Immunoglobulin G (IgG) antibody of parotitis virus, recombinant IgM antibody and application

Abstract

The invention relates to the field of in-vitro immunodiagnosis detection, in particular to an IgG antibody of mumps virus, a recombinant IgM antibody and application. Wherein the IgG antibody comprises a heavy chain constant region, a light chain constant region, and a heavy chain variable region and a light chain variable region of the IgG monoclonal antibody which is specifically combined with the mumps virus, and the IgG monoclonal antibody is of a mouse source or a rabbit source. According to the present invention, the IgG antibody is modified to obtain the recombinant IgM antibody, and the IgG antibody and the recombinant IgM antibody are not only suitable for the chemiluminescence method, but also show the expected sensitivity amplification factor in other immunological detection methods, such that the low-abundance detection of the specifically bound mumps virus can be easily achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of in vitro immunodiagnostic detection, in particular to an IgG antibody of mumps virus, a recombinant IgM antibody and application thereof. BACKGROUND

[0002] Mumps virus (MuV) belongs to Paramyxoviridae and is a single-stranded RNA virus with high infectivity. The virus only infects humans and is transmitted through droplets or direct contact. After replication in respiratory epithelial cells, MuV invades the blood to form viremia and then spreads throughout the body, mainly affecting the parotid gland and can also involve the testes, ovaries, pancreas and central nervous system.

[0003] Mumps is an acute respiratory infectious disease caused by MuV, commonly seen in children and adolescents. Typical symptoms include fever, headache, muscle soreness and parotid gland enlargement, usually affecting one side of the parotid gland first and then the other side. Some patients may have complications such as orchitis, oophoritis, meningitis or pancreatitis. Mumps is a self-limiting disease, there is no specific treatment for infection, and the main focus is prevention, with a good prognosis. Therefore, early and rapid detection of MuV not only allows for timely and effective preventive measures and treatment guidance, but also monitors the spread of the disease, and has become an important task in clinical diagnosis.

[0004] With the progress of molecular biology diagnostic techniques, the detection methods for MuV have undergone stages of pathogen isolation and culture detection, RT-PCR and immunological detection. Pathogen isolation and culture detection requires isolation of the virus from patient saliva, blood, cerebrospinal fluid or urine, which is complex and time-consuming, and there is some subjectivity in the determination of results; laboratory diagnosis can also detect RNA by RT-PCR, which has great advantages in specificity and price, but the amplification product needs to be judged by agarose gel electrophoresis, which has low sensitivity and is time-consuming; and immunological detection mainly detects specific antibodies (IgM, IgG, etc.) in patient serum to determine the infection status, although it has the advantages of simple operation, convenience and rapidness, it still has the disadvantage of insufficient sensitivity, which can cause false positive and false negative results.

[0005] In vitro diagnostic reagents are one of the basic tools for detecting whether a patient is ill and the severity of the disease, and the accuracy of the results directly affects the doctor's diagnosis and the patient's physical health and life safety. With the progress of modern medical technology, more accurate requirements are placed on medical test results, and the quality requirements for in vitro diagnostic products are also increasingly high.

[0006] In recent years, chemiluminescence detection technology has become a popular immunological technique due to its high sensitivity and good detection limit. Immunomagnetic beads, also known as magnetic beads, have good superparamagnetism, and the chemical groups on their surface can be coupled with protein molecules through covalent bonds. In addition, magnetic beads have a large specific surface area and can bind more proteins, improving the range of biological detection. Therefore, they are often combined with chemiluminescence immunoassay technology and applied in the field of biological detection.

[0007] The performance of immunoassay also depends on the epitope and affinity of the antibody selected to capture the antigen contained in the sample. The utility of IgG antibodies has been demonstrated in early pathogen detection, for example, pathogen detection is mostly IgG antibodies. IgG antibodies are typical "Y" type antibody structures, containing 2 antigen binding sites, while IgM antibodies usually exist in soluble pentamer structures containing 10 antigen binding sites and J chains, or hexamer structures containing 12 antigen binding sites without J chains. Therefore, IgM has the flexibility to bind multiple antigens and exhibits higher antigen affinity than IgG. Therefore, the inventors believe that IgMization of IgG antibodies may play a more important role in pathogen detection. Although IgM antibodies have unique characteristics, their role in clinical diagnosis and prevention has not been fully studied.

[0008] Streptavidin (SA) is a secreted protein extracted from Streptomyces culture, with a molecular weight of 65 kDa and an isoelectric point of 6.0. SA is a tetrameric protein, and one molecule of SA can specifically bind four molecules of biotin. Biotin, also known as vitamin H, has two ring structures in its molecule, with the I ring being an imidazolone ring, which is the main site for binding to SA; the II ring is a thiazole ring with a pentanoic acid side chain, the terminal carboxyl group of which is often chemically modified or modified to increase the hydrophilicity of biotin or reduce the steric effect, becoming long-chain hydrazine biotin or N-hydroxy succinimidyl ester biotin, which is used to label the thiol and amino groups of antibodies to increase the sensitivity and specificity of the biotin-streptavidin system.

[0009] The interaction between biotin-streptavidin is considered one of the most specific and stable non-covalent interactions, with a dissociation constant (KD) about 10 3 ~10 6 times that of antigen-antibody interaction. Its high affinity is mainly used to isolate and amplify the signal, improving the detection capability of low concentration analytes. At the same time, it also has high specificity, high stability and high adaptability.

[0010] Chinese patent document CN108531459A discloses an anti-human IgM monoclonal antibody, its hybridoma cell line, and its applications. The invention relates to a hybridoma cell line and the monoclonal antibody secreted by it. The antibody specifically binds to human IgM and can be used for in vitro detection of human IgM, particularly suitable for the early diagnosis of mumps virus infection. This prior art also relates to kits comprising the said hybridoma cell line or monoclonal antibody.

[0011] Chinese patent document CN 112538111 A discloses a single-chain antibody against SARS-CoV-2, its quality control, and a preparation method. The antibody comprises a light chain variable region and a heavy chain variable region sequentially from the N-terminus to the C-terminus. The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2. Chinese patent document CN117821510 A discloses a method for producing IgM antibodies and its application. When using these prior art methods, IgM pentamers can be prepared correctly, efficiently, and at higher expression levels and ratios. However, in both of these prior art solutions, when converting IgG antibodies into IgM antibodies, the heavy chain constant region of the IgM antibody typically does not involve the CH1 portion of the heavy chain constant region, or it completely uses the CH1-CH4 portion of the heavy chain constant region of the IgM antibody, resulting in an unstable structure of the obtained recombinant IgM antibody.

[0012] Chinese patent document CN 117990819 A discloses a liquid chromatography-tandem mass spectrometry (LC-MS / MS) method for detecting β-amyloid protein in plasma. Based on a triple quadrupole LC-MS / MS platform, the method quantifies β-amyloid protein in plasma samples through steps including calibration curve establishment, sample collection, antibody conjugation, enrichment, and sample loading. This prior art utilizes immunomagnetic beads to detect β-amyloid protein in plasma, primarily for the early screening and diagnosis of neurodegenerative diseases. This patent, however, mainly utilizes immunomagnetic beads to detect mumps virus.

[0013] Chinese patent document CN 119306822 A discloses an IgG antibody, an IgM antibody, and a method thereof, particularly an IgG antibody that specifically binds to Coxsackievirus. The amino acid sequences of the complementarity-determining regions (CDRH1-CDRH3) of the heavy chain variable region of the IgG antibody are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; the amino acid sequences of the complementarity-determining regions (CDRL1-CDRL3) of the light chain variable region are shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively. This prior art provides an IgG antibody that not only has good specificity, capable of specifically recognizing Coxsackievirus, but also achieves a high level of sensitivity and stability. The main focus of this prior art is the specific recognition of Coxsackievirus.

[0014] In the process of implementing this patented technical solution, it was found that among the existing methods for detecting mumps, although the immunological detection method based on the specific binding of antigen and antibody has the advantages of being simple to operate, convenient and fast, it still has disadvantages such as insufficient sensitivity and easy to cause false positive and false negative results.

[0015] Therefore, in order to improve the detection sensitivity of mumps virus, it is urgent to develop an antibody and detection reagent, as well as a method that can improve detection sensitivity, so as to expand the application scope of antigen-antibody detection. Summary of the Invention

[0016] This invention provides a gene for an IgG antibody against mumps virus, a modified recombinant IgM antibody, a detection reagent prepared from the recombinant IgM antibody, and a method and application for improving the detection sensitivity of mumps virus. The method has good specificity and can improve detection sensitivity.

[0017] To address the aforementioned technical issues, the present invention provides the following technical solutions.

[0018] In a first aspect, the present invention provides an IgG antibody that specifically binds to mumps virus, the IgG antibody comprising: a heavy chain constant region, a light chain constant region, a heavy chain variable region and a light chain variable region of the IgG monoclonal antibody that specifically binds to mumps virus, wherein the IgG monoclonal antibody is murine, and the amino acid sequences of the complementarity-determining regions CDRH1-CDRH3 of its heavy chain variable region are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; the amino acid sequences of the complementarity-determining regions CDRL1-CDRL3 of the light chain variable region are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0019] Secondly, the present invention provides an IgG antibody that specifically binds to mumps virus, the IgG antibody comprising: a heavy chain constant region, a light chain constant region, a heavy chain variable region and a light chain variable region of the IgG monoclonal antibody that specifically binds to mumps virus, wherein the IgG monoclonal antibody is murine, and the amino acid sequences of the complementarity-determining regions CDRH1-CDRH3 of its heavy chain variable region are shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively; the amino acid sequences of the complementarity-determining regions CDRL1-CDRL3 of the light chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

[0020] Thirdly, the present invention provides a gene for a specific IgG antibody that binds to mumps virus, wherein the monoclonal antibody that specifically binds to mumps virus is a murine IgG monoclonal antibody 10G4, and the amino acid sequences of the complementarity-determining regions CDRH1-CDRH3 of its heavy chain variable region are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; the amino acid sequences of the complementarity-determining regions CDRL1-CDRL3 of the light chain variable region are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0021] Fourthly, the present invention provides a gene for a specific IgG antibody that binds to mumps virus, wherein the monoclonal antibody that specifically binds to mumps virus is a murine IgG monoclonal antibody 10G6, and the amino acid sequences of the complementarity-determining regions CDRH1-CDRH3 of its heavy chain variable region are shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively; and the amino acid sequences of the complementarity-determining regions CDRL1-CDRL3 of the light chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

[0022] In order to improve the detection sensitivity of mumps virus by adopting the above technical solution, the present invention provides an IgG antibody that specifically binds to mumps virus. It not only has good specificity and can specifically recognize mumps virus, but also has high sensitivity and stability.

[0023] Preferably, the IgG antibody is coupled to magnetic beads.

[0024] Preferably, the IgG antibody is labeled with acridine ester.

[0025] Preferably, the IgG antibody is conjugated with biotin.

[0026] Fifthly, the present invention provides a recombinant IgM antibody that specifically binds to the mumps virus IgG antibody, wherein the recombinant IgM antibody is modified from the mumps virus IgG antibody of the first aspect of the present invention, and includes the heavy chain variable region, light chain variable region, light chain constant region and heavy chain constant region CH1 of the IgG antibody that specifically binds to the mumps virus, and the heavy chain constant regions CH2, CH3 and CH4 of the IgM antibody; The amino acid sequences of the complementarity-determining regions CDRH1-CDRH3 of the heavy chain variable region are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; The amino acid sequences of the complementarity-determining regions CDRL1-CDRL3 of the light chain variable region are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; The amino acid sequence of CH1, the constant region of the heavy chain of the IgG antibody, is shown in SEQ ID NO: 13; The amino acid sequence of CH2, the constant region of the heavy chain of the IgM antibody, is shown in SEQ ID NO: 14; The amino acid sequence of CH3, the constant region of the heavy chain of the IgM antibody, is shown in SEQ ID NO: 15; The amino acid sequence of CH4, the constant region of the heavy chain of the IgM antibody, is shown in SEQ ID NO: 16; The amino acid sequence of the constant region of the light chain is shown in SEQ ID NO: 17.

