CN117025548B - Mouse anti SARS-CoV-2 Spike protein hybridoma cell strain, antibody, kit and application - Google Patents
Mouse anti SARS-CoV-2 Spike protein hybridoma cell strain, antibody, kit and application Download PDFInfo
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- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The application discloses a mouse anti-SARS-CoV-2 Spike protein hybridoma cell strain, an antibody, a kit and application. The preservation number of the mouse anti-SARS-CoV-2 Spike protein hybridoma cell strain is CGMCC NO 45629, the preservation date is 2023, 07 and 13 days, the preservation classification is named as the mouse anti-SARS-CoV-2 Spike protein hybridoma cell strain, and the preservation unit is China general microbiological culture Collection center. Meanwhile, the application provides a method for detecting SARS-CoV-2 Spike protein activity. The application has the advantages that the detection method has high sensitivity and high detection accuracy, and can realize the detection of the SARS-CoV-2 Spike protein binding activity.
Description
Technical Field
The application belongs to the field of biotechnology, and in particular relates to an antibody for resisting SARS-CoV-2 Spike protein, application, a kit and a detection method.
Background
SARS-CoV-2 is very contagious and can spread even during latency, so the importance of its research and activity detection is becoming increasingly prominent.
SARS-CoV-2 is mainly composed of four structural proteins, which are Spike protein (Spike protein), nucleocapsid protein (N protein), membrane protein (M protein) and envelope protein (E protein), respectively. Wherein the Spike protein consists of two subunits, S1 and S2. The receptor binding site (RBD) in the S1 subunit is primarily responsible for binding to the angiotensin converting enzyme 2 (ACE 2) receptor on the surface of host cells to effect fusion with the host cells. Thus, spike protein is a potential target for the development of vaccines, therapeutic antibodies and clinical diagnostics.
Currently, there are many methods for detecting SARS-CoV-2 Spike protein. One of the commonly used methods is the double antibody sandwich method, using ELISA technology. The method immobilizes SARS-CoV-2 Spike protein antibody on an enzyme-linked plate, and then uses another SARS-CoV-2 Spike protein antibody labeled with horseradish peroxidase (HRP). After the addition of the test substance, an immune complex is formed, and whether SARS-CoV-2 Spike protein is contained in the test substance is judged by detecting the HRP label. Although the double antibody sandwich method can quantitatively detect the SARS-CoV-2 Spike protein content in the test object, the binding capacity of the virus to cells cannot be estimated, and the activity level cannot be reflected.
In addition, some researchers have developed a new assay using the property of SARS-CoV-2 Spike protein binding to cell surface ACE 2. This method uses a biotin ester-labeled ACE2 recombinant protein to coat magnetic particles and an alkaline phosphatase (ALP) -labeled ACE2 recombinant protein and a luminescent substrate AMPPD. The SARS-CoV-2 Spike protein detecting reagent prepared by the method omits the steps of antibody preparation and screening and uses mature marking materials, but the method can not detect RBD monomers, and because of higher cost of chemiluminescent instruments, many laboratories can not detect Spike protein by the method.
Disclosure of Invention
In order to solve at least one of the technical problems, the application develops a detection method which is low in cost, simple and convenient to operate, sensitive and accurate and can rapidly detect the content of the Spike protein of the SARS-CoV-2, and provides an antibody, application, a kit and a detection method for resisting the Spike protein of the wild strain and various mutant strains of the SARS-CoV-2.
In a first aspect, the present application provides a murine anti-SARS-CoV-2 Spike protein hybridoma cell strain, wherein the cell strain has a preservation number of CGMCC NO 45629, a preservation date of 2023, 07 and 13 days, and the preservation classification is named as a murine anti-SARS-CoV-2 Spike protein hybridoma cell strain, and the preservation unit is China general microbiological culture Collection center.
By adopting the technical scheme, the mouse anti-SARS-CoV-2 Spike protein hybridoma cell strain can contribute to research of SARS-CoV-2, and simultaneously provides a reliable experimental material for research of SARS-CoV-2.
In a second aspect, the present application provides an antibody against Spike protein of wild-type strain SARS-CoV-2 and various mutant strains, which is prepared from the mouse anti-SARS-CoV-2 Spike protein hybridoma cell strain provided herein by ascites preparation and purification.
By adopting the technical scheme, the antibody of Spike protein for resisting SARS-CoV-2 wild strain and various mutant strains can effectively identify and combine the wild strain and various mutant strains of SARS-CoV-2. Meanwhile, the antibody of Spike protein for resisting SARS-CoV-2 wild strain and various mutant strains has the following effects: in a first aspect, the present application provides an antibody against Spike proteins of wild-type strain and various mutant strains of SARS-CoV-2, which can be used to develop or improve detection methods of SARS-CoV-2, such as ELISA, immunofluorescence assay, etc. These methods can help to improve early detection and diagnostic accuracy of viruses. In a second aspect, the present application provides an antibody against Spike proteins of wild-type SARS-CoV-2 and various mutant strains, which may have the ability to neutralize viruses, and may be used to develop vaccines or therapeutic methods. Neutralizing antibodies can prevent the entry of the virus into the host cell, thereby preventing infection or alleviating symptoms of the disease. In a third aspect, the present application provides an antibody against Spike proteins of wild-type strains and various mutant strains of SARS-CoV-2, which can be used to develop therapeutic antibody drugs as an option for the treatment of SARS-CoV-2 infection. These antibodies can block viral replication and spread against critical sites of the virus, helping to reduce disease severity and recovery time. In a word, by providing an antibody against Spike proteins of wild strains and various mutant strains of SARS-CoV-2 and combining with the corresponding technical scheme, it is possible to provide important tools and strategies for detection, prevention and treatment of SARS-CoV-2. This is of great importance for protecting public health.
