CN114958774B - Anti-rabies virus monoclonal antibody, hybridoma cell strain secreting antibody and application - Google Patents

Anti-rabies virus monoclonal antibody, hybridoma cell strain secreting antibody and application Download PDF

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CN114958774B
CN114958774B CN202210493917.4A CN202210493917A CN114958774B CN 114958774 B CN114958774 B CN 114958774B CN 202210493917 A CN202210493917 A CN 202210493917A CN 114958774 B CN114958774 B CN 114958774B
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rabies virus
monoclonal antibody
variable region
chain variable
antibody
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CN114958774A (en
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宋杰
李慧
张雪梅
吴忠香
杨燕
朱文兵
张志晓
姬桐
湛小草
赵桂芳
李俊
余越
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Institute of Medical Biology of CAMS and PUMC
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Abstract

The invention relates to an anti-rabies virus monoclonal antibody, a hybridoma cell strain secreting the antibody and application thereof, and belongs to the technical fields of cell engineering and immunology. The invention prepares the monoclonal antibody by taking rabies virus as immunogen to obtain the anti-rabies virus phosphoprotein monoclonal antibody and the anti-rabies virus glycoprotein monoclonal antibody. The monoclonal antibody obtained by the invention has no cross reaction with other proteins of rabies virus and other antigens and pathogens, has good specificity and sensibility, has good application prospect in the aspect of biological diagnosis including ELISA, western-blot and immunofluorescence aiming at rabies virus, and further expands the application value of antibodies of different antigen epitopes of rabies virus.

Description

Anti-rabies virus monoclonal antibody, hybridoma cell strain secreting antibody and application
Technical Field
The invention belongs to the technical field of cell engineering and immunology, and particularly relates to an anti-rabies virus monoclonal antibody, a hybridoma cell strain secreting the antibody and application thereof.
Background
Rabies is an acute, highly lethal infectious disease of human and veterinary co-morbid origin caused by Rabies Virus (RV) infection, and human and almost all warm-blooded animals are susceptible to Rabies virus, and once the disease is almost 100% dead, no specific therapeutic drug is available until now. About 5.9 ten thousand people die annually from rabies worldwide, most of which occur in developing countries. In recent years, rabies epidemic situation rises year by year, so that the health of the whole people is seriously endangered, and the rabies epidemic situation is an important public health problem which is concerned by the world together.
Rabies virus belongs to the Rhabdoviridae family (Rhabdoviridae) and Lyssa virus belongs to the single-stranded negative strand RNA virus. The rabies virus genome contains 5 Open Reading Frames (ORFs) encoding 5 structural proteins, in turn, nucleoprotein, phosphoprotein, membrane matrix protein, glycoprotein and transcriptase macroprotein. Glycoprotein is currently considered to be the only rabies virus antigen capable of continuously inducing the virus to produce neutralizing antibodies, and is also the most widely and deeply studied antigen component. Phosphoproteins are multifunctional proteins that play a role in viral escape, viral transcriptional replication, and intracellular motility. Studies have shown that there are abundant epitopes on phosphoproteins, and more researchers have also continuously demonstrated the possibility of phosphoproteins as clinical serum detection antigens or superantigens for viral infections. Recently, it has been reported that nucleoprotein and phosphoprotein antigens of rabies virus can also induce specific immune response. Therefore, the antigen component of rabies virus plays an indispensable role in participating in the immune response of the organism to rabies virus.
The anti-rabies virus monoclonal antibody is an advantageous tool for researching a rabies virus diagnosis method, treating and preventing rabies. Since the first preparation of monoclonal antibodies to rabies virus by Wiktor and Koprowski in 1978, more and more laboratories established monoclonal antibody hybrids to rabies virus and discussed the application value of monoclonal antibodies in the aspects of immunoprotection mechanism of rabies virus, development of novel vaccines, epidemiology, protein function, diagnosis of rabies virus infection, and the like. The research of rabies antibodies aiming at different antigen epitopes is also one of the main directions of rabies antibody research. Monoclonal antibodies become optimal biological probes for detecting pathogens and antibodies due to the incomparable advantages of conventional immune serum such as high homogeneity and specificity, and are widely applied to the aspects of virus serological identification and diagnosis and treatment.
Currently, vaccination is the only method to prevent, control and destroy rabies. The safety, effectiveness and quality stability of rabies vaccine are key factors of the product for protecting immune effect. The antigen content is used as one of the main indexes for evaluating the quality of rabies vaccine, so that in the development and production process of rabies vaccine, it is important to establish a set of simple, convenient, quick, specific and sensitive rabies virus antigen detection method, so that not only can the time-consuming and labor-consuming animal experiments be replaced, the working efficiency be greatly improved, but also the antigen content of each step in the vaccine preparation process can be quickly and quantitatively detected in time, and the method is suitable for monitoring and controlling each stage of virus harvesting, concentration, inactivation, purification process and the like, thereby establishing internal control standard to guide production and quality control and ensure consistency and stability among batches. Therefore, quantitative monitoring of the antigen content in the vaccine by a simple and convenient monitoring method is of great significance for preventing the epidemic of the disease and developing a scientific immunization program.