[0027] In a sixth aspect, the present invention provides a recombinant IgM antibody that specifically binds to the mumps virus IgG antibody, wherein the recombinant IgM antibody is modified from the mumps virus IgG antibody of the second aspect of the present invention, and comprises the heavy chain variable region, light chain variable region, light chain constant region and heavy chain constant region CH1 of the IgG antibody that specifically binds to the mumps virus, and the heavy chain constant regions CH2, CH3 and CH4 of the IgM antibody; The amino acid sequences of the complementarity-determining regions CDRH1-CDRH3 of the heavy chain variable region are shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively. The amino acid sequences of the complementarity-determining regions CDRL1-CDRL3 of the light chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; The amino acid sequence of CH1, the constant region of the heavy chain of the IgG antibody, is shown in SEQ ID NO: 13; The amino acid sequence of CH2, the constant region of the heavy chain of the IgM antibody, is shown in SEQ ID NO: 14; The amino acid sequence of CH3, the constant region of the heavy chain of the IgM antibody, is shown in SEQ ID NO: 15; The amino acid sequence of CH4, the constant region of the heavy chain of the IgM antibody, is shown in SEQ ID NO: 16; The amino acid sequence of the constant region of the light chain is shown in SEQ ID NO: 17.

[0028] In some embodiments of the fifth and sixth aspects described above, the heavy chain constant region includes a first constant region, a second constant region, a third constant region, and a fourth constant region, wherein the first constant region CH1 is derived from a mouse IgG antibody.

[0029] This invention involves IgM conversion of IgG antibodies that specifically bind to the mumps virus, and then using recombinant IgM antibodies coupled with magnetic beads to capture the mumps virus. Furthermore, the recombinant IgM antibodies are coupled with biotin-streptavidin to achieve cascade amplification of detection, further increasing the sensitivity of mumps virus detection while ensuring stability and specificity. Preferably, this invention employs a double-antibody sandwich method to detect the sequences of the variable regions of the heavy and light chains of the IgG antibody.

[0030] Preferably, the second constant region CH2, the third constant region CH3, and the fourth constant region CH4 are derived from mouse IgM antibodies.

[0031] Preferably, the recombinant IgM antibody is modified from an IgG antibody that specifically binds to a biomarker.

[0032] Preferably, the heavy chain variable region and / or light chain variable region of the recombinant IgM antibody are derived from the IgG antibody that specifically binds the biomarker.

[0033] Preferably, the recombinant IgM antibody is coupled to magnetic beads.

[0034] Preferably, the coating surface of the magnetic beads is a carboxyl surface, which can be replaced by the original surface, modified surface (amino, hydroxyl and sulfate), preactivated surface (toluenesulfonyl, epoxy resin and chloromethyl group) and bioactivated surface (protein A, protein G, streptomycin, biotin).

[0035] Preferably, the recombinant IgM antibody is labeled with acridine ester.

[0036] Preferably, the recombinant IgM antibody is conjugated with biotin.

[0037] Preferably, the method for modifying recombinant IgM antibodies with IgG antibodies according to the fifth and sixth aspects above includes the following steps: S1: Animal immunization, specifically: Animal mouse immunization specifically includes a method for rapidly and efficiently activating the mouse immune system to generate an immune response: (a) the mice used for immunization are SJL mice; (b) On day 0, 25 μg of mumps antigen protein was mixed with water-soluble adjuvant at a 1:1 ratio and injected subcutaneously. (c) On day 14, 25 μg of mumps antigen protein was mixed with water-soluble adjuvant at a 1:1 ratio and injected subcutaneously; a second dose of immunization was administered. (d) On day 28, the mumps antigen protein was mixed with water-soluble adjuvant at a ratio of 1:1 and injected subcutaneously for three doses of immunization to induce the production of IgG binding antibodies. (e) Blood was collected on day 10 after the third dose of immunization. Mouse serum was serially diluted and the dilution endpoint was determined by indirect ELISA. The highest dilution that was considered positive was the serum binding antibody titer. S2: Cell screening, specifically: fusing animal B lymphocytes with myeloma cells to obtain hybridoma cells; performing positive screening on hybridoma cells, and collecting mouse or rabbit IgG monoclonal antibodies from the cultures of positive hybridoma cells; Specifically, it includes a hybridoma cell screening method: (a) Fuse spleen cells of immunized mice with myeloma cells. During cell fusion, pay attention to the state of the cells and select appropriate fusion conditions to ensure fusion efficiency and cell survival rate. (b) Hybridoma cell lines were obtained by Biacore screening; (c) Cells are preserved and expanded using liquid nitrogen preservation and periodic passage amplification to ensure long-term stability of the cell line and high efficiency of IgG antibody secretion. S3: IgG antibody sequencing, specifically: sequencing the variable region of the mouse or rabbit IgG monoclonal antibody obtained in step S2 to obtain the amino acid sequences of the heavy chain variable region and the light chain variable region of the mouse or rabbit IgG monoclonal antibody; S4: IgM modification of IgG antibodies, that is, the specific method for preparing recombinant IgM antibodies by modifying IgG antibodies; S41: Construction of expression vectors for IgM antibody heavy and light chain sequences, specifically as follows: The amino acid sequence of the variable region of the heavy chain of the IgG antibody that specifically binds to the mumps virus and the amino acid sequence of the constant region of the heavy chain of the IgG antibody CH1 were recombined with the amino acid sequences of the constant region of the heavy chain of the IgM antibody CH2, CH3, and CH4. After codon optimization, the gene was constructed into the expression vector pcDNA3.4. S42: Cell transfection and expression, specifically: Plasmids were extracted for cell transfection; the constructed heavy chain expression vector and light chain expression vector were transfected into the mammalian cell line CHO cell line expression system for expression. S43: Select cell lines that efficiently express recombinant IgM antibodies, specifically: S5: Purification of IgM antibody from IgG antibody modification and recombination, specifically: IgM antibody purification by passing cell supernatant through a Protein G chromatography column; In a seventh aspect, the present invention provides a detection reagent that specifically binds to mumps virus, comprising a first recombinant IgM antibody and a second recombinant IgM antibody, wherein the first recombinant IgM antibody and the second recombinant IgM antibody are selected from the recombinant IgM antibodies described in the fifth and sixth aspects of the present invention, and the first recombinant IgM antibody and the second recombinant IgM antibody are different.

[0038] Preferably, the mass ratio of the first antibody to the second antibody is 1:2.

[0039] Preferably, the detection reagent further includes a magnetic bead-streptavidin conjugate.

[0040] Preferably, the first recombinant IgM antibody is conjugated with biotin or with magnetic beads, the second recombinant IgM antibody is labeled with acridine ester, the mass ratio of the magnetic bead-streptavidin conjugate to the biotin-conjugated first recombinant IgM antibody is 1:2, and the feed ratio of the second recombinant IgM antibody to the acridine ester includes 1:10-30.

[0041] Preferably, the first recombinant IgM antibody and the second recombinant IgM antibody are modified from IgG antibodies that specifically bind to biomarkers.

[0042] Preferably, the first recombinant IgM antibody and the second recombinant IgM antibody include a heavy chain constant region, a light chain constant region, a heavy chain variable region, and a light chain variable region.

[0043] Preferably, the heavy chain constant region includes a first constant region, a second constant region, a third constant region, and a fourth constant region, wherein the first constant region CH1 is derived from a mouse IgG antibody.

[0044] Preferably, the second constant region CH2, the third constant region CH3, and the fourth constant region CH4 are derived from mouse IgM antibodies.

[0045] Preferably, the detection reagent comprises a first recombinant IgM antibody conjugated with magnetic beads and a second recombinant IgM antibody labeled with acridine ester.

[0046] Preferably, the preparation method of the detection reagent includes: (a) adding 50 μL of carboxyl magnetic beads to 300 μL of 0.01 M PBS, vortexing for 5 min, magnetically removing the supernatant, and repeating twice; (b) adding 150 μL of pH 6.0 MES to dissolve, vortexing for 30 s to mix; (c) adding 20 mg / mL EDC and 20 mg / mL sulfo-NHS, vortexing again to mix, and activating at 37 °C for 30 min; (d) adding 300 μL of 0.01 M PBS, vortexing for 30 s, magnetically removing the supernatant, and repeating three times; (e) adding 300 μL of... (f) Add 0.5 mg / mL of the first recombinant IgM antibody, shake and mix at 37°C for 2 h, and remove the supernatant by magnetic aspiration; (g) Add 5 mL of blocking buffer (1% BSA) to resuspend, shake at 37°C for 10 min, and remove the supernatant by magnetic aspiration. Repeat twice; (c) Add 5 mL of blocking buffer (1% BSA) to resuspend, and obtain the detection reagent. Store at 4°C for later use.

[0047] Eighthly, the present invention provides the use of an antibody described in any one of the first to sixth aspects of the present invention in the preparation of an in vitro immunodiagnostic reagent.

[0048] Preferably, the in vitro immunodiagnostic reagent is based on colloidal gold / latex immunochromatography, chemiluminescence immunoassay, enzyme-linked immunosorbent assay (ELISA), or fluorescence immunochromatography.

[0049] In a ninth aspect, the present invention provides a detection kit for specific binding to mumps virus, comprising the detection reagent of the seventh aspect of the present invention.

[0050] In a tenth aspect, the present invention provides the application of the detection reagent described in the seventh aspect of the present invention in the preparation of in vitro immunodiagnostic reagents.

[0051] Preferably, the in vitro immunodiagnostic reagent is based on colloidal gold / latex immunochromatography, chemiluminescence immunoassay, enzyme-linked immunosorbent assay (ELISA), or fluorescence immunochromatography.

[0052] Preferably, the specific steps of the colloidal gold immunochromatography method are as follows: (a) Specific antibodies are immobilized on the membrane in the form of strips (T lines), and colloidal gold labeling reagents are adsorbed onto the conjugate pad (gold pad); (b) The antigen to be tested is added to the sample pad at one end of the test strip. The sample moves forward through capillary action, dissolves the colloidal gold-labeled reagent on the conjugate pad, reacts with it, and then moves to the fixed antigen or antibody area. The conjugate of the analyte and the gold-labeled reagent binds specifically to it and is trapped and aggregated on the test strip. (c) The color development results are observed by the naked eye.

[0053] Preferably, the specific steps of the latex immunochromatography method are as follows: (a) Antibody labeled with latex microspheres; (b) Coating antibody onto an NC membrane; (c) Latex-labeled antibodies adsorbed onto the conjugate pad; (d) Chromatographic reaction; (e) The color development results are observed by the naked eye.

[0054] The specific steps of fluorescence immunochromatography are as follows: (a) Antibody labeled with fluorescent microspheres; (b) Coating antibody onto an NC membrane; (c) Fluorescently labeled antibodies adsorbed onto the binding pad; (d) Chromatographic reaction; (e) Fluorescence signal reading: Observe the fluorescence signal using an ultraviolet lamp.

