Enzyme-linked immunoassay kit for rabies virus neutralizing antibody and preparation and application thereof

Abstract

The application provides an enzyme-linked immunoassay kit for rabies virus neutralizing antibody, which comprises a rabies virus antigen coated enzyme-linked reaction plate, an enzyme-labeled anti-rabies virus monoclonal antibody, a diluent, a color developing reagent and negative and positive serum controls; wherein the rabies virus antigen is a rabies virus G antigen, the kit calculates the blocking rate by competitive ELISA to evaluate the antibody titer of a serum sample. The monoclonal antibody used in the kit of the application combines different antigen epitopes of the rabies virus G protein with high affinity, and neutralizes rabies virus strains derived from different genetic subtypes with high affinity, the antibody titer of a serum sample is evaluated by calculating the blocking rate by competitive ELISA, the sensitivity and specificity can be comparable to the existing FAVN standard determination method, and false positive and false negative detection results are basically avoided.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to antibodies against rabies virus, particularly to a method for preparing a rabies virus antibody neutralizing antibody detection kit and its preliminary application. Background Technology

[0002] Rabies virus (RABV) is a single-stranded negative-sense RNA virus belonging to the genus Lyssavirus of the family Rhabdoviridae. It is bullet-shaped with a helical nucleocapsid and an envelope containing single-stranded RNA. RABV is the pathogen that causes rabies. It is easily inactivated by sunlight, ultraviolet light, formaldehyde, quaternary ammonium compounds (such as benzalkonium chloride), lipid solvents, and 75% alcohol. Its suspension can be inactivated at 56°C for 30-60 minutes or at 100°C for 2 minutes. The virus can remain viable for several years at -80°C or after lyophilization and storage at 0-4°C. RABV contains five proteins: glycoprotein (G), nucleoprotein (N), dimeric enzyme (L), phosphoprotein (NS), and matrix (M). Glycoprotein G is the main protective antigen, inducing the body to produce neutralizing antibodies to combat viral attack.

[0003] Rabies prevention can be achieved through standardized pre-exposure and post-exposure management. Rabies vaccination is a crucial management method. Measuring the serum rabies virus neutralizing antibody titer after vaccination can assess whether the vaccine has achieved a protective level. The WHO considers a serum neutralizing antibody titer of ≥0.5 IU / mL to provide sufficient protection. If the titer is <0.5 IU / mL, multiple booster immunizations must be administered until the required antibody level is reached. The RABV-G protein is the only protein that induces the body to produce virus-neutralizing antibodies; therefore, using the RABV-G protein to detect serum neutralizing antibody titers is the most scientific and direct method.

[0004] Currently, methods for detecting rabies virus neutralizing antibodies include the Rapid Fluorescent Focus Inhibition Test (RFFIT), Fluorescent Antibody Virus Neutralization (FAVN), and Mouse Neutralization Test (MNT). The MNT method, recommended by the WHO in the 1960s for rabies virus titer determination, remains the method for determining rabies immunoglobulin titer listed in the Chinese Pharmacopoeia. However, due to its animal-based requirements, cumbersome and time-consuming operation, and non-compliance with animal testing principles, the MNT method has been gradually phased out. Since the 1980s, the WHO has recommended the RFFIT method as an alternative. The FAVN method is recommended by the International Organization for Veterinary Medicine (OIE) for detecting rabies antibody activity in animal serum.

[0005] Both the RFFIT and FAVN methods require the use of the fixed rabies virus strain CVS-11, involving live virus handling and other factors, which limits their application to high-throughput, large-scale testing. Enzyme-linked immunosorbent assay (ELISA), on the other hand, is a highly sensitive technique based on immunological reactions, combining the specific reaction of antigens and antibodies with the highly efficient catalytic action of enzymes on substrates. It is particularly suitable for high-throughput, large-scale testing. However, current ELISA kits mostly use whole rabies virus or purified viral glycoproteins as coating antigens, and the enzyme-labeled secondary antibodies are mostly polyclonal antibodies, significantly reducing the sensitivity and specificity of neutralizing antibody detection. This often leads to false positives and false negatives, resulting in unacceptable testing quality.

[0006] For example, Chinese patent application CN 102175867A discloses an enzyme-linked immunosorbent assay (ELISA) kit for detecting rabies virus antibodies, its preparation method, and detection method. This kit involves coating a recombinant rabies virus G protein as an antigen onto an ELISA plate, using horseradish peroxidase (HRP)-labeled mouse anti-human IgG Fc fragment monoclonal antibody as the enzyme marker. The kit also includes chromogenic reagent A, chromogenic reagent B, a stop solution, a sample diluent, a negative control (normal human serum), and a positive control (serum containing rabies virus antibodies).

[0007] This patented technology employs an indirect ELISA method, where recombinant rabies virus G protein is used as an antigen for coating. The serum to be tested reacts first; if antibodies (primary antibodies) are present in the serum, they will immunobind to the coated antigen. Then, a secondary antibody is added, which targets the primary antibody and immunobinds to the bound primary antibody, forming an antigen-primary antibody-secondary antibody complex. The secondary antibody is pre-labeled with HRP, and colorimetric reagents are added to develop a colorimetric result, indirectly reflecting the amount of primary antibody in the serum. However, because the primary antibodies in the serum are polyclonal antibodies, and the secondary antibody used cannot be designed to target every polyclonal antibody, this technology uses a mouse anti-human IgG Fc fragment monoclonal antibody as the secondary antibody. This cannot guarantee complete and accurate recognition of all primary antibodies in the serum, potentially leading to false positives and false negatives, resulting in inaccurate detection quality.

[0008] Chinese patent application CN 101936998A discloses a canine anti-rabies virus IgG antibody kit. The kit contains: an enzyme-labeled plate pre-coated with anti-rabies virus monoclonal antibody, which is then blocked and coated with purified rabies virus antigen; the enzyme conjugate is horseradish peroxidase-rabbit anti-canine IgG enzyme conjugate.

[0009] This patented technology employs a sandwich ELISA method. First, an anti-rabies virus monoclonal antibody is coated onto the surface. Then, an antigen (recombinant rabies virus vaccine strain) is further coated onto this surface via immunobinding. This is provided as part of the ELISA kit. The serum to be tested reacts first. If antibodies (primary antibodies) are present in the serum, they will immunobind with the coated antigen. Then, a secondary antibody is added. This secondary antibody is specific to the primary antibody and will immunobind with the already bound primary antibody, forming an antigen-primary antibody-secondary antibody complex. The secondary antibody is pre-labeled with HRP, allowing for color development with a chromogenic reagent, thus indirectly reflecting the amount of primary antibody in the serum. The difference in this technology lies in the fact that the anti-rabies virus monoclonal antibody is coated before the antigen coating, resulting in an antibody-antigen-primary antibody-secondary antibody sandwich complex, which improves signal amplification and detection sensitivity. However, because the primary antibodies in serum are polyclonal antibodies, and the secondary antibodies used cannot be designed to target every single one of these polyclonal antibodies, this technology, using rabbit anti-canine IgG antibody as the secondary antibody, cannot guarantee complete and accurate recognition of all primary antibodies in the serum, potentially leading to false positives and false negatives, and inaccurate detection quality. Furthermore, since this technology pre-coats a layer of anti-rabies virus monoclonal antibody, this secondary antibody may recognize not only the primary antibody already bound to the antigen in the serum but also the anti-rabies virus monoclonal antibody already coated on the antigen, thus increasing the amount of primary antibody present in the serum. Thirdly, the antigen used in this technology is the recombinant rabies virus vaccine strain itself, which also affects its recognition specificity and sensitivity.

[0010] Chinese patent document CN 1547028A discloses a kit and preparation method for detecting total antibodies against canine rabies virus using a competitive enzyme-linked immunosorbent assay (ELISA). The kit consists of: an ELISA plate pre-coated with rabies virus antigen (rabies virus vaccine strain), an enzyme conjugate (HRP-anti-rabies virus N protein monoclonal antibody), positive serum, negative serum, concentrated washing buffer, substrate, and stop solution.

