Rabbit-origin measles virus monoclonal antibody, related biological materials, kit and application

Abstract

The application discloses a rabbit-origin measles virus monoclonal antibody, related biological materials, a kit and application, and belongs to the field of antibody engineering. A CDR1 sequence in a heavy chain variable region of the antibody is shown in any one of SEQ ID NOs:1-6, a CDR2 sequence is shown in any one of SEQ ID NOs:7-12, and a CDR3 sequence is shown in any one of SEQ ID NOs:13-18. A CDR1 sequence in a light chain variable region is shown in any one of SEQ ID NOs:19-24, a CDR2 sequence is shown in any one of SEQ ID NOs:25-30, and a CDR3 sequence is shown in any one of SEQ ID NOs:31-36. The monoclonal antibody can specifically combine with a conserved epitope of measles virus hemagglutinin, can effectively capture low-load virus particles, realizes rapid, accurate and convenient on-site detection, and has an excellent application prospect.

Description

Technical Field

[0001] This invention belongs to the field of antibody engineering, and particularly relates to a rabbit-derived measles virus monoclonal antibody, related biological materials, reagent kits, and applications. Background Technology

[0002] Measles is an acute, exanthematous respiratory infectious disease caused by the measles virus (MV), with humans being its only known host. The virus is highly contagious, with an infection rate as high as 90% in unimmunized individuals. Current laboratory diagnosis of measles primarily relies on serum IgM antibody detection and RT-PCR nucleic acid testing. However, both have significant limitations: IgM antibodies are typically detectable several days after the rash appears, with a sensitivity of only 56.53% within 0-3 days after rash onset; while patients are infectious approximately 4 days before rash appearance, leading to a severe misalignment between the detection and infectious window periods and a high false-negative rate in the early stages. RT-PCR can achieve a sensitivity of 94.39% in the early stages of the disease, but limitations in sample collection, transportation, and specialized equipment make it difficult to meet the needs of rapid screening at the grassroots level and in the field. Therefore, developing rapid diagnostic tools suitable for the early stages of the disease is crucial for breaking the chain of transmission.

[0003] Rabbit monoclonal antibodies (RabMAbs) have demonstrated unique advantages in diagnostic applications in recent years. Due to the special nature of the rabbit immune system, RabMAbs often exhibit higher affinity and specificity, enabling them to recognize a wider range of antigenic epitopes. Their dissociation constant (KD) can be 10 to 100 times higher than that of mouse monoclonal antibodies; simultaneously, they can accurately identify subtle epitope changes. In recent years, with breakthroughs in core technologies such as hybridoma technology, phage display, and single B cell antibody screening, RabMAbs have been applied in the field of virus detection. Studies have successfully used them in the development of diagnostic methods for pathogens such as bovine herpesvirus type 1 and porcine epidemic diarrhea virus. However, existing measles virus detection methods are mostly limited to nucleoprotein (NP) targets, and there are currently no rabbit monoclonal antibody diagnostic products targeting hemagglutinin (H) protein. Summary of the Invention

[0004] Objectives of this invention: The first objective is to provide a rabbit-derived monoclonal antibody against measles virus hemagglutinin protein that exhibits strong affinity and high stability; the second objective is to provide nucleic acid molecules, recombinant vectors, recombinant cells, antibody conjugates, and reagent kits encoding this rabbit-derived measles virus monoclonal antibody; and the third objective is to provide applications of this rabbit-derived measles virus monoclonal antibody.

[0005] Technical Solution: The rabbit-derived measles virus monoclonal antibody or its antigen-binding fragment of the present invention, wherein the heavy chain CDR1 amino acid sequence of the antibody or its antigen-binding fragment is shown in any one of SEQ ID NO: 1-6, the heavy chain CDR2 amino acid sequence is shown in any one of SEQ ID NO: 7-12, the heavy chain CDR3 amino acid sequence is shown in any one of SEQ ID NO: 13-18, the light chain CDR1 amino acid sequence is shown in any one of SEQ ID NO: 19-24, the light chain CDR2 amino acid sequence is shown in any one of SEQ ID NO: 25-30, and the light chain CDR3 amino acid sequence is shown in any one of SEQ ID NO: 31-36.

[0006] Preferably, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is as shown in any one of SEQ ID NO: 37, 39, 41, 43, 45, 47 or has at least 80% similarity to the sequence shown in any one of SEQ ID NO: 37, 39, 41, 43, 45, 47, and the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO: 38, 40, 42, 44, 46, 48 or has at least 80% similarity to the sequence shown in any one of SEQ ID NO: 38, 40, 42, 44, 46, 48.