[0055] Eleventhly, the present invention provides a method for improving the sensitivity of mumps virus detection, comprising the following steps: The heavy chain expression vector is obtained by recombining the sequence of the variable region of the heavy chain of a mouse or rabbit IgG monoclonal antibody, the amino acid sequence fragment CH1 of the constant region of the heavy chain of a mouse or rabbit IgG antibody, and the amino acid sequence fragments CH2-CH4 of the constant region of the heavy chain of a mouse or rabbit recombinant IgM antibody. The heavy chain expression vector and the light chain expression vector were expressed in cells to obtain a recombinant IgM antibody; the IgG antibody is the IgG antibody that specifically binds to mumps virus as described in the first aspect and / or the second aspect of the present invention. The recombinant IgM antibody was used to prepare a detection reagent to detect the concentration of mumps virus in the sample to be tested.

[0056] Preferably, the method for improving the sensitivity of mumps virus detection includes the following steps: Animal immune response to S1'IgG antibodies: Using antigen and adjuvant, mice or rabbits were immunized by CpG immunization or LPS immunization to induce the production of IgG antibodies in the mice or rabbits. The animal mouse immunization described in step S1' specifically includes a method for rapidly and efficiently activating the mouse immune system to generate an immune response: (a) the mice used for immunization are SJL mice; (b) On day 0, 25 μg of mumps antigen protein was mixed with water-soluble adjuvant at a 1:1 ratio and injected subcutaneously. (c) On day 14, 25 μg of mumps antigen protein was mixed with water-soluble adjuvant at a 1:1 ratio and injected subcutaneously; a second dose of immunization was administered. (d) On day 28, the mumps antigen protein was mixed with water-soluble adjuvant at a ratio of 1:1 and injected subcutaneously for three doses of immunization to induce the production of IgG binding antibodies. (e) Blood was collected on day 10 after the third dose of immunization. Mouse serum was serially diluted and the dilution endpoint was determined by indirect ELISA. The highest dilution that was considered positive was the serum binding antibody titer. Alternatively, the LPS immunization method can be used, with the following specific steps: (a) First immunization: Dissolve 50 μg of antigen in 150 μL of PBS, then add 150 μL of Freund's complete adjuvant and stir to emulsify; (b) Second immunization: Dissolve 25 μg of antigen in 150 μL of PBS, then add 150 μL of Freund's incomplete adjuvant and stir to emulsify; (c) Third immunization: Dissolve 25 μg of antigen in 150 μL of PBS, then add 150 μL of Freund's incomplete adjuvant and stir to emulsify; (d) Boost immunization: 14 days after the third immunization, boost immunization was carried out in conjunction with lipopolysaccharide stimulation: 25 μg of antigen was dissolved in 50 μL of PBS, mixed well, and then mixed with 150 μL of 0.5 mg / L LPS dilution solution and injected into the peritoneal cavity of mice. The optimal dose was 0.1-0.15 mg / mouse. S2' cell screening: Animal B lymphocytes are fused with myeloma cells to obtain hybridoma cells; positive screening of hybridoma cells is performed, and mouse or rabbit IgG monoclonal antibodies are collected from the cultures of positive hybridoma cells. Alternatively, a single B cell screening method can be used, with the following steps: first, identify and isolate single B cells; then, amplify and clone antibody genes; finally, express, screen, and identify antigen-specific antibodies, i.e., mouse or rabbit IgG monoclonal antibodies. The cell screening described in step S2' specifically includes a hybridoma cell screening method: (a) Fuse spleen cells of immunized mice with myeloma cells. During cell fusion, pay attention to the state of the cells and select appropriate fusion conditions to ensure fusion efficiency and cell survival rate. (b) Hybridoma cell lines were obtained by Biacore screening; (c) Cells are preserved and expanded using liquid nitrogen preservation and periodic passage amplification to ensure long-term stability of the cell line and high efficiency of IgG antibody secretion. S3'IgG antibody sequencing: Sequencing the mouse or rabbit IgG monoclonal antibody obtained in step S2 to obtain the amino acid sequences of the heavy chain variable region and the light chain variable region of the mouse or rabbit IgG monoclonal antibody; S4' IgG antibody recombinant IgM antibody: The sequence of the variable region of the heavy chain of the mouse or rabbit IgG monoclonal antibody, the amino acid sequence fragment CH1 of the constant region of the heavy chain of the mouse or rabbit IgG antibody, and the amino acid sequence fragments CH2-CH4 of the constant region of the heavy chain of the mouse or rabbit recombinant IgM antibody are recombined to obtain a heavy chain expression vector; the heavy chain expression vector and the light chain expression vector are expressed in cells, and the culture supernatant is purified to obtain recombinant IgM antibody; Purification specifically involves passing cell supernatant through a Protein G chromatography column to purify IgM antibodies; S5' Detection of mumps virus with recombinant IgM antibody: The recombinant IgM antibody is prepared into the detection reagent described in the seventh aspect of the present invention to detect the concentration of mumps virus in the sample to be tested.

[0057] Preferably, the specific steps in step S2' are as follows: mixing the first IgM antibody with biotin, wherein the feeding ratio (mass ratio) of the first IgM antibody to the biotin includes 1:2-10.

[0058] Further, the specific steps in step S3' are as follows: mixing the second IgM antibody with acridine ester, wherein the feed ratio of the second IgM antibody to the acridine ester is 1:10-30.

[0059] Preferably, step S5' specifically comprises: (a) Select the first recombinant IgM antibody and the second recombinant IgM antibody; (b) The magnetic bead-streptavidin conjugate and the biotin-modified first recombinant IgM antibody were mixed to obtain a premix; (c) The premix is ​​mixed with the sample to be tested to obtain magnetic bead-first recombinant IgM antibody-antigen conjugate; (d) The magnetic bead-first recombinant IgM antibody-antigen conjugate is mixed with the acridinium ester-labeled second recombinant IgM antibody to obtain a first recombinant IgM antibody-antigen-second recombinant IgM antibody sandwich complex; (e) The concentration of mumps virus in the test sample is detected using a chemiluminescence method, i.e., the capture of mumps virus in test sample three.

[0060] Preferably, the recombinant IgM antibody is coupled to magnetic beads; the coating surface of the magnetic beads is a carboxyl surface or a raw surface, a modified surface (amino, hydroxyl and sulfate), a preactivated surface (toluenesulfonyl, epoxy resin and chloromethyl group) and a bioactivated surface (protein A, protein G, streptomycin, biotin).

[0061] Preferably, the recombinant IgM antibody is labeled with acridine ester.

[0062] Preferably, the recombinant IgM antibody is conjugated with biotin.

[0063] Preferably, the recombinant IgM antibody includes a first recombinant IgM antibody and a second recombinant IgM antibody.

[0064] Preferably, the first recombinant IgM antibody is coupled to magnetic beads.

[0065] Preferably, the second recombinant IgM antibody is labeled with acridine ester.

[0066] Preferably, the first recombinant IgM antibody is conjugated with avidin.

[0067] Preferably, the purification is a one-step purification method using protein G.

[0068] Preferably, the purification is performed using a hydroxyapatite (HAP) chromatography column for IgM antibody purification of cell supernatant.

[0069] Preferably, the purification process involves passing cell supernatant through an anion exchange chromatography column (QA) to purify IgM antibodies.

[0070] Preferably, the adjuvant in step S1' can be classified into water-soluble adjuvants and oil-based adjuvants according to their water solubility: oil-based adjuvants, such as commonly used Freund's adjuvant and vegetable oils, need to be emulsified to form a water-in-oil state before immunization; water-soluble adjuvants do not require a complicated emulsification process and can be directly mixed with the antigen and injected into the animal. Currently, Freund's adjuvant is mostly used in methods to activate the immune system of mice to produce an immune response. During immunization, Freund's adjuvant needs to be mixed with an equal volume of antigen aqueous solution and emulsified, while water-soluble adjuvants do not require a complex emulsification process and can be used to immunize animals simply by mixing, making the operation simpler. Although existing patents have used manganese adjuvant (manganese chloride) instead of Freund's adjuvant to immunize mice, achieving good immunization effects with less antigen load and lower injection dosage, it still has some shortcomings compared to water-based adjuvants. Aqueous adjuvants are complexes composed of fungal polysaccharides, saponins, and nucleotides. They possess strong immune cell stimulating activity, stimulating the function of various immune cells and promoting the production of multiple cytokines, making them highly effective immune-enhancing adjuvants. Using aqueous adjuvants not only reduces inflammatory responses and toxicity but also offers numerous advantages, including rapid antibody production, low antigen dosage, high antibody titers, preservation of the antigen's native conformation, high antibody affinity, and ease of use. While existing technology CN 107456575A uses manganese adjuvants for CpG immunization, this invention uses aqueous adjuvants, achieving superior immunization effects with lower antigen load and injection dosage, and highly efficiently activating immune cells.

[0071] Preferably, the adjuvant may also be manganese ions, such as manganese chloride, manganese bromide, manganese iodide, manganese sulfate, manganese nitrate, manganese perchlorate, manganese acetate, manganese carbonate, manganese borate, manganese phosphate, manganese hydrobromide, manganese tartrate, manganese fumarate, manganese maleate, manganese lactate, manganese benzenesulfonate, manganese pantothenate, manganese ascorbate, and any combination thereof.

[0072] While existing methods for detecting mumps, such as immunological detection methods based on antigen-antibody specific binding, offer advantages like simplicity, convenience, and speed, they still suffer from drawbacks such as insufficient sensitivity and susceptibility to false positives and false negatives. Furthermore, current techniques for amplifying sensitivity commonly employ IgG antibodies coupled with immunomagnetic beads or biotin-streptavidin. IgG antibodies contain two antigen-binding sites, while recombinant IgM antibodies typically contain 10 or 12. Therefore, recombinant IgM antibodies offer the flexibility required to bind multiple antigens and exhibit higher antigen affinity than IgG antibodies. This invention suggests that the recombinant IgMization of IgG antibodies may play a more significant role in mumps virus detection. This invention IgMizes IgG antibodies, using recombinant modified IgM antibodies coupled with magnetic beads and biotin-streptavidin to achieve cascade amplification detection, further amplifying the sensitivity of mumps virus detection while maintaining stability and specificity.

[0073] The IgG antibody that specifically binds to mumps virus prepared by this invention can be further amplified by biotin amplification, with a detection limit of 1:25600; while the recombinant IgM antibody modified by this invention and amplified by biotin can increase the sensitivity to 16-20 times that of the IgG antibody, with a detection limit of 1:102400.

[0074] Compared with the prior art, the beneficial effects of the present invention include at least the following aspects: (1) The present invention provides that the sensitivity of the IgG antibody that specifically binds to the mumps virus is high enough to meet the detection requirements, and the high detection sensitivity helps to achieve low abundance detection of the mumps virus that specifically binds to it.

[0075] (2) In in vitro diagnostics, affinity is an important indicator for measuring the strength of antibody-antigen binding. Ka (binding constant) and Kd (dissociation constant) are kinetic parameters describing antibody-antigen interactions. The larger the Ka value, the faster the binding rate; the smaller the Kd value, the more stable the antibody-antigen binding. In the process of preparing IgG antibodies that specifically bind to mumps virus, this invention screened IgG monoclonal antibodies 10G4 and 10G6 by combining the KD value, which reflects the binding strength between antibody and antigen, and the Ka value, which reflects the antigen-antibody binding rate. This results in IgG antibodies that not only have good specificity and can specifically recognize biomarkers, but also have high sensitivity and stability.

[0076] (3) The IgG antibody and IgM antibody provided by the present invention can achieve a purity of more than 95% by one-step purification using protein G (in actual operation, this purity often requires more than two purification steps to achieve), and can be directly used for subsequent immunological detection.

[0077] (4) In this invention, the IgG antibody that specifically binds to the mumps virus is converted to IgM, and the recombinant modified IgM antibody is coupled with magnetic beads to capture the mumps virus; furthermore, the recombinant modified IgM antibody is coupled with biotin-streptavidin to achieve cascade amplification detection, which can further amplify the sensitivity of mumps virus detection while ensuring stability and specificity.