[0011] Unlike indirect ELISA and sandwich ELISA methods, competitive ELISA does not use a secondary antibody against the primary antibody. Instead, it uses two antibodies that can both immunogenically bind to the antigen (one antibody is derived from the anti-rabies virus antibody in the serum being tested, and the other is an enzyme-labeled anti-rabies virus antibody). The antibody titer in the serum being tested is evaluated by calculating the blocking rate based on the colorimetric signal. However, this method uses the rabies virus vaccine strain itself as the antigen, which has issues with its recognition specificity and sensitivity. Furthermore, the competitive antibody used is a monoclonal antibody prepared using the rabies virus N protein as the antigen, which specifically recognizes the N protein. The N protein's efficiency in recognizing rabies virus antigens is insufficient, therefore its competitive binding ability is questionable.

[0012] This invention addresses the aforementioned technical problems by providing a method for preparing a rabies virus neutralizing antibody detection kit and its preliminary applications. This kit allows for the detection of antibody levels (0.5 IU / mL) in the serum after rabies vaccination. Based on the blocking rate results, it can determine whether the antibody level has reached 0.5 IU / mL, thereby monitoring the rabies vaccine's immunization level, indicating its effectiveness, and truly evaluating the rabies vaccine's protective effect. This ensures the safety of dogs, cats, and other animals, as well as humans, and has very promising application prospects. Summary of the Invention

[0013] To address the aforementioned technical problems, this invention provides an enzyme-linked immunosorbent assay (ELISA) kit for rabies virus neutralizing antibodies, comprising an ELISA plate coated with rabies virus antigen, an enzyme-labeled anti-rabies virus monoclonal antibody, a diluent, a chromogenic reagent, and negative and positive serum controls; wherein the rabies virus antigen is the rabies virus G antigen, and the kit evaluates the antibody titer of serum samples by calculating the blocking rate using a competitive ELISA.

[0014] Preferably, the diluent of the present invention is selected from 100% normal canine serum, 50% normal canine serum, 20% normal canine serum, 10% normal canine serum, 5% normal canine serum, 5% BSA phosphate buffer or 5% skim milk.

[0015] Preferably, the colorimetric reagent of the present invention is a tetramethylbenzidine solution, and the stop solution is a sulfuric acid or phosphoric acid solution.

[0016] This invention also provides a method for preparing the kit, the steps of which include: 1) Preparation of enzyme-labeled anti-rabies virus monoclonal antibody: Take horseradish peroxidase (HRP) solution, add HRP activation buffer and HRP coupling buffer for activation, add the anti-rabies virus monoclonal antibody to be coupled, mix well and dialyze overnight at 4°C; after dialyzing, add reducing agent to complete coupling; 2) Antigen coating: Coat rabies virus G protein with ELISA coating solution overnight at 4°C, discard the coating solution; add washing solution for washing, add blocking solution and block at room temperature for 1 hour, discard the blocking solution, and wash to obtain the antigen; wherein the blocking solution is 5% BSA PBS, 5% skim milk or 1% gelatin; 3) Prepare diluent, colorimetric reagent and negative and positive serum controls according to conventional methods.

[0017] The present invention also provides a monoclonal antibody against rabies virus for use in the kit described herein, comprising a heavy chain and a light chain, wherein the variable region of the heavy chain includes three CDR regions, the sequences of which are shown in SEQ ID NO:1-3 respectively; and the variable region of the light chain includes three CDR regions, the sequences of which are shown in SEQ ID NO:4-6 respectively.

[0018] Preferably, the full-length sequence of the heavy chain variable region of the anti-rabies virus monoclonal antibody of the present invention is shown in SEQ ID NO:7, and the full-length sequence of the light chain variable region is shown in SEQ ID NO:8.

[0019] The present invention also provides a nucleic acid molecule encoding the anti-rabies virus monoclonal antibody described herein.

[0020] The present invention also provides expression cassettes, recombinant vectors, or recombinant microorganisms containing the said nucleic acid molecules.

[0021] The present invention further provides the application of the enzyme-linked immunosorbent assay kit, the anti-rabies virus monoclonal antibody, or the nucleic acid molecule, or the expression cassette, recombinant vector, or recombinant microorganism described herein in evaluating the antibody titer of anti-rabies virus serum samples.

[0022] The present invention has the following beneficial effects:

[0023] The enzyme-linked immunosorbent assay kit of the present invention uses a competitive ELISA method to detect the antibody titer in anti-rabies serum, avoiding the problems of poor sensitivity and specificity, and the easy occurrence of false positives and false negatives in the detection of polyclonal antibodies in serum samples when using additional secondary antibodies.

[0024] The enzyme-linked immunosorbent assay (ELISA) kit of the present invention uses an anti-rabies virus monoclonal antibody as the enzyme-labeled antibody, which has high specific binding and neutralizing activity, thus improving detection sensitivity and specificity. The present invention uses rabies virus G protein as the coating antigen, which is the most effective immunogen on rabies virus, and helps the specific recognition and binding of polyclonal antibodies and enzyme-labeled monoclonal antibodies in serum samples, further improving detection sensitivity and specificity.

[0025] The recombinant monoclonal antibody against rabies virus used in this invention binds to and neutralizes rabies virus strains from different genotypes with high affinity. The affinity of most of these strains is higher than that of existing human anti-rabies virus antibodies, and they have broad-spectrum binding activity, making them suitable for the recognition and binding of G protein antigens from different strains.

[0026] This invention evaluates the antibody titer of serum samples by calculating the blocking rate using competitive ELISA. Its sensitivity and specificity are comparable to existing FAVN standard assays, effectively avoiding false positives and false positive results. Therefore, it avoids the complexity, high cost, and inconvenience of existing standard assays such as RFFIT and FAVN, as well as the inaccuracy and unreliability of existing ELISA kits. It enables convenient and readily available, portable testing of anti-rabies serum antibody titers, with simple and convenient operation. This is invaluable for individuals, dogs, and cats potentially infected with rabies virus, allowing them to see the effects of vaccinations and serum immunizations promptly. It also facilitates rapid, accurate, and low-cost evaluation of the quality of anti-rabies vaccines and serum products.

[0027] Instruction manual illustrations

[0028] Figure 1 The detection results of the recombinant plasmid for the anti-rabies virus RABV-G protein of the present invention are shown.

[0029] Figure 2 The results of the expression detection of the anti-rabies virus RABV-G protein of the present invention are shown. The left figure shows the detection results obtained by Western blotting with the His tag, where lane M represents the marker, lanes 1 and 2 represent two cell pellets, and lanes 3 and 4 represent two cell supernatants. The right figure shows the Western blotting results obtained by binding with the rabies monoclonal antibody, where lane M represents the marker, lanes 1 and 3 represent two cell pellets, and lanes 2 and 4 represent two cell supernatants.

[0030] Figure 3The image shows the SDS-PAGE results of the purified RABV-G protein (left) and the SDS-PAGE results of the RABV-G protein bound to rabies monoclonal antibody (right). Lane M is the marker, and lanes 1 and 2 are for non-reducing SDS-PAGE and reducing SDS-PAGE, respectively.

[0031] Figure 4 The EC50 of the recombinant monoclonal antibodies of the present invention with the RABV-G protein is shown. 50 Test results. The monoclonal antibody numbers represented by each legend are as follows: solid circles represent 26-12GEC. 50 =0.005363 μg / mL; solid triangles represent 1-12 g EC 50 =0.007794 μg / mL; a solid inverted triangle represents 23-5g EC 50 =0.02572 μg / mL; solid squares represent 15-3D EC 50 =0.03063 μg / mL; hollow circles represent 5-7 g EC 50 =0.0332 μg / mL; the hollow inverted triangle represents 30-9 AEC. 50 =0.04486 μg / mL; hollow rhombus represents 25-6°C EC 50 =0.05178 μg / mL; hollow squares represent 4-7 CEC 50 = 0.05503 μg / mL; gray solid circles represent 19-5C EC 50 =0.06344 μg / mL; grayscale solid squares represent 1-4 BEC 50 =0.1293 μg / mL; solid gray triangles represent 8-2B EC 50 =0.1746 μg / mL; a solid inverted triangle in grayscale represents 13-3D EC 50 =0.1888 μg / mL.

[0032] Figure 5 The results show the blocking rates of canine positive serum at different titers in this invention.