Disclosure of Invention
The invention aims to solve the defects of the prior art, and provides a monoclonal antibody capable of specifically binding with rabies virus phosphoprotein/glycoprotein and a preparation method of hybridoma cells producing the antibody, aiming at the current situation that rabies still seriously endangers the health of the whole people and various difficulties still exist in the prevention and treatment process. The monoclonal antibody of the invention has no cross reaction with other proteins of rabies virus and other antigens and pathogens, has the characteristics of high specificity and high sensitivity, can establish a novel, convenient and practical detection method, quantitatively detects the content of antigen components in rabies virus vaccine, is used for timely monitoring the virus content in the vaccine research and development and production process, and has good application prospect.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the first aspect of the invention provides a preparation method of a rabies virus phosphoprotein/glycoprotein monoclonal antibody hybridoma cell strain, comprising the following steps:
step (1), preparation of immunogen: taking purified rabies virus as immunogen;
step (2), animal immunization: four immunizations were performed at 2 week intervals on 6-8 week old Balb/c mice with the following procedure: after primary immunization, fully mixing Freund's complete adjuvant and an equal amount of immunogen for emulsification, performing subcutaneous multipoint injection on the back; the second immunization and the third immunization, the adjuvant is replaced by Freund's incomplete adjuvant, and the method and the dosage are the same as those of the first immunization; the fourth time of intraperitoneal injection of immunogen without adjuvant is adopted, and the injection quantity is the same as that of the first immunization; after 3 days, spleen cells of immunized mice were taken for preparation of hybridoma cells;
step (3), preparation and screening of hybridoma cell strains: carrying out cell fusion on SP2/0 cells in the logarithmic growth phase and spleen cells of the immunized mice in the step (2) under the action of PEG4000, culturing the fused hybridoma cells by using a 1 XHAT selective medium, detecting cell supernatants 10-14 days after cell fusion, and screening hybridoma cell strains capable of specifically secreting anti-rabies virus phosphoprotein/glycoprotein monoclonal antibodies by an enzyme-linked immunosorbent assay.
Further, it is preferable that in the step (2), the first injection amount is 200ul 10 6 FFU/ml；
In the step (3), SP2/0 cells in the logarithmic growth phase and spleen cells of the immunized mice in the step (2) are subjected to cell fusion under the action of PEG4000, and the fused hybridoma cells are cultured by using a 1 XHAT selective medium, and the specific method comprises the following steps:
selecting a Balb/c mouse with the age of 6-8 weeks, taking abdominal macrophages of the Balb/c mouse to prepare feeder cells; mixing SP2/0 cells in logarithmic growth phase with spleen cells of immunized mice in the step (2) according to the cell number ratio of 1:10, performing cell fusion by using polyethylene glycol 4000, adding hybridoma cells obtained after cell fusion into a culture plate containing feeder cells, and culturing at 37 ℃ under the condition of 5% CO2 by using a selective medium containing 1 XHAT;
also included are the cloning of hybridoma cells, specifically: subcloning and culturing the hybridoma cell strain by limiting dilution method, continuously culturing for multiple times until the antibody in the cloning hole detects 100% positive, enlarging culturing and freezing storing.
In a second aspect, the present invention provides an anti-rabies virus phosphoprotein/glycoprotein monoclonal antibody secreted by the above hybridoma cells.
The third aspect of the invention provides an anti-rabies virus phosphoprotein/glycoprotein monoclonal antibody, which comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the light chain variable region of the anti-phosphoprotein monoclonal antibody is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 2; the amino acid sequence of the light chain variable region of the anti-glycoprotein monoclonal antibody is shown as SEQ ID NO.5, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 6.
In a fourth aspect, the present invention provides a DNA encoding an anti-rabies phosphoprotein/glycoprotein monoclonal antibody comprising a heavy chain variable region and a light chain variable region; the coding DNA sequence of the light chain variable region of the anti-phosphoprotein monoclonal antibody is shown as SEQ ID NO.3, and the coding DNA sequence of the heavy chain variable region is shown as SEQ ID NO. 4; the coding DNA sequence of the light chain variable region of the anti-glycoprotein monoclonal antibody is shown as SEQ ID NO.7, and the coding DNA sequence of the heavy chain variable region is shown as SEQ ID NO. 8.
The fifth aspect of the present invention provides a kit for detecting a double-antibody sandwich antigen of rabies virus, wherein the anti-rabies virus phosphoprotein/glycoprotein monoclonal antibody is adopted, the anti-rabies virus phosphoprotein monoclonal antibody is used as a coating antibody, the enzyme-labeled anti-rabies virus glycoprotein monoclonal antibody is used as a detection antibody, or the anti-rabies virus glycoprotein monoclonal antibody is used as a coating antibody, and the enzyme-labeled monoclonal antibody phosphoprotein monoclonal antibody is used as a detection antibody.
Further, it is preferable that the enzyme is horseradish peroxidase or alkaline phosphatase.
