HSV-I IgG antibody, recombinant IgM antibody and application

Abstract

The invention relates to the field of in-vitro immunodiagnosis detection, in particular to an IgG antibody of HSV-I, 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 an IgG monoclonal antibody specifically combined with herpes simplex virus HSV-I, the IgG monoclonal antibody is of a mouse source or a rabbit source, and the IgG antibody is modified to obtain the recombinant IgM antibody. The IgG antibody and the recombinant IgM antibody are not only suitable for a chemiluminescence method, but also show expected sensitivity magnification times in other immunological detection methods, and are helpful for realizing low-abundance detection of the specific binding herpes simplex virus HSV-I.

Description

Technical Field

[0001] This invention relates to the field of in vitro immunodiagnostic testing, and more particularly to an IgG antibody against HSV-I, a recombinant IgM antibody, and their applications. Background Technology

[0002] Herpes simplex is a common viral infection caused by the herpes simplex virus (HSV), a double-stranded DNA virus. It mainly manifests as clusters of small blisters on the skin or mucous membranes, accompanied by pain, itching, or burning sensation. Herpes simplex virus belongs to the alpha subfamily of the herpesviridae family and is divided into two main types: HSV-I and HSV-II. HSV-I typically causes oral herpes (such as "cold sores"), primarily transmitted through saliva or close contact; while HSV-II primarily causes genital herpes, mostly transmitted through sexual contact. However, the sites of infection are not absolutely fixed; HSV-I can also infect the genitals through oral-genital contact, and HSV-II can occasionally cause oral infections. After infection, HSV migrates along sensory nerves to ganglia and remains latent for a long time. When the body's immunity declines, the virus can be reactivated, returning along the nerves to the skin or mucous membranes, leading to recurrent herpes. Symptoms of recurrent herpes are usually milder than the primary infection, but it can occur frequently, affecting quality of life. The key to early diagnosis lies in identifying atypical symptoms. For high-risk groups, regular screening for HSV antibodies helps to detect asymptomatic infections, thus enabling treatment to begin as early as possible. Therefore, the treatment of herpes simplex not only needs to focus on antiviral intervention after symptoms appear, but also emphasizes early diagnosis and prevention strategies to reduce the risk of infection and reduce recurrence.

[0003] The diagnosis of herpes simplex mainly relies on clinical manifestations. With the advancement of molecular biological diagnostic techniques, HSV detection methods have evolved through stages such as viral culture detection, PCR detection, and immunological detection. Viral culture detection can directly isolate and type the virus (HSV-I / HSV-II), but it has low sensitivity, is time-consuming, and has high requirements for sample preservation and transportation. PCR detection has high sensitivity and can detect extremely low viral loads, but it is costly and requires sampling when symptoms appear (such as blister fluid or ulcer secretions), limiting its detection rate for asymptomatic latent infections. Immunological detection has a short detection time and can improve detection efficiency, but its sensitivity is lower than PCR, it is greatly affected by sample quality, and cross-reactivity can easily lead to false positives.

[0004] In recent years, chemiluminescence immunoassay has become a popular immunological technique due to its high sensitivity and good detection limits. Immunomagnetic beads, also known as magnetic beads, possess excellent superparamagnetism, and their surface chemical groups can covalently couple with protein molecules. Furthermore, magnetic beads have a large specific surface area, allowing them to bind more proteins and expand the biodetection range. Therefore, they are often combined with chemiluminescence immunoassay and applied in fields such as biodetection.

[0005] In vitro diagnostic reagents are one of the fundamental tools for detecting whether a patient is ill and the severity of their condition. The accuracy of the results directly affects the doctor's diagnosis and the patient's health and safety. With the advancement of modern medical technology, there are increasingly higher demands for the precision of medical test results, leading to higher quality requirements for in vitro diagnostic products. The detection methods for pathogens (such as herpes simplex virus HSV-I) have also evolved through stages such as pathogen isolation and culture detection, PCR, and immunological detection. Pathogen isolation and culture detection requires the isolation and culture of pathogens, which is complex, time-consuming, and somewhat subjective in result interpretation. PCR detects pathogens by detecting the target DNA or RNA sequence. While it has significant advantages in specificity and price, the amplified products must be interpreted by agarose gel electrophoresis, resulting in lower sensitivity and a long processing time. Immunological detection is based on the principle of specific binding between antigens and antibodies. Although it is simple, convenient, and rapid, it still suffers from insufficient sensitivity and is prone to false positives and false negatives.

[0006] The performance of an immunoassay also depends on the epitopes and affinity of the antibodies selected to capture antigens contained in the sample. The practicality of IgG antibodies has been demonstrated in early pathogen detection; for example, pathogen detection often relies heavily on IgG antibodies. IgG antibodies have a typical "Y"-shaped antibody structure containing two antigen-binding sites, while IgM antibodies typically exist as soluble pentamers with 10 antigen-binding sites and a J-chain, or as hexamers with 12 antigen-binding sites and no J-chain. Therefore, IgM offers the flexibility required to bind multiple antigens and exhibits higher antigen affinity than IgG. Thus, the inventors believe that the IgMization of IgG antibodies may play a more significant role in pathogen detection. Despite the unique characteristics of IgM antibodies, their role in clinical diagnosis and prevention remains under-explored.

