CN111187349A - Method for preparing monoclonal antibody - Google Patents
Method for preparing monoclonal antibody Download PDFInfo
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- CN111187349A CN111187349A CN201911041699.5A CN201911041699A CN111187349A CN 111187349 A CN111187349 A CN 111187349A CN 201911041699 A CN201911041699 A CN 201911041699A CN 111187349 A CN111187349 A CN 111187349A
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- antibody
- ascites
- marker
- solid support
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Abstract
The invention discloses a rapid preparation method of a monoclonal antibody, and a hybridoma cell pair and a monoclonal antibody pair prepared by the method. In this production method, when a positive hybridoma cell or ascites antibody is produced, screening is first performed using a known antibody. The method greatly reduces the purification workload, shortens the period of preparing the monoclonal antibody by the hybridoma technology, and improves the success rate of preparation; the monoclonal antibody secreted by the hybridoma cell prepared by the method has strong specificity, high sensitivity and good stability.
Description
Technical Field
The invention relates to the field of medical inspection, in particular to a method for rapidly preparing a monoclonal antibody, a hybridoma cell pair prepared by the method and a monoclonal antibody pair.
Background
Immunodiagnosis (immunodiagnosis) is the application of immunological theories, techniques and methods to diagnose a variety of diseases and determine immune status. The immunodiagnostic reagent has the most varieties in a diagnostic kit, is widely applied to hospitals, blood stations and physical examination centers, and is mainly used for hepatitis detection, venereal disease detection, tumor detection, pregnancy detection and the like. Among them, immunodiagnosis includes radioimmunoassay, enzyme-linked immunosorbent assay, chemiluminescence, etc. The enzyme-linked immunosorbent assay has the characteristics of low cost, large-scale operation and the like; the chemiluminescence reagent has the advantages of sensitivity, rapidness, stability, strong selectivity, good reproducibility, easy operation and flexible and various methods.
The immunological detection method is a series of experimental methods for measuring antigen, antibody, immune cell and cell factor secreted by the immune cell, which are designed by applying the immunological theory. Along with the mutual infiltration among disciplines, the related range of immunology is continuously expanded, and new immunology detection methods are in the endlessly. The application range of immunological methods is also expanding day by day, which not only becomes an important method for diagnosing various clinical diseases, but also provides convenience for the research of numerous disciplines. Medically, it is an important method for determining the etiology and the lesion site of a disease, or for determining whether the immune status of the body is normal.
The main raw materials of the immunoassay comprise an antigen and an antibody, and when the antigen is detected, the antibody specifically combined with the antigen is required to be used in the detection kit. The current methods for producing antibodies are generally performed by using hybridoma technology. However, the existing hybridoma cell technology has technical defects of large workload, long time consumption, low success rate and the like in the preparation method, so that the development of a new screening method is inevitable.
Disclosure of Invention
In order to solve the above problems of the prior art, the present invention provides a method for rapidly preparing a monoclonal antibody. The method can quickly and directly screen the monoclonal antibody, and the prepared monoclonal antibody has strong specificity, high sensitivity and good stability, and can be applied to a kit for quickly and accurately detecting the antigen in a sample.
According to the invention, the monoclonal screening step is advanced from the traditional screening after monoclonal antibody purification to the preparation of positive hybridoma cells or ascites antibody preparation, so that the complex and time-consuming monoclonal antibody purification workload is reduced. And the effective monoclonal antibody can be directly screened out by using the known antibody to carry out screening work.
In one aspect, the present invention provides a method for preparing a monoclonal antibody, comprising the steps of:
(1) the antigen is used for immunizing an animal,
(2) isolating the spleen cells of the immunized animal,
(3) the cell fusion is carried out, and the cell fusion is carried out,
(4) preparing a positive hybridoma cell,
(5) preparing a monoclonal antibody;
wherein, the preparation of the monoclonal antibody comprises in vitro culture and in vivo culture; when the in vivo culture is carried out, the method further comprises the following steps:
(5-1) preparing an ascites antibody,
(5-2) purifying the prepared ascites antibody to obtain a monoclonal antibody;
wherein the screening step for positive hybridoma cells is carried out in step (4) using a known antibody, and/or the screening step for ascites antibody is carried out in step (5-1) using a known antibody.
In one embodiment, the method used in the screening step in the step (4) and the step (5-1) is a sandwich method or a competition method.
In a preferred embodiment, the sandwich method comprises the steps of:
a) marking a known antibody by a marker for later use;
b) coating the antibody in the positive hybridoma cell supernatant or the ascites antibody on a solid support, adding a known antibody marked by a marker after the antigen reaction is completed, and continuing the reaction;
adding a reaction substrate and detecting the signal value of the marker to obtain the screened positive hybridoma cells or obtain the screened ascites antibody.
In another preferred embodiment, the competition method comprises the following specific steps:
in step (4), a) labeling the known antibody with a labeling substance for use; b) coating the antigen on a solid support, adding a known antibody marked by a marker and a positive hybridoma cell supernatant at the same time, adding a reaction substrate after the reaction is finished, detecting a signal value of the marker, and screening the positive hybridoma cells according to the signal value;
in the step (5-1), a) the labeling substance labels a known antibody for use; b) coating the antigen on a solid support, adding a known antibody marked by a marker and an ascites antibody at the same time, adding a reaction substrate after the reaction is finished, detecting a signal value of the marker, and screening the ascites antibody according to the signal value.
In one embodiment, in the case of screening positive hybridoma cells using the competitive method, the known antibody labeled with the marker is present at a concentration similar or identical to the concentration of the antibody in the supernatant of the positive hybridoma cells; when the ascites antibody is screened by using the competition method, the concentration of the known antibody marked by the marker is similar or identical to that of the ascites antibody.
In a second aspect, a method for preparing a pair of monoclonal antibodies for detection by a sandwich method, the method comprising the steps of:
(1) the antigen is used for immunizing an animal,
(2) isolating the spleen cells of the immunized animal,
(3) the cell fusion is carried out, and the cell fusion is carried out,
(4) preparing a positive hybridoma cell,
(5) preparing a monoclonal antibody pair;
wherein, the preparation of the monoclonal antibody pair comprises in vitro culture and in vivo culture; when the in vivo culture is carried out, the method further comprises the following steps:
(5-1) preparing an ascites antibody,
(5-2) purifying the prepared ascites antibody to obtain a monoclonal antibody pair;
wherein the screening step of positive hybridoma cells is carried out in step (4) using antibody A and antibody B binding to different epitopes, and/or the screening step of ascites antibodies is carried out in step (5-1) using antibody A and antibody B binding to different epitopes.
In one embodiment, the antibody A and the antibody B recognizing different epitopes are paired antibodies capable of detecting antigens in a double antibody sandwich method.
In one embodiment, the method used in the screening step in the step (4) and the step (5-1) is a sandwich method or a competition method.
In a preferred embodiment, the sandwich method comprises the following specific steps:
in the step (4), a) labeling the antibody A and the antibody B with the labeling substance for standby; b) coating the antibody in the supernatant of the positive hybridoma cells on a solid support 1 and a solid support 2 respectively, and adding a marker labeled antibody A into the coated solid support 1 and adding a marker labeled antibody B into the coated solid support 2 after the antigen adding reaction is completed; c) adding a reaction substrate, detecting a signal value of the marker, and screening positive hybridoma cells according to the signal value;
in the step (5-1), a) labeling the antibody A and the antibody B with the labeling substance for standby; b) coating ascites antibodies on a solid support 1 and a solid support 2, respectively, and then adding a marker-labeled antibody A to the coated solid support 1 and a marker-labeled antibody B to the coated solid support 2; c) adding reaction substrates, detecting the signal value of the marker, and screening ascites antibodies according to the signal value.
In another preferred embodiment, the competition method comprises the following specific steps:
in the step (4), a) labeling the antibody A and the antibody B with the labeling substance for standby; b) coating the antigen on solid support 1 and solid support 2, respectively; after completion of the reaction, simultaneously adding the marker-labeled antibody A and the supernatant of the positive hybridoma cells to the coated solid support 1, and simultaneously adding the marker-labeled antibody B and the supernatant of the positive hybridoma cells to the coated solid support 2; c) adding a reaction substrate, detecting a signal value of the marker, and screening positive hybridoma cells according to the signal value;
in the step (5-1), a) labeling the antibody A and the antibody B with the labeling substance for standby; b) coating the antigen on solid support 1 and solid support 2, respectively; then, simultaneously adding antibody A labeled with a labeling substance and ascites antibody to coated solid support 1, and simultaneously adding antibody B labeled with a labeling substance and ascites antibody to coated solid support 2; c) adding reaction substrates, detecting the signal value of the marker, and screening ascites antibodies according to the signal value.
The solid support 1 and solid support 2 may be the same or different.
In another embodiment, in the case of screening positive hybridoma cells using the competitive method, the concentration of antibody a or antibody B labeled with the labeling substance is similar or identical to the concentration of antibody in the supernatant of the positive hybridoma cells; when the ascites antibody is screened by using the competition method, the concentration of the marker antibody A or antibody B is similar or identical to that of the ascites antibody.
In a third aspect, a pair of hybridoma cells prepared by the above-described preparation method;
in a preferred embodiment, the hybridoma cell pairs have a collection number of CCTCC NO: c2019231 and CCTCC NO: c2019232, or CCTCC NO: c2019233 and CCTCC NO: C2019234.
in a fourth aspect, a pair of monoclonal antibodies secreted by a pair of hybridoma cells produced by the production method according to any one of claims 5 to 7;
in a preferred embodiment, the pair of monoclonal antibodies is represented by a sequence with a accession number of CCTCC NO: c2019231 and CCTCC NO: the hybridoma cell pair of C2019232 is obtained by secretion; or the monoclonal antibody pair is prepared by the following components with the preservation number of CCTCCNO: c2019233 and CCTCC NO: the hybridoma cell pair of C2019234 was secreted.
The screening method of the invention can shorten the period of preparing the monoclonal antibody by the hybridoma technology, improve the success rate, and the prepared monoclonal antibody has strong specificity, high sensitivity and good stability.
Drawings
FIG. 1 is a photograph showing the electrophoresis of the purity test of the monoclonal antibody prepared in example 1;
FIG. 2 is a WB diagram showing the monoclonal antibodies screened in example 1;
FIG. 3 is an SDS-PAGE electrophoresis of the monoclonal antibodies screened in example 1;
FIG. 4 is the electrophoresis chart of the stability test of the monoclonal antibody obtained from the screening of example 1;
FIG. 5 is a structural diagram of the test strip used in example 1;
FIG. 6 is a photograph showing electrophoresis of purity test of the monoclonal antibody prepared in example 2;
FIG. 7 is an SDS-PAGE electrophoresis of the monoclonal antibodies screened in example 2;
FIG. 8 is the electrophoresis chart of the stability test of the monoclonal antibody obtained by screening in example 2.