The sixth aspect of the invention provides application of the anti-rabies virus phosphoprotein/glycoprotein monoclonal antibody in preparing a rabies virus vaccine antigen content detection reagent or kit, a rabies virus detection reagent or kit, and a rabies virus antibody detection reagent or kit.
The seventh aspect of the invention provides an application of the anti-rabies virus phosphoprotein/glycoprotein monoclonal antibody in preparing a quality detection reagent or a kit for rabies virus vaccine production products.
The eighth aspect of the present invention provides an application of the above-mentioned anti-rabies virus phosphoprotein/glycoprotein monoclonal antibody in preparing a medicament for preventing or treating rabies.
In the kit of the present invention, the monoclonal antibody may be a labeled complex obtained by biomarker or chemical labeling. The biomarker is an enzyme label, and the enzyme is horseradish peroxidase or alkaline phosphatase.
The monoclonal antibody capable of recognizing rabies virus phosphoprotein and the monoclonal antibody capable of recognizing rabies virus glycoprotein provided by the invention have good specificity. The recognition proteins and recognition sites among the clones have no cross reaction, and the indirect ELISA shows that the two antibodies have higher sensitivity, so that the antibodies can be used for detecting antigens in rabies viruses and vaccines. In addition, since the two monoclonal antibodies recognize different antigenic determinants on different proteins of rabies virus, respectively. Therefore, the double-antibody sandwich method can be adopted to quantitatively detect the rabies virus antigen in time by respectively utilizing two monoclonal antibodies specifically identified.
The double-antibody sandwich ELISA method constructed based on the method has good sensitivity and specificity, can be used for timely monitoring rabies virus antigens at each stage in the vaccine production process, and is used as an internal control standard for guiding the screening, determination and optimization of rabies virus vaccine preparation methods and processes.
On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.
The reagents and materials used in the present invention are commercially available.
In the invention, the anti-rabies virus phosphoprotein/glycoprotein monoclonal antibody hybridoma cell strain is two hybridoma cell strains of the anti-rabies virus phosphoprotein monoclonal antibody and the anti-rabies virus glycoprotein monoclonal antibody.
Compared with the prior art, the invention has the beneficial effects that:
the anti-rabies virus monoclonal antibody provided by the invention is based on the fact that different antigenic determinants on different proteins of the rabies virus, including rabies virus phosphoprotein and rabies virus glycoprotein, are respectively identified, so that the possibility of application of the rabies monoclonal antibody aiming at different antigenic epitopes in aspects of diagnosis, prevention and treatment of rabies is expanded, and a solid foundation is laid for establishment of a detection and quantification method of the rabies virus and research of functions of different proteins.
At present, the rabies virus detection kit based on the diabody is limited to rabies virus nucleoprotein and glycoprotein, and the rabies virus antibody detection kit based on the phosphoprotein monoclonal antibody is reported in the past. A hybridoma cell strain and a rabies virus phosphoprotein monoclonal antibody (2016115 7550. X) produced by the same are disclosed, wherein the rabies virus phosphoprotein monoclonal antibody produced by the hybridoma cell strain is screened and applied to preparation of a rabies virus detection reagent, and the detection sensitivity can reach 0.1IU/ml. The monoclonal antibody capable of recognizing rabies virus phosphoprotein and the monoclonal antibody capable of recognizing rabies virus glycoprotein have good specificity, do not have cross reaction with other rabies virus proteins and other antigens and pathogens, and are used for detecting the rabies virus, so that the double-antibody sandwich method for rapidly and quantitatively detecting the rabies virus antigen content is established, the sensitivity (0.02 IU/ml) of the rabies virus double-antibody sandwich ELISA antigen detection method is greatly improved, the method is particularly important for quality control and timely monitoring of the virus content in the research and development and production processes of rabies virus vaccines, can be widely applied to vaccine and clinical detection, and has important guiding significance for rabies detection and prevention and control.