[0033] Figure 6 The results of blocking ELISA detection of canine negative serum at different dilutions according to the present invention are shown.

[0034] Figure 7 This invention demonstrates the OD values ​​of serum samples coated with RABV-G protein under different conditions (4°C for 12h, 16h, and 37°C for 1h and 2h) in the ELISA assay. 450nm And PI results.

[0035] Figure 8This invention demonstrates the OD values ​​of serum samples blocked with different blocking solutions (5% BSA, PBS, 5% skim milk, 1% gelatin) in the ELISA assay. 450nm And PI results.

[0036] Figure 9 This invention demonstrates the OD values ​​of serum samples obtained through different dilutions (100% normal canine serum, 50% normal canine serum, 20% normal canine serum, 10% normal canine serum, 5% normal canine serum, 5% BSA PBS, 5% skim milk) in the ELISA assay. 450nm And PI results.

[0037] Figure 10 This invention demonstrates the OD values ​​of serum samples after incubation at different times (37°C for 30 min, 1 h, and 2 h) in the ELISA assay. 450nm And PI results.

[0038] Figure 11 This invention demonstrates the OD values ​​of serum samples after incubation with enzyme-labeled antibodies for different times (30 min, 1 h, and 2 h at 37°C) in the ELISA assay. 450nm And PI results.

[0039] Figure 12 This invention demonstrates the OD values ​​of serum samples treated with different color development times (5 min, 10 min, 15 min, and 20 min at room temperature in the dark) in the ELISA assay. 450nm And PI results. Detailed Implementation

[0040] The following detailed description provides further details through specific embodiments. However, it should be noted that the embodiments described below are merely for illustrating the content of the invention and do not represent that the invention is limited to the described embodiments. Therefore, non-essential improvements and adjustments made to the implementation schemes by those skilled in the art based on the above-described invention still fall within the protection scope of the invention, and the scope of protection of the appended claims shall prevail.

[0041] As is known to those skilled in the art, the rabies virus described in this invention is the pathogen that causes rabies. Biologically, this virus belongs to the genus *Rabiesvirus* within the family Rhabdoviridae. The virus particle consists of a capsid and a core. The capsid is a lipoprotein double envelope, containing glycoproteins on the outer side and matrix membrane proteins on the inner side. The nucleocapsid of the core is composed of single-stranded RNA and proteins. Rabies virus contains five proteins: glycoprotein (G), nucleoprotein (N), dimeric enzyme (L), phosphoprotein (NS), and matrix (M). Glycoprotein G can lead to the formation of neutralizing antibodies in the body, which can combat viral attack and serve as the main antigen of rabies virus. The other antigen is nucleoprotein N. Rabies virus can persist in wild animals for extended periods and is widespread among wild animals worldwide. Host animals include carnivores and bats. Foxes, wolves, jackals, badgers, raccoon dogs, skunks, raccoons, meerkats, and bats are all natural reservoir hosts of rabies and can be infected with the rabies virus, becoming sources of infection and subsequently infecting livestock such as pigs, cattle, sheep, and horses.

[0042] As is known to those skilled in the art, the polyclonal antibodies described in this invention are mixtures of antibodies against the same antigen produced by different B cells in the body, and they can recognize and bind to different antigenic epitopes on that single antigen. Monoclonal antibodies, on the other hand, are produced by the same B cells and can only recognize a specific single antigenic epitope.

[0043] As is known to those skilled in the art, the chain structure and regions of the antibody molecules described in this invention have the following meanings: Antibody molecules are generally composed of two heavy chains (i.e., H chains) and two light chains (i.e., L chains), each chain including a variable region (i.e., V region), a constant region (i.e., C region), and a hinge region. The composition and arrangement of residues near the amino terminus of the heavy and light chains vary greatly; this region is called the variable region, while the composition and arrangement of residues near the carboxyl terminus are relatively stable, hence it is called the constant region. Within the variable regions of the heavy and light chains, there are three specific segments in each chain with highly variable amino acid composition and arrangement, called hypervariable regions (i.e., HVR). These regions together constitute the antigen-binding site of the antibody molecule. This site can bind complementaryly to the corresponding antigenic epitope; therefore, the hypervariable region is also called the complementarity-determining region (i.e., CDR), denoted as CDR1, CDR2, and CDR3, respectively. The different amino acid compositions and sequences of the CDRs of different antibodies determine the specificity of the antibody's binding to the corresponding antigenic epitope, responsible for recognizing and binding the antigen, thereby exerting an immune effect. The amino acid composition and sequence of the variable regions of an antibody molecule, excluding the hypervariable region or CDR region, are relatively small and are called the backbone region (i.e., FR region).

[0044] As we know from the above common knowledge, the basic immune function of an antibody depends on three CDR regions on both the heavy and light chains. These regions are all located in the variable regions of the antibody heavy and light chains. The other amino acid sequences on the antibody molecule are generally conserved, and even if there are slight changes, they usually do not affect the basic immune function of the antibody molecule. Therefore, a sequence expressing all CDR regions can express an antibody or evaluate whether they belong to the same antibody.

[0045] As is known to those skilled in the art, ELISA is an immunoassay method, short for Enzyme-Linked Immunosorbent Assay, commonly used to measure antibodies or antigens in biological samples. ELISA assays are typically performed in 96-well plates, a format suitable for high-throughput measurement of antigens or antibodies in many samples such as serum, plasma, cell culture supernatant, and cell lysates. Several different forms of ELISA exist, but all rely on the binding of the target itself or an antibody / antigen capable of capturing the target to the coated plate surface. An assay step is then used to bind the antigen or antibody, allowing successful binding to be measured and quantified, for example, by colorimetric assay. Currently, there are four main types of ELISA: direct, indirect, sandwich, and competitive methods, all suitable for antigen or antibody detection. The main difference lies in whether the coating is an antigen or an antibody. The resulting binding complexes are respectively coated antigen-enzyme-labeled antibody, coated antigen-primary antibody-enzyme-labeled secondary antibody, coated antibody-antigen-enzyme-labeled antibody (or coated antibody-antigen-primary antibody-enzyme-labeled secondary antibody), coated antigen-antibody-enzyme-labeled competitive antibody, etc., thus each has its own advantages and disadvantages in terms of detection time, specificity, and sensitivity (signal amplification).

[0046] Other techniques, instruments, and materials related to antibody and ELISA detection are well known to those skilled in the art. Examples include immunizing animals (e.g., mice) with antigens (e.g., viruses or their immunogenic proteins), performing hybridoma fusion and screening after immunization, antibody expression and purification, and antibody structure and performance analysis. Unless otherwise specified in this invention, any other techniques, instruments, and materials known to those skilled in the art that can achieve the same purpose may be used. Even if specific techniques, instruments, and materials are specified in this invention, it does not mean that this invention can only use these techniques, instruments, and materials; it merely represents preferred embodiments of the invention. Those skilled in the art can still use any other techniques, instruments, and materials known to them that can achieve the same purpose.

[0047] Example 1: Expression and purification of RABV-G recombinant protein

[0048] 1.1 Carrier Construction

[0049] The full-length coding sequence (codons optimized for insect cells) of the rabies virus CVS-11 strain glycoprotein (GenBank: ADJ29911.1) was synthesized by Changchun Kumei Biotechnology Co., Ltd. The signal peptide sequence of the RABV-G protein was replaced with the gp67 signal peptide sequence of an insect baculovirus and added to the N-terminus of the G protein for protein secretion. A 6×His tag was added to the C-terminus to facilitate further purification. The recombinant protein sequence was then cloned into the pFastBac1 vector.