[0007] Preferably, the antigen-binding fragment includes any one or more of F(ab')2, Fab', Fab, Fv, ScFv, and dsFv.

[0008] Preferably, the antibody further includes a portion or all of the heavy chain constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, as well as a portion or all of the κ chain or λ chain constant region.

[0009] The nucleic acid molecule described in this invention encodes the aforementioned rabbit-derived measles virus monoclonal antibody or its antigen-binding fragment.

[0010] The recombinant vector of the present invention comprises the aforementioned nucleic acid molecules.

[0011] The recombinant cells described in this invention contain the aforementioned nucleic acid molecules or the aforementioned recombinant vectors.

[0012] The antibody conjugate of the present invention is obtained by conjugating the aforementioned rabbit-derived measles virus monoclonal antibody or its antigen-binding fragment with a label, wherein the label is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, radioactive labeling, and nanoparticle labeling.

[0013] The kit described in this invention contains the aforementioned rabbit-derived measles virus monoclonal antibody or its antigen-binding fragment, or antibody-drug conjugate.

[0014] The application of the rabbit-derived measles virus monoclonal antibody or its antigen-binding fragment, or antibody-drug conjugate, or kit described in this invention in the detection of measles virus.

[0015] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The rabbit-derived measles virus monoclonal antibody provided by the present invention can specifically bind to the conserved epitope of measles virus hemagglutinin, with strong affinity and high stability; 2. The rabbit-derived measles virus monoclonal antibody can effectively capture low-load virus particles and can be used in colloidal gold immunochromatographic platforms to achieve rapid, accurate and convenient on-site detection of measles virus infection, with excellent application prospects. Attached Figure Description

[0016] Figure 1 A statistical chart showing the serum antibody titer results after three immunizations of rabbits;

[0017] Figure 2 Figure showing the results of the analysis of rabbit-derived measles virus monoclonal antibody expression.

[0018] Figure 3 Figure showing the validation results of the binding ability of rabbit-derived measles virus monoclonal antibody to B cell antigen epitopes;

[0019] Figure 4 The image shows the results of the viral binding ability verification of rabbit-derived measles virus monoclonal antibodies.

[0020] Figure 5 This figure shows the results of validating the virus binding ability of rabbit-derived measles virus monoclonal antibodies based on the double-antibody sandwich method. Detailed Implementation

[0021] The technical solution of the present invention will be further described below.

[0022] Example 1: Rabbit immunization, single B cell sorting and sequencing

[0023] 1. Rabbit Immunity

[0024] Male New Zealand rabbits weighing 2 kg (purchased from Yizheng Anlimao Biotechnology Co., Ltd.) were selected. Measles live attenuated vaccine solution (based on measles virus Shanghai-191 attenuated strain, provided by Shanghai Institute of Biological Products Co., Ltd.) was used as the immunogen. The protein content in the stock solution was determined using the BCA protein detection kit (purchased from Thermo Fisher Scientific, catalog number 23225) as a reference standard for dosage.

[0025] For the initial immunization, a live attenuated measles vaccine with a protein content of 200 μg was emulsified with Freund's complete adjuvant (purchased from Merck Darmstadt, Germany, catalog number F5881-10ml) at a volume ratio of 1:1 and administered intramuscularly. The second immunization was given 28 days after the initial immunization, using a live attenuated measles vaccine with a protein content of 100 μg, emulsified with Freund's incomplete adjuvant (purchased from Merck Darmstadt, Germany, catalog number F5506-10ml) at a volume ratio of 1:1 and administered intramuscularly. The third immunization was given 28 days after the second immunization, using the same method as the second immunization.

[0026] Before the first immunization, 5 mL of blood was collected from the marginal ear vein of rabbits, and the serum was separated as a negative control serum. 28 days after the third immunization, blood was collected from the apex of the heart of rabbits, and immune serum was separated.