Drawings
FIG. 1 is a SDS-PAGE electrophoresis of an anti-rabies virus monoclonal antibody, wherein M is a protein molecular weight standard (kDa), lane 1 is an anti-rabies virus phosphoprotein monoclonal antibody 112E3, and lane 2 is an anti-rabies virus glycoprotein monoclonal antibody 119A4;
FIG. 2 is a Western blot results of anti-rabies virus phosphoprotein monoclonal antibody 112E3, wherein M is a protein molecular weight standard (kDa), lane 1 is RV G protein, and lane 2 is rabies virus;
FIG. 3 is a Western blot results of anti-rabies glycoprotein monoclonal antibody 119A4, wherein M is the protein molecular weight standard (kDa); a is a polyclonal antibody pAb control, b is an anti-rabies glycoprotein monoclonal antibody 119A4;
FIG. 4 is a graph showing the results of protein recognition localization of anti-rabies virus monoclonal antibodies; a is rabies virus glycoprotein polypeptide scanning, b is rabies virus phosphoprotein polypeptide scanning;
FIG. 5 is a gel electrophoresis diagram of the light chain-heavy chain variable region PCR of anti-rabies virus phosphoprotein monoclonal antibody 112E3, wherein a is the light chain variable region and b is the heavy chain variable region; m is the protein molecular weight standard (kDa), lane 1 is 112E3 light chain PCR product 1, lane 2 is 112E3 light chain PCR product 2;
FIG. 6 is a gel electrophoresis diagram of a light chain-heavy chain variable region PCR of an anti-rabies glycoprotein monoclonal antibody 119A4, wherein a is the light chain variable region and b is the heavy chain variable region; m is protein molecular weight standard (kDa), lane 1 is 119A4 light chain PCR product 1, lane 2 is 119A4 light chain PCR product 2;
FIG. 7 shows the antigen detection specificity of the double antibody sandwich ELISA method established in the invention;
FIG. 8 shows the results of a linear test and sensitivity of the double-antibody sandwich ELISA method established by the invention, wherein a is a linear test of the double-antibody sandwich ELISA method, the abscissa is the titer (IU/ml) of the vaccine, and the ordinate is the OD450 values corresponding to different titers of the vaccine; panel b shows the sensitivity results, human Serum Albumin (HSA) was used as the detection baseline, samples RV-1, RV-2 and RV-3 were different batches of virus harvest, and final dilution was achieved by gradient dilution to 2 -7 The corresponding diluted reference vaccine is used as a quantitative standard to quantify the antigen content; the dotted line is the detection threshold (cutoff P/N)>2.1)。
Detailed Description
The present invention will be described in further detail with reference to examples.
It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The materials or equipment used are conventional products available from commercial sources, not identified to the manufacturer.
Unless otherwise indicated herein, percentages refer to percentages by volume and ratios refer to ratios by volume.
1 XHAT selective medium: 50 XHAT (purchased from sigma), 20% (V/V) fetal bovine serum (purchased from Biological Industries), 1640 medium (purchased from Corning), 1% (V/V) diabody (penicillin-streptomycin solution);
1×ht complete medium: (50×HT (purchased from sigma), 20% (V/V) fetal bovine serum (purchased from Biological Industries), 1640 medium (purchased from Corning), 1% (V/V) diabody (penicillin-streptomycin solution);
common complete medium: 20% (V/V) fetal bovine serum (purchased from Biological Industries), 1640 medium (purchased from Corning), 1% (V/V) diabody (penicillin-streptomycin solution).
EXAMPLE 1 establishment of anti-rabies monoclonal antibody hybridoma cell lines
1. Immunogen preparation: taking purified rabies virus (rabies virus CTN cell strain) as immunogen;
2. animal immunization: four immunizations were performed at 2 week intervals on 6-8 week old Balb/c mice with the following procedure: the first immunization injection is 200ul 10 6 FFU/ml, freund's complete adjuvant and equivalent immunogen are fully mixed and emulsified, and then injected subcutaneously at the back; the second immunization and the third immunization, the adjuvant is replaced by Freund's incomplete adjuvant, and the method and the dosage are the same as those of the first immunization; the fourth time of intraperitoneal injection of immunogen without adjuvant is adopted, and the injection quantity is the same as that of the first immunization; after 3 days, spleen cells of immunized mice were taken for preparation of hybridoma cells;
3. preparation of hybridoma cell lines: the method specifically comprises the following steps: the spleen cells of immunized mice were taken 2 days before cell fusion for preparation of hybridoma cells. Selecting a Balb/c mouse with the age of 6-8 weeks, taking abdominal macrophages to prepare feeder cells, and enabling the cell concentration to be 10 5 Each ml was added to a 96-well plate at 100. Mu.l per well and placed at 37℃in 5% CO 2 Culturing under the condition for standby. SP2/0 cells (purchased from ATCC) in the logarithmic growth phase were mixed with spleen cells of immunized mice of step 2 in a cell number ratio of 1:10, and hybridoma cells obtained by fusing 45% polyethylene glycol 4000 (PEG) (purchased from sigma) cells were added to a 96-well plate containing feeder cells and cultured at 37℃under 5% CO2 using a medium containing 1 XHAT selective medium.
4. The hybridoma cell strain is screened, and specifically: the hybridoma cells obtained in the step 3 were cultured under the condition of containing 1 XHAT selective medium for 10-14 days, and half amount of the 1 XHAT selective medium was replaced every 4 days. After 12 days of culture, hybridoma cell supernatants were subjected to indirect ELISA to initially screen hybridoma cells capable of specifically secreting anti-rabies phosphoprotein/glycoprotein monoclonal antibodies. The method comprises the following specific steps: the ELISA plate is coated with purified rabies virus and RV G protein respectively, a blank control is arranged, a culture supernatant is taken as a primary antibody, SP2/0 cell culture supernatant is taken as a negative control, a secondary antibody is HRP-marked goat anti-mouse IgG (1:6000) (Thermo Scientific), after color development of TMB (Solarbio) is stopped, an ELISA meter detects an absorbance value (A450 value) at 450nm, a well with a value of more than 2.1 times of that of a Yu Yinxing control A450 is taken as a positive cell well, and the next subcloning is carried out.