[0050] 1.2 Preparation of RABV-G protein recombinant plasmid

[0051] DH10Bac TM The competent cells (D1029S, Beyotime) were thawed on ice, and 1 μg of the synthesized plasmid DNA was added to 50 μL of competent cells and mixed gently. Incubate on ice for 30 min, heat shock at 42℃ for 90 s, immediately on ice for 2 min, add 4 mL LB medium, incubate at 37℃ and shake at 180 rpm for 4 h, take an appropriate amount of bacterial culture and spread it on an LB plate (the plate contains 50 μg / mL kanamycin, 7 μg / mL gentamicin, 10 μg / mL tetracycline, 100 μg / mL Bluo-gal, and 40 μg / mL IPTG), incubate at 37℃ for 24 h to screen for blue-white clones, pick 4 white clones and expand them in LB liquid (50 μg / mL kanamycin, 7 μg / mL gentamicin, and 10 μg / mL tetracycline) for 12-16 h, take an appropriate amount of bacterial culture for PCR amplification, using the synthesized cDNA as a template, PCR amplification is performed, the 20 μL system contains: 4 μL ddH2O, 10 μL 2×Mix Buffer, 2 μL each of 10 μM forward and reverse primers, and 2 μL cDNA. PCR reaction conditions were: 98℃ for 30 sec → [98℃ for 15 sec → 57℃ for 15 sec → 72℃ for 20 sec] × 34 → 72℃ for 5 min → stored at 4℃. Electrophoresis was performed on a 1% agarose gel at 120V for 30 min, and the amplified products were identified using a DL2000 Marker (3427A, Takara). The results of the RABV-G protein recombinant plasmid detection are attached. Figure 1 As shown. Plasmid extraction was performed on the bacterial culture of positive clones, and recombinant plasmids were collected.

[0052] 1.3 Transfection of Sf9 insect cells

[0053] Inoculate 9×10⁶ cells into 6-well plates. 5Sf9 cells (80% density plated) were cultured at 27°C for at least 1 hour in Sf9 medium (10902088, Gibco) containing 4% FBS to allow them to adhere. Two 1.5 mL centrifuge tubes were prepared. 8 μL of transfection reagent and 10 μL of recombinant plasmid were added to 100 μL of Grace medium in each tube. 100 μL of transfection reagent dilution was added to 100 μL of recombinant plasmid dilution, and the mixture was gently mixed. The culture medium in the 6-well plate was discarded, and 2 mL of serum-free Grace medium was added. Then, 200 μL of the recombinant plasmid / transfection reagent mixture was slowly added. The cells were cultured at 27°C for 6 hours. The culture medium was discarded, and 2 mL of 4% FBS Sf9 medium was added. The cells were cultured at 27°C for 72 hours to harvest the first-generation seed virus. 500 μL of the first-generation seed virus supernatant was added to a 75 cm⁻¹ centrifuge tube. 2 Cells were cultured in 4% FBS Sf9 medium at 27°C for 72 hours, and the second-generation seed virus was harvested. The cell pellet and supernatant were then subjected to Western blotting (WB) to verify protein expression. Figure 2 and Figure 3 As shown, His tag detection revealed protein expression in both the precipitate and the supernatant. However, when detected with the recombinant anti-rabies virus G protein monoclonal antibody, only the protein in the supernatant could bind to the antibody. Therefore, the expression supernatant was subsequently selected for purification.

[0054] 1.4 RABV-G protein expression and purification

[0055] The validated second-generation virus was expanded and cultured. It was added to Sf9 insect cells at a 1:100 ratio and cultured in suspension at 28°C and 180 rpm for 5-7 days. Cells were harvested, centrifuged at 5000 rpm for 10 min, and the supernatant was collected for purification. Protein purification from the supernatant was performed using a nickel column. The column was first washed with 5 column volumes of ultrapure water, then equilibrated with 5 column volumes of binding buffer. The collected cell supernatant was filtered through a 0.45 μm filter and mixed with an equal volume of binding buffer, then loaded at a flow rate of 1 mL / min. The flow-through was collected. The column was washed with 10 column volumes of binding buffer until UV and conductivity were at baseline. Elution was performed with different concentrations of elution buffer (2, 15, 30, and 500 mmol / L imidazole) at a flow rate of 1 mL / min. Immediately after elution, the column was reequilibrated with 10 column volumes of binding buffer. The purified protein was then dialyzed overnight with PBS, concentrated using an ultrafiltration tube, and the concentration was adjusted to 1 mg / mL. SDS-PAGE and Western blotting were used to verify the purity and binding activity of the RABV-G protein (e.g., Figure 2 and Figure 3 (As shown).

[0056] Example 2: Screening and labeling of recombinant monoclonal antibodies

[0057] CAMouse was immunized with rabies G protein (DRA244, from nearshore organisms) and SRV9 virus (a weakened rabies virus strain preserved in our laboratory). HG Nineteen fully human anti-rabies virus monoclonal antibodies with neutralizing activity were obtained from mice (Chongqing Jinmaibo Biotechnology Co., Ltd.) through hybridoma fusion screening. Twelve antibodies with high neutralizing titers were selected for further validation and were numbered as follows: 1-4B, 4-7C, 5-7G, 8-2B, 13-3D, 15-3D, 19-5C, 25-6C, 30-9A, 1-12G, 23-5G and 26-12G.

[0058] 2.1 Recombinant monoclonal antibody EC 50 Measurement

[0059] The RABV-G protein obtained in Example 1 was diluted to 2 μg / mL using ELISA coating buffer (C1050, Solarbio). 50 μL of the solution was added to each well of a 96-well microplate (9018, Corning), and the plate was coated overnight at 4°C. 200 μL of PBST (PBS solution containing Tween-20) was added to each well, and the plate was washed 3 times. The liquid in the wells was then discarded. 200 μL of 5% BSA PBS blocking buffer was added to each well, and the plate was blocked at room temperature for 1 h. The blocking buffer was then discarded, and the plate was washed 3 times with PBST.

[0060] The above-mentioned recombinant monoclonal antibody against rabies virus was diluted with 5% BSA PBS at ratios of 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12800, respectively. A negative control group (5% BSA PBS) was set up. Each condition was repeated twice. 50 μL was added to each well and incubated at 37°C for 1 h. The liquid in the wells was discarded, and the cells were washed three times with PBST. Mouse anti-human enzyme-labeled antibody (ab99759, Abcam) was diluted with 5% BSA PBS at a ratio of 1:5000. 50 μL was added to each well and incubated at 37°C for 1 h. The liquid in the wells was discarded, and the cells were washed three times with PBST.

[0061] Add 50 μL of TMB chromogenic solution (Thermo) to each well and incubate at room temperature in the dark for 7 min. Add 50 μL of 2 mol / L sulfuric acid stop solution to each well to stop the chromogenic reaction. Detect the OD value at 450 nm using an ELISA reader.

[0062] The ELISA results were analyzed using GraphPad Prism 8.0 software. The antibody concentrations were plotted on the X-axis, corresponding to the OD values. 450nm The value is on the Y-axis. A suitable regression model is selected to fit the dose-response curve of each antibody, and the EC value is calculated. 50 .

[0063] The results are as follows Figure 4As shown, recombinant anti-rabies virus monoclonal antibodies 26-12G and 1-12G bind to the RABV-G protein in EC. 50 The lowest values ​​were 0.005363 μg / mL and 0.007794 μg / mL, respectively, indicating that the two antibodies had the strongest binding ability to RABV-G protein among the 12 antibodies. Therefore, 26-12G and 1-12G were selected as candidate marker antibodies.

[0064] 2.2 Detection of Broad-Spectrum Neutralizing Potency of Recombinant Monoclonal Antibody Against Virus

[0065] The aforementioned recombinant monoclonal antibodies were subjected to virus neutralization activity assays, using a method similar to the FAVN assay. The antibody samples were serially diluted 3-fold in a 96-well plate, with the antibody (all diluted to 0.1 mg / mL) and human rabies immunoglobulin (…). Detection was performed using a 4×6 well area (20 IU / mL); a 96-well plate was used as a control plate, and standard positive serum (0.5 IU / mL) and standard negative serum (non-immunized dog serum) were diluted as described above.

[0066] Rabies virus strains of predetermined titers, including SHJDD14 (OR122986.1), ZJZSD1601 (MG383893.1), NeiMeng927A (EU284095.2), NMALSF01 (MW411451.1), GDMM D16 (MW411450.1), HuND02 (KT221115.1), HuNPB01 (GU186386.1), and CVS-11 (EU126641.1), were diluted with MEM to 200 TCID. 50 Add 50 μL to each well, except for the cell and culture medium control wells, and neutralize in an incubator at 37°C for 60 min.