Detailed Description
In order to make those skilled in the art better understand the technical solutions in the present application, the present invention will be further described below with reference to the following embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
Definition of
Monoclonal antibodies are highly homogeneous antibodies produced by a single B cell clone that are directed against only a particular epitope. Usually, hybridoma (hybridoma) antibody technology is used to prepare hybridoma, which is a method of fusing a sensitized B cell having the ability to secrete a specific antibody and a myeloma cell having an unlimited proliferation ability into a B cell hybridoma based on cell fusion technology. The monoclonal antibodies, monoclonal antibodies and immunoglobulins are monoclonal antibodies.
The present invention is also directed to a method for producing monoclonal antibodies, which is characterized in that the method for producing monoclonal antibodies is not required to be high in terms of purification, but the high purity of the antigen is increased in the chance of producing monoclonal antibodies, which are required to be used in the procedure for producing monoclonal antibodies, which is not required to be used in the procedure for producing hybridoma cells, which is not used in the procedure for producing hybridomas, which is not used in general, which is not required, but is not used in the procedure for producing monoclonal antibodies, which is not used in the procedure for producing, which is limited in the procedure for producing, or is not used in the procedure for producing monoclonal antibodies, which is not used in the procedure for producing a hybridoma cells, which is not used in the procedure for producing a monoclonal antibodies, which is not used in the procedure for producing a single hybridoma cells, which is not used in the procedure for producing a hybridoma cells, which is not used in the source, which is limited, which is not used in the procedure for producing a hybridoma cells, which is not used in the procedure for producing a monoclonal antibodies, which is limited, which is not used in the source, which is not used in the procedure for producing a monoclonal antibodies, which is not used in the same, or not used in the procedure for producing a hybridoma cells, which is not used in the procedure for producing a hybridoma cells, which is not used in the procedure, which is not used in the method, which is not used in the same, which is not used in the procedure for producing a hybridoma cells, which is not used in the procedure for a hybridoma, which is not used in the same, which is not used in the procedure for a hybridoma cells, which is not used in the same, which is not used in the procedure for producing a hybridoma, which is not used in the same, which is not used in the method for producing a hybridoma, or is not used in the procedure for a hybridoma, which is not used in the same, or is not used in the procedure for producing a hybridoma, which is not used in the method, or is not used in the procedure for which is not used in the same, which is not used in the method, which is not used, or is not used in the same, or is not used in the procedure for the method for the procedure for the same, which is not used in the method, which is not used in the same, which is not used in the procedure for the procedure, which is not used in the same, which is not used in the procedure, which is not used in the procedure for the procedure, which is not used in the same, which is not used in the method for the same, which is not used in the method for which is not used, which is not used in the method for producing a hybridoma, which is not used in the method for which is not used in the method, which is not used in the method for producing a hybridoma, which is not used in the method, which is not used in the method for producing a rat, or is not used, which is not used in the method for producing a hybridoma, which is not used in the method for producing a, which is not used in the method for the method, which is not used in the method for producing a hybridoma cell line, which is not used in the method for the method, which is not used in the method for the method, which is not used in the method for the method, the method for the method, which is not used in the method for the method, which is not used in the method, which is not used in the method for the method, which is not used in the method for the method, which is not used, which is.
In the present invention, the solid support to be used may be selected from organic polymer compounds, inorganic compounds, biopolymers, and the like. Examples of the organic polymer compound include: latex, polystyrene, polypropylene, and the like. As the inorganic compound, there may be mentioned: magnetic materials (iron oxide, chromium oxide, cobalt, ferrite, etc.), silica, alumina, glass, and the like. Examples of the biopolymer include: insoluble agarose, insoluble dextran, gelatin, cellulose, and the like. 2 or more of these may be used in combination. The shape of the solid phase is not particularly limited, and examples thereof include: particles, microplates, microtubes, test tubes, and the like. Preferably, the present invention uses microplates as solid supports.
The label may be a substance which itself generates a signal (hereinafter, also referred to as "signal-generating substance") or a substance which catalyzes a reaction with another substance to generate a detectable signal.A signal-generating substance includes, but is not particularly limited to, fluorescent substances and radioisotopes.A preferable label is an enzyme and a fluorescent substance.A preferable example of the enzyme includes, but is not particularly limited to, alkaline phosphatase, peroxidase, β -galactosidase, glycosidase, polyphenoloxidase, tyrosinase, acid phosphatase, horseradish peroxidase, luciferase, fluorescent substances include Fluorescein Isothiocyanate (FITC), coumarin, rhodamine, Cy3, Cy5, Hoechst33342, 4', 6-diamidine-2-phenylindole (DAPI), Propidium Iodide (PI), Alexa Fluor (registered trademark), a fluorescent dye such as a phthalocyanine-based dye, a fluorescent protein such as GFP, and the like125I、35S、32P、14C and the like. The labeling substance is preferably an enzyme, more preferably horseradish peroxidase and alkaline phosphatase.
In the labeled antibody, the labeling substance is bound or immobilized to the antibody, and therefore the concentration and amount of the labeling substance in the antibody reagent reflect the number of molecules of the labeled antibody contained in the antibody reagent. The concentration or amount of the labeling substance contained in the antibody reagent can be measured by a method known in the art depending on the type of the labeling substance. Alternatively, the concentration and amount of the labeling substance in the antibody reagent can be calculated from the amount of the labeling substance used in the production of the labeled antibody.
The amount or intensity of the signal based on the labeled antibody varies depending on the number of molecules of the labeled antibody contained in the antibody reagent used, and therefore the measured value based on the signal of the labeled antibody contained in the predetermined amount of the antibody reagent reflects the number of molecules of the labeled antibody contained in the amount of the antibody reagent. The amount or intensity of the signal based on the labeled antibody can be measured by a known measurement method according to the type of the labeled substance based on the labeled antibody contained in the predetermined amount of the antibody reagent. When the concentration of the labeled antibody is high, a signal based on the labeled antibody contained in the diluted antibody reagent can be measured. In this case, the value based on the signal of the labeled antibody contained in the antibody reagent before dilution can be calculated by multiplying the measured value of the obtained signal by the dilution ratio.
In the present invention, two monoclonal antibodies secreted from the two hybridoma cells by the hybridoma cells can be used in a sandwich immunoassay project to bind or recognize different surface recognition sites of a detection object or a detection target. The monoclonal antibody pair indicates that the two monoclonal antibodies can be applied to a sandwich method immunoassay item to combine or identify different surface recognition sites of a detection object or a detection target object,
in the present invention, the positive hybridoma is a hybridoma capable of secreting an antibody. The expressions "a", "B", "c", etc. are used for descriptive purposes only to distinguish defined objects and not to define an order or primary or secondary in any way.
The present invention will be explained below by taking the preparation of monoclonal antibody in hepatitis B virus E antigen (HBeAg) detection kit as an example.
Hepatitis B virus (hepatitis B virus B), Hepatitis B Virus (HBV), belongs to DNA virus, is a hepatotropic virus, and is a partially double-stranded circular DNA virus with an envelope. The infection mode of the hepatitis B is mainly transmitted by blood transfusion, injection and mother and infant. Hepatitis B Virus (HBV) infection is a worldwide problem, with as many as 4 billion chronically infected individuals worldwide, and developing countries are the serious areas of hepatitis B. Hepatitis b usually causes chronic infection, can progress to cirrhosis and even hepatocellular carcinoma, and has a great threat to human health. Covalently closed circular deoxyribonucleic acid (covalenfly closed circular mu Lar DNA), namely cccDNA, is an important link in replication of Hepatitis B Virus (HBV), is a synthesis template of mRNA and pregenomic RNA, is also a key factor for difficult cure of HBV, and has important relation with chronic hepatitis B and relapse after antiviral treatment in long-term existence. Therefore, the detection of cccDNA is very important for understanding the change of the patient's condition and evaluating the treatment effect. However, the detection of cccDNA in liver tissue requires liver tissue biopsy, which is often unacceptable for patients, so it is necessary to find a serological marker with convenient operation and high sensitivity.
HBeAg is a major structural protein of the inner core of hepatitis B virus, and its primary structure is substantially identical to that of hepatitis B core antigen (HBcAg). HBeAg is an early marker of acute infection, and its detection serves as a marker for the presence of DNA polymerase and circular DNA molecules, indicating progressive damage and high infectivity of hepatocytes. Clinically, HBeAg positive, with HBSAg positive, HBcAb positive: this is what we commonly know as hepatitis B with three positive, strong infectivity, active virus replication in vivo, and rapid increase in virus number. If the acute hepatitis B infection stage exists, the HBeAg exists in serum for 3-6 weeks generally, and the condition can be quickly controlled by timely treatment. HBeAg is singly positive, and the condition is rare. Acute infection with hepatitis B virus is generally indicated. If the HBeAg is positive for a long time and does not generate negative conversion phenomenon, the HBeAg is possibly the expression of acute hepatitis and chronic hepatitis, and the positive HBsAg is very likely to occur. Once single positive HBeAg appears, the patient should go to a regular hospital for treatment in time to prevent chronic hepatitis B. HBeAg is positive and negative, and HBeAb is positive and negative. If this occurs, it may be a critical period for treatment of hepatitis B, and seroconversion of HBcAg may be properly achieved. The quantity of hepatitis B virus in vivo is reduced, and virus replication is controlled. Therefore, the detection of HBeAg in serum is of great clinical significance in the monitoring and treatment of HBV infection. It is necessary to establish a detection method for detecting HBeAg.
The existing methods for detecting HBeAg include enzyme-linked immunosorbent assay, chemiluminescence assay, electrochemiluminescence assay, fluorescence immunoassay, time resolution assay and microparticle chemiluminescence assay. The enzyme-linked immunosorbent assay has high sensitivity, but is complex in operation, easy to pollute, needs expensive special instruments, is more suitable for basic medical institutions, and is used for detection of suspected HBV infectors, preoperative detection of patients, screening of blood donors and the like. Chemiluminescence, electrochemiluminescence, fluorescence immunity, time resolution, microparticle chemiluminescence and the like have high sensitivity, but need to be operated by a special person and matched with expensive instruments for use, and are mainly used for dynamically monitoring the disease development, medication guidance, treatment effect, prognosis condition and the like of HBV infected patients. Aiming at screening of general physical examination people, a simple, convenient and quick method without special equipment is needed.