In a third aspect, the present application provides the use of antibodies against Spike proteins from wild-type SARS-CoV-2 strains and various mutant strains in the field of biological assays.
In a fourth aspect, the present application provides a kit for detecting SARS-CoV-2 Spike protein activity, the kit comprising an enzyme-labeled antibody, ACE2, a chromogenic solution, and a stop solution;
wherein the antibody in the enzyme-labeled antibody is an antibody of Spike protein for resisting SARS-CoV-2 wild strain and various mutant strains.
By adopting the technical scheme, the kit for detecting the activity of the SARS-CoV-2 Spike protein provides a tool for detecting the activity of the SARS-CoV-2 Spike protein, the detection of the Spike protein is realized by using antibodies of Spike proteins of anti-SARS-CoV-2 wild strains and various mutant strains and horseradish peroxidase, and the product generated by the reaction of TMB chromogenic liquid is quantitatively analyzed. The sulfuric acid solution was used to stop the reaction. In summary, the kit for detecting SARS-CoV-2 Spike protein activity has potential practical application in research and diagnosis.
Optionally, the dilution concentration of the enzyme-labeled antibody is 1×10 -4 ~1mg/mL。
Optionally, in the dilution process of the enzyme-labeled antibody, the used diluent comprises phosphate buffer solution with concentration of 1% bovine serum albumin of 10mmol/L and pH value of 7.4.
Optionally, the dilution concentration of the enzyme-labeled antibody is 1×10 -3 ~2*10 -3 mg/mL。
Optionally, in the enzyme-labeled antibody, the enzyme comprises horseradish peroxidase.
Optionally, the color development liquid comprises TMB color development liquid; the stop solution comprises sulfuric acid solution with the concentration of 2 mol/L.
Optionally, the dilute concentration of ACE2 is 2×10 -3 mg/mL。
Further alternatively, in the case of ACE2 dilution, the dilution liquid used comprises a carbonate buffer with a concentration of 50mmol/L and a pH of 9.6.
In a fifth aspect, the present application provides a method for detecting SARS-CoV-2 Spike protein activity, the method comprising the steps of:
s1, diluting ACE2 to 2 x 10 by using buffer solution -3 mg/mL, coating diluted ACE2 on the enzyme-linked plate in an amount of 100 mu L/hole, and fully incubating to combine the ACE2 with a fixing reagent on the surface of the enzyme-linked plate to prepare coated ACE2;
s2, labeling an antibody of Spike protein of the SARS-CoV-2 wild strain and various mutant strains by using HRP to prepare an enzyme-labeled antibody;
s3, adding the to-be-detected object into the enzyme-linked plate in the step S1, combining the Spike protein in the to-be-detected object with the coated ACE2, washing to remove the unbound to-be-detected sample, drying by beating to obtain a combined product of the Spike protein and the ACE2, and diluting the enzyme-labeled antibody to 1X 10 by using a diluent -4 1mg/mL, adding 100 mu L/hole of the mixture into a conjugate of Spike protein and ACE2, and reacting at 37 ℃ to form immune complexes of the ACE2, the Spike protein and the enzyme-labeled antibody;
s4, washing and removing unbound substances in the compound in the step S3, drying, adding a color development liquid, reacting for not less than 5min at 37 ℃ in a dark condition, adding a sulfuric acid solution with the volume of 50 mu L and the concentration of 2mol/L to terminate the reaction, measuring the optical density of the color reaction in the ELISA plate at the wavelength of 450nm by using an ELISA reader, and judging the binding activity between Spike protein and ACE2 according to the optical density value.
By adopting the technical scheme, the method for detecting the activity of the SARS-CoV-2 Spike protein is successfully developed. Compared with the prior art, the method has the following advantages: in the first aspect, the conventional double antibody sandwich method can only detect the content of Spike protein, and cannot evaluate the binding capacity and activity level with cells. The detection method provided by the application can directly reflect the binding activity between Spike protein and ACE2 by measuring the optical density value. In a second aspect, the detection method provided by the present application can detect RBD (receptor binding domain) monomers, which is a key region for binding SARS-CoV-2 Spike protein to host cells, and has important significance for research and diagnosis. In the third aspect, compared with some existing methods, the detection method provided by the application is low in cost, can reduce detection cost, and improves feasibility and implementation of detection. In a fourth aspect, the detection method provided by the present application employs high quality antibody and enzyme-labeled technology, which can provide high sensitivity and high specificity detection results. In the fifth aspect, by measuring the optical density value, an accurate result can be obtained, and the accuracy and reliability of detection are improved. Therefore, the SARS-CoV-2 Spike protein activity detection method has potential to be applied in the research and diagnosis fields, and can provide important technical support for disease monitoring, virus screening and clinical diagnosis.