[0007] Streptavidin (SA) is a secretory protein extracted from Streptomyces cultures, with a molecular weight of 65 kDa and an isoelectric point of 6.0. SA is a tetrameric protein; one molecule of SA can specifically bind four molecules of biotin. Biotin, also known as vitamin H, has two ring structures in its molecule. Ring I is an imidazolide ring, which is the main binding site for SA; Ring II is a thiazole ring with a valerate side chain. Its terminal carboxyl group is often chemically modified or altered to increase the hydrophilicity of biotin or reduce steric hindrance, transforming it into long-chain hydrazide biotin or N-hydroxysuccinimide ester biotin, used to label the thiol and amino groups of antibodies to increase the sensitivity and specificity of the biotin-streptavidin system.

[0008] The biotin-streptavidin interaction is considered one of the most specific and stable non-covalent interactions, with a dissociation constant (KD) approximately 10 times that of antigen-antibody interactions. 3 ~10 6 Its high affinity is mainly used to isolate and amplify signals, improving the detection capability of low-concentration analytes. It also has high specificity, high stability, and high adaptability.

[0009] Currently, antigen-antibody detection plays an important role in disease diagnosis. However, current antigen-antibody detection methods still have limitations for applications requiring high sensitivity, such as early-stage pathogen infection and biomarkers that are low in blood due to the influence of the blood-brain barrier.

[0010] Chinese patent document CN 112538111 A discloses a single-chain antibody against the novel coronavirus, 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 the method described in this patent, IgM pentamers can be prepared correctly, efficiently, and at higher expression levels and ratios. In these two 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.

[0011] 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 technique utilizes immunomagnetic beads to detect β-amyloid protein in plasma and is primarily applied to the early screening and diagnosis of neurodegenerative diseases. This invention, however, primarily utilizes immunomagnetic beads to detect herpes simplex virus.

[0012] 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.

[0013] While existing methods for detecting herpesviruses, such as immunological detection methods based on the specific binding of antigens and antibodies, have advantages such as being simple to operate, convenient, and rapid, they still have disadvantages such as insufficient sensitivity and a tendency to produce false positive and false negative results.

[0014] Therefore, in order to improve the detection sensitivity of herpes simplex virus HSV-I, it is urgent to develop an antibody and detection reagent, as well as a method that can improve detection sensitivity, thereby expanding the application scope of antigen-antibody detection. Summary of the Invention

[0015] This invention provides an IgG antibody that specifically binds to herpes simplex virus HSV-I, along with the antibody gene, a modified recombinant IgM antibody, a detection reagent prepared from the recombinant IgM antibody, and the application of the antibody. These improvements enhance detection sensitivity and facilitate the detection of low abundance of herpes simplex virus HSV-I that specifically binds to it.

[0016] To address the aforementioned technical issues, the present invention provides the following technical solutions.

[0017] In a first aspect, the present invention provides a first IgG antibody that specifically binds to herpes simplex virus HSV-I, 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 herpes simplex virus HSV-I, 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.

[0018] The second type is an IgG antibody that specifically binds to herpes simplex virus HSV-I. 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 IgG monoclonal antibody that specifically binds to herpes simplex virus HSV-I. The IgG monoclonal antibody is murine in origin. 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.

[0019] Furthermore, the IgG monoclonal antibody of the present invention can also be derived from rabbits.

[0020] Secondly, the present invention provides a gene for a specific IgG antibody that binds to herpes simplex virus HSV-I, wherein the monoclonal antibody that specifically binds to herpes simplex virus HSV-I is a murine IgG monoclonal antibody 1G9, 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] Thirdly, the present invention provides a gene for a specific IgG antibody that binds to herpes simplex virus HSV-I, wherein the monoclonal antibody that specifically binds to herpes simplex virus HSV-I is a murine IgG monoclonal antibody 4D7, 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] Preferably, the IgG antibody is coupled to magnetic beads.

[0023] Preferably, the IgG antibody is labeled with acridine ester.

[0024] Preferably, the IgG antibody is conjugated with biotin.

[0025] Fourthly, the present invention provides a recombinant IgM antibody that specifically binds to IgG antibodies against herpes simplex virus HSV-I, wherein the recombinant IgM antibody is modified from the first IgG antibody that specifically binds to herpes simplex virus HSV-I 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 herpes simplex virus HSV-I, 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.

[0026] Fifthly, the present invention provides a recombinant IgM antibody that specifically binds to IgG antibodies against herpes simplex virus HSV-I, wherein the recombinant IgM antibody is modified from the second type of IgG antibody that specifically binds to herpes simplex virus HSV-I in 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 herpes simplex virus HSV-I, 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.

[0027] In some embodiments of the fourth and fifth 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.

[0028] Preferably, the second constant region CH2, the third constant region CH3, and the fourth constant region CH4 are derived from mouse IgM antibodies.

[0029] Preferably, the recombinant IgM antibody is modified from an IgG antibody that specifically binds to a biomarker.

[0030] 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.

[0031] Preferably, the recombinant IgM antibody is coupled to magnetic beads.

[0032] 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).

[0033] Preferably, the recombinant IgM antibody is labeled with acridine ester.

[0034] Preferably, the recombinant IgM antibody is conjugated with biotin.

[0035] Preferably, the preparation method of the recombinant IgM antibody in the fourth and fifth aspects described above is as follows: (1) Construction of expression vectors for IgM antibody heavy and light chain sequences: The amino acid sequence of the variable region of the heavy chain of the IgG antibody that specifically binds to HSV-I and the amino acid sequence fragment CH1 of the constant region of the heavy chain of the IgG antibody were recombined with the amino acid sequences of CH2, CH3, and CH4 of the constant region of the heavy chain of the IgM antibody. After codon optimization, the gene was constructed into the expression vector pcDNA3.4. (2) Cell transfection and expression: 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. (3) Select cell lines that express IgM antibodies efficiently; (4) The IgM antibody is then purified.