In the specific embodiment of the invention, HBeAg (New Material technology Co., Ltd., Sichuan Mike, No. 091614) is used for immunizing Balb/C mice, the tail serum antibody titer of the mice is screened by an ELISA indirect method, and the mice with higher antibody titer are selected for cell fusion.
Example 1
The immunochromatography takes a microporous membrane as a solid phase carrier, sample liquid is electrophoresed on the membrane through capillary action, simultaneously an object to be detected in the sample is combined with a corresponding antigen or antibody on the membrane, and a gold or selenium marker deposition line directly observed by naked eyes is formed in a certain area, so that an experimental result is obtained, and the unbound marker moves forwards and does not form the deposition line. The method is simple and convenient to operate, a certain amount of serum is only needed to be sucked, no professional is needed, the macroscopic bright color can appear anywhere within 5 to 15 minutes at any time according to the specification, and the result can be interpreted without a special instrument.
The immunochromatography requires two antibodies for labeling the colloidal gold particles and coating the microporous nitrocellulose membrane, respectively. Generally, a monoclonal antibody and a polyclonal antibody are selected by an immunochromatography method for coating and labeling, and because the specificity of the polyclonal antibody is not high as that of the monoclonal antibody, the technical defects of high false positive, low sensitivity and the like exist when clinical serum is detected by using the pair, in order to solve the problems, two monoclonal antibodies are selected for coating and labeling, and the false positive rate and the sensitivity are obviously higher than those of the pair.
In order to obtain the two paired monoclonal antibodies, the two monoclonal antibodies aiming at different antigenic determinants of HBeAg are prepared by adopting a hybridoma technology, while the monoclonal antibodies are prepared by adopting the traditional hybridoma technology, an ELISA indirect method is mostly adopted when positive cell strains are screened after fusion, and the method has the technical defects of large workload, long time consumption, low success rate and the like, so that the development of a new screening method becomes a necessary trend.
The idea of the invention is as follows: screening a pair of commercial paired antibodies (known antibodies and different epitope), carrying out gradient dilution on the commercial paired antibodies, detecting the antibodies and positive hybridoma cell supernatants after the gradient dilution in parallel by using a commercial kit, assigning the concentration of the antibodies in the positive hybridoma cell supernatants according to the OD value of the antibodies, wherein the concentration of a marker antibody (known antibody) is consistent with the concentration of the antibodies in the positive hybridoma cell supernatants in order to achieve fair competition, and carrying out gradient dilution to coat the antigen after the concentrations of the antibodies and the enzyme-labeled antibody are determined, so as to determine the optimal antigen coating amount; and finally, after coating the optimal antigen amount, adding a positive hybridoma cell supernatant and a complete culture medium as positive and negative controls, and determining the cut off value. Through the research, the antigen coating amount, the enzyme-labeled antibody dilution ratio and the cut off value of the competition method can be determined, and the determined values are repeatedly used for detection for 3 times, so that the feasibility of the method is proved, and the method can be used for screening positive cell strains after fusion.
Determination of antibodies binding to different epitopes
1. Screening monoclonal antibodies with different epitopes
The document reports that HBeAg has 5 epitopes, 4 linear epitopes and 1 conformational epitope, wherein the conformational epitope is formed by the mutual formation of 4 linear epitopes, and when the clinical screening of hepatitis B is carried out, the HBeAg in serum is detected by a double antibody sandwich method generally, so that in order to ensure the accuracy of detection, a conformational antibody is required to be provided for the paired antibodies, which provides an idea for developing reagent raw materials, namely starting from the initial screening work, the epitope property can be started, and only two antibodies recognizing different antigenic determinants can be paired, so that the invention provides a new screening method for developing the raw material preparation of the clinical detection reagent, and a competition method is used for screening the positive supernatant after hybridoma fusion. Firstly, determining a pair of standard paired antibodies, carrying out horseradish peroxidase labeling on the paired antibodies, estimating the antibody concentration in the supernatant of the hybridoma, ensuring that the concentrations of the paired antibodies are consistent when the paired antibodies compete with the enzyme-labeled antibodies for binding the antigen, after determining the coating amount of the competitive binding antigen, simultaneously adding the enzyme-labeled antibody and the supernatant of the hybridoma for competitive binding, wherein the competitive binding is competitive with the enzyme-labeled antibody, the epitope of the competitive binding is consistent with that of the enzyme-labeled antibody, and the reverse is true.
In order to reduce the screening workload, the primary qualitative analysis is carried out on the epitope recognized by the antibody (the method is patented), whether the epitope recognized by each antibody is a linear epitope or a conformational epitope is determined, if the conformational epitope is recognized, the reactivity is greatly reduced after the antigen is denatured, the activity inhibition rate is more than 50%, if the linear epitope is recognized, the activity reduction range is small, and the activity inhibition rate is less than 50%. Recombinant HBeAg (Sichuan Mike New Material technology Co., Ltd., batch No. 091614) was subjected to denaturation treatment (boiling for 10 minutes), and the antigens before and after denaturation were respectively designated as rHBeAg and drHBeAg, and were coated at a concentration of 5. mu.g/mL and 100. mu.L/well with 4 ℃ carbonate buffer overnight. 0.5% casein (prepared in 0.1M PBS) 150. mu.L/well, blocking at 37 ℃ for 2h, pouring off the blocking solution, and draining for later use.
Purchasing a plurality of commercialized antibodies binding different antigen epitopes, adding 1 mu g/mL of the commercialized antibodies binding different antigen epitopes into an ELISA plate coated with the antigen, incubating for 40min at 37 ℃, pouring liquid in the hole, washing the plate with ELISA washing liquid for five times, adding 100 mu L/hole of horseradish peroxidase-labeled goat anti-mouse IgG (New Sichuan material production, lot number 091516) diluted by 10000 times, incubating for 30min at 37 ℃ and washing the plate, adding 100 mu L of buffer solution containing 0.1% (M/V) o-phenylenediamine and 0.1% (V/V) hydrogen peroxide into each hole, pH5.0 citric acid phosphate buffer solution, incubating for 10min at 37 ℃, adding 50 mu L of 2M sulfuric acid solution into each hole to terminate the reaction, and detecting the absorption value of 450nm by a multifunctional reading instrument. Finally, paired antibodies 9010 and 9017 were screened. The specific results are shown in the data analysis of table 1, wherein 9010 is a conformational epitope, and 9017 is a linear epitope.
Table 1:
| name of antibody | rHBeAg(OD450) | drHBeAg(OD450) | Antigen-antibody reactivity (%) |
| 9010 | 3.058 | 0.846 | 72 |
| 9017 | 2.321 | 1.678 | 27 |
2. Labelling of monoclonal antibodies with different epitopes
This example uses horseradish peroxidase to label the 9010 and 9017 antibodies.
Solution preparation:
a.2mg/mL HRP: dissolving 1mg of Horse Radish Peroxidase (HRP) in 0.5mL of purified water (for use in the present preparation)
60mM sodium metaperiodate: 80.2mg sodium metaperiodate was dissolved in 6.25mL purified water (ready for use)
c.160mM ethylene glycol: 8.93 μ L of ethylene glycol was added to 1mL of purified water (ready to use)
d.1mg/mL sodium borohydride: 1mg sodium borohydride dissolved in 1mL purified water (ready for use)
e. Saturated ammonium sulfate
f.0.05M carbonate buffer (pH 9.6): sodium carbonate 1.59g/L, sodium bicarbonate 2.93g/L
Slowly dripping 0.5mL of sodium metaperiodate into 0.5mL of HRP, uniformly mixing while adding, standing at a temperature of 2-8 ℃ in a dark place for 30min, slowly adding 0.5mL of ethylene glycol into the solution while adding, uniformly mixing, reacting at room temperature in a dark place for 30min, slowly adding the activated HRP into 0.5mg of antibody (the mark ratio is 1: 2), dialyzing 0.05M CB overnight, changing the solution once during the period, taking out the antibody in a dialysis belt, calculating the volume, adding 0.2mL of sodium borohydride into each mg of antibody (the sodium borohydride is added when the reaction with water begins to generate bubbles), reacting at a temperature of 2-8 ℃ for 2h, adding an isovolumetric saturated ammonium sulfate solution into the solution, standing for 2h at the temperature of 2-8 ℃, centrifuging for 10min at 10000rpm, discarding the supernatant, redissolving the precipitate with 20mM PBS, dialyzing overnight with 20mM PBS, taking out the labeled 9010-HRP and 9017-HRP, calculating the corresponding concentration according to the volume, and adding isovolumetric glycerol for storage.
3. Detection of whether 9010 and 9017 are paired or not by using double-antibody sandwich method
Table 2:
antigen-immunized mice
1. Recombinant HBeAg (Sichuan Mike New Material technology Co., Ltd., batch No. 091614) immunized mice recombinant HBeAg was diluted to 2.0mg/mL with physiological saline and MagicTMMouse Adjuvant (Creative Diagnostics, Cat. CDN-A001) was mixed in equal volume, gently pipetted up and down 5 times with 100. mu.L pipette, leg intramuscular injection was performed with 50-100. mu.L final volume of each BALB/c Mouse (Chengdouda center of the Experimental animals, 6 weeks old females, 4 mice), and recombinant HBeAg (40. mu.g/BALB/c Mouse) was prepared with Adjuvant. After 3 weeks (21 days), the second immunization was performed as above, and after 2 weeks (14 days), tail blood was collected 35 days after the first immunization, and the antibody titer in the serum was measured by the ELISA indirect method, as shown in Table 3, and the results in Table 3 show that 4 mice had titers in the order of: 8X 106、8×106、103、104. Wherein the serum titer of 2 mice is more than 1: 106And the fusion protein can be used for cell fusion.
Table 3: immune mouse serum titer detection
Preparation of hybridoma cell line
1. Cell fusion
1.1 preparation of feeder cells
Normal 12-week-old BALB/c mice peritoneal macrophages were used as feeder cells. 1 day before the fusion, BALB/c was killed by pulling eye blood and neck, 0.1% benzalkonium bromide was soaked for 1 min, then 75% was addedSoaking in ethanol for 1 min, lifting abdominal skin from posterior abdomen with sterilizing scissors and forceps in a super clean bench, and exposing peritoneum. Wiping peritoneum with alcohol cotton ball for sterilization. 2mL of RPMI1640 medium was injected into the peritoneal cavity with a syringe, taking care to avoid penetration into the intestinal tract. The syringe was fixed with the right hand, the needle was left in the abdominal cavity, the abdomen was gently massaged with an alcohol cotton ball held with the left hand for 1 minute, and then the injected culture solution was aspirated. Centrifuging at 1000r/min for 5-10 min, and discarding the supernatant. Resuspending with RPMI1640 culture medium containing 20% newborn calf serum and 1% double antibody, and adjusting cell concentration to 2-5 × 105Adding into 96-well plate at 100 μ L/well, 37 deg.C and 5% CO2And (5) culturing.