Wherein, RV G protein is the existing protein, which can be seen in Wei, yan Liwei, cinnamomum, zhang Xuemei, xu, wu Zhongxiang, lu Kongjie, sun Ming and Dong Shaozhong. Prokaryotic expression of rabies virus CTN-1 strain G protein and basic function evaluation [ J ]. J.J.J.of Chinese biologicals, 2016,29 (10): 1032-1036.DOI:10.13200/j.cnki.cjb.001471.
5. Cloning of hybridoma cells, specifically: selecting positive cell holes obtained in the step 4, cloning cells in the positive holes by adopting a limiting dilution method, sucking 200 mu l of cells in each hole into a sample adding groove, diluting the cells to 20ml by using 1 XHT complete culture medium, uniformly adding the cells into a new 96-well plate, and carrying out 200 mu l of cells in each hole at 37 ℃ and 5% CO (carbon monoxide) 2 After 5 days of culture, indirect ELISA detection is carried out again, cell holes which are more than 2.1 times higher than the negative control A450 value and have good cell growth state are selected again, the cells are cloned to another 96-well plate, 1 XHAT selective culture medium is replaced for culture, and three to four rounds of culture are repeated until 100% of antibodies in the cloning holes are detected positive. Selecting cells with good growth state, gradually expanding and culturing by using a common complete culture medium, and freezing and storing by liquid nitrogen.
In the subcloning culture screening process, an ELISA plate is coated with purified rabies virus and RV G protein respectively, hybridoma fine culture supernatant is taken after sealing for indirect ELISA, blank control is set, SP2/0 cell culture supernatant is negative control, goat anti-mouse IgG (1:6000) is marked by HRP and is used as secondary antibody, monoclonal cells capable of identifying rabies virus phosphoprotein and glycoprotein are screened according to the reading of the A450 value of the ELISA plate, and further subcloning and amplification culture are carried out on hybridoma cell strains.
The result is that 2 cell lines with stable secretion antibodies are obtained by subcloning positive hybridoma cells for 4 times, the serial numbers are RV 112E3 and RV 119A4, and the obtained hybridoma cell lines can be stably passaged and can continuously and stably secrete the monoclonal antibodies of rabies virus phosphoproteins/glycoproteins.
EXAMPLE 2 Large-scale preparation and identification of monoclonal antibodies against rabies virus phosphoproteins/glycoproteins
1. Large-scale preparation of monoclonal antibodies against rabies virus phosphoproteins/glycoproteins
Taking 8-10 week old healthy Balb/c mice, injecting 500ul liquid paraffin oil into the abdominal cavity, and injecting the positive cloned cells obtained by the step expansion culture into the abdominal cavity of the mice after two weeks, wherein the injection amount of each mouse cell is 10 6 -10 7 And collecting ascites samples after 10-14 days, sub-packaging, freezing or purifying. The above-mentioned mouse ascites is centrifuged at 8000rpm/min for 30 minutes to remove cellular components and other sediments, the collected supernatant is purified by Protein A column (purchased from full-scale gold), the purity and integrity of the antibody are analyzed by 12% SDS-PAGE, the heavy chain and light chain of the 2 monoclonal antibodies are about 55KD and 28KD respectively, and finally the obtained monoclonal antibody of the mouse anti-rabies virus phosphoprotein/glycoprotein is concentrated to 1mg/ml by ultrafiltration centrifuge tubes, the serial numbers are 112E3 and 119A4 respectively, and the obtained monoclonal antibody is split-packed and frozen for standby.
2. Analysis and identification of biological properties of monoclonal antibodies
1. Monoclonal antibody type identification
The type of antibody was identified using Mouse Monoclonal Antibody Isotyping Kit (Roche), see instructions. The results demonstrate that Mab 112E3 is of the IgG2b subtype, mab 119A4 is of the IgG1 subtype and the light chains are of the Kappa type.
2. Monoclonal antibody specificity identification
Purified RV G protein and rabies virus samples were taken separately, 5 XSDS-PAGE protein loading buffer [250mM Tris-HCl (pH 6.8), 10% (W/V) SDS,0.5% (W/V) BPB,50% (V/V) glycerol, 5% (W/V) beta-mercaptoethanol ] was added in a ratio of 4:1, and the final concentration of the buffer was 1×, heated at 95℃for 10 minutes to sufficiently denature the protein. And (3) after cooling to room temperature, respectively loading the RV G protein and rabies virus treated samples directly into SDS-PAGE gel sample loading holes. 80V for 30 min, 150V for 50 min. The proteins after electrophoresis were transferred to PVDF membrane (purchased from Millipore) using a semi-dry transfer device (BIO-RAD) with a transfer current of 100mA and a transfer time of 45min according to the instructions of the transfer device. Immediately after the completion of the transfer, the PVDF membrane after the completion of the transfer was placed in 5% (W/V) of skimmed milk powder TBS [20mM Tris-HCl,500mM NaCl (pH 7.5) ] prepared in advance, slowly shaken (40 rpm/min) on a shaker, and blocked at room temperature for 60 minutes. The purified monoclonal antibody was used as a primary antibody, diluted 1:1000 with TBST wash [20mM Tris-HCl,500mM NaCl (pH 7.5) 0.01% Tween-20 (V/V) ] and added to PVDF membrane after completion of blocking for overnight incubation. An anti-dilution solution is discarded. 5ml of TBST membrane washing solution was added. Shaking (speed 80 rpm/min) on a side shaking table was repeated 5 times. Incubation was then performed with HRP-labeled goat anti-mouse IgG as secondary antibody (diluted 1:10000 with TBST wash) for 1h at room temperature. Discarding the secondary anti-dilution solution, repeating TBST film washing for 5 times, adding ECL (purchased from Millipore) reagent, tabletting in a darkroom, performing film washing or machine exposure through a developer fixing solution, and detecting the identification effect of the monoclonal antibody. As a result, as shown in FIG. 2, the 112E3 monoclonal antibody screened by ELISA was able to specifically recognize phosphoprotein of rabies virus. The result of WB identification with respect to 119A4 monoclonal antibody by using non-denatured Native-Page is shown in FIG. 3, and 119A4 and polyclonal antibody were consistent with specific recognition of natural rabies virus glycoprotein.