Optionally, the incubation conditions in step S1 are as follows: phosphate buffer (10 mmol/L, pH 7.4) containing 2% bovine serum albumin and 5% sucrose was prepared and added to the plate described in step S1 in an amount of 200. Mu.L/well, and incubated at 37℃for 2 hours to prepare an ACE 2-coated plate.
Optionally, step S3 is as follows:
s3, adding the to-be-detected object into the enzyme-linked plate in the step S1, combining the Spike protein in the to-be-detected object with the coated ACE2, washing to remove the unbound to-be-detected sample, drying by beating to obtain a combined product of the Spike protein and the ACE2, and diluting the enzyme-labeled antibody to 1X 10 by using a diluent -3 1mg/mL, and adding 100 mu L/hole of the mixture into a conjugate of Spike protein and ACE2, and reacting at 37 ℃ to form immune complexes of the ACE2, the Spike protein and the enzyme-labeled antibody.
Preferably, step S3 is as follows:
s3, adding the to-be-detected object into the enzyme-linked plate in the step S1, combining the Spike protein in the to-be-detected object with the coated ACE2, washing to remove the unbound to-be-detected sample, drying by beating to obtain a combined product of the Spike protein and the ACE2, and diluting the enzyme-labeled antibody to 1X 10 by using a diluent -3 ~4*10 -3 mg/mL and added to Spike protein bind ACE2 at 100. Mu.L/wellIn the combined conjugates, the reaction was performed at 37℃to form an immune complex of ACE2, spike protein and enzyme-labeled antibody.
Preferably, step S3 is as follows:
s3, adding the to-be-detected object into the enzyme-linked plate in the step S1, combining the Spike protein in the to-be-detected object with the coated ACE2, washing to remove the unbound to-be-detected sample, drying by beating to obtain a combined product of the Spike protein and the ACE2, and diluting the enzyme-labeled antibody to 1X 10 by using a diluent -3~ 2*10 -3 mg/mL and 100. Mu.L/well of Spike protein to ACE2 binding conjugate was reacted at 37℃to form an immune complex of ACE2, spike protein and enzyme-labeled antibody.
Further preferably, step S3 is as follows:
s3, adding the to-be-detected object into the enzyme-linked plate in the step S1, combining the Spike protein in the to-be-detected object with the coated ACE2, washing to remove the unbound to-be-detected sample, drying by beating to obtain a combined product of the Spike protein and the ACE2, and diluting the enzyme-labeled antibody to 2X 10 by using a diluent -3 mg/mL and 100. Mu.L/well of Spike protein to ACE2 binding conjugate was reacted at 37℃to form an immune complex of ACE2, spike protein and enzyme-labeled antibody.
By adopting the technical scheme, the concentration provided by the application is 2 x 10 -3 The accuracy of the assay is further improved by the use of an HRP-labeled antibody against the Spike protein of wild-type SARS-CoV-2 and various mutants in mg/mL. In step S3, the concentration of the enzyme-labeled antibody used was 2×10 -3 At the concentration of mg/mL, the enzyme-labeled antibody and different SARS-CoV-2 Spike proteins can effectively react and have good color development effect. This is shown at 2 x 10 -3 Binding between the enzyme-labeled antibody and Spike protein is most effective at mg/mL concentrations. The concentration of the enzyme-labeled antibody can fully and specifically react with target proteins to provide accurate detection results. Thus, by concentration of the enzyme-labeled antibody at 2×10 -3 The experiment is carried out under the condition of mg/mL, so that the detection accuracy can be improved, and the binding reaction with SARS-CoV-2 Spike protein can be effectively carried out. This will beProviding reliable technical support for subsequent research and diagnosis work.
In summary, the present invention includes at least one of the following beneficial technical effects:
the mouse anti-SARS-CoV-2 Spike protein hybridoma cell strain can contribute to research of SARS-CoV-2, and simultaneously provides a reliable experimental material for research of SARS-CoV-2.
The antibody of Spike protein for resisting SARS-CoV-2 wild strain and various mutant strains can effectively identify and combine the wild strain and various mutant strains of SARS-CoV-2. Meanwhile, the antibody of Spike protein for resisting SARS-CoV-2 wild strain and various mutant strains has the following effects: in a first aspect, the present application provides an antibody against Spike proteins of wild-type strain and various mutant strains of SARS-CoV-2, which can be used to develop or improve detection methods of SARS-CoV-2, such as ELISA, immunofluorescence assay, etc. These methods can help to improve early detection and diagnostic accuracy of viruses. In a second aspect, the present application provides an antibody against Spike proteins of wild-type SARS-CoV-2 and various mutant strains, which may have the ability to neutralize viruses, and may be used to develop vaccines or therapeutic methods. Neutralizing antibodies can prevent the entry of the virus into the host cell, thereby preventing infection or alleviating symptoms of the disease. In a third aspect, the present application provides an antibody against Spike proteins of wild-type strains and various mutant strains of SARS-CoV-2, which can be used to develop therapeutic antibody drugs as an option for the treatment of SARS-CoV-2 infection. These antibodies can block viral replication and spread against critical sites of the virus, helping to reduce disease severity and recovery time. In a word, by providing an antibody against Spike proteins of wild strains and various mutant strains of SARS-CoV-2 and combining with the corresponding technical scheme, it is possible to provide important tools and strategies for detection, prevention and treatment of SARS-CoV-2. This is of great importance for protecting public health.