1.2 preparation of immune spleen cells
Taking immune serum to reach the titer of 1: 106The BALB/c mice were subjected to blood sampling by removing eyeballs, and serum was isolated as a positive control serum for antibody detection. Meanwhile, a lethal mouse is dislocated through the neck, 0.1% benzalkonium bromide is soaked for 1 minute, the mouse is soaked in 75% alcohol for 1 minute, the left abdominal skin is lifted on a sterile plate in a super clean bench, the spleen can be seen, the forceps are changed, the peritoneum is cut off by using sterile scissors, the spleen is taken out and placed in a plate filled with 10mL RPMI1640 culture solution, the washing is carried out lightly, and the surrounding connective tissues are carefully stripped. The spleen was transferred to another plate containing 10mL of RPMI1640 medium and gently squeezed (or squeezed with a plunger) using an elbow forceps or a bent needle mounted on a 1mL syringe to force the spleen cells into the RPMI1640 medium in the plate. The mixture is blown and beaten for several times by a pipette to prepare single cell suspension. To remove large clumps from the spleen cell suspension, filtration through a 200 mesh copper mesh was used. Harvesting spleen cell suspension, centrifuging for 5-10 minutes at 1000r/min, centrifuging and washing for 1-2 times by using RPMI1640 culture solution, then suspending the cells in 10mL of RPMI1640 culture solution, uniformly mixing, taking the suspension, and adding a phloroglucinol blue staining solution for counting living cells for later use. Usually 1X 10 per mouse8～2.5×108And (4) spleen cells.
1.3 preparation of myeloma cells
The manner in which the myeloma cells are maintained prior to fusion is of paramount importance to the successful acquisition of the hybridoma. The aim is to keep the cells in logarithmic growth for as long a time as possible, fuseIt must not be less than 1 week before. The cryopreserved cells were not in a state suitable for fusion until 2 weeks after recovery, and the longer myeloma cells were likely to recover for at least 5 days. Myeloma cells that are in logarithmic growth during culture are maintained in medium containing 10% calf serum by inoculating 10-fold serial dilutions of myeloma cells with 6 flasks of 5mL medium. After 1 week, the cells were re-cultured in flasks after the culture reached a relatively dense and non-growing flask. Typical doubling times are 14-16 hours. The preparation method of the myeloma cell suspension comprises the following steps: myeloma cells are expanded and cultured 48 to 36 hours before fusion (2 to 3 bottles and 25cm are generally needed for a fusion experiment with a 96-well plate)2Preparation of cells cultured in flasks). On the day of fusion, cells were gently blown down from the vial wall using a glass pipette and collected in a 50mL centrifuge tube. Centrifuging at 1000r/min for 5-10 min, and discarding the supernatant. 30mL of RPMI1640 culture medium was added, and the mixture was centrifuged and washed once in the same manner. The cells were then resuspended in 10mL of RPMI1640 medium and mixed well. Taking myeloma cell suspension, adding 0.4% of fetuin blue for counting living cells for later use. When counting cells, 0.1mL of the cell suspension was added to 0.9mL, mixed well and counted on a hemocytometer. Cell density (one/mL) ═ 4 cells/4 × 104X dilution factor.
1.4 cell fusion and Selective culture of hybridoma cells
Spleen cells and myeloma cells were mixed as 7: 1, adding the mixture into a 50mL centrifuge tube, supplementing the RPMI1640 culture solution to 30mL, and fully and uniformly mixing. Centrifuging at 1000r/min for 5-10 min, and sucking the supernatant as clean as possible. Flicking the fusion tube bottom on the palm to loosen and uniform the precipitated cells; preheating in 37 deg.C water bath. Add 1mL of 50% PEG (pH 7.4) preheated to 40 ℃ in 1 minute (optimally 45 seconds) using a 1mL pipette, gently shaking while adding. Adding 20-30 mL of RPMI1640 culture solution preheated to 37 ℃ within 90 seconds by using a glass dropper; standing for 10 minutes at 20-37 ℃. Centrifuging at 700r/min for 5-10 min, and discarding the supernatant. Resuspended in RPMI1640 medium containing 1% HAT and 20% newborn bovine serum, and evenly divided into 10 96-well cell culture plates. 37 ℃ and 5% CO2Culturing, and supplementing RPMI1640 culture solution containing 1% HAT and 20% newborn calf serum to the wells the next day90% of the volume. After 5 days, 1/2 medium was replaced with HAT medium in wells, and after 7 days, 1/2 medium was replaced in wells.
Fourthly, preparing positive hybridoma cells
1. Hybridoma cell supernatant antibody concentration and cut off model for the competition method in this example
1.1 search of supernatant antibody concentration
After the supernatant of hybridoma cells (positive cell strains screened by a traditional indirect method) and the antibodies generated by the cell strains are diluted in a gradient way (10ng/mL, 100ng/mL, 200ng/mL, 500ng/mL and 1 mu g/mL), the reaction results are detected according to the instruction of a hepatitis B virus E antibody kit (Kowa V201601) (competition method), a multifunctional reading instrument detects the 450nm absorption value, and the concentration range of the antibodies in the supernatant of the hybridoma cells is judged by comparing the 450nm absorption value, and the detection results are shown in Table 4. The result shows that the concentration of the antibody secreted by the hybridoma supernatant is more than or equal to 1 mu g/mL.
Table 4: detection of gradient diluted antibody and hybridoma supernatant by Kohua hepatitis B virus E antibody detection kit (competition method)
Remarking: the supernatant and the antibody in the table are both from the same strain of positive hybridoma; positive and negative controls are provided for the kit
Cut off values for this kit were calculated from the positive and negative controls in table 4. The calculation method is that the cutoff value (positive OD value + negative OD value)/2 is 1.455, the OD value is negative when it is greater than 1.455, and the OD value is positive when it is less than 1.455, and it can be seen from the table that the difference between the supernatant of the hybridoma 96-well plate and the supernatant of the hybridoma culture bottle is not obvious, so that the concentration of the hybridoma supernatant is not changed according to the culture mode. Since the OD value of the supernatant of the hybridoma 96-well plate is close to the 2. mu.g/mLOD value of the antibody, the antibody concentration secreted from the supernatant of the hybridoma 96-well plate can be determined to be about 2. mu.g/mL.
1.2 determination of the amount of antigen coating
The antigen (recombinant HBeAg antigen) was diluted with a carbonate buffer gradient to 0. mu.g/mL, 1. mu.g/mL, 3. mu.g/mL, 5. mu.g/mL, 7. mu.g/mL, 9. mu.g/mL, 100. mu.L/well overnight at 4 ℃. 0.5% casein (prepared in 0.1M PBS) 150. mu.L/well, blocking at 37 ℃ for 2h, pouring off the blocking solution, and draining for later use. 9010-HRP and 9017-HRP are diluted to 2 mu g/mL, added with hybridoma supernatant respectively, incubated for 40min at 37 ℃, liquid in the holes is poured out, 100 mu L of 0.1% (M/V) o-phenylenediamine, 0.1% (V/V) hydrogen peroxide and pH5.0 citric acid phosphate buffer are added into each hole after the ELISA washing liquid is washed for five times, incubation is carried out for 10min at 37 ℃, 50 mu L of 2M sulfuric acid solution is added into each hole to stop the reaction, a multifunctional reading instrument detects the absorption value of 450nm, the result is shown in Table 5, the saturated antigen concentration of the combined antibody, namely the concentration of the antigen when the OD value is stable, is the optimal concentration of the antigen, the table 5 shows that the OD value is not increased any more when the antigen coating amount is 5 mu g/mL, and the optimal coating amount of the antigen in the competition method is 5 mu g/mL.
Table 5: reaction results of different antigen coating amounts competing for binding to antibody
1.3 determination of cut off value by Competition method
There are many methods for evaluating the cut off value, and the present invention is not limited to the use of this one method for evaluating the cut off value. When the hybridoma supernatant after fusion is detected by a direct competition method, the result judgment method is that the epitope which has competition with the enzyme-labeled antibody is the same as the epitope of the enzyme-labeled antibody, and vice versa. Therefore, the cut off value is determined first. According to the above experimental results, recombinant HBeAg was diluted to 5. mu.g/mL with carbonate buffer and 100. mu.L/well was coated overnight at 4 ℃. 0.5% casein (prepared in 0.1M PBS) 150. mu.L/well, blocking at 37 ℃ for 2h, pouring off the blocking solution, and draining for later use. Adding HRP-9017(1:8K), HRP-9010(1:4K) and hybridoma supernatant, adding antibodies 9017 and 9010 and complete culture medium as negative and positive control, incubating at 37 ℃ for 30min, pouring out liquid in the wells, washing the plate with ELISA washing liquid for five times, adding 100 mu L of solution containing 0.1% (M/V) o-phenylenediamine, 0.1% (V/V) hydrogen peroxide, pH5.0 citric acid phosphate buffer, incubating at 37 ℃ for 10min, adding 50 mu L of 2M sulfuric acid solution into each well to stop the reaction, and detecting the absorption value at 450nm by using a multifunctional reader, wherein the result is shown in Table 6.
Table 6:
since 9010 is a conformational epitope antibody and is also an antibody in which 4 other linear epitopes are mutually formed, 100% of the antibodies having the same epitope cannot compete off in the reaction by the competition method, the OD value was 0.673. From the data analysis, it was also known that the antibody in the hybridoma supernatant was a linear epitope antibody. The formula cut off was calculated as 1/2 (mean positive control + mean negative control) according to ELISA competition cut off values. The 9010 antibody is used as an enzyme-labeled antibody, the cut off value is 1.327, the result is negative when the detection result is larger than the cut off value, and the result is positive otherwise; the 9017 antibody is used as an enzyme-labeled antibody, namely the cut off value is 0.992, the antibody with the detection result larger than the cut off value is negative, and the antibody with the detection result larger than the cut off value is positive otherwise.