[0036] In a sixth aspect, the present invention provides a detection reagent that specifically binds to herpes simplex virus HSV-I, comprising a first IgM antibody and a second IgM antibody, wherein the first IgM antibody and the second IgM antibody are selected from the recombinant IgM antibodies described in the fourth and fifth aspects of the present invention, and the first IgM antibody and the second IgM antibody are different.

[0037] Preferably, the mass ratio of the first antibody to the second antibody is 1:2.

[0038] Preferably, the detection reagent further includes a magnetic bead-streptavidin conjugate.

[0039] Preferably, the first IgM antibody is conjugated with biotin or with magnetic beads, and the second IgM antibody is labeled with acridine ester. The mass ratio of the magnetic bead-streptavidin conjugate to the biotin-conjugated first IgM antibody is 1:2. The feed ratio of the second IgM antibody to the acridine ester includes 1:10-30.

[0040] Preferably, the first IgM antibody and the second IgM antibody are modified from IgG antibodies that specifically bind to biomarkers.

[0041] Preferably, the first IgM antibody and the second IgM antibody include a heavy chain constant region, a light chain constant region, a heavy chain variable region, and a light chain variable region.

[0042] 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.

[0043] Preferably, the second constant region CH2, the third constant region CH3, and the fourth constant region CH4 are derived from mouse IgM antibodies.

[0044] Preferably, the detection reagent comprises a first IgM antibody conjugated to magnetic beads and a second IgM antibody labeled with acridine ester.

[0045] 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 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. (g) Add 5 mL of blocking buffer (1% BSA) to resuspend, and obtain the detection reagent. Store at 4 °C for later use.

[0046] In a seventh aspect, the present invention provides the use of an antibody described in any one of the first to fifth aspects of the present invention in the preparation of an in vitro immunodiagnostic reagent.

[0047] Preferably, the in vitro immunodiagnostic reagent is based on colloidal gold / latex immunochromatography, chemiluminescence immunoassay, enzyme-linked immunosorbent assay (ELISA), or fluorescence immunochromatography.

[0048] Eighthly, the present invention provides a detection kit for specific binding of herpes simplex virus HSV-I, comprising the detection reagent described in the sixth aspect of the present invention.

[0049] Ninthly, the present invention provides the application of the detection reagent described in the sixth aspect of the present invention in the preparation of in vitro immunodiagnostic reagents.

[0050] Preferably, the in vitro immunodiagnostic reagent is based on colloidal gold / latex immunochromatography, chemiluminescence immunoassay, enzyme-linked immunosorbent assay (ELISA), or fluorescence immunochromatography.

[0051] In a tenth aspect, the present invention provides a method for improving the sensitivity of herpes simplex virus detection, comprising the following steps: The heavy chain variable region sequence of a mouse or rabbit IgG monoclonal antibody, the amino acid sequence fragment CH1 of the heavy chain constant region of a mouse or rabbit IgG antibody, and the amino acid sequence fragments CH2-CH4 of the heavy chain constant region of a mouse or rabbit recombinant IgM antibody are genetically recombined to obtain a heavy chain expression vector; the heavy chain expression vector and the light chain expression vector are expressed in cells to obtain a recombinant IgM antibody; the IgG antibody is the first or / and second type of IgG antibody specifically binding to herpes simplex virus HSV-I as described in the first aspect of the present invention; The recombinant IgM antibody was used to prepare a detection reagent to detect the concentration of herpes simplex virus HSV-I in the sample to be tested.

[0052] Preferably, the method for improving the sensitivity of herpes simplex virus detection includes the following steps: Animal immune response to S1'IgG antibodies: Animals were immunized with antigen, CpG and adjuvant to induce the production of IgG antibodies; S2' cell screening: B lymphocytes of animals are fused with myeloma cells to obtain hybridoma cells; positive screening of hybridoma cells is performed, and IgG monoclonal antibodies are collected from the cultures of positive hybridoma cells; S3'IgG antibody sequencing: The IgG monoclonal antibody obtained in step S2 is sequenced to obtain the amino acid sequences of the heavy chain variable region and the light chain variable region of the IgG monoclonal antibody; S4' IgG antibody recombinant IgM antibody: The sequence of the variable region of the heavy chain of the 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; S5' Recombinant IgM antibody detection of herpes simplex virus: The recombinant IgM antibody is prepared into the detection reagent described in the sixth aspect of the present invention to detect the concentration of herpes simplex virus HSV-I in the sample to be tested.

[0053] The specific steps of step S5' are as follows: The magnetic bead-streptavidin conjugate and the biotin-modified first IgM antibody were mixed to obtain a premix; The premix and the sample to be tested are mixed to obtain a magnetic bead-first IgM antibody-antigen conjugate; The magnetic bead-first IgM antibody-antigen conjugate is mixed with the acrid ester-labeled second IgM antibody to obtain a first IgM antibody-antigen-second IgM antibody sandwich complex. The concentration of herpes simplex virus HSV-I in the sample to be tested was detected using a chemiluminescence method.

[0054] Preferably, the recombinant IgM antibody is coupled to magnetic beads.

[0055] Preferably, the recombinant IgM antibody is labeled with acridine ester.

[0056] Preferably, the recombinant IgM antibody is conjugated with biotin.

[0057] Preferably, the recombinant IgM antibody includes a first IgM antibody and a second IgM antibody.

[0058] Preferably, the first IgM antibody is coupled to a magnetic bead.