The kit for detecting SARS-CoV-2 Spike protein activity has potential practical application in research and diagnosis.
The method for detecting SARS-CoV-2 Spike protein activity successfully develops a method for detecting SARS-CoV-2 Spike protein activity. Compared with the prior art, the method has the following advantages: in the first aspect, the conventional double antibody sandwich method can only detect the content of Spike protein, and cannot evaluate the binding capacity and activity level with cells. The detection method provided by the application can directly reflect the binding activity between Spike protein and ACE2 by measuring the optical density value. In a second aspect, the detection method provided by the present application is capable of detecting RBD (receptor binding domain) monomers, which is a critical region for binding of coronavirus Spike protein to host cells, and has important significance for research and diagnosis. In the third aspect, compared with some existing methods, the detection method provided by the application is low in cost, can reduce detection cost, and improves feasibility and implementation of detection. In a fourth aspect, the detection method provided by the present application employs high quality antibody and enzyme-labeled technology, which can provide high sensitivity and high specificity detection results. In the fifth aspect, by measuring the optical density value, an accurate result can be obtained, and the accuracy and reliability of detection are improved. Therefore, the SARS-CoV-2 Spike protein activity detection method has potential to be applied in the research and diagnosis fields, and can provide important technical support for disease monitoring, virus screening and clinical diagnosis.
The concentration provided herein is 2 x 10 -3 The accuracy of the assay is further improved by the use of an HRP-labeled antibody against the Spike protein of wild-type SARS-CoV-2 and various mutants in mg/mL. In step S3, the concentration of the enzyme-labeled antibody used was 2×10 - 3 At the concentration of mg/mL, the enzyme-labeled antibody and different SARS-CoV-2 Spike proteins can effectively react and have good color development effect. This is shown at 2 x 10 -3 Binding between the enzyme-labeled antibody and Spike protein is most effective at mg/mL concentrations. The concentration of the enzyme-labeled antibody can fully and specifically react with target proteins to provide accurate detection results. Thus, by concentration of the enzyme-labeled antibody at 2×10 -3 The experiment is carried out under the condition of mg/mL, the detection accuracy can be improved, and the binding reaction with SARS-CoV-2 Spike protein can be effectively carried outAnd (3) row. This will provide reliable technical support for subsequent research and diagnostic work.
Drawings
FIG. 1 is a graph showing the results of Spike protein activity test for different antigens in example 9.
FIG. 2 is a graph showing the results of Spike protein activity test for different samples in example 10.
Detailed Description
The present application is described in further detail below with reference to the drawings and examples.
In a first aspect, the present application designs a mouse anti-SARS-CoV-2 Spike protein hybridoma cell strain, the cell strain has a preservation number of CGMCC NO 45629, a preservation date of 2023, 07 and 13 days, and the preservation classification is named as a mouse anti-SARS-CoV-2 Spike protein hybridoma cell strain, and the preservation unit is China general microbiological culture Collection center.
In a second aspect, the present application contemplates an antibody against Spike protein of wild-type strain SARS-CoV-2 and various mutant strains, said antibody being prepared from the murine anti-SARS-CoV-2 Spike protein hybridoma cell line provided herein by ascites preparation followed by purification.
In a third aspect, the present application contemplates the use of antibodies against Spike proteins from wild-type strains and various mutant strains of SARS-CoV-2 in the field of biological assays.
In a fourth aspect, the present application contemplates a kit for detecting SARS-CoV-2 Spike protein activity, the kit comprising an enzyme-labeled antibody, ACE2, a chromogenic solution, and a stop solution;
wherein the antibody in the enzyme-labeled antibody is an antibody of Spike protein for resisting SARS-CoV-2 wild strain and various mutant strains.
In a fifth aspect, the present application contemplates a method for detecting SARS-CoV-2 Spike protein activity, the method comprising the steps of:
s1, diluting ACE2 to 2 x 10 by using buffer solution -3 mg/mL, coating diluted ACE2 on the enzyme-linked plate in an amount of 100 mu L/hole, and fully incubating to combine the ACE2 with a fixing reagent on the surface of the enzyme-linked plate to prepare coated ACE2;
s2, labeling an antibody of Spike protein of the SARS-CoV-2 wild strain and various mutant strains by using HRP to prepare an enzyme-labeled antibody;
s3, adding the to-be-detected object into the enzyme-linked plate in the step S1, combining the Spike protein in the to-be-detected object with the coated ACE2, washing to remove the unbound to-be-detected sample, drying by beating to obtain a combined product of the Spike protein and the ACE2, and diluting the enzyme-labeled antibody to 1X 10 by using a diluent -4 1mg/mL, adding 100 mu L/hole of the mixture into a conjugate of Spike protein and ACE2, and reacting at 37 ℃ to form immune complexes of the ACE2, the Spike protein and the enzyme-labeled antibody;
s4, washing and removing unbound substances in the compound in the step S3, drying, adding a color development liquid, reacting for not less than 5min at 37 ℃ in a dark condition, adding a sulfuric acid solution with the volume of 50 mu L and the concentration of 2mol/L to terminate the reaction, measuring the optical density of the color reaction in the ELISA plate at the wavelength of 450nm by using an ELISA reader, and judging the binding activity between Spike protein and ACE2 according to the optical density value.