2. Screening for Positive hybridoma cells
2.1 Primary screening of Positive hybridoma cells
Recombinant HBeAg was diluted to 5. mu.g/mL with 0.06M pH9.6 carbonate buffer, 100. mu.L/well coated overnight at 4 ℃. 0.5% casein (prepared in 0.1M PBS) 150. mu.L/well, blocking at 37 ℃ for 2h, pouring off the blocking solution, and draining for later use. And on the ninth day after cell fusion, 50 mu L of cell supernatant and enzyme-labeled antibody (HRP-9017 or HRP-9010) with similar concentrations are added into the 96-well enzyme-labeled detection plate at the same time, incubation is carried out for 30min at 37 ℃, after the plate is washed by ELISA washing liquid for five times, 100 mu L of solution containing 0.1% (M/V) o-phenylenediamine, 0.1% (V/V) hydrogen peroxide, pH5.0 citric acid hydrogen peroxide phosphate buffer solution is added into each well, incubation is carried out for 10min at 37 ℃, 50 mu L of 2M sulfuric acid solution is added into each well to terminate the reaction, and a multifunctional reading instrument detects the absorption value of 450 nm. Taking an unlabeled enzyme antibody of a corresponding enzyme-labeled antibody as a positive control, taking an RPMI1640 complete culture solution as a negative control, judging whether the enzyme-labeled antibody is negative or positive according to the cut off value of different enzyme-labeled antibodies, and judging whether the OD value is less than the cut off value is positive or negative otherwise in the detection result of the supernatant. The positive result shows that the secreted antibody has the same or similar epitope with the enzyme-labeled antibody, and the same antibody can be detected through specificity, stability, sensitivity, detection of clinical samples or epitope sequencing; negative results show that the secreted antibody is opposite to or matched with the enzyme-labeled antibody, and whether the secreted antibody is different from the enzyme-labeled antibody can be detected by an ELISA double-antibody sandwich method. After fusion, 12 positive cell strains are detected for the first time and are respectively named as: 20D2, 56G1, 80B3, 100C3, 121D4, 156B9, 178C6, 194E3, 201H4, 211A1, 222F1 and 231C10, 186 positive cell strains are detected after primary screening by an indirect method, screening positivity is reduced by more than 80% by a competition method, workload is reduced by 80%, cycle is shortened, and success rate is improved.
2.2 cloning of Positive hybridoma cells
The above positive hybridoma cell suspension was prepared by plating feeder cells according to the method for preparing feeder cells before the fusion, diluting the suspension to a dilution of 1 cell per ml with HT medium containing 20% serum, and screening the positive wells for 4 or more successive clones as described above until 100% single clones were obtained.
2.3 cryopreservation of Positive hybridoma cells
Cloning the cells to 100% of monoclone and detecting 12 positive cells by a direct competition method, transferring the cells into 24 holes for continuous culture, transferring the cells into a cell bottle for expanded culture when the cells are full of 80%, performing bottle division and passage when the cells are full of 80%, dispersing the cells in the cell bottle by using a proper amount of serum-free RPMI-1640 medium when the passage cells grow to logarithmic phase, collecting the cell suspension in a conical centrifuge tube, recording the volume V of the cell suspension, taking a proper amount of cell suspension for cell counting to obtain the density (one/mL) of the cell suspension, centrifuging the rest at 1000rpm for 5min, discarding supernatant, calculating the number of the precipitated cells according to the cell density and the volume before centrifugation, adding a proper amount of freezing medium into the cell precipitate to adjust the cell density to 4-8 × 106Counting the cells per mL (if the number of cells is not within the range, re-centrifuging the cells according to the total cell count, re-adding an appropriate amount of the frozen stock solution to finally make the cell concentration be 4-8 × 106one/mL), then subpackaged in sterile freezing tubes, and each freezing tube is subpackaged with 0.5mL of resuspended cell freezing solution. 12 hybridoma cell strains capable of stably secreting the antibody are obtained by cell fusion once, and the clone numbers of the hybridoma cell strains are 20D2, 56G1, 80B3, 100C3, 121D4, 156B9, 178C6, 194E3, 201H4, 211A1, 222F1 and 231C 10. (the secreted antibodies are numbered 25#, 52#, 64#, 80#, 96#, 102#, 114#, respectively,156#, 189#, 201#, 224#, 262#), and was deposited in new material bio-technology ltd, sikawa, 12 months, 2018.
Fifth, prepare monoclonal antibody
1. Preparation of monoclonal antibodies
Selecting 12-14 weeks healthy BALB/c mice, injecting 0.3-0.5 mL adjuvant (KX 0210048, Peking Boolong, cat number) into the abdominal cavity of each mouse for about 15 days (12-18 days), then injecting positive hybridoma cells, and injecting 0.5-1 × 10 into the abdominal cavity of each mouse6And (3) hybridoma cells. Ascites can be generated after 9-12 days of cell inoculation, the ascites generation condition of the mice is observed every day, if the abdomen is obviously enlarged and the skin is tense when the mice are touched by hands, the neck can be pulled to kill the mice, the ascites is sucked into a 15mL centrifuge tube by a dropper, and 1-5 mL ascites can be obtained by one mouse. Centrifuging the collected ascites to obtain supernatant, and storing the small sample in a refrigerator below-20 deg.C for use.
2. Purification of monoclonal antibodies
The anti-HBeAg monoclonal antibody ascites frozen below-20 ℃ is placed in a refrigerator at 2-8 ℃ for thawing overnight one day in advance. The next day, ascites fluid was mixed, centrifuged at 12000rpm for 20 minutes at 2-8 ℃ to remove oil and precipitate, the supernatant was diluted 5-10 times with Mab loading buffer and filtered with 0.22 μm filter. The above-mentioned post-filtrate was loaded with 5mL-mabselectsure media and the breakthrough was collected using AKTA purification apparatus. And (3) after the sample loading is finished, balancing the chromatographic column by using a balance liquid until the baseline is stable, eluting the target protein by using an eluent, collecting an elution peak of more than 100mAu, cleaning the chromatographic column by using 0.1M sodium hydroxide after the elution is finished, and then storing the chromatographic column. The eluted target protein is neutralized, and 0.1mL of a neutralizing solution is added dropwise to each mL of the eluate. After mixing and neutralization, the protein was dialyzed in 5L of dialysis solution, and the solution was changed every two hours for 3 times. And (4) centrifuging the dialyzed target protein at 12000rpm for 20 minutes to obtain a supernatant, namely a final finished product, and measuring the purity and the concentration.
3. Purity testing of monoclonal antibodies
The electrophoresis glass plate is assembled, and SDS-PAGE gel of 12% separating gel at the lower layer and 5% concentrated gel at the upper layer is prepared. The gel electrophoresis tank is assembled, and a proper amount of 1 × electrophoresis buffer solution is added. After the antibody concentration was measured, a small amount of the antibody was diluted with 20mM PBS to a concentration of about 1mg/mL, 40. mu.l of the diluted antibody was added to 10. mu.l of 5 Xsample buffer, mixed well, boiled for 10 minutes, and centrifuged at 5000rpm for 10 minutes for use. Remove 10. mu.L of supernatant, electrophoresis at 70V to the boundary between the concentrated gel and the separation gel (about 15 min), and electrophoresis at 140V until bromophenol blue comes out of the gel. After electrophoresis, the SDS-PAGE gel is stripped from the electrophoresis glass plate, put into a staining solution, and the glass plate is cleaned and dried for later use. The SDS-PAGE gel was dip-stained with Coomassie Brilliant blue stain for 30 minutes and then eluted with Coomassie Brilliant blue stain until the background was colorless (which was suitably heat-destained). The PAGE gel was photographed with a gel imager and the image intensity was analyzed by software to estimate antibody purity. And (4) detecting whether the molecular weight and the protein band type of each band of the antibody electrophoresis chart are correct. The electrophoretogram is shown as 1, and the purity is more than 90%.
4. Detection of specificity of monoclonal antibodies
The specificity of the antibody prepared above was examined by WB assay. Adding 10 mu L of treated BSA and HBeAg into two sample adding holes with glue concentration of 7%, adding pre-staining marker, voltage of 100v, 2h, 10% serum (TBST dilution), shaking table incubation at room temperature for 1h, diluting antibody with 10% calf serum (TBST) respectively, diluting ratio of 1:2000, placing two membranes into diluted primary antibody diluent respectively, and standing overnight at 4 ℃. Washing the membrane with BST for 3 times, adding IgG-HRP (1: 10K), shaking-culturing for 1h at room temperature, washing the membrane with TBST for 3 times, and exposing. The results are shown in FIG. 2, and FIG. 2 shows: only 25# and 262# of all antibodies have specific reaction with HBeAg, the rest antibodies have no specific reaction with HBeAg, and all antibodies have no reaction with BSA;
the 25# hybridoma cell strain is preserved, and specifically preserved in the China center for type culture Collection in 2019, 10 months and 24 days, wherein the preservation number is CCTCC NO: C2019231.
the 262# hybridoma cell strain is preserved, and specifically, the 262# hybridoma cell strain is preserved in a China center for type culture Collection in 2019, 10 months and 24 days, wherein the preservation number of the Wuhan university in Wuhan, China is CCTCC NO: C2019232.
sixthly, verification of monoclonal antibody
The method for labeling the antibody 25# and 262# by horseradish peroxidase is the same as that of 9010 and 9017.
1. The double antibody sandwich method detects the pairing reaction of 25# and 262 #.
Table 7:
2. stability verification of monoclonal antibody 25# and 262# prepared by the invention
Stability of monoclonal antibodies of the invention in harsh environments monoclonal antibodies 25# and 262# of the invention were treated under the following conditions: repeatedly freezing and thawing at a-20 ℃ for 2 times; repeated freeze thawing at the temperature of between 20 and b for 3 times; repeatedly freezing and thawing at c-20 deg.C for 4 times; repeated freeze thawing at d-20 deg.c for 5 times; e-20 deg.C for 7 months; f thermal acceleration at 37 ℃ for 7 days; g 37 ℃ heat accelerated for 14 days, other conditions are not changed, the antibody under the above conditions is subjected to SDS-PAGE, whether degradation is caused or not is detected, the result is shown in figure 3, and the result in figure 3 shows that the antibody under all conditions is not degraded. Similarly, the antibody under the conditions is prepared into an immunochromatography detection reagent for respectively detecting 15 parts of positive reference substances and 15 parts of negative reference substances, the coincidence rate is 100%, and the antibody is good in stability and the detection reagent prepared by the antibody is good in precision. And simultaneously selecting commercial antibodies 9017 and 9010, processing under the same conditions as above, performing SDS-PAGE, preparing detection reagents, and respectively detecting 15 parts of positive reference substances and 15 parts of negative reference substances, wherein the results show that the detection reagents begin to be partially degraded after being repeatedly frozen and thawed 5 times or being stored at-20 ℃ for 7 months and being thermally accelerated at 37 ℃ for 14 days, and the detection results are not completely consistent with the reference substances. The results of the electrophoresis are shown in FIG. 4, which shows that the commercial antibody had begun to partially degrade after repeated freeze-thawing 5 times or storage at-20 ℃ for 7 months at 37 ℃ with heat acceleration for 14 days. The results of the assays are shown in Table 8, and the results in Table 8 show that the antibodies prepared according to the present invention have better stability than the commercial antibodies.