[0067] BHK-21 cells were resuspended at 4 × 10⁻⁶. 5 Add 50 μL of antibody per well (except for the culture medium control) and incubate for 72 h using standard methods. Discard the culture medium, add 80% cold acetone, and fix at -20°C for 1 h. Discard the fixative, wash 3 times with PBST, add 1:1000 diluted FITC-anti-rabies virus immunoglobulin (FUJIREBIO) to each well, and incubate at 37°C for 1 h. Discard the antibody, wash 3 times with PBST, add 50 μL / well of 90% glycerol buffer, and observe the fluorescence signal in the wells under a fluorescence microscope. Wells showing specific green fluorescence are considered positive, and the virus neutralization titer of the sample is calculated according to the Spearman-Karber formula.

[0068] Six rabies virus street strains of prevalent genotypes in my country (Asian type I: SHJDD14, ZJZSD1601, and HuND02; Asian type II: HuNPB01 and GDMM D16; Arctic-associated group: NeiMeng927A; and World group grassland type: NMALSF01) were selected for testing, with the CVS-11 fixed strain used as a standard control. As shown in Table 1, the antibodies 26-12G, 5-7G, 19-5C, 25-6C, and 30-9A of this invention exhibited significant neutralizing activity against various genotypes of rabies virus street strains prevalent in my country. Therefore, 26-12G, 5-7G, 19-5C, 25-6C, and 30-9A were selected as candidate marker antibodies.

[0069] In summary, according to EC 50 After verifying the broad-spectrum neutralizing activity against the virus, a total of 6 antibodies, namely 1-12G, 26-12G, 5-7G, 19-5C, 25-6C, and 30-9A, were selected as candidate antibodies for HRP labeling.

[0070] Table 1 Broad-spectrum neutralizing titer of recombinant antibodies

[0071]

[0072] Note: "+" indicates <50 IU / mL, "++" indicates 50-100 IU / mL, and "++++" indicates >100 IU / mL.

[0073] 2.3 Quality Identification of HRP-Labeled Antibodies and Enzyme-Labeled Antibodies

[0074] Six recombinant monoclonal antibodies (1-12G, 26-12G, 5-7G, 19-5C, 25-6C, and 30-9A) screened by HRP conjugation kit (D601047, Shanghai Sangon Biotech) were conjugated using the following method:

[0075] Take 500 μL of HRP solution into a 5 mL centrifuge tube, add 200 μL of HRP activation buffer, and react slowly at room temperature for 30 minutes on a rotary suspending apparatus. Add 200 μL of HRP coupling buffer, let stand for 30 minutes, and the solution will slowly turn back to brownish-yellow. Add 1 mg of the target protein to be coupled (volume controlled at about 1 mL), gently mix with a pipette, and transfer the liquid to a dialysis bag. First, fix one end of the dialysis bag with a dialysis clamp, fill it with liquid, and then fix the other end. Finally, place the dialysis bag in 2 L of coupling dialysis solution and dialyze overnight at 4 °C. After dialysis, transfer the liquid in the dialysis bag to a 5 mL centrifuge tube, add 100 μL of reducing agent, let stand at room temperature for 2 hours, and gently mix with a pipette 2-3 times every half hour. Coupling is complete.

[0076] The concentration of HRP-labeled enzyme-labeled antibody was determined using the CBB (Coomassie Blue) method, as follows: Twelve 1.5 mL centrifuge tubes were used, and 200 μL of CBB Staining Solution (PA102, Beijing Tiangen) was added to each tube. Six centrifuge tubes were then filled with 0, 2, 4, 6, 8, and 10 μL of protein standard BSA (1 mg / mL), respectively. The other six centrifuge tubes were filled with the corresponding enzyme-labeled antibody, and the tubes were gently tapped to mix. 150 μL of the liquid from each centrifuge tube was then added to each of the twelve wells of a 96-well plate (42592, Costa). The OD value at 595 nm was measured using a microplate reader. A scatter plot was created in Excel with OD value on the x-axis and protein concentration on the y-axis, with a trend line and formulas added. The OD value of the enzyme-labeled antibody was then substituted into the formulas to calculate the corresponding concentration.

[0077] Add an appropriate amount of enzyme-labeled antibody and TMB for a suitable incubation time, then stop the reaction with 2 mol / L sulfuric acid. Read the OD values ​​at 403 nm and 280 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the molar ratio using the formula:

[0078] Enzyme amount (mg / mL) = OD 403nm ×0.4; IgG content (mg / mL) = (OD 280nm -OD 403nm ×0.3)×0.62; Molar ratio = enzyme amount / IgG amount ×4.

[0079] According to the results, a molar ratio of 0.7 resulted in poor efficacy, 1.0 in good efficacy, and 1.5-2.0 in the best efficacy. As shown in Table 2, the molar ratios of the six enzyme-labeled antibodies ranged from 1.7 to 3.1, indicating good HRP conjugation efficacy.

[0080] Table 2. Effects of enzyme-labeled antibody labeling

[0081]

[0082] 2.4 Selection of the best enzyme-labeled antibody

[0083] Dilute RABV-G protein to 0.25 μg / mL using ELISA coating buffer, add 50 μL to each well of a 96-well microplate, and incubate overnight at 4°C. Discard the coating buffer. Add 200 μL of PBST washing buffer to each well, wash three times, and discard the liquid in the wells. Add 5% BSA to each well. 200 μL of PBS blocking buffer was added and incubated at room temperature for 1 h. The blocking buffer was discarded, and the cells were washed three times. 50 μL of canine RABV antibody-positive serum (4 IU / mL), canine RABV antibody-negative serum and their serially diluted five times, canine CDV (canine distemper virus) antibody-positive serum, mouse RABV antibody-positive serum, and mouse RABV antibody-negative serum were added to each well, and the cells were incubated at 37°C for 1 h. The liquid in the wells was discarded, and the cells were washed three times with PBST. 50 μL of normal canine serum was added to each well to its optimal working concentration, and the cells were incubated at 37°C for 1 h. The liquid in the wells was discarded, and the cells were washed three times. 50 μL of TMB chromogenic buffer was added to each well, and the cells were incubated at room temperature in the dark for 10 min. 50 μL of 2 mol / L sulfuric acid stop solution was added to each well to stop the chromogenic reaction. The OD450 nm value was measured using a microplate reader, and the blocking rate was calculated using the formula: PI = 1 - P / N = 1 - (positive OD450 nm) / (positive OD450 nm). 450nm / Negative OD 450nm Antibodies with high blocking rates in RABV antibody-positive serum and low blocking rates in CDV antibody-positive serum were selected as the optimal antibodies for blocking ELISA.

[0084] Table 3. Blocking rate of serum at different dilutions (OD) 450nm and PI

[0085]

[0086] Table 4. Serum positive for CDV antibody and OD with different enzyme-labeled antibodies. 450nm and PI

[0087]

[0088] From Table 3 and Figure 5 The results showed that the PI value increased with increasing serum antibody titer; when the serum antibody titer was greater than 0.25 IU / mL, the blocking rate PI value of HRP-25-6C was higher than that of other antibodies. The OD values ​​of the negative sera for all six antibodies were relatively stable, with HRP-5-7G showing the highest OD value. (Table 4 and...) Figure 6The results showed that for CDV antibody-positive sera, the blocking rates (PIs) of all six antibodies were relatively low, while the specificity of all six antibodies was very good, with HRP-19-5C exhibiting the lowest blocking rate. Looking at the blocking rate (PI) of mouse positive sera, HRP-26-12G, HRP-25-6C, and HRP-30-9A showed higher PIs, with HRP-25-6C showing the highest blocking rate. Considering both the blocking rate and specificity results, HRP-25-6C was ultimately selected as the enzyme-labeled secondary antibody.