[0027] 2. Serum antibody titer determination

[0028] The aforementioned negative control serum (non-immunized group) and immune serum (immunized group) were used to detect antibody specificity using an indirect enzyme-linked immunosorbent assay (ELISA). The steps were as follows: The measles live attenuated vaccine solution with the previously determined protein concentration was diluted with 0.05M carbonate buffer (pH 9.6) to a protein concentration of 1 μg / mL. 50 μL was added to each well of the ELISA plate and incubated overnight at 4°C. After coating, the plate was washed four times with PBS buffer (PBST) containing 0.5% (v / v) Tween 20. 200 μL of PBST containing 1 g / 100 mL casein was added to each well, and the plate was blocked at 37°C for 2 h. After blocking, the plate was washed four times with PBST, and then 100 μL of PBST containing 1 g / 100 mL casein was added to each well. Incubate negative control serum or immune serum diluted with PBST at ratios of 1:25600, 1:51200, 1:102400, 1:204800, 1:409600, 1:819200, 1:1638400, or 1:3276800 for 1 h at 37°C. After incubation, wash four times with PBST, then add HRP-labeled goat anti-rabbit secondary antibody diluted 1:5000 and incubate at 37°C for 1 h. After incubation, wash four times with PBST, and develop the color using TMB chromogenic reagent solution (purchased from Thermo Fisher Scientific, catalog number 002023) at room temperature in the dark for 20 min. Then, add 50 μL of 1 M H2SO4 to stop the color development. Measure the absorbance (OD) at 450 nm using a microplate reader. 450 ), and plot the absorbance-dilution ratio curve.

[0029] The results are as follows Figure 1 As shown, the antibody titer in the rabbit serum after the third immunization was greater than 3,276,800, indicating that a high-titer rabbit polyclonal antibody serum against measles virus had been obtained.

[0030] 3. Prediction of conserved epitopes of measles virus hemagglutinin

[0031] The conservation and secondary structure of peptides were predicted using the NCBI online database, and the hydrophilicity of peptides was predicted using the Thermi Fisher peptide analysis tool. Finally, three highly conserved peptides across genotypes were identified in the H protein: ES (positions 235-250 of B3 H protein, sequence as shown in SEQ ID NO: 49), EY (positions 379-410 of B3 H protein, sequence as shown in SEQ ID NO: 50), and GL (positions 190-205 of B3 H protein, sequence as shown in SEQ ID NO: 51).

[0032] The B-cell epitope prediction of the peptides was analyzed using IEDB software. The results confirmed the identification of the conserved epitope LSSKRSEL (SEQ ID NO: 52) in the ES peptide; the conserved epitope CVPLKDNR (SEQ ID NO: 53) in the EY peptide; and the conserved epitope GQFSNM (SEQ ID NO: 54) in the GL peptide.

[0033] 4. Specific single B cell sorting

[0034] Peripheral blood was collected from rabbits 28 days after the third immunization. Peripheral blood was separated from rabbit peripheral blood lymphocyte separation medium (purchased from Wuhan Saiwei Biotechnology Co., Ltd., catalog number G2099) at a volume ratio of 3:1 by density gradient centrifugation at 400 × g for 30 min at room temperature. The mononuclear cell layer (white membrane layer) was aspirated, and PBS buffer containing 1% (w / v) bovine serum albumin (BSA) was added. The cells were centrifuged at 200 × g for 10 min at 4°C, and the supernatant was discarded. Washing was repeated 3 times. The cells were resuspended in PBS buffer and counted to obtain a concentration of 1.5 × 10⁻⁶ cells / mL. 6 A suspension of peripheral blood mononuclear cells (PBMCs) per mL was used for subsequent sorting.

[0035] Beijing Zexiyuan Biotechnology Co., Ltd. was commissioned to synthesize N-terminal biotinylated ES, EY, and GL peptides. These were then conjugated with fluorescently labeled streptavidin to prepare antigen tetramers. Specifically, 5 μg of N-terminal biotinylated ES, EY, or GL peptides were mixed with either APC-labeled streptavidin (SA-APC, purchased from Thermo Fisher Scientific, catalog number SA1005) or FITC-labeled streptavidin (SA-FITC, purchased from Thermo Fisher Scientific, catalog number SA1001) at a molar ratio of 1:4. The mixtures were incubated on ice in the dark for 10 minutes. The incubation was repeated 10 times to allow the fluorescently labeled streptavidin to fully bind with the biotinylated peptide, ultimately yielding six fluorescently labeled antigen monomers: APC-labeled ES peptide tetramer (ES-SA-APC), APC-labeled EY peptide tetramer (EY-SA-APC), APC-labeled GL peptide tetramer (GL-SA-APC), FITC-labeled ES peptide tetramer (ES-SA-FITC), FITC-labeled EY peptide tetramer (EY-SA-FITC), and FITC-labeled GL peptide tetramer (GL-SA-FITC).