Table 8:
3. preparation of the kit
3.125 # colloidal gold labeling
Preparing colloidal gold particle solution within the range of 40nm by adopting a trisodium citrate reduction method, and adjusting the pH value to 9.0 for later use. Taking a certain amount of colloidal gold particle solution, placing the colloidal gold particle solution on a magnetic stirrer, diluting 25# with PBS according to the proportion of 1:2000, adding the diluted solution into the colloidal gold solution, stirring and reacting for 60min, adding PEG20000 solution into the mixed reaction solution to enable the final concentration of PEG to be about 1%, placing the mixed reaction solution on a refrigerated centrifuge (4 ℃) to centrifuge at a slow speed (the speed is 11000 r/min-13000 r/min), centrifuging for 10min, and then removing the supernatant. The lower pellet was washed repeatedly with PBS and centrifuged again to discard the excess supernatant. The precipitate was resuspended in PBS buffer to a final protein concentration of about 50. mu.g/mL and stored at 2-8 ℃ for future use.
3.2 preparation of gold-labeled pad
The gold label pad is made of polyester film, treated with PBS containing 1% BSA and 1% Tween-20, sprayed with the prepared colloidal gold-labeled 25# antibody according to 40 μ L, and dried at 37 ℃ for 2h for later use.
3.3 preparation of sample pad
The sample pad is a polyester fiber pad, and is soaked in a self-made treatment solution containing a surfactant and then dried for later use.
3.4262 # antibody coating detection line and quality control line coated goat anti-rabbit IgG
The 262# antibody and the goat anti-rabbit IgG were diluted to 1.5mg/mL and 1.2mg/mL solutions, respectively, in PBS containing 1% BSA, and then sprayed on a nitrocellulose membrane using a striping machine, and dried at 37 ℃ for 1h for use.
3.5 kit Assembly
The assembly of the test strips in the kit is performed according to the scheme of fig. 5. Wherein the PVC back plate and the water absorption pad are made of conventional materials. The assembled large plate is cut into bare strips with the diameter of 7mm by a slitter for standby.
3.6 detection of clinical samples with the kit
The two anti-HBeAg monoclonal antibodies 25# and 262# prepared by the invention are matched to prepare a kit for detecting clinical samples, the kit is used for detecting clinical samples simultaneously with a commercial new kit, the detection results are compared in parallel, 1000 random samples are detected, the coincidence rate is 100%, 65 positive samples are detected, 65 samples are detected, 63 samples are detected, 2 samples are missed, the missed samples are retested by an yapei kit, the results are still positive, the detection results are shown in table 9-1, the detection results of the random samples are shown in table 9, and the kit is superior to the new kit.
Table 9:
table 9-1:
in conclusion, the clinical sample is detected on the colloidal gold platform, the false positive rate is low, the sensitivity is high, the detection kit is continuously prepared to detect the clinical sample after repeated freeze thawing, heat acceleration, 1-8 ℃ storage for 1 month and-20 ℃ storage for 3 months, and the result proves that the anti-HBeAg monoclonal antibody prepared by the invention can be specifically combined with HBeAg in serum, and the high-sensitivity, high-specificity and high-stability monoclonal antibody can accurately and quickly detect whether the HBeAg exists in the serum on the colloidal gold platform (sandwich method).
Example 2
The chemiluminescence method can more sensitively and accurately reflect the changes of viruses in hepatitis B patients, including the level changes of cccDNA and HBV DNA in vivo, can dynamically reflect the natural process of diseases and the response condition to antiviral treatment, and can also predict the curative effect, so the chemiluminescence method is widely applied clinically at present, but most of the market of HBeAg detection kits (chemiluminescence methods) is monopolized by international companies such as Roche, Yapeh and the like. The invention prepares the HBeAg quantitative detection kit based on the self-developed full-automatic chemiluminescence immunodetection system and carries out comparative evaluation with Roche.
The traditional hybridoma technology is used for preparing monoclonal antibody, and the process comprises the following steps: immunogen screening, animal immunization, cell fusion, positive cell strain cloning, ascites preparation, ascites purification and antibody screening, and has the advantages of complex process, long period and low success rate. If one of the key steps is optimized, the period can be shortened, and the success rate can be improved, so that the prepared monoclonal antibody has higher quality. In order to solve the technical problems, one-step pairing screening is added before the traditional purification step, the prepared ascites antibody is directly paired, and purification is carried out according to the active pairing result, so that the purification workload is reduced, and the success rate is improved.
The specific idea of this embodiment is:
the hybridoma is used for preparing the monoclonal antibody, positive cell strains are screened by an indirect method, 10 96-well plates are fused to obtain at most about 900 positive cell strains, the downstream purification workload is increased, but the pairing reaction can be really generated very little at last, if the ascites antibody is screened for one time before being purified, only the ascites antibody which can generate the pairing reaction with a standard antibody is purified, so that the workload is reduced, the period is shortened, and the success rate is improved. In order to solve the problems, the invention aims to screen a pair of standard paired antibodies, and carry out horseradish peroxidase labeling on the standard paired antibodies, an ELISA indirect method simultaneously carries out gradient dilution to detect the standard antibodies and ascites antibodies, the specific binding of the antigen antibodies, under the condition that the concentrations of the antigen and the enzyme labeled antibodies are the same, the concentrations of primary antibodies are similar or the same if the OD values are the same, the concentration of the ascites antibodies is estimated according to the concentration of the standard antibodies (the concentration of the antibodies with the OD value of about 2.0 is selected in the research), the ascites antibodies are coated according to the estimated concentration, the pairing condition of the ascites antibodies and the standard antibodies is detected by a sandwich ELISA method, finally, pairwise pairing is carried out on the pairwise mateable antibodies, and the mateable antibodies are prepared into a kit to be compared with a kit of Roche for detection.
Determination of antibodies binding to different epitopes
The specific procedure is the same as in example 1.
Second, immunizing mouse
1. Recombinant HBeAg (Sichuan Mike New Material technology Co., Ltd., batch No. 091614) immunized mice
Diluting the recombinant HBeAg to 2.0mg/mL with physiological saline, and mixing with MagicTMMouse Adjuvant (creative diagnostics, product number CDN-A001) is mixed in equal volume, gently blown up and down 5 times by a 100-L pipette, leg muscle injection is carried out by 50-100-L of the final volume of each BALB/c Mouse (achievement reaches the center of the great laboratory animal, female in 6 weeks, 4 mice) and the recombinant HBeAg (40-g/BALB/c Mouse) is matched with the Adjuvant. After 3 weeks (21 days), the second immunization was performed as above, and after 2 weeks (14 days), tail blood was collected 35 days after the first immunization, and the antibody titer in the serum was measured by the ELISA indirect method, as shown in Table 10, and as shown in the results of Table 10, the titers of 4 mice were in the order of: 2X 106、4×106、8×106、4×106. The serum titers of 4 mice were all greater than 1: 106And the fusion protein can be used for cell fusion.
Table 10:
preparation of hybridoma cell line
The specific procedure is the same as in example 1.
Fourthly, preparing positive hybridoma cells
1. Screening for Positive hybridoma cells
The recombinant HBeAg is diluted to 5 mu g/mL by 0.06M carbonate buffer solution with pH9.6, and each hole of a 96-hole enzyme label plate is coated with 100 mu L for detecting the cell culture supernatant after fusion. Placing the mixture in a refrigerator at 2-8 ℃ overnight, discarding the liquid in the holes the next day, washing the plate with ELISA washing liquid three times, patting the plate dry, sealing the plate dry with 0.01M PBS (pH7.2) containing 10% calf serum at 150 mu L/hole at 37 ℃ for 2 hours, patting the plate dry, and carrying out vacuum packaging for later use. On the ninth day after cell fusion, 100 μ L of cell supernatant is taken by an eight-channel discharge gun and placed in the 96-well enzyme-labeled assay plate, incubation is carried out for 40min at 37 ℃, the ELISA assay plate is washed for five times, 100 μ L/well of horseradish peroxidase-labeled goat anti-mouse IgG (produced by Sichuan new materials, batch number 011816, 1mg/mL) diluted by 10000 times is added, incubation is carried out for 30min at 37 ℃ with the plate washed, 100 μ L of buffer solution containing 0.1% (M/V) o-phenylenediamine, 0.1% (V/V) hydrogen peroxide and pH5.0 citric acid phosphate is added into each well, incubation is carried out for 10min at 37 ℃, 50 μ L of 2M sulfuric acid solution is added into each well to terminate the reaction, and a multifunctional reading instrument is used for detecting the absorption value of 450 nm. The mouse serum (eyeball) is diluted to 100 times during fusion to be used as a positive control, RPMI1640 complete culture solution is used as a negative control, the OD value of the negative control is less than 0.2, the OD value of the positive control is more than 1.8, the detection system is effective, when the OD value of the sample is more than or equal to 2 times the OD value of the negative control, the sample is positive, and otherwise the sample is negative. According to statistics, 625 of 960 wells are fusion wells, and the fusion rate is 65.1%. The anti-HBeAg monoclonal antibody is detected by ELISA method, 625 holes are detected, wherein the number of positive holes is 26, and the positive rate is 4.16%.
2. Cloning of Positive hybridoma cells
The above positive hybridoma cell suspension was prepared by plating feeder cells according to the method for preparing feeder cells before the fusion, diluting the suspension to a dilution of 1 cell per ml with HT medium containing 20% serum, and screening positive wells by the indirect ELISA method described above, and cloning was continued for 4 times or more until 100% single cloning was achieved.
