CN116731978A - Hybridoma cell strain of anti-human hepatitis B surface antigen monoclonal antibody and preparation method of anti-human hepatitis B surface antigen monoclonal antibody - Google Patents
Hybridoma cell strain of anti-human hepatitis B surface antigen monoclonal antibody and preparation method of anti-human hepatitis B surface antigen monoclonal antibody Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
- C07K16/082—Hepadnaviridae, e.g. hepatitis B virus
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention relates to the technical field of bioengineering, in particular to an anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain and a preparation method of the anti-human hepatitis B surface antigen monoclonal antibody, wherein the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain comprises an anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain 1 and an anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain 2, the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain 1, the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain 2 and passage cell strains thereof can respectively stably secrete the anti-human hepatitis B surface antigen monoclonal antibody 1 and the anti-human hepatitis B surface antigen monoclonal antibody 2, and impurities can be effectively removed after the monoclonal antibodies secreted by the hybridoma cell strains are respectively purified, so that the monoclonal antibodies with strong specificity, high purity, high titer, good stability, good uniformity and strong repeatability are obtained.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a hybridoma cell strain of an anti-human hepatitis B surface antigen monoclonal antibody and a preparation method of the anti-human hepatitis B surface antigen monoclonal antibody.
Background
Hepatitis B surface antigen (HBsAg) is a coat protein of hepatitis B virus, which is an expression of hepatitis B virus S gene, which belongs to the category of viral envelope proteins, and although it is not infectious per se, it belongs to the first-occurring serum virus antigen in the later disease intervention, and the basic structure is closely related to HBV templates, and there is a large correlation with DNA level, so that the value of research on HBsAg is analyzed.
HBsAg itself is not infectious, but its appearance is often accompanied by the presence of hepatitis b virus, so it is a marker of infected hepatitis b virus. Hepatitis B surface antigen examination is used to determine whether hepatitis B virus is infected. It may be present in the patient's blood, saliva, milk, sweat, tear, nasopharyngeal secretions, semen, and vaginal secretions. HBsAg positivity only indicates that the person is infected with HBV, but it is not possible to determine whether the person is infectious. If it is desired to determine whether the hepatitis B virus in the patient is infectious, other items are also tested. The phenotype of hepatitis B virus is its serotype, which is determined by a few residues on the outer membrane major protein.
The most commonly used methods for measuring hepatitis B surface antigen in China are enzyme-linked immunosorbent assay (ELISA) and colloidal gold chromatography (GIA). ELISA has high sensitivity, strong specificity and good repeatability, can be used for quantitative and semi-quantitative analysis, but has relatively complex operation steps, needs special equipment and is not suitable for emergency treatment and blood screening. The GIA is simple, convenient and quick to operate, can be detected by a single person, does not need special equipment, is limited by methodology, and has lower accuracy than ELISA.
The main biological raw materials of the human hepatitis B surface antigen detection reagent are a pair of HBsAg monoclonal antibodies, and the performance of the HBsAg monoclonal antibodies plays a vital role in the accuracy, sensitivity and specificity of the human hepatitis B surface antigen detection reagent.
The preparation of monoclonal antibody against human hepatitis B surface antigen mainly includes in vivo induction of ascites in mouse, in vitro culture of hybridoma cells, genetic engineering and other methods. The method for preparing the in-vivo induced ascites of the mice has low cost, high yield and relatively simple production process, and can realize mass preparation in a short period, so the method is widely applied. However, the monoclonal antibody against human hepatitis B surface antigen obtained by the method contains various impurities, and in order to obtain the antibody with high titer, high purity and high specificity, a proper purification method is required to be screened for separation and purification so as to meet the application in the aspects of industry, scientific research or medical diagnosis.