[0059] Preferably, the second IgM antibody is labeled with acridine ester.

[0060] Preferably, the first IgM antibody is conjugated with avidin.

[0061] Preferably, the purification is a one-step purification method using protein G.

[0062] Preferably, the adjuvant may be one or more of the following: mineral adjuvants (e.g., aluminum adjuvants, calcium phosphate adjuvants), oil adjuvants, microbial adjuvants (e.g., Corynebacterium pumilus, BCG), biodegradable particulate immune adjuvants, lipid-containing substances (e.g., liposomes, immunostimulatory complexes), molecular adjuvants (e.g., cytokines, co-stimulatory molecules), and chemical substances (e.g., levamisole, cimetidine). In some embodiments, the adjuvant may be Freund's adjuvant (made from liquid paraffin oil, anhydrous lanolin, and phosphate buffer) and / or novel adjuvants (e.g., Super quick adjuvant, Magic™ Mouse Adjuvant, Quick Antibody series of immune adjuvants, Ribi Adjuvant, Titermax, MF59, AS03, and AF3, etc.).

[0063] 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.

[0064] The HSV-I IgG antibody 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 by biotin amplification can be 16-20 times more sensitive than the IgG antibody, with a detection limit of 1:102400.

[0065] Compared with the prior art, the beneficial effects of the present invention include at least the following aspects: (1) The present invention provides an IgG antibody that specifically binds to herpes simplex virus HSV-I with a high sensitivity level, which meets the detection requirements and has high detection sensitivity, which helps to realize the detection of low abundance of herpes simplex virus HSV-I that specifically binds to it.

[0066] (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 herpes simplex virus HSV-I, this invention screened IgG monoclonal antibodies 1G9 and 4D7 by combining the KD value, which reflects the binding strength between the antibody and the antigen, and the Ka value, which reflects the antigen-antibody binding rate. This results in IgG antibodies that not only have good specificity but can also specifically recognize biomarkers, and their sensitivity and stability can also reach a high level.

[0067] (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.

[0068] (4) The recombinant IgM antibody is based on the antibody that specifically binds to herpes simplex virus HSV-I. At the same time, the present invention found that the heavy chain constant region CH1 of the IgM antibody is actually crucial for the expression, purification, spatial structure and affinity of the IgM antibody to the 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 the IgG antibody, so that the structure of the recombinant IgM antibody obtained by modification 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 the IgG antibody that specifically binds to the biomarker according to the different antigens, and the application range is wide.

[0069] (5) 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 specific binding of herpes simplex virus HSV-I by 16-20 times compared with IgG antibodies. That is, it has high sensitivity and accuracy, and low non-specific adsorption and detection limit, avoiding the occurrence of false positive and false negative results, and is suitable for detecting low abundance biomarkers.

[0070] (6) 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

[0071] 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.

[0072] Figure 1 A schematic diagram of the heavy chain expression vector for a recombinant IgM antibody that specifically binds to IgG antibodies against herpes simplex virus HSV-I; Figure 2 A schematic diagram of the structure of a light chain expression vector for a recombinant IgM antibody that specifically binds to IgG antibodies against herpes simplex virus HSV-I. Figure 3 Figure showing the results of thermally accelerated stability verification of recombinant IgM antibody against IgG antibody specifically binding to herpes simplex virus HSV-I. Detailed Implementation

[0073] 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.

[0074] 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.

[0075] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0076] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0077] 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.

[0078] 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%.

[0079] 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.

[0080] Example: This IgG antibody specifically binds to herpes simplex virus HSV-I, 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 specifically binding to herpes simplex virus HSV-I, 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.

[0081] This IgG antibody specifically binds to herpes simplex virus HSV-I, 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 specifically binding to herpes simplex virus HSV-I, 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.

[0082] In some embodiments, the IgG monoclonal antibody may also be derived from rabbits.

[0083] The gene for the specific binding IgG antibody against herpes simplex virus HSV-I, wherein the monoclonal antibody that specifically binds to the IgG antibody against herpes simplex virus HSV-I is a murine IgG monoclonal antibody 1G9, 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.

[0084] The gene for the specific binding IgG antibody against herpes simplex virus HSV-I, wherein the monoclonal antibody that specifically binds to the IgG antibody against herpes simplex virus HSV-I is a murine IgG monoclonal antibody 4D7, 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.

[0085] In some embodiments, the IgG antibody is coupled to magnetic beads.

[0086] In some embodiments, the IgG antibody is labeled with acridine ester.

[0087] In some embodiments, the IgG antibody is conjugated with biotin.

[0088] The recombinant IgM antibody that specifically binds to the IgG antibody of herpes simplex virus HSV-I is modified from the IgG antibody that specifically binds to the herpes simplex virus HSV-I 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 herpes simplex virus HSV-I, 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.

[0089] The recombinant IgM antibody that specifically binds to the IgG antibody of herpes simplex virus HSV-I is modified from the IgG antibody that specifically binds to the herpes simplex virus HSV-I 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 herpes simplex virus HSV-I, 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: 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.

[0090] 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.

[0091] 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.

[0092] In some embodiments, the recombinant IgM antibody is modified from an IgG antibody that specifically binds to a biomarker.

[0093] 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.

[0094] In some embodiments, the recombinant IgM antibody is coupled to magnetic beads.

[0095] 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).

[0096] In some embodiments, the recombinant IgM antibody is labeled with acridine ester.

[0097] In some embodiments, the recombinant IgM antibody is conjugated with biotin.