Preferably, the concentration of the enzyme-labeled antibody in step S3 is 2×10 -3 mg/mL。
In studying the SARS-CoV-2 Spike protein, applicants have found that there are a number of methods for detecting this protein. One common method is the double antibody sandwich method, which is performed using ELISA technology. The method immobilizes SARS-CoV-2 Spike protein antibody on an enzyme-linked plate, and then uses another SARS-CoV-2 Spike protein antibody labeled with horseradish peroxidase (HRP). And (3) adding the test object, forming immune complex, and judging whether the test object contains SARS-CoV-2 Spike protein by detecting the HRP label. Although the double antibody sandwich method can quantitatively detect the SARS-CoV-2 Spike protein content in the test object, the binding capacity of the virus to cells cannot be estimated, and the activity level cannot be reflected. Another approach was developed based on the binding properties of the SARS-CoV-2 Spike protein to cell surface ACE 2. The method is carried out by coating a biotin ester-labeled ACE2 recombinant protein on magnetic particles and detecting using alkaline phosphatase (ALP) labeled ACE2 recombinant protein and a luminescent substrate AMPPD. This method eliminates the antibody preparation and screening steps and utilizes already mature labeling materials. However, this method cannot detect RBD monomers, and many laboratories cannot use this method for Spike protein detection due to the high cost of chemiluminescent instrumentation. In summary, although there are a number of methods for detecting SARS-CoV-2 Spike protein, each method has its limitations.
Accordingly, the applicant provides a murine anti-SARS-CoV-2 Spike protein hybridoma cell line having a collection number of CGMCC NO 45629, a collection date of 2023, 07 month 13, a collection classification named murine anti-SARS-CoV-2 Spike protein hybridoma cell line, and a collection unit of China general microbiological culture Collection center, with the objective of evaluating the binding ability and activity level of SARS-CoV-2 Spike protein to cells for the purpose of developing a more accurate, sensitive, and economical method.
On the basis of designing a mouse anti-SARS-CoV-2 Spike protein hybridoma cell strain, the antibody of Spike protein of SARS-CoV-2 wild strain and various mutant strains with stronger specificity and sensitivity is prepared by ascites preparation and purification. Such antibodies are capable of recognizing and binding to the Spike protein of SARS-CoV-2, and are useful for detecting and studying the function and properties of the protein. Through research, the antibodies developed by the application against Spike proteins of wild strains and various mutant strains of SARS-CoV-2 can effectively identify and bind to Spike proteins of both wild-type and various mutant strains. During the evolution of SARS-CoV-2, the Spike protein may have been mutated, so it is important to be able to use antibodies with a broad recognition capacity for the detection and study of different strains. The application of the antibody can provide powerful support for SARS-CoV-2 related research, vaccine development, medicine screening and other fields. Through evaluation of the binding capacity and activity level of Spike proteins, the transmission mechanism and infection characteristics of viruses can be better known, and scientific basis and strategic guidance are provided for disease prevention and control and treatment. The antibodies provided in the present application have certain advantages, and are expected to provide a reliable, convenient and efficient tool for researchers, accelerate the progress of SARS-CoV-2 Spike protein-related research, and promote the development of related vaccines and therapeutic strategies.
The following are specific examples of the present application
Preparation example
The preparation example provides a kit for detecting SARS-CoV-2 Spike protein activity, which comprises an enzyme-labeled antibody, ACE2, a chromogenic solution and a stop solution;
in the kit, the antibody in the enzyme-labeled antibody is an antibody of Spike protein for resisting SARS-CoV-2 wild strain and various mutant strains.
In the enzyme-labeled antibody, the preparation method of the Spike protein antibody for resisting SARS-CoV-2 wild strain and various mutant strains comprises the following steps:
the mouse anti SARS-CoV-2 Spike protein hybridoma cell strain is prepared through ascites and purification to obtain anti SARS-CoV-2 wild strain and various mutant strain Spike protein antibody.
Specific information of the mouse anti-SARS-CoV-2 Spike protein hybridoma cell strain is as follows: a mouse anti SARS-CoV-2 Spike protein hybridoma cell strain, the cell strain preserving number is CGMCC NO 45629, the preserving date is 2023 and 07 and 13, the preserving classification is named as mouse anti SARS-CoV-2 Spike protein hybridoma cell strain, and the preserving unit is China general microbiological culture Collection center. The address of the preservation unit is Beijing city Chaoyang area North Chen West Lu No. 1 and 3.
Wherein the color development liquid is TMB color development liquid.
Wherein the stop solution is sulfuric acid solution with the concentration of 2 mol/L.