[0078] (5) Recombinant IgM antibody modified based on antibody that specifically binds to mumps virus. At the same time, the present invention found that the heavy chain constant region CH1 of IgM antibody is actually crucial for the expression, purification, spatial structure and affinity of IgM antibody to antigen. The recombinant IgM antibody provided by the present invention not only retains the heavy chain constant region CH1, but also creatively replaces it with the heavy chain constant region CH1 of IgG antibody, so that the structure of the modified recombinant IgM antibody is stable and will not affect the specificity and affinity of the heavy chain variable region and the light chain variable region. The heavy chain variable region and the light chain variable region can be obtained by modifying IgG antibody that specifically binds biomarkers according to different antigens, and have a wide range of applications.

[0079] (6) Experimental verification shows that, compared with recombinant IgM antibodies prepared by existing methods (e.g., CN118772272A), the recombinant IgM antibody provided by the present invention can improve the sensitivity of detecting biomarkers by 4-5 times compared with IgG antibodies while ensuring the original stability, specificity and affinity of IgG antibodies. The recombinant IgM antibody conjugated with biotin can improve the sensitivity of detecting mumps virus specifically bound by IgG antibodies by 16-20 times compared with IgG antibodies. That is, it has high sensitivity and accuracy, low non-specific adsorption and detection limit, avoids false positive and false negative results, and is suitable for detecting low abundance biomarkers.

[0080] (7) The IgG antibody and recombinant IgM antibody provided by the present invention are not only applicable to chemiluminescence method, but also show the expected sensitivity amplification in other immunological detection methods (such as colloidal gold / latex immunochromatography, enzyme-linked immunosorbent assay, and fluorescence immunochromatography), which helps to realize the detection of low abundance of biomarkers. Attached Figure Description

[0081] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. The elements or parts in the drawings are not necessarily drawn to scale. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0082] Figure 1 A schematic diagram of the heavy chain expression vector for a recombinant IgM antibody that specifically binds to the mumps virus IgG antibody; Figure 2 A schematic diagram of the structure of a light chain expression vector for a recombinant IgM antibody that specifically binds to the mumps virus IgG antibody; Figure 3 The figure shows the results of thermally accelerated stability verification of recombinant IgM antibody against IgG antibody that specifically binds to mumps virus. Detailed Implementation

[0083] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0084] In this document, the terms "upper," "lower," "inner," "outer," "front," "rear," "one end," and "the other end," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the present invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the present invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0085] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0086] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0087] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0088] As used in this specification, the term "about" typically means + / -5% of the value, more typically + / -4%, more typically + / -3%, more typically + / -2%, even more typically + / -1%, even more typically + / -0.5%.

[0089] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered as having specifically disclosed all possible subranges and the individual numerical values ​​within those ranges. For example, a description of the range 1-6 should be considered as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and the individual numbers within those ranges, such as 1, 2, 3, 4, 5, and 6. This rule applies regardless of the breadth of the range.

[0090] Definition of noun: DMEM: Dulbecco's Modified Eagle Medium. Biacore detection: a biomolecular interaction analysis method based on surface plasmon resonance (SPR) technology; HAT: HAT Medium, a selective culture medium used for screening hybridoma cells; ELISA method: Enzyme-Linked Immunosorbent Assay; Antibody fragments refer to the antigen-binding region, light chain, and heavy chain variable regions of an antibody.

[0091] Example: The first specific IgG antibody or antibody fragment thereof that binds to mumps virus, wherein the IgG antibody comprises: a heavy chain constant region, a light chain constant region, a heavy chain variable region and a light chain variable region of the specific IgG monoclonal antibody binding to mumps virus, wherein the IgG monoclonal antibody is murine, and the amino acid sequences of the complementarity-determining regions CDRH1, CDRH2, and CDRH3 of its heavy chain variable region are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; the amino acid sequences of the complementarity-determining regions CDRL1, CDRL2, and CDRL3 of the light chain variable region are shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

[0092] The second type is a specific IgG antibody that binds to the mumps virus. The IgG antibody includes: a heavy chain constant region, a light chain constant region, a heavy chain variable region, and a light chain variable region of the specific IgG monoclonal antibody that binds to the mumps virus. The IgG monoclonal antibody is murine in origin. The amino acid sequences of the complementarity-determining regions CDRH1, CDRH2, and CDRH3 of the heavy chain variable region are shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively. The amino acid sequences of the complementarity-determining regions CDRL1, CDRL2, and CDRL3 of the light chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively.

[0093] In some embodiments, the IgG monoclonal antibody of the present invention may also be derived from rabbits.

[0094] The gene of the IgG antibody or antibody fragment thereof that specifically binds to the mumps virus, wherein the monoclonal antibody that specifically binds to the IgG antibody of the mumps virus is a murine IgG monoclonal antibody 10G4, the amino acid sequences of the complementarity-determining regions CDRH1-CDRH3 of its heavy chain variable region are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively; the amino acid sequences of the complementarity-determining regions CDRL1-CDRL3 of the light chain variable region are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 respectively.

[0095] The gene of the IgG antibody or antibody fragment thereof that specifically binds to the mumps virus, wherein the monoclonal antibody that specifically binds to the IgG antibody of the mumps virus is a murine IgG monoclonal antibody 10G6, the amino acid sequences of the complementarity-determining regions CDRH1, CDRH2, and CDRH3 of the heavy chain variable region are shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively; the amino acid sequences of the complementarity-determining regions CDRL1, CDRL2, and CDRL3 of the light chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively.

[0096] In some embodiments, the IgG antibody is coupled to magnetic beads.

[0097] In some embodiments, the IgG antibody is labeled with acridine ester.

[0098] In some embodiments, the IgG antibody is conjugated with biotin.

[0099] The recombinant IgM antibody or its antibody fragment specifically binding to the mumps virus IgG antibody is modified from the mouse or rabbit IgG antibody specifically binding to the mumps virus described in this invention, and includes the heavy chain variable region, light chain variable region, light chain constant region and heavy chain constant region CH1 of the IgG antibody specifically binding to the mumps virus, and the heavy chain constant regions CH2, CH3 and CH4 of the IgM antibody.

[0100] The recombinant IgM antibody or its antibody fragment specifically binding to the mumps virus IgG antibody is modified from the mumps virus IgG antibody of the present invention, and includes the heavy chain variable region, light chain variable region, light chain constant region and heavy chain constant region CH1 of the IgG antibody specifically binding to the mumps virus, and the heavy chain constant regions CH2, CH3 and CH4 of the IgM antibody. The amino acid sequences of the complementarity-determining regions CDRH1, CDRH2, and CDRH3 of the heavy chain variable region are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. The amino acid sequences of the complementarity-determining regions CDRL1, CDRL2, and CDRL3 of the light chain variable region are shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively. The amino acid sequence of CH1, the constant region of the heavy chain of the IgG antibody, is shown in SEQ ID NO: 13; The amino acid sequence of CH2, the constant region of the heavy chain of the IgM antibody, is shown in SEQ ID NO: 14; The amino acid sequence of CH3, the constant region of the heavy chain of the IgM antibody, is shown in SEQ ID NO: 15; The amino acid sequence of CH4, the constant region of the heavy chain of the IgM antibody, is shown in SEQ ID NO: 16; The amino acid sequence of the constant region of the light chain is shown in SEQ ID NO: 17.

[0101] The recombinant IgM antibody or its antibody fragment specifically binding to the mumps virus IgG antibody is modified from the mumps virus IgG antibody of the present invention, and includes the heavy chain variable region, light chain variable region, light chain constant region and heavy chain constant region CH1 of the IgG antibody specifically binding to the mumps virus, and the heavy chain constant regions CH2, CH3 and CH4 of the IgM antibody. The amino acid sequences of the complementarity-determining regions CDRH1, CDRH2, and CDRH3 of the heavy chain variable region are shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively. The amino acid sequences of the complementarity-determining regions CDRL1, CDRL2, and CDRL3 of the light chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively. The amino acid sequence of CH1, the constant region of the heavy chain of the IgG antibody, is shown in SEQ ID NO: 13; The amino acid sequence of CH2, the constant region of the heavy chain of the IgM antibody, is shown in SEQ ID NO: 14; The amino acid sequence of CH3, the constant region of the heavy chain of the IgM antibody, is shown in SEQ ID NO: 15; The amino acid sequence of CH4, the constant region of the heavy chain of the IgM antibody, is shown in SEQ ID NO: 16; The amino acid sequence of the constant region of the light chain is shown in SEQ ID NO: 17.

[0102] In some embodiments of the recombinant IgM antibody, the heavy chain constant region includes a first constant region, a second constant region, a third constant region, and a fourth constant region, wherein the first constant region CH1 is derived from a mouse IgG antibody.

[0103] In some embodiments, the second constant region CH2, the third constant region CH3, and the fourth constant region CH4 are derived from mouse IgM antibodies.

[0104] In some embodiments, the recombinant IgM antibody is modified from an IgG antibody that specifically binds to a biomarker.

[0105] In some embodiments, the heavy chain variable region and / or light chain variable region of the recombinant IgM antibody are derived from the IgG antibody that specifically binds the biomarker.

[0106] In some embodiments, the recombinant IgM antibody is coupled to magnetic beads.

[0107] In some embodiments, the coating surface of the magnetic beads is a carboxyl surface, which can be replaced by a raw surface, a modified surface (amino, hydroxyl and sulfate), a pre-activated surface (toluenesulfonyl, epoxy resin and chloromethyl group) and a bio-activated surface (protein A, protein G, streptomycin, biotin).

[0108] In some embodiments, the recombinant IgM antibody is labeled with acridine ester.

[0109] In some embodiments, the recombinant IgM antibody is conjugated with biotin.

[0110] In some embodiments, the method for modifying recombinant IgM antibodies with IgG antibodies described above includes the following steps: S1: Animal immunization, specifically: Animal mouse immunization specifically includes a method for rapidly and efficiently activating the mouse immune system to generate an immune response: (a) the mice used for immunization are SJL mice; (b) On day 0, 25 μg of mumps antigen protein was mixed with water-soluble adjuvant at a 1:1 ratio and injected subcutaneously. (c) On day 14, 25 μg of mumps antigen protein was mixed with water-soluble adjuvant at a 1:1 ratio and injected subcutaneously; a second dose of immunization was administered. (d) On day 28, the mumps antigen protein was mixed with water-soluble adjuvant at a ratio of 1:1 and injected subcutaneously for three doses of immunization to induce the production of IgG binding antibodies. (e) Blood was collected on day 10 after the third dose of immunization. Mouse serum was serially diluted and the dilution endpoint was determined by indirect ELISA. The highest dilution that was considered positive was the serum binding antibody titer. S2: Cell screening, specifically: fusing animal B lymphocytes with myeloma cells to obtain hybridoma cells; performing positive screening on hybridoma cells, and collecting mouse or rabbit IgG monoclonal antibodies from the cultures of positive hybridoma cells; Specifically, it includes a hybridoma cell screening method: (a) Fuse spleen cells of immunized mice with myeloma cells. During cell fusion, pay attention to the state of the cells and select appropriate fusion conditions to ensure fusion efficiency and cell survival rate. (b) Hybridoma cell lines were obtained by Biacore screening; (c) Cells are preserved and expanded using liquid nitrogen preservation and periodic passage amplification to ensure long-term stability of the cell line and high efficiency of IgG antibody secretion. S3: IgG antibody sequencing, specifically: sequencing the mouse or rabbit IgG monoclonal antibody obtained in step S2 to obtain the amino acid sequences of the heavy chain variable region and the light chain variable region of the mouse or rabbit IgG monoclonal antibody. S4: IgM modification of IgG antibodies, that is, the specific method for preparing recombinant IgM antibodies by modifying IgG antibodies; S41: Construction of expression vectors for IgM antibody heavy and light chain sequences, specifically as follows: The amino acid sequence of the variable region of the heavy chain of the IgG antibody that specifically binds to the mumps virus and the amino acid sequence of the constant region of the heavy chain of the IgG antibody CH1 were recombined with the amino acid sequences of the constant region of the heavy chain of the IgM antibody CH2, CH3, and CH4. After codon optimization, the gene was constructed into the expression vector pcDNA3.4. S42: Cell transfection and expression, specifically: Plasmids were extracted for cell transfection; the constructed heavy chain expression vector and light chain expression vector were transfected into the mammalian cell line CHO cell line expression system for expression. S43: Select cell lines that efficiently express recombinant IgM antibodies, specifically: S5: Purification of IgM antibody modified and recombined with IgG antibody; the purification process specifically involves passing cell supernatant through a Protein G chromatography column to purify IgM antibody. The specific mumps virus detection reagent includes a first recombinant IgM antibody and a second recombinant IgM antibody, wherein the first recombinant IgM antibody and the second recombinant IgM antibody are selected from the above-mentioned recombinant IgM antibodies, and the first recombinant IgM antibody and the second recombinant IgM antibody are different.