[0098] The specific method for preparing recombinant IgM antibodies is as follows: (1) Construction of expression vectors for IgM antibody heavy and light chain sequences: The amino acid sequence of the variable region of the heavy chain of the IgG antibody that specifically binds to HSV-I and the amino acid sequence fragment CH1 of the constant region of the heavy chain of the IgG antibody were recombined with the amino acid sequences of CH2, CH3, and CH4 of the constant region of the heavy chain of the IgM antibody. After codon optimization, the gene was constructed into the expression vector pcDNA3.4. (2) Cell transfection and expression: 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. (3) Select cell lines that express IgM antibodies efficiently; (4) The IgM antibody is then purified.

[0099] The detection reagent that specifically binds to herpes simplex virus HSV-I includes a first IgM antibody and a second IgM antibody, wherein the first IgM antibody and the second IgM antibody are selected from the above-mentioned recombinant IgM antibody, and the first IgM antibody and the second IgM antibody are different.

[0100] In some embodiments, the mass ratio of the first antibody to the second antibody is 1:2.

[0101] In some embodiments, the detection reagent further includes a magnetic bead-streptavidin conjugate.

[0102] In some embodiments, the first IgM antibody is conjugated with biotin or with magnetic beads, the second IgM antibody is labeled with acridine ester, and the mass ratio of the magnetic bead-streptavidin conjugate to the biotin-conjugated first IgM antibody is 1:2; the feed ratio of the second IgM antibody to the acridine ester includes 1:10-30.

[0103] In some embodiments, the first IgM antibody and the second IgM antibody are modified from IgG antibodies that specifically bind to biomarkers.

[0104] In some embodiments, the first IgM antibody and the second IgM antibody include a heavy chain constant region, a light chain constant region, a heavy chain variable region, and a light chain variable region.

[0105] 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.

[0106] 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.

[0107] In some embodiments, the detection reagent includes a first IgM antibody conjugated to magnetic beads and a second IgM antibody labeled with acridine ester.

[0108] 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 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. (g) Add 5 mL of blocking buffer (1% BSA) to resuspend, and obtain the detection reagent. Store at 4 °C for later use.

[0109] This invention employs the use of antibodies described in any one of the first to fifth aspects of the present invention in the preparation of in vitro immunodiagnostic reagents.

[0110] 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.

[0111] This detection kit for specific binding to herpes simplex virus HSV-I includes the detection reagent described in the sixth aspect of this invention.

[0112] This describes the application of the aforementioned detection reagent in the preparation of in vitro immunodiagnostic reagents.

[0113] 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.

[0114] The method for improving the sensitivity of herpes simplex virus detection includes the following steps: The heavy chain variable region sequence of a mouse or rabbit IgG monoclonal antibody, the amino acid sequence fragment CH1 of the heavy chain constant region of a mouse or rabbit IgG antibody, and the amino acid sequence fragments CH2-CH4 of the heavy chain constant region of a mouse or rabbit recombinant IgM antibody are genetically recombined to obtain a heavy chain expression vector; the heavy chain expression vector and the light chain expression vector are expressed in cells to obtain a recombinant IgM antibody; the IgG antibody is the first or / and second type of IgG antibody specifically binding to herpes simplex virus HSV-I as described in the first aspect of the present invention; The recombinant IgM antibody was used to prepare a detection reagent to detect the concentration of herpes simplex virus HSV-I in the sample to be tested.

[0115] In some specific embodiments, the method for improving the sensitivity of herpes simplex virus detection includes the following steps: Animal immune response to S1'IgG antibodies: Animals were immunized with antigen, CpG and adjuvant to induce the production of IgG antibodies; S2' cell screening: B lymphocytes of animals are fused with myeloma cells to obtain hybridoma cells; positive screening of hybridoma cells is performed, and IgG monoclonal antibodies are collected from the cultures of positive hybridoma cells; S3'IgG antibody sequencing: The IgG monoclonal antibody obtained in step S2 is sequenced to obtain the amino acid sequences of the heavy chain variable region and the light chain variable region of the IgG monoclonal antibody; S4' IgG antibody recombinant IgM antibody: The sequence of the variable region of the heavy chain of the 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; S5' Recombinant IgM antibody detection of herpes simplex virus: The recombinant IgM antibody is prepared into the detection reagent described in the sixth aspect of the present invention to detect the concentration of herpes simplex virus HSV-I in the sample to be tested.

[0116] The specific steps of step S5' are as follows: The magnetic bead-streptavidin conjugate and the biotin-modified first IgM antibody were mixed to obtain a premix; The premix and the sample to be tested are mixed to obtain a magnetic bead-first IgM antibody-antigen conjugate; The magnetic bead-first IgM antibody-antigen conjugate is mixed with the acrid ester-labeled second IgM antibody to obtain a first IgM antibody-antigen-second IgM antibody sandwich complex. The concentration of herpes simplex virus HSV-I in the sample to be tested was detected using a chemiluminescence method.

[0117] In some embodiments, the recombinant IgM antibody is coupled to magnetic beads.

[0118] In some embodiments, the recombinant IgM antibody is labeled with acridine ester.

[0119] In some embodiments, the recombinant IgM antibody is conjugated with biotin.

[0120] In some embodiments, the recombinant IgM antibody includes a first IgM antibody and a second IgM antibody.

[0121] In some embodiments, the first IgM antibody is coupled to a magnetic bead.

[0122] In some embodiments, the second IgM antibody is labeled with acridine ester.

[0123] In some embodiments, the first IgM antibody is conjugated with avidin.

[0124] In some embodiments, the purification is a one-step purification of protein G.