[0036] The aforementioned PBMC suspension was stained with 1% (v / v) PE-labeled goat anti-rabbit IgG cross-adsorption secondary antibody (Thermo Fisher Scientific, catalog number P2771MP), 1% (v / v) live / dead cell dye (Thermo Fisher Scientific, catalog number L34994), 2% (v / v) APC-labeled antigen tetramer, and 2% (v / v) FITC-labeled antigen tetramer. The mixture was incubated on ice in the dark for 30 min. Sorting was performed using a 4-laser 16-color flow cytometer, with the following gate sequence: single cell population, live cell population, PE... + Cell population, APC detected within this phylum + FITC + The proportion of double-positive cell populations was determined, and these double-positive cell populations were collected to obtain single B cells.

[0037] 5. B-cell receptor sequencing

[0038] Rabbit single B cells obtained by flow cytometry sorting were used as starting material. The cDNA of the variable region of the heavy chain of B cell receptor IgG and the variable region of the light chain Kappa was specifically amplified using a commercial single B cell amplification kit (purchased from Nanjing Detai Biotechnology Co., Ltd., catalog number DTT07). The cDNA was then sent to Sangon Biotech Co., Ltd. for sequencing to obtain the variable region gene sequence.

[0039] Six rabbit-derived measles virus monoclonal antibodies were obtained, including: GL-A2, with the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 37 and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 38; GL-A11, with the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 39 and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 40; GL-B7, with the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 41 and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 42; ES-A8, with the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 43 and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 44; ES-A13, with the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 45 and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 46; and EY-E14, with the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 47 and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 48.

[0040] Example 2: Expression of rabbit-derived measles virus monoclonal antibody

[0041] 1. Construction of rabbit-derived measles virus monoclonal antibody plasmid

[0042] Through PrimeSTAR ®The Max DNA Polymerase kit (purchased from Baori Biotechnology Co., Ltd., catalog number R045) was used as a template to amplify the cDNA of the B cell receptor IgG heavy chain variable region and light chain Kappa variable region obtained by the above-mentioned amplification and sequencing. The kit was used with TY-A2-HF (SEQ ID NO: 55) and TY-A2-HR (SEQ ID NO: 56) to amplify the GL-A2 heavy chain variable region coding sequence fragment with homologous arms, with TY-A2-HF and TY-A11-HR (SEQ ID NO: 57) to amplify the GL-A11 heavy chain variable region coding sequence fragment with homologous arms, with TY-A1A12B5B7-HF (SEQ ID NO: 58) and TY-A1A8A12B-HR (SEQ ID NO: 59) to amplify the GL-B7 heavy chain variable region coding sequence fragment with homologous arms, and with TY-B2B3B9-HF (SEQ ID NO: 58) to amplify the GL-B7 heavy chain variable region coding sequence fragment with homologous arms. The ES-A8 heavy chain variable region coding sequence fragment with homologous arms was obtained by amplification using TY-A1A8A12B-HR. The ES-A13 heavy chain variable region coding sequence fragment with homologous arms was obtained by amplification using TY-A1A12B5B7-HF and TY-A1A8A12B-HR. The EY-E14 heavy chain variable region coding sequence fragment with homologous arms was obtained by amplification using TY-E13-HF (SEQ ID NO: 61) and TY-A1A8A12B-HR. The GL-A2 and GL-B7 light chain Kappa variable region coding sequence fragments with homologous arms were obtained by amplification using TY-LF (SEQ ID NO: 62) and TY-LR (SEQ ID NO: 63). The GL-A11 light chain Kappa variable region coding sequence fragment with homologous arms was obtained by amplification using TY-A11-LF (SEQ ID NO: 64) and TY-LR. The ES-A8 heavy chain variable region coding sequence fragment with homologous arms was obtained by amplification using TY-ESA8-LF (SEQ ID NO: 60). NO: 65) TY-LR amplification yielded an ES-A8 light chain Kappa variable region coding sequence fragment with a homologous arm. TY-ESA13-LF (SEQ ID NO: 66) TY-LR amplification yielded an ES-A13 light chain Kappa variable region coding sequence fragment with a homologous arm. TY-E14-LF (SEQ ID NO: 67) TY-LR amplification yielded an EY-E14 light chain Kappa variable region coding sequence fragment with a homologous arm.Simultaneously, using the eukaryotic expression vector containing the rabbit monoclonal antibody heavy chain constant region (SEQ ID NO: 72) as a template, amplification was performed using CHF (SEQ ID NO: 68) and SP-HR (SEQ ID NO: 69) to obtain the eukaryotic expression vector fragment containing the rabbit monoclonal antibody heavy chain constant region; using the eukaryotic expression vector containing the rabbit monoclonal antibody light chain constant region (SEQ ID NO: 73) as a template, amplification was performed using CLF (SEQ ID NO: 70) and SP-LR (SEQ ID NO: 71) to obtain the eukaryotic expression vector fragment containing the rabbit monoclonal antibody light chain constant region.

