.

3. The kit according to claim 1 or 2, characterized in that, The kit further comprises one or more components selected from the following: (a) A container or package b, in which a standard product (positive standard) of enterokinase protein is contained; and / or (b) A container or package c, in which a quality control product of enterokinase protein is contained; preferably, the quality control product includes a positive quality control product and a negative quality control product; (c) A container or package d, in which a substrate corresponding to the detectable label is contained; (d) A container or package e, in which a chromogenic agent is contained; (e) A container or package f, in which a coating solution is contained; (f) A container or package g, in which a washing solution is contained; (g) A container or package f, in which a termination solution is contained.

4. The kit according to claim 2, wherein The detectable label includes (but is not limited to): horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-D-galactosidase, urease, catalase, or glucose amylase.

5. The kit according to claim 2, wherein The solid-phase carrier includes: a microplate, magnetic beads, microspheres, a chip, a glass slide, a test strip.

6. The kit according to claim 1 or 2, characterized in that, The linear range value of the kit is 0.0312 - 32 ng / ml; and / or The detection sensitivity of the kit is 0.043 ng / mL.

7. A monoclonal antibody that specifically binds to enterokinase protein, selected from: (i) a monoclonal antibody secreted by a hybridoma cell line with the preservation number CCTCC NO: C2023161; (ii) a monoclonal antibody secreted by a hybridoma cell line with the preservation number CCTCC NO: C2023162.

8. Use of the monoclonal antibody according to claim 7 for preparing a kit for detecting enterokinase protein; preferably, the detection includes quantitative detection, semi-quantitative detection or qualitative detection.

9. A hybridoma cell line that produces monoclonal antibodies specifically binding to enterokinase protein, selected from: The hybridoma cell line with the preservation number of CCTCC NO: C2023161; The hybridoma cell line with the preservation number of CCTCC NO: C2023162.

10. A method for detecting enterokinase protein, the method comprising the following steps: (a) Loading the sample to be tested onto a solid-phase carrier coated with a first monoclonal antibody, so that the enterokinase protein in the sample to be tested binds to the first monoclonal antibody on the solid-phase carrier, forming a solid-phase carrier with a "enterokinase protein - first monoclonal antibody" binary complex; (b) Loading a second monoclonal antibody onto the solid-phase carrier obtained in (a), so as to form a solid-phase carrier with a "second monoclonal antibody - enterokinase protein - first monoclonal antibody" ternary complex; and the second monoclonal antibody carries a marker; (c) Detecting the marker in the ternary complex, so as to determine the presence or absence and the amount of enterokinase protein in the sample to be tested; The additional condition is that steps (a) and (b) can be carried out sequentially or simultaneously; wherein, the first monoclonal antibody and the second monoclonal antibody are selected from: monoclonal antibodies secreted by the hybridoma cell line with the preservation number of CCTCC NO: C2023161, monoclonal antibodies secreted by the hybridoma cell line with the preservation number of CCTCC NO: C2023162; preferably, the first monoclonal antibody is a monoclonal antibody secreted by the hybridoma cell line with the preservation number of CCTCC NO: C2023161, and the second monoclonal antibody is a monoclonal antibody secreted by the hybridoma cell line with the preservation number of CCTCC NO: C2023162.

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