[0125] In some embodiments, the adjuvant may be one or more of the following: mineral adjuvants (e.g., aluminum adjuvants, calcium phosphate adjuvants), oil adjuvants, microbial adjuvants (e.g., Corynebacterium pumilus, BCG), degradable particulate immune adjuvants, lipid-containing substances (e.g., liposomes, immunostimulatory complexes), molecular adjuvants (e.g., cytokines, co-stimulatory molecules), and chemical substances (e.g., levamisole, cimetidine). In some embodiments, the adjuvant may be Freund's adjuvant (made from liquid paraffin oil, anhydrous lanolin, and phosphate buffer) and / or novel adjuvants (e.g., Superquick adjuvant, Magic™ Mouse Adjuvant, QuickAntibody series of immune adjuvants, RibiAdjuvant, Titermax, MF59, AS03, and AF3, etc.).

[0126] In some embodiments, 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.

[0127] Specific application example: This method for improving the sensitivity of herpes simplex virus detection includes the following steps: S1: Animal immune response, specifically: S11 immunized animals: 4 female SJL mice; S12 Immunization Protocol: 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 second immunization was given 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. Specific procedures for implementing S13 immunization: On day 0, mice were immunized for the first time with 10 μg CpG, 50 μg manganese chloride and 50 μg HSV-I antigen, 100 μL / mouse was injected subcutaneously. On day 14, mice were immunized twice with 10 μg CpG, 50 μg manganese chloride and 25 μg HSV-I 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 HSV-I antigen, with 100 μL / mouse injected subcutaneously. Immunization Program 2: On day 0, 25 μg of HSV-I antigen protein was mixed with water-soluble adjuvant at a 1:1 ratio and injected subcutaneously. On day 14, the water-soluble adjuvant was mixed with 25 μg of HSV-I antigen protein at a 1:1 ratio and injected subcutaneously for a second dose of immunization. On day 28, 25 μg of HSV-I antigen protein was mixed with water-soluble adjuvant at a 1:1 ratio and injected subcutaneously for three doses of immunization. Ten days after immunization, mouse serum was collected and serially diluted. The dilution endpoint was determined using an indirect ELISA method, and the highest dilution considered positive was defined as the serum binding antibody titer. The results of three HSV-I immunohistometry tests are shown in Table 1. This invention differs from the prior art (CN 107456575 A) in its immunization protocol. The prior art uses manganese adjuvant for CpG immunization, while this invention uses an aqueous adjuvant, achieving superior immunization effects with less antigen load and lower injection dosage, and highly efficiently activating immune cells.

[0128] Table 1. Antibody titers in serum after three immunizations of HSV-I mice under two immunization regimens (×10⁻¹⁰) 4 ) Advantages: While existing patents mention using manganese adjuvant (manganese chloride) instead of Freund's adjuvant to immunize mice, achieving good immunization effects with lower antigen load and 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, preservation of the antigen's native conformation, high antibody affinity, and ease of use. In immunization against HSV-I antigen, antibody titers produced using aqueous adjuvants were significantly higher than those produced using manganese adjuvants.

[0129] S2: Cell screening, specifically: S21: Sorting mouse B lymphocytes; a. The ELISA results of mouse serum antibody titers are shown in Table 2, indicating that the serum titers of all immunized mice were greater than 1:10. 4 This achieves the requirements for cell fusion. Mice with the required immunotiter were used for B lymphocyte sorting. 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 (synthetic cell culture medium), and pass it through a sieve to prepare a spleen cell suspension; Alternatively, A: Gently scrape the cells with tweezers, aspirate the cell suspension into a centrifuge tube, allow it to settle naturally for 5 minutes, transfer the suspension to another centrifuge tube, discard any larger tissue fragments, and centrifuge to precipitate the cells; B: Place the spleen on a 200-mesh stainless steel mesh, 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 through a nylon mesh to obtain a single-cell suspension, and centrifuge 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; S22 fusion of myeloma cells (SP2 / 0) and 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 expanded to a 1:5 scale. c. When SP2 / 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 using HAT. 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. 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; S23-selective 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 test. The HSV-I antibody affinity data are shown in Table 2. Table 2. Results of antibody affinity tests on the HSV-I10 monoclonal cell line 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 HSV-I-1G9, HSV-I-4D7, HSV-II-8C4, and HSV-II-11C9 for expanded subculture; f. Centrifuge, collect the supernatant, and perform affinity chromatography on a Protein G column to obtain 95% high-purity IgG monoclonal antibody from the hybridoma culture supernatant sample.

[0130] S3: IgG antibody sequencing; specifically: sequencing the variable region of mouse HSV-I IgG antibody, and obtaining the amino acid sequences of the complementarity-determining regions CDRH1-CDRH3 of the heavy chain variable region of the monoclonal antibody 1G9 that specifically binds to HSV-I, as shown in SEQ ID NO: 1-SEQ ID NO: 3; the amino acid sequences of the complementarity-determining regions CDRL1-CDRL3 of the light chain variable region, as shown in SEQ ID NO: 4-SEQ ID NO: 6; SEQ ID NO: 1: DYEMH SEQ ID NO: 2: AIHPGSGGTAYNQKFKG SEQ ID NO: 3: GSADAMDY SEQ ID NO: 4: KAGQSLLNSGNQSKAYLT SEQ ID NO: 5: WASSGYS SEQ ID NO: 6: AGDSSYPLT The amino acid sequences of the complementarity-determining regions CDRH1-CDRH3 of the heavy chain variable region of the obtained monoclonal antibody 4D7 that specifically binds to HSV-I 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.