[0089] 2.5 Determination of enzyme-labeled antibody titer

[0090] Dilute RABV-G protein to 2 μg / mL using ELISA coating buffer. Add 50 μL to each well of a 96-well microplate and incubate overnight at 4°C. Discard the coating buffer. Add 200 μL of PBST washing buffer to each well and wash three times. Discard the liquid in the wells. Add 200 μL of 5% BSA PBS blocking buffer to each well and block at room temperature for 1 h. Discard the blocking buffer and wash three times with PBST. Dilute the enzyme-labeled antibody with 5% BSA PBS at serial dilutions of 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, ..., 1:3276800, for a total of 16 dilutions. Perform two replicates for each condition. Set up negative control wells with 5% BSA PBS, with two replicates. Add 50 μL to each well and incubate at 37°C for 1 h. Discard the liquid in the wells and wash three times with PBST. Add 50 μL of TMB chromogenic solution to each well and incubate at room temperature in the dark for 10 min. Add 50 μL of 2 mol / L sulfuric acid stop solution to each well to stop the chromogenic reaction. Measure the OD value at 450 nm using an ELISA reader and calculate the absorbance ratio (P / N) between the wells containing the diluted antibody and the negative control wells. A P / N > 2.1 is considered positive, and the positive result with the highest dilution ratio is taken as the measured antibody titer.

[0091] The P / N value was calculated based on the results in Table 5. As shown in Table 6, the potency of HRP-25-6C is 1:6400.

[0092] Table 5. Results of ELISA detection using enzyme-labeled antibody and negative control.

[0093]

[0094] Table 6 Mean P / N values ​​of enzyme-labeled antibodies

[0095]

[0096] 2.6 Determination of the optimal working concentrations of RABV-G protein and enzyme-labeled antibody using checkerboard titration method

[0097] 2.6.1 Chessboard titration method - direct ELISA method

[0098] RABV-G protein was diluted to 0.5 μg / mL, 0.25 μg / mL, and 0.125 μg / mL using ELISA coating buffer. 50 μL was added to each well of a 96-well microplate and incubated overnight at 4°C. The coating buffer was then discarded. 200 μL of PBST washing buffer was added to each well, and the plates were washed three times. The liquid in the wells was then discarded. 200 μL of 5% BSA PBS blocking buffer was added to each well, and the plates were blocked at room temperature for 1 hour. The blocking buffer was then discarded, and the plates were washed three times with PBST. Finally, 5% BSA PBS blocking buffer was added to each well. The enzyme-labeled antibody was diluted with PBS at ratios of 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, and 1:6400, and 50 μL was added to each well. The mixture was incubated at 37°C for 1 h, and the liquid in the wells was discarded. The mixture was washed three times with PBST. 50 μL of TMB chromogenic solution was added to each well, and the mixture was incubated at room temperature in the dark for 10 min. 50 μL of 2 mol / L sulfuric acid stop solution was added to each well to stop the chromogenic reaction. The absorbance value at a wavelength of 450 nm was measured using an ELISA reader.

[0099] The results are shown in Table 7. Based on the distribution of OD values, RABV-G protein at concentrations of 0.5 μg / mL and 0.25 μg / mL, and enzyme-labeled antibody at dilutions of 200, 400, and 800 times were selected to further test the optimal dilutions of RABV-G protein and antibody for blocking ELISA.

[0100] Table 7 Results of RABV-G protein and enzyme-labeled antibody detection by checkerboard ELISA

[0101]

[0102] 2.6.2 Checkerboard titration method - blocking ELISA

[0103] RABV-G protein was diluted to 0.5 μg / mL and 0.25 μg / mL using ELISA coating buffer. 50 μL was added to each well of a 96-well microplate and incubated overnight at 4°C. The coating buffer was then discarded. 200 μL of PBST washing buffer was added to each well, and the plates were washed three times. The liquid in each well was then discarded. 5% BSA was added to each well. 200 μL of PBS blocking buffer was added and blocked at room temperature for 1 h. The blocking buffer was discarded, and the sample was washed 3 times. Standard canine positive serum (5 IU / mL) and standard canine negative serum were added to the stock solution, along with a blank control well. 50 μL of the stock solution was added to each well, and the sample was incubated at 37°C for 1 h. The liquid in the wells was discarded, and the sample was washed 3 times. The enzyme-labeled antibody was diluted with normal canine serum at ratios of 1:200, 1:400, and 1:800. 50 μL of the diluted solution was added to each well, and the sample was incubated at 37°C for 1 h. The liquid in the wells was discarded, and the sample was washed 3 times. 50 μL of TMB chromogenic buffer was added to each well, and the reaction was incubated at room temperature in the dark for 10 min. 50 μL of 2 mol / L sulfuric acid stop solution was added to each well to stop the reaction. The OD value at 450 nm was measured using an ELISA reader, and the blocking rate was calculated using Excel. The blocking rate formula is: PI = 1 - (standard positive OD value) / (standard positive OD value). 450nmStandard negative OD 450nm 。 )

[0104] A higher blocking rate (PI) value indicates a better combination of antigen coating concentration and antibody dilution. Based on the results in Tables 8 and 9, the PI value was higher at a RABV-G protein coating concentration of 0.25 μg / mL than at 0.5 μg / mL; therefore, the G protein coating concentration was determined to be 0.25 μg / mL. The HRP-25-6C dilution factor of 1:800 was determined based on the G protein concentration.

[0105] Table 8. Results of ELISA detection of RABV-G protein and enzyme-labeled antibody at different dilutions.

[0106]

[0107] Table 9. Blocking efficiency (PI) results of different dilutions of RABV-G protein and enzyme-labeled antibody combinations.

[0108]

[0109] Example 3: Optimization of ELISA detection conditions for anti-rabies virus neutralizing antibody blocking

[0110] The coating conditions, blocking buffer type, enzyme-labeled antibody dilution buffer type, sample incubation time, enzyme-labeled antibody incubation time, and TMB color development time of the blocking ELISA were optimized using a single-factor variable method.

[0111] 3.1 Optimization of RABV-G protein coating conditions

[0112] Dilute RABV-G protein to 0.25 μg / mL using ELISA coating buffer, add 50 μL to each well of a 96-well microplate, and incubate at 4°C for 12 h, 16 h, and 37°C for 1 h and 2 h, respectively, then discard the coating buffer. Add 200 μL of PBST washing buffer to each well, wash 3 times, and discard the liquid in the wells. Add 5% BSA to each well. 200 μL of PBS blocking buffer was added and incubated at room temperature for 1 h. The blocking buffer was then discarded, and the sample was washed three times with PBST. Canine standard positive serum (5 IU / mL) and canine standard negative serum were added in stock solutions, with three replicates and a blank control well. 50 μL of HRP-25-6C was added to each well and incubated at 37°C for 1 h. The liquid in the wells was discarded, and the sample was washed three times with PBST. 50 μL of normal canine serum was diluted 1:800 with HRP-25-6C and added to each well. The sample was incubated at 37°C for 1 h, and the liquid in the wells was discarded, and the sample was washed three times with PBST. 50 μL of TMB chromogenic buffer was added to each well and incubated at room temperature in the dark for 10 min. 50 μL of 2 mol / L sulfuric acid stop solution was added to each well to terminate the chromogenic reaction. The OD value at 450 nm was measured using a microplate reader, and the blocking rate was calculated using Excel. The blocking rate formula is: PI = 1 - (standard positive OD value) / (standard positive OD value). 450nm Standard negative OD 450nm).

[0113] The results are as follows Figure 7 As shown, the negative serum OD value was the highest after coating at 4℃ for 16h; the PI values ​​of coating at 4℃ for 12h and 4℃ for 16h were similar and both very high; overall, the optimal coating condition is coating at 4℃ for 16h.

[0114] 3.2 Optimization of Sealing Fluid

[0115] Dilute RABV-G protein to 0.25 μg / mL using ELISA coating buffer, add 50 μL to each well of a 96-well microplate, incubate overnight at 4°C, and discard the coating buffer. Add 200 μL of PBST washing buffer to each well, wash three times, discard the liquid in the wells, and blot dry. Then wash with 5% BSA. PBS, 5% skim milk, and 1% gelatin were used as blocking solutions. 200 μL was added to each well, and the mixture was incubated at room temperature for 1 hour. The blocking solution was discarded, and the samples were washed three times with PBST. Canine standard positive serum (5 IU / mL) and canine standard negative serum were loaded in stock solutions, with three replicates. A blank control well was also included. 50 μL of HRP-25-6C was added to each well, and the mixture was incubated at 37°C for 1 hour. The liquid in the wells was discarded, and the samples were washed three times with PBST. 50 μL of TMB chromogenic solution was added to each well, and the mixture was incubated at room temperature in the dark for 10 minutes. 50 μL of 2 mol / L sulfuric acid stop solution was added to each well to terminate the chromogenic reaction. The OD value at 450 nm was measured using a microplate reader, and the blocking rate was calculated using Excel. The blocking rate formula is: PI = 1 - (standard positive OD value) / (standard positive OD value). 450nm Standard negative OD 450nm ).