3. Cryopreservation of positive hybridoma cells
Cloning the cells to 100% of monoclone and detecting the cells to be positive by an indirect method, transferring the cells to 24 holes (26 positive clones) for continuous culture, transferring the cells to a cell bottle for expanded culture when the cells are full of 80%, performing bottle division and passage when the cells of the positive hybridoma are full of 80% of the cell bottle, dispersing the cells in the cell bottle by using a proper amount of serum-free RPMI-1640 medium when the passage cells grow to a logarithmic phase, collecting the cell suspension in a conical centrifuge tube, recording the volume V of the cell suspension, taking a proper amount of the cell suspension for cell counting to obtain the density (one/mL) of the cell suspension, centrifuging the cell suspension at the rest speed of 1000rpm for 5min, discarding supernatant, calculating the number of the precipitated cells according to the cell density and the volume before centrifugation, adding a proper amount of freezing medium into the cell precipitate to adjust the cell density to 48 multiplied by6Counting the cells per mL (if the number of cells is not within the range, re-centrifuging the cells according to the total cell count, re-adding an appropriate amount of the frozen stock solution to finally make the cell concentration be 4-8 × 106one/mL), then subpackaged in sterile freezing tubes, and each freezing tube is subpackaged with 0.5mL of resuspended cell freezing solution. 26 cell strains capable of stably secreting antibodies are obtained by cell fusion once, and the clone numbers are (289G6, 281C5, 284D2, 289A1, 290F3, 292C7, 286D2, 293F4, 296H4, 337C11, 301D4, 301A5, 303C4, 308A5, 309H2, 310D3, 311F3, 311C6, 315B5, 315D9, 316D9, 319E8, 318H4, 322C9, 325D8 and 330B9), and are preserved in Skywa New Material Biotech limited company on 12, 16 days in 2017.
Fifth, prepare monoclonal antibody
1. Preparation of ascites antibody
Sensitization: healthy BALB/c mice of 12-14 weeks are selected, and each mouse is injected with 0.3-0.5 mL adjuvant (KX 0210048, product number: Beijing Boolong) in the abdominal cavity for about 15 days (12-18 days). Cells were raised to log phase and were full, smooth, and well defined. Then injecting the hybridoma cells, wherein the volume of one injection of the mouse is 0.5mL, and the inoculation quantity is 0.75-1.25 multiplied by 106Individual hybridoma cells/individual. Ascites can be generated after inoculating the cells for 9-12 days, and the generation of the ascites of the mice is observed every dayIn case of obvious abdominal distension and skin tension when touched by hands, the neck can be pulled to kill the mouse, the ascites is sucked into a 15mL centrifuge tube by a dropper, and 1-5 mL ascites can be obtained from one mouse. Centrifuging the collected ascites to obtain supernatant, and storing the small sample in a refrigerator below-20 deg.C for use.
2. Dilution of ascites antibody
Group HBeAg (Sichuan Mike New Material technology Co., Ltd., lot No. 091614) was diluted 5. mu.g/mL with 0.06M carbonic acid buffer solution pH9.6, and 100. mu.L/well was coated overnight at 4 ℃. 0.5% casein (prepared in 0.1M PBS) 150. mu.L/well, blocking at 37 ℃ for 2h, pouring off the blocking solution, and draining for later use. Diluting 26 ascites antibodies, 9010(2.8mg/mL) and 9017(3.1mg/mL) in a gradient manner by 10 times, adding 100 mu L/hole into the enzyme label plate, incubating at 37 ℃ for 40min, washing the plate with ELISA washing liquid for five times, adding 100 mu L/hole of horseradish peroxidase-labeled goat anti-mouse IgG (New Sichuan material production, batch No. 011816), incubating at 37 ℃ for 30min with the plate washed, adding 100 mu L of buffer solution containing 0.1% (M/V) o-phenylenediamine, 0.1% (V/V) hydrogen peroxide, pH5.0 citric acid phosphate buffer solution, incubating at 37 ℃ for 10min, adding 50 mu L of 2M sulfuric acid solution into each hole to terminate the reaction, and detecting the absorption value at 450nm by a multifunctional reading instrument. The mouse serum is diluted to 100 times during fusion to be used as a positive control, 0.1M PBS is used as a negative control, the OD value of the negative control is less than 0.2, the OD value of the positive control is more than 1.8, the detection system is effective, when the OD value of the sample is more than or equal to 2 multiplied by the OD value of the negative control, the sample is positive, otherwise, the sample is negative. The results are shown in Table 11, and therefore, the antibody concentration of the ascites antibody was selected to have an OD of about 2.0, and the pairing reaction was carried out.
Table 11:
3. pairing reaction of ascites antibody and standard antibody
The ascites antibodies were coated by diluting 26 strains of ascites antibodies with 0.06M carbonic acid buffer solution (pH9.6) at corresponding times according to the dilutions of the ascites antibodies examined in Table 11. 100 u L/hole 4 ℃ coated overnight. 0.5% casein (prepared by 0.1 MPBS) 150 μ L/well, sealing at 37 deg.C for 2 hr, pouring off the sealing solution, and draining. Recombinant HBeAg (Sichuan Mike New Material technology Co., Ltd., batch No. 091614) was diluted to 10ng/mL with 0.1M PBS, 100. mu.L/well was added to the above-mentioned drained ELISA plate, incubating at 37 ℃ for 40min, pouring out liquid in the wells, washing the plate with ELISA washing liquid for five times, adding 100 mu L of enzyme-labeled antibodies (9010 and 9017-labeled enzymes) labeled in the above example 2 into each well, incubating at 37 ℃ for 30min, pouring out liquid in the wells, washing the plate with ELISA washing liquid for five times, adding 100 mu L of solution containing 0.1% (M/V) o-phenylenediamine, 0.1% (V/V) hydrogen peroxide, pH5.0 citric acid phosphate buffer solution, incubating at 37 ℃ for 10min, adding 50 mu L of 2M sulfuric acid into each well to terminate the reaction, detecting the absorption value at 450nm by using a multifunctional reading instrument, and obtaining results in Table 12, wherein the results in Table 12 show that only ascites antibody 400# can be paired with 9017, and ascites antibody 432# can be paired with 9010.
Table 12:
the 400# hybridoma cell strain is preserved, and specifically preserved in the China center for type culture Collection in 2019, 10 months and 24 days, wherein the preservation number is CCTCC NO: C2019233.
the 432# hybridoma cell strain is preserved, and specifically, the 432# hybridoma cell strain is preserved in a China center for type culture Collection in 2019, 10 months and 24 days, wherein the preservation number of the university of Wuhan, China is CCTCC NO: C2019234.
4. purification of ascites antibodies
And thawing the 26 ascites antibodies frozen below-20 ℃ in a refrigerator at 2-8 ℃ overnight one day in advance. The next day, the ascites fluid was mixed well, centrifuged at 12000rpm for 20 minutes at 2-8 ℃ to remove oil and precipitate, the supernatant was diluted 5-10 times with Mab loading buffer and filtered with 0.22 μm filter. The above-mentioned post-filtrate was loaded with 5mL-mabselectsure media and the breakthrough was collected using AKTA purification apparatus. After the sample loading is finished, the chromatographic column is balanced by using a balance liquid until the baseline is stable, the target protein is eluted by using eluent, the elution peak which is more than 100mAu is collected, the chromatographic column is cleaned by using 0.1M sodium hydroxide after the elution is finished, and then the chromatographic column (bocglon, product number: B002) is stored. The eluted target protein is neutralized, and 0.1mL of a neutralizing solution is added dropwise to each mL of the eluate. After mixing and neutralization, the protein was dialyzed in 5L of dialysis solution, and the solution was changed every two hours for 3 times. And (4) centrifuging the dialyzed target protein at 12000rpm for 20 minutes to obtain a supernatant, namely a final finished product, and measuring the purity and the concentration.
5. Purity testing of monoclonal antibodies
The electrophoresis glass plate is assembled, and SDS-PAGE gel of 12% separating gel at the lower layer and 5% concentrated gel at the upper layer is prepared. The gel electrophoresis tank is assembled, and a proper amount of 1 × electrophoresis buffer solution is added. After the antibody concentration was measured, a small amount of the antibody was diluted with 20mM PBS to a concentration of about 1mg/mL, 40. mu.l of the diluted antibody was added to 10. mu.l of 5 Xsample buffer, mixed well, boiled for 10 minutes, and centrifuged at 5000rpm for 10 minutes for use. Remove 10. mu.L of supernatant, electrophoresis at 70V to the boundary between the concentrated gel and the separation gel (about 15 min), and electrophoresis at 140V until bromophenol blue comes out of the gel. After electrophoresis, the SDS-PAGE gel is stripped from the electrophoresis glass plate, put into a staining solution, and the glass plate is cleaned and dried for later use. The SDS-PAGE gel was dip-stained with Coomassie Brilliant blue stain for 30 minutes and then eluted with Coomassie Brilliant blue stain until the background was colorless (which was suitably heat-destained). The PAGE gel was photographed with a gel imager and the image intensity was analyzed by software to estimate antibody purity. And (4) detecting whether the molecular weight and the protein band type of each band of the antibody electrophoresis chart are correct. The electrophoretogram is shown in figure 6, and the purity is more than 90%.
Sixthly, verification of monoclonal antibody
1. Pairing reaction of screened ascites purified monoclonal antibody and commercial monoclonal antibody
The purified 26 antibodies were diluted to 2.5. mu.g/mL each with 0.06M carbonate buffer pH9.6 and coated overnight at 4 ℃ with 100. mu.L/well. 0.5% casein (prepared in 0.1M PBS) 150. mu.L/well, blocking at 37 ℃ for 2h, pouring off the blocking solution, and draining for later use. Diluting the recombinant HBeAg (Sichuan Mike new material technology Co., Ltd., batch No. 091614) to 10ng/mL with 0.1M PBS, adding 100 muL/well into the flap-dried ELISA plate, incubating at 37 ℃ for 40min, pouring off the liquid in the well, washing the plate with ELISA washing liquid for five times, adding 100 muL labeled standard enzyme-labeled antibody into each well, incubating at 37 ℃ for 30min, pouring off the liquid in the well, washing the plate with ELISA washing liquid for five times, adding 100 muL of 0.1% (M/V) o-phenylenediamine, 0.1% (V/V) hydrogen peroxide, pH5.0 citric acid phosphate buffer solution, incubating at 37 ℃ for 10min, adding 50 muL 2M sulfuric acid solution into each well to terminate the reaction, detecting the 450nm absorption value with a multifunctional reading instrument, the results are shown in Table 13, the results of Table 13 show that the pairing results of the coated ascites antibody and the coated and the purified antibody are consistent with the standard antibody, which shows that the scheme of preliminary screening is firstly carried out through the pairing of the ascites antibody and the standard antibody, the purification workload can be greatly reduced.
Table 13:
2. pairing reaction between screened ascites antibodies
2.1 Horseradish peroxidase-labeled monoclonal antibodies 400# and 432#, respectively
The procedure of the experiment was the same as in example 1.