[0043] The products were subjected to 1% agarose gel electrophoresis and purified using a DNA recovery and purification kit (purchased from Sangon Biotech Co., Ltd., catalog number B610353-0200) to obtain heavy chain variable region nucleic acid fragments and light chain variable region nucleic acid fragments with homologous arms GL-A2, GL-A11, GL-B7, ES-A8, ES-A13, and EY-E14, as well as heavy chain and light chain expression vectors with homologous arms.

[0044] Using homologous recombinase (purchased from Ibotek Biotechnology Co., Ltd., catalog number RK21020), the coding sequence fragments of the heavy chain variable region of GL-A2, GL-A11, GL-B7, ES-A8, ES-A13, and EY-E14 were homologously recombined into eukaryotic expression vector fragments containing the constant region of the rabbit monoclonal antibody heavy chain, resulting in 6 recombinant rabbit monoclonal antibody heavy chain eukaryotic expression plasmids; the coding sequence fragments of the light chain variable region of GL-A2, GL-A11, GL-B7, ES-A8, ES-A13, and EY-E14 were homologously recombined into eukaryotic expression vector fragments containing the constant region of the rabbit monoclonal antibody light chain, resulting in 6 recombinant rabbit monoclonal antibody light chain eukaryotic expression plasmids.

[0045] The resulting recombinant rabbit monoclonal antibody heavy chain eukaryotic expression plasmid and recombinant rabbit monoclonal antibody light chain eukaryotic expression plasmid were transformed into [various transfection methods]. E. coli DH5α competent cells were cultured at 37°C for 1 h and then plated on LB agar plates containing 100 μg / mL ampicillin. After culturing at 37°C for 12 h, three single clones were selected and sent to Jiangsu Kangwei Century Biotechnology Co., Ltd. for sequencing to verify the sequence correctness. After verification, the cells were expanded and further extracted to obtain recombinant rabbit monoclonal antibody heavy chain eukaryotic expression plasmids expressing the complete heavy chains of GL-A2, GL-A11, GL-B7, ES-A8, ES-A13, and EY-E14, as well as recombinant rabbit monoclonal antibody light chain eukaryotic expression plasmids expressing the complete light chains.

[0046] 2. Expression of rabbit-derived measles virus monoclonal antibodies

[0047] First, transfection was performed using a 1 mL system, specifically: using ExpiFectamine.TM The 293 transfection kit (purchased from Thermo Fisher Scientific, catalog number A14524) was used to transfect 1000 ng each of the aforementioned recombinant rabbit monoclonal antibody eukaryotic expression plasmids expressing the heavy chain and corresponding light chain into 3 × 10⁻⁶ cells. 6 Expi293F cells with a cell count / mL and a viability ≥95% were selected. On day 4 after transfection, 10 μL of cell culture supernatant was collected. After centrifugation at 2000 rpm, the supernatant was added to 6× protein electrophoresis buffer (purchased from Beijing TransGen Biotech Co., Ltd., catalog number DL101) and denatured at 95℃ for 10 min. The mixture was then loaded onto a 12.5% ​​SDS-PAGE gel and electrophoresed at a constant voltage of 160 V for 45 min. After electrophoresis, Coomassie Brilliant Blue G-250 rapid staining solution (purchased from Shanghai Shenger Biotechnology Co., Ltd., catalog number SB-WB207) was used for staining, and the results were imaged.

[0048] The results are as follows Figure 2 As shown, SDS-PAGE analysis confirmed that the main band was located at 180 kDa, indicating successful expression of the rabbit IgG antibody. Based on this result, the transfection was performed in a 10 mL system, and the cell culture supernatant was collected on day 6 for subsequent experiments.

[0049] 3. Purification of rabbit-derived measles virus monoclonal antibody

[0050] The cell culture supernatant collected on day 6 was centrifuged at 4°C and 2000 rpm for 10 min. The supernatant was collected, filtered through a 0.45 μm filter membrane, and loaded onto a pre-packed rProtein A / G Agarose 4FF column (purchased from Jiangsu Qianzhusong Biotechnology Co., Ltd., catalog number QS01011A) and incubated overnight at 4°C. Non-specific binding proteins were removed by washing three times with PBS buffer. Then, the cells were eluted three times with 4 mL of 0.1 M glycine solution at pH=3. Immediately after each elution, 1 M Tris-HCl buffer at pH=8.8 was added at a ratio of 10:1 (elution buffer: neutralization buffer) for neutralization. The cells were then analyzed by SDS-PAGE gel electrophoresis according to the aforementioned method.