[0111] In some embodiments, the mass ratio of the first antibody to the second antibody is 1:2.

[0112] In some embodiments, the detection reagent further includes a magnetic bead-streptavidin conjugate.

[0113] In some embodiments, the first recombinant IgM antibody is conjugated with biotin or with magnetic beads, the second recombinant IgM antibody is labeled with acridinium ester, the mass ratio of the magnetic bead-streptavidin conjugate to the biotin-conjugated first recombinant IgM antibody is 1:2, and the feed ratio of the second recombinant IgM antibody to the acridinium ester includes 1:10-30.

[0114] In some embodiments, the first recombinant IgM antibody and the second recombinant IgM antibody are modified from IgG antibodies that specifically bind biomarkers.

[0115] In some embodiments, the first recombinant IgM antibody and the second recombinant IgM antibody include a heavy chain constant region, a light chain constant region, a heavy chain variable region, and a light chain variable region.

[0116] In some embodiments, the heavy chain constant region includes a first constant region, a second constant region, a third constant region, and a fourth constant region, wherein the first constant region CH1 is derived from a mouse IgG antibody.

[0117] In some embodiments, the second constant region CH2, the third constant region CH3, and the fourth constant region CH4 are derived from mouse IgM antibodies.

[0118] In some embodiments, the detection reagent comprises a first recombinant IgM antibody conjugated to magnetic beads and a second recombinant IgM antibody labeled with acridine ester.

[0119] In some embodiments, the preparation method of the detection reagent includes: (a) adding 50 μL of carboxyl magnetic beads to 300 μL of 0.01 M PBS, vortexing for 5 min, magnetically removing the supernatant, and repeating twice; (b) adding 150 μL of pH 6.0 MES to dissolve, vortexing for 30 s to mix; (c) adding 20 mg / mL EDC and 20 mg / mL sulfo-NHS, vortexing again to mix, and activating at 37 °C for 30 min; (d) adding 300 μL of 0.01 M PBS, vortexing for 30 s, magnetically removing the supernatant, and repeating three times; (e) adding 300 μL of... (f) Add 0.5 mg / mL of the first recombinant IgM antibody, shake and mix at 37°C for 2 h, and remove the supernatant by magnetic aspiration; (g) Add 5 mL of blocking buffer (1% BSA) to resuspend, shake at 37°C for 10 min, and remove the supernatant by magnetic aspiration. Repeat twice; (c) Add 5 mL of blocking buffer (1% BSA) to resuspend, and obtain the detection reagent. Store at 4°C for later use.

[0120] This detection kit specifically binds to the mumps virus and includes the detection reagent containing the recombinant IgM antibody of this invention.

[0121] This invention utilizes the IgG antibody and recombinant IgM antibody described in the present invention in the preparation of in vitro immunodiagnostic reagents.

[0122] This invention relates to the application of the detection reagent containing recombinant IgM antibody in the preparation of in vitro immunodiagnostic reagents.

[0123] In some embodiments, the in vitro immunodiagnostic reagent is based on colloidal gold / latex immunochromatography, chemiluminescence immunoassay, enzyme-linked immunosorbent assay (ELISA), or fluorescence immunochromatography.

[0124] In some embodiments, the specific steps of colloidal gold immunochromatography are as follows: (a) Specific antibodies are immobilized on the membrane in the form of strips (T lines), and colloidal gold labeling reagents are adsorbed onto the conjugate pad (gold pad); (b) The antigen to be tested is added to the sample pad at one end of the test strip. The sample moves forward through capillary action, dissolves the colloidal gold-labeled reagent on the conjugate pad, reacts with it, and then moves to the fixed antigen or antibody area. The conjugate of the analyte and the gold-labeled reagent binds specifically to it and is trapped and aggregated on the test strip. (c) The color development results are observed by the naked eye.

[0125] In some embodiments, the specific steps of latex immunochromatography are as follows: (a) Antibody labeled with latex microspheres; (b) Coating antibody onto an NC membrane; (c) Latex-labeled antibodies adsorbed onto the conjugate pad; (d) Chromatographic reaction; (e) The color development results are observed by the naked eye.

[0126] The specific steps of fluorescence immunochromatography are as follows: (a) Antibody labeled with fluorescent microspheres; (b) Coating antibody onto an NC membrane; (c) Fluorescently labeled antibodies adsorbed onto the binding pad; (d) Chromatographic reaction; (e) Fluorescence signal reading: Observe the fluorescence signal using an ultraviolet lamp.

[0127] The method for improving the sensitivity of mumps virus detection includes the following steps: The heavy chain expression vector is obtained by recombining the sequence of the variable region of the heavy chain of a mouse or rabbit IgG monoclonal antibody, the amino acid sequence fragment CH1 of the constant region of the heavy chain of a mouse or rabbit IgG antibody, and the amino acid sequence fragments CH2-CH4 of the constant region of the heavy chain of a mouse or rabbit recombinant IgM antibody. The heavy chain expression vector and the light chain expression vector were expressed in cells to obtain a recombinant IgM antibody; the IgG antibody is the IgG antibody that specifically binds to mumps virus as described in the first aspect and / or the second aspect of the present invention. The recombinant IgM antibody was used to prepare a detection reagent to detect the concentration of mumps virus in the sample to be tested.

[0128] In some specific embodiments, the method for improving the sensitivity of mumps virus detection includes the following steps: Animal immune response to S1'IgG antibodies: Using antigen and adjuvant, mice or rabbits were immunized by CpG immunization or LPS immunization to induce the production of IgG antibodies in the mice or rabbits. The animal mouse immunization described in step S1' specifically includes a method for rapidly and efficiently activating the mouse immune system to generate an immune response: (a) the mice used for immunization are SJL mice; (b) On day 0, 25 μg of mumps antigen protein was mixed with water-soluble adjuvant at a 1:1 ratio and injected subcutaneously. (c) On day 14, 25 μg of mumps antigen protein was mixed with water-soluble adjuvant at a 1:1 ratio and injected subcutaneously; a second dose of immunization was administered. (d) On day 28, the mumps antigen protein was mixed with water-soluble adjuvant at a ratio of 1:1 and injected subcutaneously for three doses of immunization to induce the production of IgG binding antibodies. (e) Blood was collected on day 10 after the third dose of immunization. Mouse serum was serially diluted and the dilution endpoint was determined by indirect ELISA. The highest dilution that was considered positive was the serum binding antibody titer. Alternatively, the LPS immunization method can be used, with the following specific steps: (a) First immunization: Dissolve 50 μg of antigen in 150 μL of PBS, then add 150 μL of Freund's complete adjuvant and stir to emulsify; (b) Second immunization: Dissolve 25 μg of antigen in 150 μL of PBS, then add 150 μL of Freund's incomplete adjuvant and stir to emulsify; (c) Third immunization: Dissolve 25 μg of antigen in 150 μL of PBS, then add 150 μL of Freund's incomplete adjuvant and stir to emulsify; (d) Boost immunization: 14 days after the third immunization, boost immunization was carried out in conjunction with lipopolysaccharide stimulation: 25 μg of antigen was dissolved in 50 μL of PBS, mixed well, and then mixed with 150 μL of 0.5 mg / L LPS dilution solution and injected into the peritoneal cavity of mice. The optimal dose was 0.1-0.15 mg / mouse. S2' cell screening: Animal B lymphocytes are fused with myeloma cells to obtain hybridoma cells; positive screening of hybridoma cells is performed, and mouse or rabbit IgG monoclonal antibodies are collected from the cultures of positive hybridoma cells. Alternatively, a single B cell screening method can be used, with the following steps: first, identify and isolate single B cells; then, amplify and clone antibody genes; finally, express, screen, and identify antigen-specific antibodies, i.e., mouse or rabbit IgG monoclonal antibodies. The cell screening described in step S2' specifically includes a hybridoma cell screening method: (a) Fuse spleen cells of immunized mice with myeloma cells. During cell fusion, pay attention to the state of the cells and select appropriate fusion conditions to ensure fusion efficiency and cell survival rate. (b) Hybridoma cell lines were obtained by Biacore screening; (c) Cells are preserved and expanded using liquid nitrogen preservation and periodic passage amplification to ensure long-term stability of the cell line and high efficiency of IgG antibody secretion. S3'IgG antibody sequencing: Sequencing the mouse or rabbit IgG monoclonal antibody obtained in step S2 to obtain the amino acid sequences of the heavy chain variable region and the light chain variable region of the mouse or rabbit IgG monoclonal antibody; S4' IgG antibody recombinant IgM antibody: The sequence of the variable region of the heavy chain of the mouse or rabbit IgG monoclonal antibody, the amino acid sequence fragment CH1 of the constant region of the heavy chain of the mouse or rabbit IgG antibody, and the amino acid sequence fragments CH2-CH4 of the constant region of the heavy chain of the mouse or rabbit recombinant IgM antibody are recombined to obtain a heavy chain expression vector; the heavy chain expression vector and the light chain expression vector are expressed in cells, and the culture supernatant is purified to obtain recombinant IgM antibody; Purification specifically involves passing cell supernatant through a Protein G chromatography column to purify IgM antibodies; S5' Detection of mumps virus with recombinant IgM antibody: The recombinant IgM antibody is prepared into the detection reagent described in the sixth aspect of the present invention to detect the concentration of mumps virus in the sample to be tested.

[0129] In some embodiments, step S2' specifically involves mixing the first IgM antibody with biotin, wherein the feeding ratio (mass ratio) of the first IgM antibody to the biotin includes 1:2-10.

[0130] Further, the specific steps in step S3' are as follows: mixing the second IgM antibody with acridine ester, wherein the feed ratio of the second IgM antibody to the acridine ester is 1:10-30.

[0131] In some embodiments, step S5' specifically comprises: (a) Select a first recombinant IgM antibody and a second recombinant IgM antibody; the mass ratio of the first antibody to the second antibody is 1:2; (b) The magnetic bead-streptavidin conjugate and the biotin-modified first recombinant IgM antibody were mixed to obtain a premix; (c) The premix is ​​mixed with the sample to be tested to obtain magnetic bead-first recombinant IgM antibody-antigen conjugate; (d) The magnetic bead-first recombinant IgM antibody-antigen conjugate is mixed with the acridinium ester-labeled second recombinant IgM antibody to obtain a first recombinant IgM antibody-antigen-second recombinant IgM antibody sandwich complex; (e) The concentration of mumps virus in the sample to be tested was detected using a chemiluminescence method.