Wherein the dilution concentration of ACE2 is 2 x 10 -3 mg/mL。
Examples 1 to 4, optimization of the concentration of the enzyme-labeled antibody
Example 1
This example uses a kit for detecting SARS-CoV-2 Spike protein activity provided by the preparation example to detect binding activity (OD value) between the relevant antigens Spike protein and ACE2 for different antigens. The detection method comprises the following steps:
s1, coating: using carbonate buffer solution50mmol/L, pH 9.6) of ACE2 to 2X 10 -3 mg/mL, and coating the diluted ACE2 on an enzyme-linked plate in an amount of 100 μl/hole, and overnight at 4 ℃;
s2, sealing: phosphate buffer (10 mmol/L, pH 7.4) containing 2% bovine serum albumin and 5% sucrose was prepared and added to the enzyme-linked plate in step S1 in an amount of 200. Mu.L/well, and incubated at 37℃for 2 hours to prepare a coated enzyme-linked plate of ACE2;
s3, adding the ACE 2-coated enzyme-linked plate with the concentration of 2 x 10 -3 mg/mL of different antigens are reacted for 1h at 37 ℃, so that Spike proteins in the antigens are combined with ACE2 coated on an enzyme-linked plate, and unbound antigens are removed by using a washing solution, so that a Spike protein and ACE2 conjugate is prepared;
s4, performing HRP (high-rate) labeling on antibodies for resisting SARS-CoV-2 wild strains and various mutant strain Spike proteins by adopting a sodium periodate oxidation method to prepare enzyme-labeled antibodies, and diluting the enzyme-labeled antibodies to 1mg/mL; the diluent used in the step S4 is phosphate buffer solution containing 1% bovine serum albumin with the concentration of 10mM and the pH value of 7.4;
s5, adding enzyme-labeled antibody with the concentration of 1mg/mL into the conjugate of Spike protein and ACE2, and reacting for 1h at 37 ℃ to form immune complex of the ACE2, the Spike protein and the enzyme-labeled antibody;
s6, washing to remove unbound substances on the ELISA plate, adding TMB color development liquid, reacting for 10min at 37 ℃ in the dark, adding 50 mu L of sulfuric acid solution with the concentration of 2mol/L to terminate the reaction, measuring the optical density of the color reaction in a reagent disk at the wavelength of 450nm by using an ELISA reader, and judging the binding activity between Spike protein and ACE2 according to the optical density value, wherein the binding activity of the ACE2 and the Spike protein is positively correlated with the color development of Elisa.
Example 2
The difference between this example and example 1 is that the concentration of the enzyme-labeled antibody in this example is 1×10 -2 mg/mL, the remainder remaining consistent with example 1.
Example 3
The difference between this example and example 1 is that the concentration of the enzyme-labeled antibody in this example is 1×10 -3 mg/mL, the remainder remaining consistent with example 1.
Example 4
The difference between this example and example 1 is that the concentration of the enzyme-labeled antibody in this example is 1×10 -4 mg/mL, the remainder remaining consistent with example 1.
The results of the detection of binding activity (OD value) between different antigens Spike protein and ACE2 in examples 1 to 4 are shown in table 1.
TABLE 1 detection summary of binding Activity (OD value) between different antigens Spike proteins and ACE2 in examples 1-4
Information on antigens used in examples 1 to 4 is shown in Table 2.
Table 2 information on antigens used in examples 1 to 4 is shown in Table
Analysis of results:
examples 2 to 4 differ from example 1 in the concentration of the enzyme-labeled antibody used. The applicants have found from the results of examples 1-4 that when the concentration of enzyme-labeled antibodies against Spike proteins of the wild-type strain of SARS-CoV-2 and the various mutants is 1X 10 -3 At mg/mL, the results of color development with different antigens are better.
In order to determine the concentration of the enzyme-labeled antibody at which the reaction between the enzyme-labeled antibody and SARS-CoV-2 Spike protein reached saturation, the concentration of the enzyme-labeled antibody was further diluted to a concentration of 1mg/mL in examples 5 to 8.
Examples 5 to 8 of the present application are as follows
Example 5
The difference between this example and example 3 is that the concentration of the enzyme-labeled antibody in this example is 4×10 -3 mg/mL, the choice of antigen was also different.
Example 6
The difference between this example and example 3 is that in this example the enzyme-labeled antibodyThe concentration of the body is 2 x 10 -3 mg/mL, the choice of antigen was also different.
Example 7
The difference between this example and example 3 is that the concentration of the enzyme-labeled antibody in this example is 1×10 -3 mg/mL, the choice of antigen was also different.
Example 8
The difference between this example and example 3 is that the concentration of the enzyme-labeled antibody in this example is 5×10 -4 mg/mL, the choice of antigen was also different.
The results of the detection of the binding activity (OD value) between different antigens Spike proteins and ACE2 in examples 4 to 8 are shown in tables 3 to 4.