2.2400 # and 432# antibodies pairing reaction results
The purified 400# and 432# antibodies were each diluted to 2.5. mu.g/mL with 0.06M carbonated buffer pH9.6 and coated overnight at 100. mu.L/well at 4 ℃. 0.5% casein (prepared in 0.1M PBS) 150. mu.L/well, blocking at 37 ℃ for 2h, pouring off the blocking solution, and draining for later use. Diluting the recombinant HBeAg (Sichuan Mike new material technology Co., Ltd., batch No. 091614) to 10ng/mL with 0.1M PBS, adding 100 uL/well into the drained ELISA plate, incubating at 37 ℃ for 40min, pouring off the liquid in the well, washing the plate with ELISA washing liquid for five times, adding 100 uL of the enzyme-labeled antibody marked in the example 9 into each well, incubating at 37 ℃ for 30min, pouring off the liquid in the well, washing the plate with ELISA washing liquid for five times, adding 100 uL of the solution containing 0.1% (M/V) o-phenylenediamine, 0.1% (V/V) hydrogen peroxide into each well, incubating at 37 ℃ for 10min, adding 50 uL of 2M sulfuric acid solution into each well to terminate the reaction, detecting the absorption value at 450nm by a multifunctional reading instrument, and the results are shown in Table 14 and Table 14, wherein the results show that the monoclonal antibodies 400# and 432# prepared by the invention can be paired.
Table 14:
3. stability verification of monoclonal antibodies 400# and 432#
The stability of the monoclonal antibody of the invention in a severe environment, the monoclonal antibodies 400# and 432# of the invention are treated under the following conditions: repeatedly freezing and thawing at a-20 ℃ for 2 times; repeated freeze thawing at the temperature of between 20 and b for 3 times; repeatedly freezing and thawing at c-20 deg.C for 4 times; repeated freeze thawing at d-20 deg.c for 5 times; e-20 deg.C for 7 months; f thermal acceleration at 37 ℃ for 7 days; g 37 ℃ heat accelerated for 14 days, other conditions were unchanged, the antibodies under the above conditions were subjected to SDS-PAGE to determine whether they were degraded, the results are shown in FIG. 7, and the results in FIG. 7 show that the antibodies under all conditions were not degraded. Similarly, the antibody under the conditions is prepared into a chemiluminescence detection reagent for respectively detecting 15 parts of positive reference substances and 15 parts of negative reference substances, the coincidence rate is 100%, and the antibody has good stability and the detection reagent prepared by the antibody has good precision. And simultaneously selecting commercial antibodies 9017 and 9010, processing under the same conditions as above, performing SDS-PAGE, preparing detection reagents, and respectively detecting 15 parts of positive reference substances and 15 parts of negative reference substances, wherein the results show that the detection reagents begin to be partially degraded after being repeatedly frozen and thawed 5 times or being stored at-20 ℃ for 7 months and being thermally accelerated at 37 ℃ for 14 days, and the detection results are not completely consistent with the reference substances. The results of the electrophoresis are shown in FIG. 8, which shows that the commercial antibody had begun to partially degrade after repeated freeze-thawing 5 times or storage at-20 ℃ for 7 months at 37 ℃ with heat acceleration for 14 days. The results are shown in Table 15, and the results in Table 15 show that the antibodies prepared by the invention have better stability than the commercial antibodies.
Table 15:
4. preparation of monoclonal antibody kit
4.1 acridinium ester-labeled antibody 432#
Marking acridine ester on an antibody 432# and storing at-20 ℃ after adding glycerol; the labeled antibody was diluted with a buffer containing BSA.
4.2 Biotin-labeled antibody 400#
The antibody 400# is labeled with biotin, and the labeled antibody is stored at-20 ℃ after being added with glycerol. After labeling, the magnetic beads were coated with the antibody, and after coating was completed, the beads were diluted with a buffer containing BSA.
4.3 kit validation
And (3) testing the calibrator and the quality control product by using the newly configured reagent, and adjusting the dilution to ensure that the testing result of the quality control product is within the range of the marked value.
5. Testing clinical samples in contrast to third party reagents
And (3) testing 200 clinical samples by using the newly prepared reagents, wherein 50 positive samples and 150 negative samples are compared with the corresponding testing results of the comparison reagents, and calculating the sample coincidence rate. The details are shown in Table 16; it can be seen that the chemiluminescence kit prepared by using the monoclonal antibody pair obtained by screening has high consistency of detection results compared with the existing kit.
Table 16:
it is to be understood that the invention disclosed is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
Claims (10)
1. A method for preparing a monoclonal antibody, which is characterized by comprising the following steps: the preparation method comprises the following steps:
(1) the antigen is used for immunizing an animal,
(2) isolating the spleen cells of the immunized animal,
(3) the cell fusion is carried out, and the cell fusion is carried out,
(4) preparing a positive hybridoma cell,
(5) preparing a monoclonal antibody;
wherein, the preparation of the monoclonal antibody comprises in vitro culture and in vivo culture; when the in vivo culture is carried out, the method further comprises the following steps: (5-1) preparing an ascites antibody,
(5-2) purifying the prepared ascites antibody to obtain a monoclonal antibody;
wherein the screening step for positive hybridoma cells is carried out in step (4) using a known antibody, and/or the screening step for ascites antibody is carried out in step (5-1) using a known antibody.
2. The method of claim 1, wherein: the method used in the screening step in the step (4) and the step (5-1) is a sandwich method or a competition method.
3. The method of claim 2, wherein: the sandwich method comprises the following steps:
a) marking a known antibody by a marker for later use;
b) coating the antibody in the positive hybridoma cell supernatant or the ascites antibody on a solid support, adding a known antibody marked by a marker after the antigen reaction is completed, and continuing the reaction;
c) adding a reaction substrate and detecting the signal value of the marker to obtain the screened positive hybridoma cells or obtain the screened ascites antibody.
4. The method of claim 2, wherein: the competition method comprises the following steps:
in step (4), a) labeling the known antibody with a labeling substance for use; b) coating the antigen on a solid support, adding a known antibody marked by a marker and a positive hybridoma cell supernatant at the same time, adding a reaction substrate after the reaction is finished, detecting a signal value of the marker, and screening the positive hybridoma cells according to the signal value;
in the step (5-1), a) the labeling substance labels a known antibody for use; b) coating the antigen on a solid support, adding a known antibody marked by a marker and an ascites antibody at the same time, adding a reaction substrate after the reaction is finished, detecting a signal value of the marker, and screening the ascites antibody according to the signal value;
preferably, in the case of screening positive hybridoma cells using the competitive method, the known antibody labeled with the marker is present at a concentration similar or identical to that of the antibody in the supernatant of the positive hybridoma cells; when the ascites antibody is screened by using the competition method, the concentration of the known antibody marked by the marker is similar or identical to that of the ascites antibody.
5. A preparation method of a monoclonal antibody pair for detection by a sandwich method is characterized by comprising the following steps: the preparation method comprises the following steps:
(1) the antigen is used for immunizing an animal,
(2) isolating the spleen cells of the immunized animal,
(3) the cell fusion is carried out, and the cell fusion is carried out,
(4) preparing a positive hybridoma cell,
(5) preparing a monoclonal antibody pair;
wherein, the preparation of the monoclonal antibody pair comprises in vitro culture and in vivo culture; when the in vivo culture is carried out, the method further comprises the following steps:
(5-1) preparing an ascites antibody,
(5-2) purifying the prepared ascites antibody to obtain a monoclonal antibody pair;
wherein the screening step of positive hybridoma cells is carried out in step (4) using antibody A and antibody B binding to different epitopes, and/or the screening step of ascites antibodies is carried out in step (5-1) using antibody A and antibody B binding to different epitopes.
6. The method of claim 5, wherein: the method used in the screening step in the step (4) and the step (5-1) is a sandwich method or a competition method.
7. The method of claim 6, wherein: the sandwich method comprises the following steps:
in the step (4), a) labeling the antibody A and the antibody B with the labeling substance for standby; b) coating the antibody in the supernatant of the positive hybridoma cells on a solid support 1 and a solid support 2 respectively, and adding a marker labeled antibody A into the coated solid support 1 and adding a marker labeled antibody B into the coated solid support 2 after the antigen adding reaction is completed;
c) adding a reaction substrate, detecting a signal value of the marker, and screening positive hybridoma cells according to the signal value;
in the step (5-1), a) labeling the antibody A and the antibody B with the labeling substance for standby; b) coating ascites antibodies on a solid support 1 and a solid support 2, respectively, and then adding a marker-labeled antibody A to the coated solid support 1 and a marker-labeled antibody B to the coated solid support 2; c) adding reaction substrates, detecting the signal value of the marker, and screening ascites antibodies according to the signal value.
8. The method of claim 7, wherein: the competition method comprises the following steps:
in the step (4), a) labeling the antibody A and the antibody B with the labeling substance for standby; b) coating the antigen on solid support 1 and solid support 2, respectively; after completion of the reaction, simultaneously adding the marker-labeled antibody A and the supernatant of the positive hybridoma cells to the coated solid support 1, and simultaneously adding the marker-labeled antibody B and the supernatant of the positive hybridoma cells to the coated solid support 2; c) adding a reaction substrate, detecting a signal value of the marker, and screening positive hybridoma cells according to the signal value;
in the step (5-1), a) labeling the antibody A and the antibody B with the labeling substance for standby; b) coating the antigen on solid support 1 and solid support 2, respectively; then, simultaneously adding antibody A labeled with a labeling substance and ascites antibody to coated solid support 1, and simultaneously adding antibody B labeled with a labeling substance and ascites antibody to coated solid support 2; c) adding reaction substrates, detecting the signal value of the marker, and screening ascites antibodies according to the signal value.
Preferably; when a positive hybridoma is screened by using a competition method, the concentration of the antibody A or the antibody B marked by the marker is similar or identical to that of the antibody in the supernatant of the positive hybridoma; when the ascites antibody is screened by using the competition method, the concentration of the marker antibody A or antibody B is similar or identical to that of the ascites antibody.
9. A pair of hybridoma cells characterized by: the hybridoma cell pair is prepared by the preparation method of any one of claims 5 to 7; preferably, the preservation number of the hybridoma cell pair is CCTCC NO: c2019231 and CCTCCNO: c2019232, or CCTCC NO: c2019233 and CCTCC NO: C2019234.
10. a monoclonal antibody pair characterized by: the monoclonal antibody pair is obtained by secreting a hybridoma cell pair prepared by the preparation method of any one of claims 5 to 7;
preferably, the monoclonal antibody pair consists of a monoclonal antibody with a collection number of CCTCC NO: c2019231 and CCTCC NO: the hybridoma cell pair of C2019232 is obtained by secretion; or the monoclonal antibody pair is prepared by the following components in a collection number of CCTCC NO: c2019233 and CCTCCNO: the hybridoma cell pair of C2019234 was secreted.
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