[0132] In some embodiments, the recombinant IgM antibody is coupled to magnetic beads; the magnetic beads are coated with a carboxyl surface or a pristine surface, a modified surface (amino, hydroxyl, and sulfate), a preactivated surface (toluenesulfonyl, epoxy resin, and chloromethyl groups), and a bioactivated surface (protein A, protein G, streptomycin, biotin).

[0133] In some embodiments, the recombinant IgM antibody is labeled with acridine ester.

[0134] In some embodiments, the recombinant IgM antibody is conjugated with biotin.

[0135] In some embodiments, the recombinant IgM antibody includes a first recombinant IgM antibody and a second recombinant IgM antibody.

[0136] In some embodiments, the first recombinant IgM antibody is coupled to magnetic beads.

[0137] In some embodiments, the second recombinant IgM antibody is labeled with acridine ester.

[0138] In some embodiments, the first recombinant IgM antibody is conjugated with avidin.

[0139] In some embodiments, the purification is a one-step purification of protein G.

[0140] In some embodiments, the purification is performed using a hydroxyapatite (HAP) chromatography column for IgM antibody purification from cell supernatant.

[0141] In some embodiments, the purification involves passing cell supernatant through an anion exchange chromatography (QA) column to purify IgM antibodies.

[0142] In some embodiments, the adjuvant in step S1' can be classified into water-soluble adjuvants and oil-based adjuvants according to their water solubility: oil-based adjuvants, such as commonly used Freund's adjuvant and vegetable oils, need to be emulsified to form a water-in-oil state before immunization; water-soluble adjuvants do not require a complicated emulsification process and can be directly mixed with the antigen and injected into the animal. Currently, Freund's adjuvant is mostly used in methods to activate the mouse immune system to produce an immune response. During immunization, Freund's adjuvant needs to be mixed with an equal volume of antigen aqueous solution and emulsified, while water-soluble adjuvants do not require a complex emulsification process and can be used to immunize animals simply by mixing, making the operation simpler. Although existing patents have described using manganese adjuvant (manganese chloride) instead of Freund's adjuvant to immunize mice, achieving better immunization effects with less antigen load and lower injection dosage, it still has some shortcomings compared to water-based adjuvants. Aqueous adjuvants are complexes composed of fungal polysaccharides, saponins, and nucleotides. They possess strong immune cell stimulating activity, capable of stimulating the function of various immune cells and promoting the production of multiple cytokines, making them a highly effective immune-enhancing adjuvant. Using aqueous adjuvants not only reduces inflammatory responses and toxicity but also offers numerous advantages, including rapid antibody production, low antigen dosage, high antibody titers, preservation of the antigen's native conformation, high antibody affinity, and ease of use.

[0143] Preferably, the adjuvant may also be manganese ions, such as manganese chloride, manganese bromide, manganese iodide, manganese sulfate, manganese nitrate, manganese perchlorate, manganese acetate, manganese carbonate, manganese borate, manganese phosphate, manganese hydrobromide, manganese tartrate, manganese fumarate, manganese maleate, manganese lactate, manganese benzenesulfonate, manganese pantothenate, manganese ascorbate, and any combination thereof.

[0144] While existing methods for detecting mumps, such as immunological detection methods based on antigen-antibody specific binding, offer advantages like simplicity, convenience, and speed, they still suffer from drawbacks such as insufficient sensitivity and susceptibility to false positives and false negatives. Furthermore, current techniques for amplifying sensitivity commonly employ IgG antibodies coupled with immunomagnetic beads or biotin-streptavidin. IgG antibodies contain two antigen-binding sites, while recombinant IgM antibodies typically contain 10 or 12. Therefore, recombinant IgM antibodies offer the flexibility required to bind multiple antigens and exhibit higher antigen affinity than IgG antibodies. This invention suggests that the recombinant IgMization of IgG antibodies may play a more significant role in mumps virus detection. This invention IgMizes IgG antibodies, using recombinant modified IgM antibodies coupled with magnetic beads and biotin-streptavidin to achieve cascade amplification detection, further amplifying the sensitivity of mumps virus detection while maintaining stability and specificity.

[0145] The IgG antibody against mumps virus prepared by this invention can be further amplified by biotin amplification, with a detection limit of 1:25600; while the recombinant IgM antibody modified by this invention and amplified by biotin can increase the sensitivity to 16-20 times that of the IgG antibody, with a detection limit of 1:102400.

[0146] Specific application example: This method for improving the sensitivity of mumps virus detection uses a mouse-derived drug and includes the following steps: S1: Animal immunization, specifically: (1) Immunized animals: 4 female SJL mice; (2) Immunization regimen: Before immunization, 0.5 mL of blood was collected from mice, and serum was separated as a pre-immunization control. On day 0, mice were given a primary immunization via subcutaneous injection in the axilla and back. A secondary immunization was administered 14 days later via subcutaneous injection in the axilla and back, for a total of two immunizations. The dose for each immunization was 100 μL per mouse. (3) Specific steps for implementing immunization: Immunization Plan 1: On day 0, mice were immunized with 10 μg CpG, 50 μg manganese chloride and 50 μg mumps antigen by subcutaneous injection of 100 μL / mouse. On day 14, mice were immunized twice with 10 μg CpG, 50 μg manganese chloride and 25 μg mumps antigen, 100 μL / mouse was injected subcutaneously. On day 28, mice were immunized three times with 10 μg CpG, 50 μg manganese chloride and 12.5 μg mumps antigen, 100 μL / mouse was injected subcutaneously. Immunization Program 2: On day 0, 25 μg of mumps antigen protein was mixed with water-soluble adjuvant at a 1:1 ratio and injected subcutaneously. On day 14, a second dose of immunization was administered by subcutaneous injection of a water-soluble adjuvant mixed with 25 μg of mumps antigen protein at a 1:1 ratio. On day 28, the mumps antigen protein was mixed with water-soluble adjuvant at a 1:1 ratio and injected subcutaneously for three doses of immunization. After immunizing mice, blood was collected 10 days later. Mouse serum was serially diluted, and the dilution endpoint was determined by indirect ELISA. The highest dilution that was considered positive was the serum binding antibody titer. The results of three immunization titers are shown in Table 1. Table 1. Antibody titers in the serum of mice after three immunizations under two immunization regimens (×10⁻⁶) 4 ) (4) Advantages: While existing patents mention using manganese adjuvant (manganese chloride) instead of Freund's adjuvant to immunize mice, achieving good immunization effects with less antigen load and lower injection dosage, they still have some shortcomings compared to aqueous adjuvants. Aqueous adjuvants are complexes composed of fungal polysaccharides, saponins, and nucleotides, possessing strong immune cell stimulating activity. They can stimulate the function of various immune cells and promote the production of various cytokines, making them a highly effective immune-enhancing adjuvant. Using aqueous adjuvants not only reduces inflammatory responses and toxicity but also offers advantages such as rapid antibody production, lower antigen dosage, higher antibody titers, no disruption of the antigen's natural conformation, high antibody affinity, and ease of operation. In immunization against mumps antigen, the antibody titers produced using aqueous adjuvants are significantly higher than those produced using manganese adjuvants.

[0147] S2: Cell screening, specifically; (1) Sorting mouse B lymphocytes: a. The ELISA results of mouse serum antibody titers are shown in Table 1, indicating that the serum titers of immunized mice were all greater than 1:10. 4 This achieves the requirements for cell fusion. B lymphocytes are sorted from mice with the required immunotiter. b. After collecting blood from the orbital cavity, the serum was separated and used as a positive control; c. To avoid splenic infarction, the mice were quickly euthanized by cervical dislocation and then immersed in 75% alcohol. d. Under aseptic conditions, remove the spleen, remove the surrounding fat and connective tissue, and separate the spleen; e. Grind the spleen using a grinder, mix it with DMEM, and pass it through a sieve to prepare a spleen cell suspension; (Or, A: Gently scrape with tweezers, aspirate the cell suspension into a centrifuge tube and let it settle naturally for 5 minutes. Transfer the suspension to another centrifuge tube, discard larger tissue pieces, and centrifuge to precipitate the cells; B: Place the spleen on a 200-mesh stainless steel mesh and gently crush the spleen with a syringe needle core to obtain a cell suspension; C: Crush the spleen with tweezers, add 5 ml of 400 u / mL collagenase (type III) per spleen, digest at 37 degrees Celsius for 20 minutes, filter with a nylon mesh to obtain a single-cell suspension), and centrifuge the cell suspension at 2000 rpm for 10 minutes; f. Discard the supernatant, wash with DMEM, and centrifuge at 2000 rpm for 10 min; g. Discard the supernatant, resuspend in DMEM, and store at 37°C; (2) Fusion of myeloma cells (SP2 / 0) with B lymphocytes: a. Resuscitate SP2 / 0 cells one week prior to fusion; b. When SP2 / 0 cells are cultured until they are plump, numerous, and in good condition, they are then expanded to a 1:5 scale-up culture. c. When SP 2 / 0 cells are cultured to the logarithmic growth phase, cell fusion is performed; d.SP2 / 0 cells and mouse B lymphocytes were mixed at a ratio of 1:2 and centrifuged at 1000 rpm for 10 min; e. Discard the supernatant and gently break up the cell clumps at the bottom to allow the SP2 / 0 cells and mouse B lymphocytes to mix and spread evenly at the bottom of the tube. f. Add 1 mL of preheated 50% PEG (pH=8.0) dropwise evenly over 1 min, and react at 37°C for 1 min; g. Add 10 mL of DMEM to terminate the reaction; h. Centrifuge at 1000 rpm for 10 min, discard the supernatant, and resuspend the cells in HAT medium; i. Take 200 μL of cell suspension per well and add it to a 96-well culture plate containing feeder cells. Then, transfer the plate to a 37°C, 5% CO2 incubator for further culture and observation.