3. Monoclonal antibody protein recognition localization
RVP protein and G protein fragment polypeptides (15-16 aa/peptide) were synthesized by Kunming Zanana Biotechnology Co., ltd, see Table 1. All polypeptides were dissolved and diluted to 10. Mu.g/ml with PBS buffer, and 100. Mu.l per well was coated on a 96-well ELISA plate and incubated overnight at 4 ℃; removing the solution in the wells, washing the plates 3 times with 0.05% PBST, adding 200 μl of 5% skimmed milk powder PBST into the enzyme-labeled wells, incubating at 37deg.C for 2h, and sealing; the solution in the wells is discarded, the plate is washed 3 times by 0.05% PBST, the obtained purified ascites monoclonal antibody is diluted to 10 mug/ml by PBS buffer solution, 100 mug of the purified ascites monoclonal antibody is respectively added into the coated reaction wells, the reaction wells are incubated for 1h at 37 ℃, blank wells and negative control wells are simultaneously made, the blank wells are non-coated wells, and the negative control wells replace the ascites monoclonal antibody by PBS solution as primary antibodies. The solution in the wells was discarded, the plate was washed 3 times with 0.05% PBST, fresh diluted HPR-goat anti-mouse IgG (1:6000) was added to each reaction well, 100. Mu.l per well was incubated for 1h at 37 ℃; the solution in the wells was discarded, the plate was washed 3 times with 0.05% PBST, 100. Mu.l of TMB substrate developing solution was added to each well, the reaction was stopped by adding 100. Mu.l of 2M sulfuric acid after developing at 37℃for 10 minutes in the absence of light, and the absorbance at 450nm (A450 value) was measured. As a result, as shown in FIG. 4, 112E3 was able to specifically recognize rabies virus phosphoprotein P3 (NIEDNQAHLQGEPIE), while 119A4 was able to specifically recognize rabies virus glycoprotein G15 (RGLYKSLKGACKLKL), 112E3 and 119A4 recognized different epitopes on different proteins of rabies virus, respectively, and neither recognition protein nor recognition site was cross-reacted between clones for detection of the advantage of high specificity and sensitivity.
TABLE 1 RVP protein and G protein fragment polypeptide (15-16 aa/peptide)
4. Monoclonal antibodies 112E3 and 119A4 variable region sequencing
1) RNA extraction: extracting RNA of selected hybridoma cell recognizing rabies virus phosphoprotein/glycoprotein by TRNzol-A+ (TIANGEN) with cell number of 10 5 。
2) Reverse transcription to obtain cDNA: the obtained RNA was reverse transcribed into cDNA using PrimeScript II 1st Strand cDNA Synthesis Kit, see kit (Takara, dalia).
3) Nested PCR amplification of antibody heavy chain variable region: the nested PCR amplification system was 56ul total:
PrimeSTAR Max premixM(Takara,Dalian))28ul；
5ul of the cDNA obtained in the step 2);
VH Forward (2 pM per primer concentration): each 1ul, 4 total, 4ul;
VH Reverse (2 pM per primer concentration): each 1ul, 19 total, 19ul.
The amplification procedure was: pre-denaturation at 98 ℃ for 1 min; denaturation at 98℃for 10 sec, renaturation at 65℃for 10 sec, extension at 72℃for 10 sec, and cycle 35 times; extension was carried out at 72℃for 5 minutes.
4) Nested PCR amplification of antibody light chain variable region: nested PCR amplification system 52ul:
PrimeSTAR Max premixM(Takara,Dalian)26ul；
5ul of the cDNA obtained in the step 2);
VL (κ) Forward (2 pM per primer concentration): each 1ul, 4 total, 4ul;
VL (kappa) Reverse (2 pM per primer concentration): each 1ul, 17 total, 17ul.