TABLE 3 summary of the (OD value) test results for wild type strains (43047P) of examples 5 to 8
TABLE 4 summary of the results of delta (OD value) measurements for the mutants of examples 5-8 (43052P)
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Referring to tables 3-4, applicants have found that when the concentration of enzyme-labeled antibodies against Spike proteins of wild-type SARS-CoV-2 and various mutants is 2X 10 -3 The results of the color development of the antigen with different concentrations are better when mg/mL, and the concentration of the enzyme-labeled antibody is 2 x 10 -3 When mg/mL, the enzyme-labeled antibody and different SARS-CoV-2 Spike proteins can effectively react, and a good color development effect is presented. This is shown at 2 x 10 -3 Binding between the enzyme-labeled antibody and Spike protein is most effective at mg/mL concentrations. The concentration of the enzyme-labeled antibody can fully and specifically react with target proteins to provide accurate detection results. Thus, by concentration of the enzyme-labeled antibody at 2×10 -3 The experiment is carried out under the condition of mg/mL, so that the detection accuracy can be improved, and the binding reaction with SARS-CoV-2 Spike protein can be effectively carried out. By detecting the OD value, it is possible toTo assess the interaction and binding capacity of SARS-CoV-2 Spike protein with other substances (e.g., antibodies). The OD value represents the intensity of the reaction in the test sample and thus indirectly reflects the activity level of Spike protein. The main purposes of the present application for detecting the activity level of Spike proteins include the following: in a first aspect, spike protein is a critical structure for the entry of new coronaviruses into human cells and vaccine development requires assessment of Spike protein activity levels of vaccine candidates. By detecting the binding capacity of Spike protein to antibodies, vaccine candidates can be evaluated for the immune response induced, including the intensity and efficacy of antibody production. In a second aspect, activity level detection of Spike protein can be used to screen and evaluate potential antiviral drugs. Certain drugs may interfere with binding of Spike proteins to receptors, thereby blocking viral entry into cells. The inhibition effect of these drugs can be evaluated by detecting the activity level of Spike protein, thereby guiding drug development and treatment selection. In a third aspect, the infection of a new coronavirus in a different population can be understood by monitoring the activity level of Spike protein. According to the activity level of Spike protein, the infection degree in different areas or people can be estimated, and further important basis is provided for disease monitoring and epidemiological investigation. Thereby providing reliable technical support for subsequent research and diagnosis work. Thus, the applicant has found that the optimal dilution concentration of the enzyme-labelled antibodies against Spike proteins of wild-type SARS-CoV-2 and of various mutants is 2X 10 -3 mg/mL。
Example 9 sensitivity detection
This example differs from example 6 in that this example performs Spike protein activity tests on antigens at different concentrations. See table 5 for antigen information.
The results of the Spike protein activity test for the different antigens in this example are shown in figure 1.
TABLE 5 information summary of different antigens in example 9
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Referring to FIG. 1, FIG. 1 shows the relationship between the S1/RBD concentration and the OD value, and it can be seen that the OD value increases as the S1/RBD concentration increases. According to the result, the method for detecting SARS-CoV-2 Spike protein activity provided by the application has the advantage of detecting SARS-CoV-2 Spike protein activity. By detecting the OD value, the interaction and binding capacity of SARS-CoV-2 Spike protein with other substances (e.g., antibodies) can be assessed. The OD value represents the intensity of the reaction in the test sample and thus indirectly reflects the activity level of Spike protein. Meanwhile, the method for detecting the activity of the SARS-CoV-2 Spike protein has the advantage of being capable of rapidly and accurately detecting the activity of the Spike protein. By measuring OD values, researchers can understand the function and properties of Spike proteins, and further understand the mechanism of infection and antibody-antigen interactions. This is important for antiviral drug development, vaccine evaluation and virology research. Therefore, the detection method for SARS-CoV-2 Spike protein activity provided by the application can provide information about SARS-CoV-2 Spike protein activity, which has important significance for related research and application.
Example 10 specificity experiments
The difference between this example and example 6 is that this example separately detects the activity of SARS-CoV-2 Spike protein on 7 different samples to be tested, which are recombinant SARS-CoV-2 Spike S1, recombinant SARS-CoV Spike S1, recombinant MERS-CoV Spike S1, recombinant HCoV-229E Spike S1, recombinant HCoV-NL63 Spike S1, recombinant HCoV-OC43 Spike S1 and recombinant HCoV-HKU1 Spike S1 proteins, respectively. The test results of the example show that the enzyme-labeled antibodies against the Spike proteins of the wild strain of SARS-CoV-2 and various mutant strains provided by the application can effectively react with the Spike S1 protein of SARS-CoV-2 and can not carry out binding reaction with the Spike S1 proteins of the other 6 viruses, which indicates that the antibodies against the Spike proteins of the wild strain of SARS-CoV-2 and various mutant strains provided by the application have strong specificity.
The results of the Spike protein activity test for the different samples in this example are shown in fig. 2.
According to the test results shown in examples 1-10, the method for detecting SARS-CoV-2 Spike protein activity provided by the present application was successfully developed. Compared with the prior art, the method has the following advantages: in the first aspect, the conventional double antibody sandwich method can only detect the content of Spike protein, and cannot evaluate the binding capacity and activity level with cells. The detection method provided by the application can evaluate the binding activity between Spike protein and ACE2 by comparing the optical density values obtained by measurement. The higher the optical density value, the more tightly bound Spike protein binds to ACE2, and the stronger the binding activity.
In a second aspect, the detection method provided by the present application is capable of detecting RBD (receptor binding domain) monomers, which is a critical region for binding of coronavirus Spike protein to host cells, and has important significance for research and diagnosis.
In the third aspect, compared with some existing methods, the detection method provided by the application is low in cost, can reduce detection cost, and improves feasibility and implementation of detection.
In a fourth aspect, the detection method provided by the present application employs high quality antibody and enzyme-labeled technology, which can provide high sensitivity and high specificity detection results.