[0148] After culturing j.HAT for 3-7 days, replace the culture medium with HT, 200 μL per well; k. Depending on the culture progress, perform fusion-positive screening using ELISA after 4-7 days; (3) Screening for positive hybridoma cells: a. Dilute hybridoma cells multiple times and seed them onto 96-well cell culture plates; b. Use ELISA to detect the antibodies secreted by cells in the supernatant of each well. Culture wells in which the supernatant can bind to specific antigens are positive wells. c. Use the limiting dilution method to subclone cells in positive wells, and check whether each culture well is a positive well. Repeat 3-4 times until the positive rate of subcloning reaches 100%. d. The supernatant of the selected monoclonal cell clusters was subjected to Biacore testing. The antibody affinity data are shown in Table 2. Table 2. Results of antibody affinity testing for 10 MUV monoclonal cell lines in Biacore. In the field of in vitro diagnostics, affinity is an important indicator for measuring the strength of antibody-antigen binding. Ka (binding constant) and Kd (dissociation constant) are kinetic parameters describing antibody-antigen interactions. A larger Ka value indicates a faster binding rate, while a smaller Kd value indicates a more stable antibody-antigen binding. KD is the dissociation equilibrium constant, which is the ratio of Ka to Kd. A smaller KD value indicates a stronger affinity between the antibody and the antigen. In in vitro diagnostics, high detection sensitivity and short detection time are required, and faster antigen-antibody binding times enable quicker capture of pathogens. Therefore, when screening antibodies, it is important to consider not only the KD value, which reflects the binding strength between the antibody and the antigen, but also the Ka value, which reflects the antigen-antibody binding rate. e. Select MUV-10G4 and MUV-10G6 for extended subculture; f. Centrifuge, collect the supernatant, and perform affinity chromatography using a Protein G column to obtain 95% high-purity IgG monoclonal antibody from the hybridoma culture supernatant sample; S3: IgG antibody sequencing: Sequencing of the variable region of mouse IgG antibodies; The amino acid sequences of the complementarity-determining regions CDRH1-CDRH3 of the heavy chain variable region of the obtained monoclonal antibody 10G4 are shown in SEQ ID NO: 1-SEQ ID NO: 3, respectively; the amino acid sequences of the complementarity-determining regions CDRL1-CDRL3 of the light chain variable region are shown in SEQ ID NO: 4-SEQ ID NO: 6, respectively. SEQ ID NO: 1: DYWMN SEQ ID NO: 2: QIRNKPYNYETYYSESVKG SEQ ID NO: 3: GTWFAY SEQ ID NO: 4: ASGQSIVHSNGNTYLE SEQ ID NO: 5: KVSNRFS SEQ ID NO: 6: GQCSHVPET The amino acid sequences of the complementarity-determining regions CDRH1-CDRH3 of the heavy chain variable region of the obtained monoclonal antibody 10G6 are shown in SEQ ID NO: 7-SEQ ID NO: 9, respectively; the amino acid sequences of the complementarity-determining regions CDRL1-CDRL3 of the light chain variable region are shown in SEQ ID NO: 10-SEQ ID NO: 12, respectively. SEQ ID NO: 7: DYWMN SEQ ID NO: 8: QIAKSPYNYETYYSESVKG SEQ ID NO: 9: SGTWFGS SEQ ID NO: 10: RGGQSIVHSNGSTYLE SEQ ID NO: 11: KVGSAFS SEQ ID NO: 12: FQGSHVAAT S4: IgM modification of IgG antibodies; The heavy chain variable region sequence of mouse IgG antibodies (monoclonal antibodies 10G4 and 10G6 mentioned above) and the CH1 fragment of the heavy chain constant region sequence of mouse IgG antibodies were recombined with the CH2, CH3, and CH4 segments of the heavy chain constant region of mouse IgM antibodies. After codon optimization, the resulting sequence was constructed into the expression vector pcDNA3.4. The structure of the heavy chain expression vector is shown below. Figure 1 As shown. Figure 1 The diagram shows the regions from the 5' to the 3' end, including L (light chain), IGHV (immunoglobulin heavy chain variable region gene), D (diversity region), J (connector region), mlgG1-CH1 (constant region 1 of membrane-bound IgG1), CH2, CH3, and CH4 (constant regions 2, 3, and 4 of membrane-bound IgM). mlgG1-CH1, together with mIgM-CH2, CH3, and CH4, constitutes the heavy chain constant region of the modified IgM antibody. The structure of the light chain expression vector is as follows Figure 2 As shown; Figure 2 The various regions from the 5' end to the 3' end are shown, including V (immunoglobulin light chain variable region gene), J (linker region) and IGKC (constant region gene). Plasmids were extracted for cell transfection; in, The amino acid sequence of mlgG1-CH1 is: AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCG (SEQ ID NO: 13); The amino acid sequence of mIgM-CH2 is: AVAEMNPNVNVFVPPRDGFSGPAPRKSKLICEATNFTPKPITVSWLKDGKLVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNLNVYTCRVDHRGLTFLKNVSSTCA (SEQ ID NO: 14). The amino acid sequence of mIgM-CH3 is: ASPSTDILTFTIPPSFADIFLSKSANLTCLVSNLATYETLNISWASQSGEPLETKIKIMESHPNGTFSAKGVASVCVEDWNNRKEFVCTVTHRDLPSPQKKFISKPN (SEQ ID NO: 15). The amino acid sequence of mIgM-CH4 is: EVHKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADISVQWLQRGQLLPQEKYVTSAPMPEPGAPGFYFTHSILTVTEEEWNSGETYTCVVGHEALpHLVTERTVDKSTGGTCY (SEQ ID NO: 16). The amino acid sequence of the light chain constant region is: RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 17). S5: Recombinant antibody expression in CHO cells: (1) Resuscitate CHO cells: a. Thaw frozen CHO cells in a 37°C water bath; b. Centrifuge at 1200 rpm for 2 minutes; c. Remove the supernatant and resuspend the CHO cells in fresh culture medium; d. Collect 1 mL of cells daily for counting and viability testing; e. Control the density at 3.0 × 10⁻⁶ before transfection. 6 cells / mL; (2) Extraction of transfection-grade plasmids: a. Add 80 ng of expression plasmid to 200 μL of competent cell suspension; b. The competent cells with added plasmid were heat-shocked at 42 °C, transformed for 90 s, and cooled on ice for 5 min; c. Add 1 mL of LB liquid medium, incubate at 37°C with shaking at 200 rpm for 30 min; d. Spread 100 μL of cell suspension onto a selection plate containing ampicillin resistance and incubate statically at 37 °C for 24 h; e. Pick single clones and seed them into 200 mL of LB medium, and incubate at 37 ℃ and 200 rpm for 16 h; f. Collect the bacterial culture, centrifuge, remove the supernatant, add 250 μL of RB solution, and suspend the bacteria thoroughly in a shaker; g. Add 250 μL of LB solution, incubate at 37 °C for 2 min; h. Add 250 μL of NB solution. Incubate at 37 ℃ for 2 min, then centrifuge at 15000 rpm for 15 min; i. Collect the supernatant into the adsorption column and centrifuge at 15000 rpm for 30 s; g. Discard the supernatant, add 700 μL of WB solution to the adsorption column, centrifuge at 15000 rpm for 30 s, and repeat twice; k. Place the adsorption column into a 1.5 mL centrifuge tube, add 50 μL of Elution Buffer, and incubate at 37 °C for 2 min; Centrifuge at 15000 rpm for 1 min and collect the plasmid; (3) Transfection a. Add light chain and heavy chain expression plasmids to the transfection buffer, mix and dilute them at a mass ratio of 1:1, then mix with the transfection reagent and incubate at room temperature for 5-20 min. b. Add the transfection reagent mixture to the cells to be transfected and incubate at 37 ℃ and 120 rpm for 5-6 days with shaking. c. Centrifuge and collect the supernatant; S6: Purification of IgM antibody derived from IgG antibody modification and recombinant synthesis, specifically as follows: (1) Cell supernatant was purified for antibody via Protein G column: a. Filter all buffer solutions used in the chromatography using a 0.45 μm filter membrane; b. Centrifuge the sample at 12,000 rpm for 15 min to remove larger cells and cell debris; c. Dilute the sample 1:1 with loading buffer, then filter the diluted sample through a 0.45 μm filter membrane; d. Use 0.1M NaOH to rinse 2CV, then pause for 15 min to remove impurities; e. Equilibrate the chromatography column with 5-10 column volumes of loading buffer at a flow rate of 1 mL / min until the pH of the eluent is 7.4. f. Load the filtered diluted sample onto the chromatography column at a flow rate of 1 mL / min, and wash with 5 to 10 column volumes of loading buffer until the baseline is stable. g. Elute the column with elution buffer. When the elution peak appears, collect 3 mL of effluent from each tube until the elution peak returns to the baseline. Immediately adjust the pH of the collected effluent from each tube to 7.0 with 1.0 mol / L Tris-HCl buffer (pH 9.0). h. Elute the column with 3 to 5 column volumes of regeneration buffer at a flow rate of 1 mL / min; i. Reequilibrate the chromatography column with 5 to 10 column volumes of loading buffer until the pH of the eluent is 7.4, and then equilibrate with 10 column volumes of pure water. j. Collect the eluent from each tube; The purity of the purified antibody was determined using SDS-PAGE. a. The accelerated stability of the modified IgM antibody solution was investigated at 37 °C for 0, 3, 7, and 14 days. b. The sample is diluted or concentrated to the specified concentration with PBS, mixed with SDS (1:1) to adjust the loading volume, mixed well and then denatured, and the purity is identified by SDS-PAGE. c. Heat in a water bath for 5 minutes, cool to room temperature, centrifuge at 12000 rpm for 1 minute, and collect the supernatant for later use; d. Load 10 μL of the prepared protein sample into each well of the electrophoresis tank; e. After loading the sample, add buffer solution and cap it; f. Use 90V for the stacking gel section and 15-20 min for the separating gel section; use 180V for the separating gel section and 30-50 min for the separating gel section. g. Stain the gel with Coomassie Brilliant Blue R250 for 15 min; h. Add the decolorizing solution, microwave and boil for 1 minute to accelerate decolorization, repeat 3 times until the gel base color is almost colorless; The results are as follows Figure 3 (Lane 1-9 show the thermally accelerated stability verification results of IgM antibody 10G4 on days 0, 3, 7, and 14, and the thermally accelerated stability verification results of marker and IgM antibody 10G6 on days 0, 3, 7, and 14, respectively.) As shown, the IgM antibody obtained by combining the CH1 fragment of the mouse IgG1 constant region with the CH2 / 3 / 4 of the mouse IgM constant region not only has good thermal stability, but also can achieve a purity of over 95% in a one-step purification method based on protein G. S7: Performance verification of the modified IgM antibody, specifically as follows; (1) Affinity test of IgM antibody after IgG antibody modification: The supernatants of IgG-10G4, IgG-10G6, IgM-10G4, and IgM-10G6 cells were used for Biacore assays. The results are shown in Table 3. Table 3. Biacore test results for MUV IgG and IgM antibodies. The modified IgM antibody has a higher Ka value than the IgG antibody, indicating that it binds to the antigen faster and can capture pathogens more quickly, making it more suitable for in vitro diagnostic applications. (2) Sensitivity detection results of IgG antibody and modified IgM antibody: This experiment validated the conventional IgG system (specifically, magnetic bead-IgG 10G4 / acridone ester-IgG 10G6, referred to as IgG antibody) and the IgM system (specifically, magnetic bead-IgM 10G4 / acridone ester-IgM 10G6, referred to as IgM antibody).

[0149] The IgG antibody was diluted with Tris buffer at 100, 300, 900, 2700, 8100, 24300, 72900, 218700, and 656100 times, respectively; the modified IgM antibody was diluted with Tris buffer at 1000, 3000, 9000, 27000, 81000, 243000, 729000, 2187000, and 6561000 times, respectively. The sensitivity was detected on a chemiluminescence platform, and the results are shown in Table 4. Table 4. Sensitivity detection results of IgG antibody and modified IgM antibody The results showed that the highest measured value of the IgG antibody raw material reached 1,300,000, and the highest measured value of the IgM antibody raw material reached 700,000; the linear peak of the IgG antibody raw material was approximately 7,000,000; and the linear peak of the IgM antibody raw material was approximately 2,800,000. In other words, the above results indicate that the IgG antibody prepared by this invention can achieve a high level of sensitivity, which is sufficient for detection needs. The sensitivity of the IgM antibody obtained by the modification of this invention is further improved, and can be controlled to be 4-5 times that of the IgG antibody.

[0150] (3) Precision detection of the modified IgM antibody: The modified IgM antibody was diluted 4500-fold, 20000-fold, and 50000-fold with Tris buffer, and measured 10 times on one negative reference, one positive reference, and one limit-of-detection sample, respectively. The coefficient of variation (CV (%) = SD (standard deviation) / mean (mean) × 100%) should not exceed 10.0%. The results are shown in Table 5. Table 5. Precision testing of the modified IgM antibody The results are shown in Table 5. The IgM antibody modified by this invention showed positive results for both the positive repeatability reference and the limit of detection reference, and negative results for the negative repeatability reference. The coefficient of variation (CV) could be controlled to be less than 5%. (4) Inter-batch variation detection of the modified IgM antibody: Three batches of small-scale samples were produced, and 25 negative samples were simultaneously tested using chemiluminescence immunoassay. The relative deviation was calculated. The results are shown in Table 6. Table 6. Inter-batch variation detection of the modified IgM antibody The results, as shown in Table 6, indicate that the batch-to-batch variation of the IgM antibody obtained by the modification of this invention is small, and the relative deviation can be controlled within ±5%. (5) Stability test of the modified IgM antibody: The stability of the modified IgM antibody was assessed on day 0, day 7 at 4°C, and day 7 at 37°C. The results are shown in Table 7.