[0051] SDS-PAGE gel electrophoresis analysis showed that the target antibody content was highest in the liquid sample after the first elution. Therefore, the liquid after the first elution was used as a dialysis bag with a molecular weight cutoff of 3 kDa and dialyzed in 4 L of PBS buffer at 4°C for 4 h, with fresh PBS buffer replaced every hour during dialysis. The liquid obtained after dialysis was added to a 100 kDa ultrafiltration tube (purchased from Merck Darmstadt, Germany, catalog number UFC910008), centrifuged at 4000 × g for 10 min, and the final volume was concentrated to 1 mL by ultrafiltration. After filtration through a 0.22 μm filter membrane, the protein was quantified using a BCA protein detection kit and diluted with PBS buffer to obtain rabbit-derived measles virus monoclonal antibodies GL-A2, GL-A11, GL-B7, ES-A8, ES-A13, and EY-E14 with a final protein concentration of 1 mg / mL.

[0052] According to the above experimental method, the antibody expression positive control vector (purchased from Thermo Fisher Scientific, catalog number A14662) was transfected into Expi293F cells, and the negative rabbit monoclonal antibody was obtained by expression and purification.

[0053] Example 3: Functional Validation of Rabbit-Derived Measles Virus Monoclonal Antibody

[0054] 1. Validation of antigenic epitope binding of rabbit-derived measles virus monoclonal antibody

[0055] Streptavidin (Thermo Fisher Scientific, catalog number 22832) was diluted to 1 μg / mL with PBS buffer. 50 μL was added to each well of an ELISA plate and incubated overnight at 4°C. After coating, the plate was washed four times with PBST, and 50 μL of 10 μg / mL biotinylated ES, EY, or GL peptide as described in Example 1 was added to each well. The plate was incubated at 37°C for 1 h. Afterward, the plate was washed four times with PBST, and then 200 μL of PBS buffer containing 10% (w / v) BSA and 0.00022 mg / mL D-biotin (purchased from Changzhou Tiandi Renhe Biotechnology Co., Ltd., catalog number SLR00601) was added to each well. The plate was blocked at 37°C for 2 h. After blocking, the plate was washed four times with PBST, and then 50 μL of 10 μg / mL biotinylated ES, EY, or GL peptide as described in Example 1 was added to each well. Solution of rabbit-derived measles virus monoclonal antibody GL-A2, GL-A11, GL-B7, ES-A8, ES-A13, or EY-E14 at a concentration of μg / mL was prepared, with a separate well containing 50 μL of PBS buffer as a negative control. Incubation was performed at 37°C for 1 h. After incubation, the sample was washed four times with PBST, and then HRP-labeled goat anti-rabbit secondary antibody diluted 1:5000 was added. Incubation was performed at 37°C for 1 h. After incubation, the sample was washed four times with PBST, and then developed using TMB chromogenic agent solution at room temperature in the dark for 20 min. The development was then stopped by adding 50 μL of 1 M H2SO4. The absorbance at 450 nm (OD) was measured using a microplate reader.450 ), and plot the absorbance-dilution ratio curve.

[0056] The results are as follows Figure 3 As shown, all rabbit-derived measles virus monoclonal antibodies can effectively bind to their respective antigenic epitopes.

[0057] 2. Verification of virus particle binding of rabbit-derived measles virus monoclonal antibody

[0058] Take the previously determined protein concentration of the measles attenuated live vaccine solution and dilute it to 1 μg / mL with 0.05M carbonate buffer (pH 9.6). Add 50 μL to each well of the ELISA plate and coat overnight at 4°C. After coating, wash four times with PBST, add 200 μL of PBST containing 1 g / 100 mL casein to each well, and block at 37°C for 2 h. After blocking, wash four times with PBST, then add 50 μL of rabbit-derived measles virus monoclonal antibody solution (GL-A2, GL-A11, GL-B7, ES-A8, ES-A13, or EY-E14) at a concentration of 1 μg / mL to each well. A well containing 50 μL of PBS buffer is used as a negative control. Incubate at 37°C for 1 h. After incubation, wash four times with PBST, then add HRP-labeled goat anti-rabbit secondary antibody diluted 1:5000, and incubate at 37°C for 1 h. h; After incubation, wash 4 times with PBST, develop color with TMB chromogenic agent solution at room temperature in the dark for 20 min, then add 50 μL of 1 M H2SO4 to stop the color development, and measure the absorbance at 450 nm (OD) using a microplate reader. 450 ), and plot the absorbance-dilution ratio curve.