[0131] SEQ ID NO: 7: DYEAH SEQ ID NO: 8: AIHAGSGGTAYNQKFKG SEQ ID NO: 9: GSGDAYDY SEQ ID NO: 10: KGGQSLLNSGNQKAYLT SEQ ID NO: 11: WASTGYS SEQ ID NO: 12: ANDSSYPLT S4: IgM conversion of IgG antibodies, specifically: The heavy chain variable region sequence of mouse IgG antibodies that specifically bind to HSV-I (monoclonal antibodies 1G9 and 4G7 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 recombinant 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 recombinant IgM antibody. The structure of the light chain expression vector for mouse IgG antibodies that specifically bind to HSV-I 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 then 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, specifically: S51 resuscitates CHO cells: a. Thaw cryopreserved 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; S52 transfection-grade plasmid extraction: a. Add 80 ng of expression plasmid to 200 μL of competent cell suspension; b. Heat shock competent cells with added plasmid at 42°C for 90 seconds, transform them, and then cool them on ice for 5 minutes. 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-clonal cells and seed them into 200 mL of LB medium, then incubate at 37°C 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 minutes; h. Add 250 μL of NB solution, incubate at 37°C for 2 min, and 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; S53 transfection: a. Add the light chain and heavy chain expression plasmids of HSV-I 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°C with shaking at 120 rpm for 5-6 days; c. Centrifuge and collect the supernatant; S6: Purification of recombinant IgM antibody derived from IgG antibody modification and recombination, specifically as follows: S61 HSV-I cell supernatant was purified for antibody purification using a 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 minutes 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.

[0132] S62 used SDS-PAGE to identify the purity of the purified HSV-I antibody: a. The accelerated stability of the modified recombinant IgM antibody solution was investigated at 37℃ for 0, 3, 7, and 14 days.

[0133] 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. f. Use 90V for the stacking gel section and 15-20min for the separating gel section; use 180V for the separating gel section and 30-50min for the separating gel section. g. Stain the gel with Coomassie Brilliant Blue R250 for 15 min; h. Add decolorizing solution, microwave and boil for 1 minute to accelerate decolorization, repeat 3 times until the gel background color is nearly colorless; the thermal accelerated stability verification results of the modified HSV-I IgM antibody are as follows. Figure 3 As shown; like Figure 3Lanes 1-9 in the diagram show the thermally accelerated stability verification results of the marker, IgM antibody 1G9 on days 0, 3, 7, and 14, and IgM antibody 4D7 on days 0, 3, 7, and 14, respectively. The recombinant IgM antibody obtained by combining the CH1 fragment of the mouse IgG1 constant region with the CH2 / CH3 / CH4 constant region of mouse IgM 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 validation of the modified HSV-I recombinant IgM antibody, specifically: S71: Affinity test of recombinant IgM antibody against HSV-I modified from IgG antibody; The supernatant of IgG-1G9, IgG-4D7, IgM-1G9, and IgM-4D7 cells was used for Biacore assay. The results are shown in Table 3. Table 3. Biacore test results of HSV-I IgG and IgM antibodies. The modified recombinant IgM antibody for HSV-I has a higher Ka value than its corresponding IgG antibody, indicating that it binds to the antigen faster and can capture HSV-I more quickly, making it more suitable for in vitro diagnostics.

[0134] S72: Sensitivity detection results of HSV-I IgG antibody and recombinant IgM antibody of modified HSV-I; This embodiment verifies the conventional IgG system (specifically, magnetic beads-IgG1G9 / acridone ester-IgG4D7, referred to as IgG antibody) and the IgM system (specifically, magnetic beads-IgM1G9 / acridone ester-IgM4D7, referred to as IgM antibody). The IgG antibody was diluted with Tris buffer at 100, 300, 900, 2700, 8100, 24300, 72900, 218700, and 656100 times, respectively; the modified recombinant 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 HSV-I IgG antibody and recombinant IgM antibody against modified HSV-I. The results showed that the highest measured value of the HSV-I IgG antibody raw material reached 1,100,000, and the highest measured value of the HSV-I recombinant IgM antibody raw material reached 5,900,000; the linear peak of the HSV-I IgG antibody raw material was approximately 650,000, and the linear peak of the HSV-I recombinant IgM antibody raw material was approximately 2,700,000. In other words, the above results indicate that the sensitivity of the HSV-I IgG antibody prepared by this invention can reach a high level, which is sufficient for detection needs. The sensitivity of the HSV-I recombinant IgM antibody obtained by the modification of this invention is further improved, and can be controlled to be 4-5 times that of the HSV-I IgG antibody.

[0135] S73: Precision detection of the modified recombinant IgM antibody; The modified HSV-I recombinant IgM antibody was diluted 4500 times, 20000 times, and 50000 times with Tris buffer, and was repeatedly measured 10 times for one negative reference, one positive reference, and one limit of detection sample. The coefficient of variation (CV (%) = SD (standard deviation) / mean (mean) × 100%) should not be greater than 10.0%. The results are shown in Table 5. Table 5. Precision testing of recombinant IgM antibodies against modified HSV-I The results are shown in Table 5. The recombinant IgM antibody of HSV-I obtained by the present invention was positive for both the positive repeatability reference and the limit of detection reference, and negative for both the negative repeatability reference. The coefficient of variation (CV) could be controlled to be less than 5%.

[0136] S74: Inter-batch variation detection of the modified recombinant IgM antibody; Three batches of HSV-I pilot samples were produced, and 25 negative samples were simultaneously detected using chemiluminescence immunoassay. The relative deviation was calculated. The results are shown in Table 6.