[0151] Table 7. Stability test of the modified IgM antibody The results are shown in Table 7. The IgM antibody obtained by the modification of this invention meets the stability requirements. S8: Magnetic bead-conjugated antibody; (1) Component 1: Preparation of magnetic bead-streptavidin conjugate, the specific steps are as follows: a. Select JSR Carboxyl MS160 magnetic beads, mix the magnetic beads, take 50 μL of carboxyl magnetic beads into a 1 mL centrifuge tube, add 300 μL of 0.01 M PBS buffer and vortex for 5 min; magnetically remove the supernatant, repeat 2 times. b. Add 150 μL of 100 mM pH 6.5 MES to dissolve, and vortex for 30 seconds to mix. c. Add 100 μL of freshly prepared 10 mg / mL EDC and 100 μL of 10 mg / mL sulfo-NHS, vortex again to mix, and activate at 25-37℃ for 20 min-1 h, preferably at 25℃ for 20 min; d. Add 300 μL of 0.01M PBS buffer and vortex for 30 s; e. Use magnetic attraction to remove the supernatant, repeat 3 times; f. Add 50 μg-300 μg streptavidin, add 500 μL 0.01M PBS buffer, add 0.05% Tween 20, mix gently, mix by inverting using a vertical mixer, and couple at 25°C for 2 hours. g. Magnetic adsorption, remove the supernatant, add 200 μL PBST solution (pH 7.2, containing 1% BSA) to resuspend, react at 25℃ for 1 h to block; h. Remove the supernatant by magnetic adsorption. Wash three times with 200 μL PBS solution (pH 7.2) each time, then resuspend in 0.01 M PBS, 0.05% Tween 20, and 5% sucrose solution and store at 4°C. (2) Component 2: Preparation of IgM antibody 10G4-biotin conjugate, the specific steps are as follows: a. Antibody preparation: Cut open the bottom of the Zeba desalting column, place it in a 2mL EP tube, loosen the column cap, centrifuge at 1500g for 1min, and remove the liquid in the tube; add 300μL PBS buffer, centrifuge, and repeat this process 3 times, removing the liquid in the tube each time; add the sample to the column; centrifuge at 1500g for 2min, and collect the solution in the 1.5mL EP tube, which is the target product; b. Replace the buffer with 0.1M PBS pH 7.2 using a Zeba desalting column and adjust the IgM antibody 10G4 concentration to 2.0 mg / mL; c. Take 500 μL of IgM antibody 10G4 and 0.33-6.66 μL of 10mM / L NHS-Biotin (feed ratio of 1:2-1:10), vortex mix, and then rotate mix at 25℃-37℃ for 30min-2h; preferably at 25℃ for 30min. d. Add 300 μL of 0.1 M PBS buffer and dialyze, wash twice to obtain biotin-modified antibody IgM antibody 10G4; (3) Component 3: Preparation of IgM antibody 10G6-acridone ester conjugate, the specific steps are as follows: a. Dilute IgM antibody 10G6 to 2 mg / mL using 0.01 M pH 7.2 PBS; b. Take 1 mL of IgM antibody 10G6 and add it to 400 μL of 0.25 mg / mL acridinium ester solution (feed ratio of 1:10-1:30). React for 30 min, then add lysine to terminate the reaction for 20 min. c. Add to a dialysis bag and dialyze in 0.01M pH7.2 PBS, changing the dialysate every 2-3 hours during the process; d. The IgM antibody 10G6-acridone ester conjugate was stored in a storage solution of 0.05 mol / L Tris pH 7.2, 2% BSA, 0.05% Tween-20, and 0.2% PC300.

[0152] (4) Capture of mumps virus in samples: a. Based on antibody mass, component 1 (i.e., magnetic bead-streptavidin conjugate) and component 2 (i.e., IgM antibody 10G4-biotin conjugate) are premixed in a 1:2 ratio to form a magnetic bead-IgM antibody 10G4 complex. b. Add 20 μL of magnetic bead-IgM antibody 10G4 complex to 100 μL of sample and incubate at 37℃ for 10 min. c. Magnetic attraction to remove supernatant; d. Add 200 μL of 0.1 M PBS buffer and wash twice; e. Magnetic attraction to remove supernatant; f. Add 20 μL of pure water to resuspend and obtain magnetic bead-IgM antibody 10G4-antigen conjugate; mix the magnetic bead-IgM antibody 10G4-antigen conjugate with 20 μL of component 3 (i.e. IgM antibody 10G6-acrididine ester conjugate) at 37°C and incubate for 15 min to form a solid-phase antibody-antigen-antibody sandwich complex. g. Use magnetic adsorption to remove the supernatant, add 200 μL of PBST solution (pH 7.2, containing 1% BSA) to resuspend, and wash repeatedly 3 times; h. Add the pre-excitation solution and the excitation solution, and use the instrument to detect the number of photons and interpret the numerical value; i. The collected clinical samples were diluted 25600, 51200, and 102400 times using sample dilution buffer, and three repeated measurements were performed. The average value was calculated, and the biotin signal amplification ratio was then calculated based on these data. The results are shown in Table 8.

[0153] Table 8. Comparison of biotin signal amplification between IgG antibody and modified IgM antibody. The above results show that the sensitivity of the IgG antibody prepared by the present invention can be further amplified by biotin amplification, with a detection limit of 1:25600; while the sensitivity of the IgM antibody modified by the present invention by biotin amplification can be increased to 16-20 times that of the IgG antibody, with a detection limit of 1:102400.

[0154] The IgG antibody and recombinant IgM antibody prepared by this invention, as well as the detection reagent prepared using the recombinant IgM antibody, can be used not only to detect clinical samples, but also to detect non-clinical samples (i.e., for non-diagnostic purposes).

[0155] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. An IgG antibody that specifically binds to mumps virus, characterized in that, The IgG antibody comprises: a heavy chain constant region, a light chain constant region, a heavy chain variable region, and a light chain variable region of an IgG monoclonal antibody that specifically binds to mumps virus. The IgG monoclonal antibody is murine in origin, and the amino acid sequences of the complementarity-determining regions CDRH1-CDRH3 of its heavy chain variable region are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; the amino acid sequences of the complementarity-determining regions CDRL1-CDRL3 of the light chain variable region are shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

2. An IgG antibody that specifically binds to mumps virus, characterized in that, The IgG antibody comprises: a heavy chain constant region, a light chain constant region, a heavy chain variable region, and a light chain variable region of an IgG monoclonal antibody that specifically binds to mumps virus. The IgG monoclonal antibody is murine in origin, and the amino acid sequences of the complementarity-determining regions CDRH1-CDRH3 of its heavy chain variable region are shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively; the amino acid sequences of the complementarity-determining regions CDRL1-CDRL3 of the light chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively.

3. A gene for a specific binding IgG antibody against mumps virus, characterized in that, The monoclonal antibody that specifically binds to the mumps virus IgG is a murine IgG monoclonal antibody 10G4, and the amino acid sequences of the complementarity-determining regions CDRH1-CDRH3 of its heavy chain variable region are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; the amino acid sequences of the complementarity-determining regions CDRL1-CDRL3 of the light chain variable region are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

4. A gene for a specific binding IgG antibody against mumps virus, characterized in that, The monoclonal antibody that specifically binds to the mumps virus is a murine IgG monoclonal antibody 10G6, and the amino acid sequences of the complementarity-determining regions CDRH1-CDRH3 of its heavy chain variable region are shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively; the amino acid sequences of the complementarity-determining regions CDRL1-CDRL3 of the light chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

5. A recombinant IgM antibody that specifically binds to mumps virus IgG, characterized in that, The recombinant IgM antibody is modified from the IgG antibody that specifically binds to the mumps virus according to claim 1, and includes the heavy chain variable region, light chain variable region, light chain constant region and heavy chain constant region CH1 of the IgG antibody that specifically binds to the mumps virus, as well as the heavy chain constant regions CH2, CH3 and CH4 of the IgM antibody. The amino acid sequences of the complementarity-determining regions CDRH1-CDRH3 of the heavy chain variable region are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively. The amino acid sequences of the complementarity-determining regions CDRL1-CDRL3 of the light chain variable region are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; The amino acid sequence of CH1, the constant region of the heavy chain of the IgG antibody, is shown in SEQ ID NO: 13; The amino acid sequence of CH2, the constant region of the heavy chain of the IgM antibody, is shown in SEQ ID NO: 14; The amino acid sequence of CH3, the constant region of the heavy chain of the IgM antibody, is shown in SEQ ID NO: 15; The amino acid sequence of CH4, the constant region of the heavy chain of the IgM antibody, is shown in SEQ ID NO: 16; The amino acid sequence of the constant region of the light chain is shown in SEQ ID NO:

17.

6. A recombinant IgM antibody that specifically binds to mumps virus IgG, characterized in that, The recombinant IgM antibody is modified from the IgG antibody that specifically binds to the mumps virus according to claim 2, and includes the heavy chain variable region, light chain variable region, light chain constant region and heavy chain constant region CH1 of the IgG antibody that specifically binds to the mumps virus, as well as the heavy chain constant regions CH2, CH3 and CH4 of the IgM antibody. The amino acid sequences of the complementarity-determining regions CDRH1-CDRH3 in the heavy chain variable region are shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively. The amino acid sequences of the complementarity-determining regions CDRL1-CDRL3 of the light chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; The amino acid sequence of CH1, the constant region of the heavy chain of the IgG antibody, is shown in SEQ ID NO: 13; The amino acid sequence of CH2, the constant region of the heavy chain of the IgM antibody, is shown in SEQ ID NO: 14; The amino acid sequence of CH3, the constant region of the heavy chain of the IgM antibody, is shown in SEQ ID NO: 15; The amino acid sequence of CH4, the constant region of the heavy chain of the IgM antibody, is shown in SEQ ID NO: 16; The amino acid sequence of the constant region of the light chain is shown in SEQ ID NO:

17.

7. A detection reagent that specifically binds to mumps virus, characterized in that, It includes a first recombinant IgM antibody and a second recombinant IgM antibody, wherein the first recombinant IgM antibody and the second recombinant IgM antibody are selected from the recombinant IgM antibodies of claims 5 and / or 6, and the first recombinant IgM antibody and the second recombinant IgM antibody are different.

8. A detection kit for specific binding to mumps virus, characterized in that, It includes the detection reagent as described in claim 7.

9. The use of the detection reagent according to claim 7 in the preparation of in vitro immunodiagnostic reagents.

10. A method for improving the sensitivity of mumps virus detection, characterized in that, Includes the following steps: The heavy chain expression vector is obtained by recombining the sequence of the variable region of the heavy chain of a mouse or rabbit IgG monoclonal antibody, the amino acid sequence fragment CH1 of the constant region of the heavy chain of a mouse or rabbit IgG antibody, and the amino acid sequence fragments CH2-CH4 of the constant region of the heavy chain of a mouse or rabbit recombinant IgM antibody. The heavy chain expression vector and the light chain expression vector were expressed in cells to obtain a recombinant IgM antibody; the IgG antibody is the IgG antibody that specifically binds to mumps virus as described in claim 1 and / or 2. The recombinant IgM antibody was used to prepare a detection reagent to detect the concentration of mumps virus in the sample to be tested.

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