[0059] The results are as follows Figure 4 As shown, all rabbit-derived measles virus monoclonal antibodies have the ability to specifically bind to intact virus particles.

[0060] 3. Functional validation of rabbit-derived measles virus monoclonal antibodies using a double-antibody sandwich method

[0061] Rabbit-derived measles virus monoclonal antibodies GL-A2, GL-A11, GL-B7, ES-A8, ES-A13, and EY-E14, along with a negative rabbit monoclonal antibody as an isotype control, were diluted to 1 μg / mL with 0.05 M carbonate buffer (pH 9.6). 50 μL of each antibody was added to each well of an ELISA plate and incubated overnight at 4°C. After coating, the plates were washed four times with PBST, and 200 μL of PBST containing 1 g / 100 mL casein was added to each well. The plates were then blocked at 37°C for 2 h. After blocking, the plates were washed four times with PBST, and 50 μL of 1 μg / mL live attenuated measles vaccine solution was added to each well. The plates were incubated at 37°C for 1 h. After incubation, the plates were washed four times with PBST, and 50 μL of human immunoglobulin (purchased from Chengdu Rongsheng Pharmaceutical Co., Ltd., National Drug Approval Number S20237008) was added to each well. The plates were incubated at 37°C for 1 h. h; After incubation, wash 4 times with PBST, then add HRP-labeled anti-human IgG antibody diluted 1:2500 (purchased from Thermo Fisher Scientific, catalog number 62-8420), and incubate at 37°C for 1 h; after incubation, wash 4 times with PBST, develop color with TMB chromogenic reagent solution at room temperature in the dark for 20 min, then add 50 μL of 1 M H2SO4 to stop the color development, and measure the absorbance at 450 nm (OD) using a microplate reader. 450 ), and plot the absorbance-dilution ratio curve.

[0062] The results are as follows Figure 5 As shown, the double-antibody sandwich method proves that the obtained rabbit-derived measles virus monoclonal antibody also has antigen capture function, which can provide core raw materials for constructing a double-antibody sandwich detection system.

[0063] Example 4: Practical application of rabbit-derived measles virus monoclonal antibody

[0064] 1. Prepare colloidal gold immunochromatographic test strips for rapid on-site screening of measles virus.

[0065] The obtained rabbit-derived measles virus monoclonal antibodies were used as gold-labeled antibodies and detection line-coating antibodies, respectively, and assembled into colloidal gold test strips. These test strips can directly detect throat swabs, nasopharyngeal secretions, or saliva samples, and the results can be visually interpreted. They are suitable for on-site screening in primary healthcare institutions and disease control centers, as well as for rapid response to related situations.

[0066] 2. Assemble an enzyme-linked immunosorbent assay (ELISA) kit for batch detection in the laboratory.

[0067] A double-antibody sandwich ELISA kit was constructed using a rabbit-derived measles virus monoclonal antibody as the capture antibody, combined with an enzyme-labeled detection antibody (such as another anti-measles virus antibody recognizing a different epitope or an anti-H protein polyclonal antibody). This kit is suitable for laboratory testing of batch samples and can perform quantitative or semi-quantitative analysis of measles virus antigens, for use in epidemiological surveillance and evaluation of vaccine immunization efficacy.

Claims

1. A rabbit-derived measles virus monoclonal antibody or its antigen-binding fragment, characterized in that, The antibody or its antigen-binding fragment is selected from any of the following: The GL-A2 type has the heavy chain CDR1 sequence as shown in SEQ ID NO: 1, the CDR2 sequence as shown in SEQ ID NO: 7, and the CDR3 sequence as shown in SEQ ID NO: 13, and the light chain CDR1 sequence as shown in SEQ ID NO: 19, the CDR2 sequence as shown in SEQ ID NO: 25, and the CDR3 sequence as shown in SEQ ID NO:

31. The GL-A11 type has the heavy chain CDR1 sequence as shown in SEQ ID NO: 2, the CDR2 sequence as shown in SEQ ID NO: 8, and the CDR3 sequence as shown in SEQ ID NO: 14, and the light chain CDR1 sequence as shown in SEQ ID NO: 20, the CDR2 sequence as shown in SEQ ID NO: 26, and the CDR3 sequence as shown in SEQ ID NO:

32. The GL-B7 type has the heavy chain CDR1 sequence as shown in SEQ ID NO: 3, the CDR2 sequence as shown in SEQ ID NO: 9, and the CDR3 sequence as shown in SEQ ID NO: 15, and the light chain CDR1 sequence as shown in SEQ ID NO: 21, the CDR2 sequence as shown in SEQ ID NO: 27, and the CDR3 sequence as shown in SEQ ID NO:

33. The ES-A8 type has the heavy chain CDR1 sequence as shown in SEQ ID NO: 4, the CDR2 sequence as shown in SEQ ID NO: 10, and the CDR3 sequence as shown in SEQ ID NO: 16, and the light chain CDR1 sequence as shown in SEQ ID NO: 22, the CDR2 sequence as shown in SEQ ID NO: 28, and the CDR3 sequence as shown in SEQ ID NO:

34. The ES-A13 type has the heavy chain CDR1 sequence as shown in SEQ ID NO: 5, the CDR2 sequence as shown in SEQ ID NO: 11, and the CDR3 sequence as shown in SEQ ID NO: 17, and the light chain CDR1 sequence as shown in SEQ ID NO: 23, the CDR2 sequence as shown in SEQ ID NO: 29, and the CDR3 sequence as shown in SEQ ID NO:

35. EY-E14 type, whose heavy chain CDR1 sequence is shown as SEQ ID NO: 6, CDR2 sequence is shown as SEQ ID NO: 12, and CDR3 sequence is shown as SEQ ID NO: 18, and whose light chain CDR1 sequence is shown as SEQ ID NO: 24, CDR2 sequence is shown as SEQ ID NO: 30, and CDR3 sequence is shown as SEQ ID NO:

36.

2. The rabbit-derived measles virus monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region sequence of the GL-A2 type is as shown in SEQ ID NO: 37 or has at least 80% similarity to the sequence shown in SEQ ID NO: 37, and the light chain variable region sequence is as shown in SEQ ID NO: 38 or has at least 80% similarity to the sequence shown in SEQ ID NO: 38; the heavy chain variable region sequence of the GL-A11 type is as shown in SEQ ID NO: 39 or has at least 80% similarity to the sequence shown in SEQ ID NO: 39, and the light chain variable region sequence is as shown in SEQ ID NO: 40 or has at least 80% similarity to the sequence shown in SEQ ID NO: 40; the heavy chain variable region sequence of the GL-B7 type is as shown in SEQ ID NO: 41 or has at least 41% similarity to the sequence shown in SEQ ID NO: 37, and the light chain variable region sequence is as shown in SEQ ID NO: 42 or has at least 80% similarity to the sequence shown in SEQ ID NO: 42; the heavy chain variable region sequence of the ES-A8 type is as shown in SEQ ID NO:

37. The sequence shown in NO: 43 or has at least 80% similarity to the sequence shown in SEQ ID NO: 43, and the light chain variable region sequence shown in SEQ ID NO: 44 or has at least 80% similarity to the sequence shown in SEQ ID NO: 44; the heavy chain variable region sequence of the ES-A13 type is shown in SEQ ID NO: 45 or has at least 80% similarity to the sequence shown in SEQ ID NO: 45, and the light chain variable region sequence shown in SEQ ID NO: 46 or has at least 80% similarity to the sequence shown in SEQ ID NO: 46; the heavy chain variable region sequence of the EY-E14 type is shown in SEQ ID NO: 47 or has at least 80% similarity to the sequence shown in SEQ ID NO: 47, and the light chain variable region sequence shown in SEQ ID NO: 48 or has at least 80% similarity to the sequence shown in SEQ ID NO:

48.

3. The rabbit-derived measles virus monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, The antigen-binding fragments include any one or more of F(ab')2, Fab', Fab, Fv, ScFv, and dsFv.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes a rabbit-derived measles virus monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 3.

5. A recombinant vector, characterized in that, It includes the nucleic acid molecule as described in claim 4.

6. A recombinant cell, characterized in that, It comprises the nucleic acid molecule of claim 4 or the recombinant vector of claim 5.

7. An antibody conjugate, characterized in that, The antibody conjugate is obtained by conjugating the rabbit-derived measles virus monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 3 with a label, wherein the label is selected from one of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, and radioactive labeling.

8. A reagent kit, characterized in that, Contains a rabbit-derived measles virus monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 3, or an antibody conjugate as described in claim 7.

9. The use of the rabbit-derived measles virus monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 3, or the antibody-drug conjugate as described in claim 7, or the kit as described in claim 8, in the detection of measles virus for purposes other than disease diagnosis and treatment.

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