Disclosure of Invention
The invention aims to provide a preparation method of an anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain, an anti-human hepatitis B surface antigen monoclonal antibody, wherein the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain 1, the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain 2 and passage cell strains thereof can respectively and stably secrete the anti-human hepatitis B surface antigen monoclonal antibody 1 and the anti-human hepatitis B surface antigen monoclonal antibody 2, and the monoclonal antibodies secreted by the hybridoma cell strains can be subjected to saturated ammonium sulfate salt and blue gel affinity chromatography treatment or protein-G chromatography and ion exchange chromatography treatment, so that impurities can be effectively removed, and the monoclonal antibodies with strong specificity, high purity, high titer, good stability, good uniformity and strong repeatability can be obtained.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: the preparation method of the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain comprises the steps of:
(1) Immunization of animals
Taking 3 male BALB/c mice of 6 weeks old, immunizing with natural protein of human hepatitis B surface antigen as immunogen, performing tail-breaking blood collection on the mice, detecting serum titer to more than 10 ten thousand, and performing cell fusion;
(2) Cell fusion
Killing mice, soaking the mice in 75% alcohol for 5min, picking spleen of immunized mice in a sterile environment, grinding dispersed cells, washing and centrifuging to obtain B lymphocytes, mixing B lymphocytes and myeloma cells SP2/0 according to the ratio of cell number to 5:1, centrifuging, washing cells with serum-free DMEM culture medium to ensure that the mixed cells are serum-free, centrifuging to discard supernatant, slowly adding 1mL of 50% PEG solution with mass concentration in a water bath at 37 ℃ at a constant speed, dropwise adding and mixing the supernatant, slowly adding 40mL of serum-free DMEM culture medium at a pre-temperature of 37 ℃ after the reaction of the supernatant for 90s, ensuring that PEG is thoroughly diluted to terminate fusion, centrifuging at 1000rpm for 10min, discarding the supernatant, re-suspending cell sediment with HAT culture medium at the pre-temperature of 37 ℃, fully mixing the supernatant, placing the supernatant into a 96-hole cell culture plate, and culturing in a culture box with 5% carbon dioxide;
(3) Hybridoma cell screening and subcloning
The third day and the eighth day after fusion, 1/2 of culture solution in the 96-hole cell culture plate is replaced by HAT culture medium respectively, and the tenth day absorbs cell supernatant, namely cell supernatant to be detected;
the method comprises the steps of screening cells to be detected by an indirect ELISA method, after the first screening, completely replacing culture solution with HAT culture medium, repeatedly screening for three times, selecting cells with strong positive results from the three times, subcloning the cells for 2-3 times generally until the positive rate of a 96-well plate reaches 100%, and ending subcloning to obtain two anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strains, namely an anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain 1 and an anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain 2.
As an optimization scheme for this purpose,
the preparation method of the natural protein of the human hepatitis B surface antigen comprises the following steps:
(1) Low concentration PEG precipitation: collecting serum of asymptomatic human hepatitis B surface antigen carrier, precipitating with 5% final concentration PEG6000 to remove large particles and lipoprotein, standing at 4deg.C for 2 hr, centrifuging at 4000rpm for 30 min, discarding precipitate, and collecting supernatant;
(2) High concentration PEG precipitation: precipitating the supernatant with PEG6000 with final concentration of 10%, standing at 4deg.C for 2h, centrifuging at 4000rpm for 30 min, discarding the precipitate, and collecting supernatant to obtain primary pure natural protein of human hepatitis B surface antigen;
(3) Preparing an immunoaffinity chromatographic column;
(4) The affinity chromatography purification of the human hepatitis B surface antigen comprises the following steps:
connecting the immunoaffinity chromatography column prepared in the step (3) to a computer ultraviolet detector, and when the immunoaffinity chromatography column is used for purifying, monitoring the real-time change of a sample OD280 in the purifying process, and collecting a purified effective sample according to an OD280 monitoring result;
(1) balance: washing the immunoaffinity chromatographic column with a binding solution until the first peak detected by the computer ultraviolet detector OD280 returns to the baseline, namely equilibrium is reached, wherein the binding solution is 10mM PBS and 150mM NaCl;
(2) loading: the immune affinity chromatographic column after balancing the natural protein of the primary pure human hepatitis B surface antigen obtained in the step (2) is used for collecting effluent liquid until the baseline of detection of the computer ultraviolet detector OD280 is stable;
(3) eluting: adding eluent which is glycine-hydrochloric acid solution with pH of 3.0, collecting when the baseline detected by the computer ultraviolet detector OD280 rises, regulating the pH of the collected eluent to 7.4 by using a neutralizing solution, desalting by using a Sephadex G25 column, and packaging and storing at-20 ℃, wherein the neutralizing solution is 1M Tris-HCl with pH of 8.5.
As an optimization scheme for this purpose,
the preparation method of the immunoaffinity chromatographic column comprises the following steps:
(1) coupling: placing the purchased finished medium-cyanogen bromide preactivated agarose gel in a sand core funnel, washing with a precooled coupling solution A at 0-4 ℃ for at least 30min;
coupling solution A was 1mM HCl;
(2) a ligand: placing outsourced anti-human hepatitis B surface antigen monoclonal antibody into a coupling solution B for washing, wherein the coupling concentration of the anti-human hepatitis B surface antigen monoclonal antibody is 5-10mg/mL of filler;
coupling solution B was 0.1M NaHCO3, 0.5M NaCl, pH8.3;
(3) diluting the medium washed in the step (1) by using a coupling solution A, mixing the diluted medium with the monoclonal antibody of the anti-human hepatitis B surface antigen washed in the step (2) in an equal volume, and uniformly mixing the mixture at the room temperature of 20-25 ℃ for 2h or overnight at the temperature of 4 ℃ to obtain gel;
(4) the gel was blocked and washed:
closing: pumping out the coupling supernatant and adding a blocking solution, wherein the blocking solution is 0.1M Tris-HCl, the pH value is 8.0, and the room temperature blocking is carried out for 2-4 hours;
cleaning: the immunoaffinity chromatography column is obtained by alternately washing with 0.1M Tris-HCl and 0.5M NaCl mixed solution with pH8-9, 0.1M acetate buffer solution with pH3-4 and 0.5M NaCl mixed solution for 3-6 times, washing with 5 times of medium volume liquid each time, and washing with PBS buffer solution.