[0116] from Figure 8 The results showed that the PI values ​​of the three blocking solutions were not significantly different. 1% gelatin had the highest PI value, 5% skim milk had a similar PI value to 1% gelatin, and 5% BSA PBS had a slightly lower PI value. Since the PI values ​​of the three blocking solutions were not significantly different, the more commonly used 5% BSA PBS was selected as the blocking solution.

[0117] 3.3 Optimization of enzyme-labeled antibody dilution solution

[0118] RABV-G protein was diluted to 0.25 μg / mL using ELISA coating buffer. 50 μL was added to each well of a 96-well microplate and incubated overnight at 4°C. The coating buffer was then discarded. 200 μL of PBST washing buffer was added to each well, and the plate was washed three times. The liquid in the wells was then discarded. 200 μL of 5% BSA PBS blocking buffer was added to each well and the plate was blocked at room temperature for 1 h. The blocking buffer was then discarded, and the plate was washed three times with PBST. Canine standard positive serum (5 IU / mL) and canine standard negative serum were loaded in duplicate, with 50 μL added to each well as a blank control. The plate was incubated at 37°C for 1 h. The liquid in the wells was discarded, and the plate was washed three times with PBST. The plate was then incubated with 100% normal canine serum, 50% normal canine serum, 20% normal canine serum, 10% normal canine serum, 5% normal canine serum, and 5% BSA, respectively. HRP-25-6C was diluted 1:800 with PBS and 5% skim milk, and 50 μL was added to each well. The mixture was incubated at 37°C for 1 hour, and the liquid in the wells was discarded. The cells were washed three times with PBST. 50 μL of TMB chromogenic solution was added to each well, and the mixture was incubated at room temperature in the dark for 10 minutes. 50 μL of 2 mol / L sulfuric acid stop solution was added to each well to terminate the reaction. The OD value at 450 nm was measured using a microplate reader, and the blocking rate was calculated using Excel. The blocking rate formula is: PI = 1 - (standard positive OD value) 450nm Standard negative OD 450nm ).

[0119] The results are as follows Figure 9 As shown, the OD value of negative serum gradually increases as the concentration of normal canine serum in the antibody diluent decreases. The PI value of normal canine serum as the enzyme-labeled antibody diluent is higher than that of 5% BSA, PBS, and 5% skim milk, with 50% normal canine serum exhibiting the highest PI. Therefore, 50% normal canine serum was selected as the enzyme-labeled antibody diluent.

[0120] 3.4 Optimization of Sample Incubation Time

[0121] Dilute RABV-G protein to 0.25 μg / mL using ELISA coating buffer, add 50 μL to each well of a 96-well microplate, and incubate overnight at 4°C. Discard the coating buffer. Add 200 μL of PBST washing buffer to each well, wash three times, and discard the liquid in the wells. Add 5% BSA to each well. 200 μL of PBS blocking buffer was added and blocked at room temperature for 1 h. The blocking buffer was discarded, and the sample was washed three times with PBST. Canine standard positive serum (5 IU / mL) and canine standard negative serum were loaded in three replicates, with blank control wells included. 50 μL of each solution was added to each well and incubated at 37°C for 30 min, 1 h, and 2 h, respectively. The liquid in the wells was discarded, and the sample was washed three times with PBST. HRP-25-6C was diluted 1:800 with 50% normal canine serum and 50 μL of the solution was added to each well. The sample was incubated at 37°C for 1 h, and the liquid in the wells was discarded. The sample was washed three times with PBST. 50 μL of TMB chromogenic buffer was added to each well and incubated at room temperature in the dark for 10 min. 50 μL of 2 mol / L sulfuric acid stop solution was added to each well to terminate the chromogenic reaction. The OD value at 450 nm was measured using a microplate reader, and the blocking rate was calculated using Excel. The blocking rate formula is: PI = 1 - (standard positive OD value) / (standard positive OD value). 450nm Standard negative OD 450nm ).

[0122] The results are as follows Figure 10 As shown, the negative serum OD value was the highest after 1 hour of serum incubation. The PI values ​​were not significantly different between 1 hour and 2 hours of incubation, with the PI value being slightly higher after 2 hours. Considering all factors, serum incubation at 37°C for 1 hour was selected.

[0123] 3.5 Optimization of enzyme-labeled antibody incubation time

[0124] Dilute RABV-G protein to 0.25 μg / mL using ELISA coating buffer, add 50 μL to each well of a 96-well microplate, and incubate overnight at 4°C. Discard the coating buffer. Add 200 μL of PBST washing buffer to each well, wash three times, and discard the liquid in the wells. Add 5% BSA to each well. 200 μL of PBS blocking buffer was added and blocked at room temperature for 1 h. The blocking buffer was discarded, and the sample was washed three times with PBST. Canine standard positive serum (5 IU / mL) and canine standard negative serum were loaded in three replicates, with a blank control well. 50 μL of HRP-25-6C was added to each well and incubated at 37°C for 1 h. The liquid in the wells was discarded, and the sample was washed three times with PBST. 50 μL of HRP-25-6C was diluted 1:800 with 50% normal canine serum and added to each well. The sample was incubated at 37°C for 30 min, 1 h, and 2 h, respectively. The liquid in the wells was discarded, and the sample was washed three times with PBST. 50 μL of TMB chromogenic solution was added to each well and incubated at room temperature in the dark for 10 min. 50 μL of 2 mol / L sulfuric acid stop solution was added to each well to stop the chromogenic reaction. The OD value at 450 nm was measured using a microplate reader, and the blocking rate was calculated using Excel. The blocking rate formula is: PI = 1 - (standard positive OD value) / (standard positive OD value). 450nm Standard negative OD450nm ).

[0125] The results are as follows Figure 11 As shown, the serum OD value was highest after 2 hours of enzyme-labeled antibody incubation, but the PI value was lowest. The serum OD value was lowest after 30 minutes of enzyme-labeled antibody incubation, but the PI value was highest. Therefore, 37℃ and 30 minutes were chosen as the incubation time for enzyme-labeled antibodies.

[0126] 3.6TMB color development time

[0127] Dilute RABV-G protein to 0.25 μg / mL using ELISA coating buffer, add 50 μL to each well of a 96-well microplate, and incubate overnight at 4°C. Discard the coating buffer. Add 200 μL of PBST washing buffer to each well, wash three times, and discard the liquid in the wells. Add 5% BSA to each well. 200 μL of PBS blocking buffer was added and incubated at room temperature for 1 h. The blocking buffer was then discarded, and the sample was washed three times with PBST. Canine standard positive serum (5 IU / mL) and canine standard negative serum were loaded in stock solutions, with three replicates and a blank control well. 50 μL of HRP-25-6C was added to each well and incubated at 37°C for 1 h. The liquid in the wells was discarded, and the sample was washed three times with PBST. 50 μL of HRP-25-6C was diluted 1:800 with 50% normal canine serum and added to each well. The sample was incubated at 37°C for 30 min, and the liquid in the wells was discarded. The sample was washed three times with PBST. 50 μL of TMB chromogenic solution was added to each well and incubated at room temperature in the dark for 5 min, 10 min, 15 min, and 20 min, respectively. 50 μL of 2 mol / L sulfuric acid stop solution was added to each well to terminate the chromogenic reaction. The OD value at 450 nm was read using a microplate reader, and the blocking rate was calculated using Excel. The blocking rate formula is: PI = 1 - (standard positive OD value) / (standard positive OD value). 450nm Standard negative OD 450nm ).

[0128] The results are as follows Figure 12 As the development time increased, the OD values ​​of both positive and negative serum gradually increased. The PI value was the highest at 10 min, and considering all factors, a development time of 10 min was selected.