The amplification method comprises the following steps: pre-denaturation at 98 ℃ for 1 min; denaturation at 98℃for 10 seconds, renaturation at 60℃for 10 seconds, extension at 72℃for 10 seconds, and cycle 35 times; extending at 72 ℃ for 5 minutes; the PCR primers are shown in Table 2.
5) Identification of nested PCR products by agarose gel electrophoresis: sample loading: marker (DL 2000), VH, VL; the results are shown in FIGS. 5 and 6 using a 1% agarose gel, 100mA, for 20 min.
6) Antibody variable region sequencing and sequence analysis: sequencing the gel recovery product of step 5) in a T vector fed to Tsingke company, comparing the sequencing result with IgBLAST to analyze the sequence, and translating the obtained sequence into an amino acid sequence of protein. As a result, 112E3 has an amino acid sequence comprising a light chain variable region as shown in SEQ ID No.1 and a heavy chain variable region as shown in SEQ ID No. 2; the coding DNA sequence of the light chain variable region is shown as SEQ ID NO. 3; the coding DNA sequence of the heavy chain variable region is shown as SEQ ID NO. 4; 119A4 comprises a light chain variable region as shown in SEQ ID No.5 and a heavy chain variable region as shown in SEQ ID No. 6; the coding DNA sequence of the light chain variable region is shown as SEQ ID NO. 7; the coding DNA sequence of the heavy chain variable region is shown as SEQ ID NO. 8; the sequence was aligned to show no identical sequence, indicating that the obtained sequence was specific for each of the 2 clones.
TABLE 2 PCR primers
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Example 3 establishment of rabies virus double antibody sandwich ELISA antigen detection method
1. Establishment of rabies virus double-antibody sandwich ELISA antigen detection method
1) Coating: 100 μl/well of anti-rabies virus monoclonal antibody was diluted with 0.05M carbonate buffer at pH9.6 and coated overnight at 4deg.C;
2) Washing: the solution in the wells was discarded and the plate was washed 3 times with 0.05% pbst;
3) Closing: adding a blocking solution, namely a sample diluent, 200 μl/hole, and blocking at 37deg.C for 2h;
4) Washing: the solution in the wells was discarded and the plate was washed 3 times with 0.05% pbst;
5) Adding an antigen: the antigen is properly diluted by a sample diluent, 100 μl/well is incubated for 1h at 37 ℃;
6) Washing: the solution in the wells was discarded and the plate was washed 3 times with 0.05% pbst;
7) Adding the ELISA monoclonal antibody: adding horseradish peroxidase-labeled antibody into the reaction hole, and incubating for 50min at 37 ℃ with 100 μl/hole;
8) Washing: the solution in the wells was discarded and the plate was washed 3 times with 0.05% pbst;
9) Adding a substrate: 100 μl of TMB substrate chromogenic solution (from Solarbio) per well was developed in the absence of light at 37deg.C for 10min;
10 Adding a stop solution: the reaction was terminated with 100. Mu.l of 2M sulfuric acid, and the absorbance at 450nm (A450 value) was measured.
2. Selection of coating monoclonal antibody and ELISA monoclonal antibody
The preparation method comprises the steps of respectively coating 96-well ELISA plates with 2 anti-rabies virus monoclonal antibodies 112E3 and 119A4 (10 mug/ml) prepared by the method as coating antibodies, adding positive control (rabies virus sample) and negative control (cell culture supernatant) as to-be-detected, respectively diluting the 2 monoclonal antibodies to 0.5 mug/ml by horseradish peroxidase labeling, operating according to the ELISA steps, selecting a pairing combination of the coating monoclonal antibodies and the ELISA monoclonal antibodies according to OD450, and selecting one group of detection systems for evaluation, specifically using 112E3 strain as the coating antibody and enzyme labeled monoclonal antibody 119A4 strain as the detection antibody.
3. Selection of working concentration of coating monoclonal antibody and ELISA monoclonal antibody
The use concentration of each monoclonal antibody is discussed respectively by taking the anti-rabies virus phosphoprotein coating monoclonal antibody 112E3 and the HRP marked anti-rabies virus glycoprotein ELISA monoclonal antibody 119A4 as the combination. 112E3 to 20, 10, 5, 2.5, 0.6 and 0.16 mug/ml, respectively coating an ELISA plate, adding positive control (rabies virus sample) and negative control (PBS) as samples to be detected, diluting an ELISA antibody to 0.5 mug/ml, and detecting according to the ELISA steps; the detection working concentration of the ELISA monoclonal antibody 119A4 is determined by comparing antibody detection results with different concentrations including 1, 0.5, 0.1 and 0.01 mug/ml, the coating concentration of the coating monoclonal antibody 112E3 is selected to be 2.5 mug/ml by comprehensive evaluation, and the working concentration of the ELISA monoclonal antibody 119A4 is 0.5 mug/ml.
4. Specificity experiments
Inactivated poliovirus type I (inactivated poliovirus type I, IPV-I), IPV-II, IPV-III, hepatitis A virus (hepatitis A virus, HAV), enterovirus (Enterovirus 71, EV71), rabies virus reference vaccine, rabies virus preparation sample and PBS were detected as positive controls, respectively, and PBS was used as negative control to evaluate the specificity of the method according to the above-established double-antibody sandwich ELISA antigen detection method. The results show that the OD450 values of the other 5 virus antigens detected by the double-antibody sandwich ELISA method are lower than the critical value of yin and yang, namely the detection results are all negative, which indicates that the method has good detection specificity on rabies virus antigens, and the result is shown in figure 7.