[0137] Table 6. Inter-batch variation detection of recombinant IgM antibody with modified HSV-I The results are shown in Table 6. The recombinant IgM antibody obtained by the present invention has small batch-to-batch variation and the relative deviation can be controlled within ±5%.

[0138] S75: Stability test of the modified recombinant IgM antibody; The stability of the modified HSV-I recombinant 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.

[0139] Table 7. Stability test of recombinant IgM antibody against modified HSV-I The results, as shown in Table 7, indicate that the recombinant IgM antibody obtained by the present invention meets the stability requirements.

[0140] S8: Magnetic bead-conjugated antibody, specifically: Component 1: Preparation of magnetic bead-streptavidin conjugate; 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 0.01 M PBS buffer and vortex for 30 seconds; 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, and containing 1% BSA) to resuspend, react at 25℃ for 1 h to block; h. Use magnetic adsorption to remove the supernatant. Wash three times with 200µL PBS solution (pH 7.2) each time, then resuspend in 0.01M PBS, 0.05% Tween 20, and 5% sucrose solution and store at 4°C.

[0141] Component 2: Preparation of recombinant IgM antibody 1G9-biotin conjugate of HSV-I; 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 1G9 concentration to 2.0 mg / mL; c. Take 500 μL of recombinant IgM antibody 1G9 and 0.33-6.66 μL of 10 mM / L NHS-Biotin (feed ratio of 1:2-1:10), vortex mix, and then rotate mix at 25℃-37℃ for 30 min-2 h; preferably at 25℃ for 30 min. d. Add 300 μL of 0.1 M PBS buffer and dialyze, wash twice to obtain biotin-modified IgM antibody 1G9.

[0142] Component 3: Preparation of recombinant IgM antibody 4D7-acrididine ester conjugate of HSV-I; a. Dilute IgM antibody 4D7 to 2 mg / mL using 0.01 M pH 7.2 PBS; b. Take 1 mL of recombinant IgM antibody 4D7 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 4D7-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.

[0143] S9: HSV-I capture in the sample; a. Based on antibody mass, component 1 (i.e., magnetic bead-streptavidin conjugate) and component 2 (i.e., HSV-I recombinant IgM antibody 1G9-biotin conjugate) are premixed at a ratio of 1:2 to form magnetic bead-IgM antibody 1G9 complex. b. Add 20 μL of magnetic bead-IgM antibody 1G9 complex to 100 μL of sample, and incubate at 37°C 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. Resuspend the conjugate in 20 μL of purified water to obtain the magnetic bead-IgM antibody 1G9-antigen conjugate. Mix the magnetic bead-IgM antibody 1G9-antigen conjugate with 20 μL of component 3 (i.e., IgM antibody 4D7-acrididine ester conjugate) by rotation and incubate at 37°C 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.

[0144] Table 8. Comparison of biotin signal amplification between HSV-I IgG antibody and recombinant IgM antibody modified with HSV-I. The above results show that the sensitivity of the HSV-I 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 recombinant 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.

[0145] 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).

[0146] 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 herpes simplex virus HSV-1, 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 herpes simplex virus HSV-I, wherein the IgG monoclonal antibody is murine; 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.

2. An IgG antibody that specifically binds to herpes simplex virus HSV-1, 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 herpes simplex virus HSV-I, wherein the IgG monoclonal antibody is murine; 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.

3. A gene that specifically binds to IgG antibodies against herpes simplex virus (HSV-I), characterized in that, The monoclonal antibody that specifically binds to the IgG antibody against herpes simplex virus HSV-I is a murine IgG monoclonal antibody 1G9. 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.

4. A gene that specifically binds to IgG antibodies against herpes simplex virus (HSV-I), characterized in that, The monoclonal antibody that specifically binds to the IgG antibody against herpes simplex virus HSV-I is a murine IgG monoclonal antibody 4D7. 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.

5. A recombinant IgM antibody that specifically binds to IgG antibodies against herpes simplex virus (HSV-I), characterized in that, The recombinant IgM antibody is modified from the IgG antibody that specifically binds to herpes simplex virus HSV-I 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 herpes simplex virus HSV-I, 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 IgG antibodies against herpes simplex virus (HSV-I), characterized in that, The recombinant IgM antibody is modified from the IgG antibody that specifically binds to herpes simplex virus HSV-I 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 herpes simplex virus HSV-I, 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 herpes simplex virus HSV-I, characterized in that, It includes a first IgM antibody and a second IgM antibody, wherein the first IgM antibody and the second IgM antibody are selected from the recombinant IgM antibodies of claims 5 and / or 6, and the first IgM antibody and the second IgM antibody are different.

8. A detection kit that specifically binds to herpes simplex virus HSV-I, 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 herpes simplex virus detection, characterized in that, Includes the following steps: The heavy chain variable region sequence of a mouse or rabbit IgG monoclonal antibody, the amino acid sequence fragment CH1 of the heavy chain constant region of a mouse or rabbit IgG antibody, and the amino acid sequence fragments CH2-CH4 of the heavy chain constant region of a mouse or rabbit recombinant IgM antibody are genetically recombined to obtain a heavy chain expression vector; the heavy chain expression vector and the light chain expression vector are expressed in cells to obtain a recombinant IgM antibody; the IgG antibody is the IgG antibody that specifically binds to herpes simplex virus HSV-I as described in claim 1 or / and 2. The recombinant IgM antibody was used to prepare a detection reagent to detect the concentration of herpes simplex virus HSV-I in the sample to be tested.

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