In the fifth aspect, by measuring the optical density value, an accurate result can be obtained, and the accuracy and reliability of detection are improved.
Therefore, the SARS-CoV-2 Spike protein activity detection method has potential to be applied in the research and diagnosis fields, and can provide important technical support for disease monitoring, virus screening and clinical diagnosis.
In conclusion, the antibodies against Spike proteins of SARS-CoV-2 wild strains and various mutant strains provided by the application have the advantages of strong specificity, high sensitivity, high detection accuracy and the like. Meanwhile, the method for detecting SARS-CoV-2 Spike protein activity has the characteristics of rapidness, simplicity, convenience, low cost, high accuracy, high sensitivity and high specificity.
The foregoing are all preferred embodiments of the present application, and are not intended to limit the scope of the present application in any way, therefore: all equivalent changes according to the principles of this application should be covered by the protection scope of this application.
Claims (10)
1. The mouse anti SARS-CoV-2 Spike protein hybridoma cell strain is characterized in that the cell strain has a preservation number of CGMCC NO 45629, a preservation date of 2023, 07 and 13 days, and the preservation classification is named as the mouse anti SARS-CoV-2 Spike protein hybridoma cell strain, and the preservation unit is China general microbiological culture Collection center.
2. An antibody against Spike protein of SARS-CoV-2 wild strain and delta strain, omikovin strain, beta strain, gamma strain, characterized in that the antibody is prepared by preparing mouse anti SARS-CoV-2 Spike protein hybridoma cell strain of claim 1 through ascites and purifying.
3. Use of an antibody against Spike protein of SARS-CoV-2 wild strain and delta strain, omnikow strain, beta strain, gamma strain as claimed in claim 2 in the field of biological detection.
4. A kit for detecting SARS-CoV-2 Spike protein activity, which is characterized by comprising an enzyme-labeled antibody, ACE2, a chromogenic solution and a stop solution; wherein the antibody in the enzyme-labeled antibody is an antibody of Spike protein of SARS-CoV-2 wild strain and delta strain, omikovia strain, beta strain and gamma strain.
5. The kit for detecting SARS-CoV-2 Spike protein activity as claimed in claim 4, wherein the dilution concentration of the enzyme-labeled antibody is 1X 10 -4 ~ 1mg/mL。
6. A reagent for detecting SARS-CoV-2 Spike protein activity as claimed in claim 5The kit is characterized in that the dilution concentration of the enzyme-labeled antibody is 1 x 10 -3 ~ 2*10 -3 mg/mL。
7. The kit for detecting SARS-CoV-2 Spike protein activity as claimed in claim 4, wherein the enzyme comprises horseradish peroxidase in the enzyme-labeled antibody.
8. The kit for detecting SARS-CoV-2 Spike protein activity as claimed in claim 4, wherein the color development liquid comprises TMB color development liquid; the stop solution comprises sulfuric acid solution with the concentration of 2 mol/L.
9. A method for detecting SARS-CoV-2 Spike protein activity is characterized in that the method comprises the following steps:
s1, diluting ACE2 to 2 x 10 by using buffer solution -3 mg/mL, coating diluted ACE2 on the enzyme-linked plate in an amount of 100 mu L/hole, and fully incubating to combine the ACE2 with a fixing reagent on the surface of the enzyme-linked plate to prepare coated ACE2;
s2, using HRP to mark an antibody of Spike protein of the SARS-CoV-2 wild strain and delta strain, omikovin strain, beta strain and gamma strain to prepare enzyme-labeled antibody;
s3, adding the to-be-detected object into the enzyme-linked plate in the step S1, combining the Spike protein in the to-be-detected object with the coated ACE2, washing to remove the unbound to-be-detected sample, drying by beating to obtain a combined product of the Spike protein and the ACE2, and diluting the enzyme-labeled antibody to 1X 10 by using a diluent -4 1mg/mL, adding 100 mu L/hole of the mixture into a conjugate of Spike protein and ACE2, and reacting at 37 ℃ to form immune complexes of the ACE2, the Spike protein and the enzyme-labeled antibody;
s4, washing and removing unbound substances in the compound in the step S3, drying, adding a color development liquid, reacting for not less than 5min at 37 ℃ in a dark condition, adding a sulfuric acid solution with the volume of 50 mu L and the concentration of 2mol/L to terminate the reaction, measuring the optical density of the color reaction in the ELISA plate at the wavelength of 450nm by using an ELISA reader, and judging the binding activity between Spike protein and ACE2 according to the optical density value.
10. The method for detecting SARS-CoV-2 Spike protein activity as claimed in claim 9, wherein step S3 is as follows:
s3, adding the to-be-detected object into the enzyme-linked plate in the step S1, combining the Spike protein in the to-be-detected object with the coated ACE2, washing to remove the unbound to-be-detected sample, drying by beating to obtain a combined product of the Spike protein and the ACE2, and diluting the enzyme-labeled antibody to 1X 10 by using a diluent -3 1mg/mL, and adding 100 mu L/hole of the mixture into a conjugate of Spike protein and ACE2, and reacting at 37 ℃ to form immune complexes of the ACE2, the Spike protein and the enzyme-labeled antibody.
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| CN117025548A (en) | 2023-11-10 |
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