5. Sensitivity and linear range testing
Rabies viruses are respectively treatedVaccine references were diluted to 2.0, 1.75, 1.5, 1.25, 0.625, 0.313, 0.156IU/ml, and tested according to the rabies virus double antibody sandwich ELISA antigen detection method of example 3, and the final linear results are shown in fig. 8 a. By subjecting different batches of rabies virus harvest solutions RV-1, RV-2, RV-3 and rabies virus vaccine to 2-time gradient dilution to 2 -7 The initial concentration of the vaccine is 1.25IU/mL, the sensitivity of the method is 0.02IU/mL, the linear range is 0.15-2.0IU/mL, and as shown in figure 8b, the experimental result shows that the kit has good sensitivity and linear range.
The results show that the anti-rabies glycoprotein/phosphoprotein monoclonal antibody has strong specificity and good sensitivity, can be further developed and can be used for detecting, preventing, controlling and investigating epidemic diseases and the like of rabies, and has important practical significance. The double-antibody sandwich ELISA antigen detection method constructed based on the monoclonal antibody has no cross reaction to other epidemic virus antigens, has good specificity and sensitivity, carries out rapid quantitative detection on the antigen content of each stage in the vaccine preparation process in time by establishing an internal control standard, is suitable for guiding the monitoring and control of the steps of virus harvesting, concentration, inactivation and purification processes, and provides basis for screening, determining and optimizing the rabies virus vaccine preparation method and process.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
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Claims (9)

1. A double-antibody sandwich antigen detection kit for rabies virus is characterized in that an anti-rabies virus phosphoprotein monoclonal antibody and an anti-rabies virus glycoprotein monoclonal antibody are adopted, the anti-rabies virus phosphoprotein monoclonal antibody is taken as a coating antibody, the enzyme-labeled anti-rabies virus glycoprotein monoclonal antibody is taken as a detection antibody, or the anti-rabies virus glycoprotein monoclonal antibody is taken as a coating antibody, and the enzyme-labeled monoclonal antibody phosphoprotein monoclonal antibody is taken as a detection antibody;
the anti-rabies virus phosphoprotein monoclonal antibody comprises a heavy chain variable region and a light chain variable region;
the anti-rabies glycoprotein monoclonal antibody also comprises a heavy chain variable region and a light chain variable region;
the amino acid sequence of the light chain variable region of the rabies virus resistance phosphoprotein monoclonal antibody is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 2; the amino acid sequence of the light chain variable region of the anti-rabies glycoprotein monoclonal antibody is shown as SEQ ID NO.5, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 6.
2. The kit for detecting the rabies virus double-antibody sandwich antigen according to claim 1, wherein the enzyme is horseradish peroxidase or alkaline phosphatase.
3. The anti-rabies virus phosphoprotein monoclonal antibody is characterized by comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the light chain variable region of the anti-rabies virus phosphoprotein monoclonal antibody is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 2.
4. The anti-rabies virus glycoprotein monoclonal antibody is characterized by comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the light chain variable region of the anti-rabies virus glycoprotein monoclonal antibody is shown as SEQ ID NO.5, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 6.
5. A DNA encoding an anti-rabies virus phosphoprotein monoclonal antibody, wherein the anti-rabies virus phosphoprotein monoclonal antibody comprises a heavy chain variable region and a light chain variable region; the coding DNA sequence of the light chain variable region of the anti-rabies virus phosphoprotein monoclonal antibody is shown as SEQ ID NO.3, and the coding DNA sequence of the heavy chain variable region is shown as SEQ ID NO. 4.
6. A DNA encoding an anti-rabies virus glycoprotein monoclonal antibody, wherein said anti-rabies virus glycoprotein monoclonal antibody comprises a heavy chain variable region and a light chain variable region; the coding DNA sequence of the light chain variable region of the anti-rabies glycoprotein monoclonal antibody is shown as SEQ ID NO.7, and the coding DNA sequence of the heavy chain variable region is shown as SEQ ID NO. 8.
7. Use of the anti-rabies virus phosphoprotein monoclonal antibody of claim 3 or the anti-rabies virus glycoprotein monoclonal antibody of claim 4 in the preparation of a rabies virus vaccine antigen content detection reagent or kit, a rabies virus antibody detection reagent or kit.
8. Use of the anti-rabies virus phosphoprotein monoclonal antibody of claim 3 or the anti-rabies virus glycoprotein monoclonal antibody of claim 4 in the preparation of a rabies virus vaccine production product quality detection reagent or kit.
9. Use of the anti-rabies virus phosphoprotein monoclonal antibody of claim 3 or the anti-rabies virus glycoprotein monoclonal antibody of claim 4 in the preparation of a medicament for preventing or treating rabies.
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