[0129] Example 4: Determination of the Criterion Value for Rabies Virus Blocking ELISA Detection

[0130] RABV-G protein was diluted to 0.25 μg / mL using ELISA coating buffer. 50 μL was added to each well of a 96-well microplate and incubated overnight at 4°C. The coating buffer was then discarded. 200 μL of PBST washing buffer was added to each well, and the plate was washed three times. The liquid in the wells was then discarded. 200 μL of 5% BSA PBS blocking buffer was added to each well, and the plate was blocked at room temperature for 1 h. The blocking buffer was then discarded, and the plate was washed three times with PBST. Eight serially diluted samples of canine and feline RABV antibody-negative serum (antibody titer <0.5 IU / mL) and canine RABV antibody-positive serum (4 IU / mL) were used, along with canine standard positive serum (5 IU / mL), canine standard negative serum, and a blank control. 50 μL of each sample was added to each well and the plate was incubated at 37°C for 1 h. The liquid in the wells was discarded, and the plate was washed three times with PBST. Finally, 50% normal canine serum was used to... HRP-25-6C was diluted 1:800, and 50 μL was added to each well. The mixture was incubated at 37°C for 30 min, and the liquid in the wells was discarded. The cells were washed three times with PBST. 50 μL of TMB chromogenic buffer was added to each well, and the mixture was incubated at room temperature in the dark for 10 min. 50 μL of 2 mol / L sulfuric acid stop solution was added to each well to terminate the reaction. The OD value at 450 nm was measured using a microplate reader, and the blocking rate was calculated using Excel. The blocking rate formula is: PI = 1 - S / N (PI: blocking rate, S: OD value of sample serum). 450nm N: Standard negative serum OD 450nm ).

[0131] Regarding the determination of the cutoff value, the OD values ​​and PI of 88 canine and feline negative sera were selected and are shown in Table 10. The mean and standard deviation of the PI of the 88 negative sera were calculated. The cutoff value was calculated according to the formula Cutoff = mean PI + 2 × SD. The mean PI value was 0.1845 and the standard deviation was 0.1963. The preliminary PI cutoff value was determined to be 0.577, that is, when PI ≥ 0.577, the result was judged as positive (RABV neutralizing antibody ≥ 0.5 IU / mL); when PI < 0.577, the result was judged as negative (RABV neutralizing antibody < 0.5 IU / mL).

[0132] Table 10. OD values ​​and PI values ​​of 88 RABV antibody-negative serum samples from dogs and cats

[0133]

[0134] Example 5: Preliminary Application of Rabies Virus Neutralizing Antibody Blocking ELISA Kit

[0135] 5.1 Concordance rate between this kit and FAVN for canine and feline serum sample testing

[0136] Dilute RABV-G protein to 0.25 μg / mL using ELISA coating buffer, add 50 μL to each well of a 96-well microplate, and incubate overnight at 4°C. Discard the coating buffer. Add 200 μL of PBST washing buffer to each well, wash three times, and discard the liquid in the wells. Add 5% BSA to each well. 200 μL of PBS blocking buffer was added and incubated at room temperature for 1 h. The blocking buffer was then discarded, and the sample was washed three times with PBST. 50 μL of each of 265 canine and feline serum samples was added, along with canine standard positive serum (5 IU / mL), canine standard negative serum, and a blank control. The sample was incubated at 37°C for 1 h. The liquid in the wells was discarded, and the sample was washed three times with PBST. 50 μL of HRP-25-6C was diluted 1:800 with 50% normal canine serum and added to each well. The sample was incubated at 37°C for 30 min. The liquid in the wells was discarded, and the sample was washed three times with PBST. 50 μL of TMB chromogenic buffer was added to each well and incubated at room temperature in the dark for 10 min. 50 μL of 2 mol / L sulfuric acid stop solution was added to each well to stop the chromogenic reaction. The OD value at 450 nm was measured using a microplate reader, and the blocking rate was calculated using Excel. The blocking rate formula is: PI = 1 - S / N (PI: blocking rate, S: OD of sample serum). 450nm N: Standard negative serum OD 450nm ).

[0137] The 265 collected serum samples were subjected to FAVN detection and blocking ELISA detection, and their antibody titers and PI values ​​are shown in Table 11. The blocking rate of each serum sample was compared with the cutoff value to determine its positive or negative status. The results of this kit were compared with the FAVN results, as shown in Table 12. According to the cutoff value determination, 260 out of the 265 serum samples were consistent with the FAVN results, with a concordance rate of 98.11% and a false negative rate of 1.9%. No false positive samples were found. Compared with the FAVN detection method recommended by OIE, the anti-rabies virus blocking ELISA detection kit of this invention is simple to operate, has a short time consumption, a high concordance rate, no false positive samples, and does not require rabies virus culture. It is expected to replace the existing FAVN detection method.

[0138] Table 11. PI and antibody titers of 265 serum samples

[0139]

[0140]

[0141]

[0142] Table 12 Comparison of ELISA and FAVN test results

[0143]

[0144] The above descriptions are merely embodiments of the present invention. Commonly known technical knowledge in the solutions is not described in detail here. Those skilled in the art are aware of all common technical knowledge in the field prior to the filing date, are aware of all existing technologies in that field, and have the ability to apply conventional experimental methods prior to that date. Those skilled in the art can, under the guidance of this application, improve and implement this solution in combination with their own capabilities. Some typical well-known technologies should not be obstacles for those skilled in the art to implement this application. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the structure of the present invention. These should also be considered within the scope of protection of the present invention, and will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. An enzyme-linked immunosorbent assay (ELISA) kit for neutralizing rabies virus antibodies, comprising an ELISA plate coated with rabies virus antigen, an enzyme-labeled anti-rabies virus monoclonal antibody, a diluent, a stop solution, a chromogenic reagent, and negative and positive serum controls; wherein the rabies virus antigen is rabies virus G antigen, and the kit evaluates the antibody titer of serum samples by calculating the blocking rate using a competitive ELISA. The anti-rabies virus monoclonal antibody is composed of a heavy chain and a light chain, wherein the full-length sequence of the variable region of the heavy chain is shown in SEQ ID NO:7, and the full-length sequence of the variable region of the light chain is shown in SEQ ID NO:

8.

2. The enzyme-linked immunosorbent assay kit according to claim 1, characterized in that: The diluent is selected from phosphate buffer of 100% normal canine serum, 50% normal canine serum, 20% normal canine serum, 10% normal canine serum, 5% normal canine serum, 5% BSA, or 5% skim milk.

3. The enzyme-linked immunosorbent assay kit according to claim 1 or 2, characterized in that: The colorimetric reagent is a tetramethylbenzidine solution, and the stop solution is a sulfuric acid or phosphoric acid solution.

4. A method for preparing the kit according to any one of claims 1-3, comprising the steps of: 1) Preparation of enzyme-labeled anti-rabies virus monoclonal antibody: Take horseradish peroxidase (HRP) solution, add HRP activation buffer and HRP coupling buffer for activation, add the anti-rabies virus monoclonal antibody to be coupled, mix well and dialyze overnight at 4°C; after dialyzing, add reducing agent to complete coupling; 2) Antigen coating: Coat rabies virus G protein with ELISA coating solution overnight at 4°C, discard the coating solution; add washing solution for washing, add blocking solution and block at room temperature for 1 hour, discard the blocking solution, and wash to obtain the antibody; wherein the blocking solution is 5% BSA PBS, 5% skim milk or 1% gelatin; 3) Preparation of diluent, colorimetric reagent and negative and positive serum controls.

5. A monoclonal antibody against rabies virus for use in the kit according to any one of claims 1-3, comprising a heavy chain and a light chain, wherein the full-length sequence of the variable region of the heavy chain is shown in SEQ ID NO:7, and the full-length sequence of the variable region of the light chain is shown in SEQ ID NO:

8.

6. A nucleic acid molecule encoding the antibody of claim 5.

7. An expression cassette, recombinant vector, or recombinant microorganism containing the nucleic acid molecule of claim 6.

8. The use of the enzyme-linked immunosorbent assay kit according to any one of claims 1-3, the anti-rabies virus monoclonal antibody according to claim 5, the nucleic acid molecule according to claim 6, or the expression cassette, recombinant vector, or recombinant microorganism according to claim 7 in evaluating the antibody titer of anti-rabies virus serum samples.

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