As an optimization scheme for this purpose,
the subcloning operation method comprises the following steps: transferring the screened positive holes to a new 96-hole cell culture plate, subcloning by adopting a double-ratio dilution method, and after seven days, changing the culture medium into a DMEM complete culture medium containing 20v/v% FBS, and continuing to culture; after 8-12 days, detecting culture supernatant by using an indirect ELISA method, continuously screening and subcloning twice, selecting single positive holes of cell clusters, continuing subcloning, and simultaneously expanding culture and freezing the rest cells cloned each time.
As an optimization scheme for this purpose,
the method for screening the cells to be detected by using an indirect ELISA method comprises the following specific steps:
(1) coating: diluting the natural protein of the hepatitis B surface antigen into 1 mug/mL by using a coating liquid, coating 100 mug/hole into an ELISA plate, coating at 37 ℃ for 2 hours or 4 ℃ overnight, washing the plate for 5 times by using a PBST buffer solution, removing unbound antigen and impurities, and drying the ELISA plate by beating;
the coating liquid is carbonate buffer solution with pH of 9.6;
(2) closing: adding a PBST buffer solution containing 1w/v% BSA into each hole for sealing, incubating for 2 hours at 37 ℃ with 150 mu L/hole, washing the plate with the PBST buffer solution for 5 times, and drying the ELISA plate;
(3) adding cell supernatant to be detected: adding the cell supernatant to be detected into an ELISA plate, using mouse immune serum as positive control, using HAT culture medium as blank control, washing the plate with PBST buffer solution for 5 times in water bath at 37 ℃ for 30min, and drying the ELISA plate;
(4) Adding enzyme-labeled secondary antibodies: diluting the HRP-labeled goat anti-mouse antibody 1000 times by using PBS buffer solution, adding the diluted goat anti-mouse antibody into an ELISA plate, 50 mu L/hole, washing the plate for 5 times by using PBST buffer solution in water bath at 37 ℃ for 30min, and drying the ELISA plate by beating;
(5) color development: adding TMB color development liquid to develop color, and incubating for 10min at 37 ℃ with 100 mu L/hole; stopping with 2mol/L sulfuric acid, detecting 50 mu L/hole at 450nm wavelength of enzyme label instrument, and detecting sample OD value/negative control OD value not less than 2.1 to obtain positive result, thus completing one-time screening.
As an optimization scheme for this purpose,
the method for immunizing by using the natural protein of the human hepatitis B surface antigen as immunogen comprises the following steps: the first immunization dose is 80 mug/one, the second immunization is carried out after one week, the immunization dose is 40 mug/one, the third immunization is carried out after one week, and the immunization dose is 40 mug/one; mixing immunogen with Freund's complete adjuvant with equal volume, emulsifying thoroughly, and performing subcutaneous multipoint immunization on dorsal inguinal region; performing the fourth immunization after one week, wherein the immunization dose is 50 mug/dose, mixing with an equal volume of sterile physiological saline, and performing intraperitoneal injection immunization; after 5 days, mice were bled for tail-breaking.
The purification method of the anti-human hepatitis B surface antigen monoclonal antibody 1 prepared by the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain comprises the following steps:
(1) Preparation of ascites: selecting balb/c male mice of about 12 weeks of age, injecting 0.8mL liquid paraffin into each mouse, and injecting screened anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain 1 into the abdominal cavity after 5 days, wherein the number of the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strains is 10 6 Closely observing the state of the mice after injection, collecting ascites after the abdomen of the mice swells obviously, centrifuging for 10min at 8000rpm and 4 ℃, removing lipid by adsorption of silicon dioxide, filtering by qualitative filter paper, and collecting supernatant;
(2) Purification of anti-human hepatitis B surface antigen monoclonal antibody 1
Taking out the ascites from the refrigerator at the temperature of minus 20 ℃, centrifuging for 10min at 9000r/min after complete melting, filtering with a 0.45 mu M filter membrane, pretreating the ascites by an ammonium sulfate salting-out method, purifying by a blue gel affinity chromatography column, collecting the purified antibody, dialyzing with a 0.01M PBS buffer, and filtering with a 0.22 mu M filter to obtain the purified anti-human hepatitis B surface antigen monoclonal antibody 1.
The specific operation of the ammonium sulfate salting-out method purification is as follows:
a. taking 1 part by volume of ascites, adding 1 part by volume of saturated ammonium sulfate solution to ensure that the saturation of the ascites is 50 percent, stirring the ascites while fully mixing the ascites, centrifuging the ascites for 20 minutes at 9000r/min after stirring the ascites for 30 minutes, discarding the supernatant, and collecting the precipitate;
b. Adding 0.8 volume part of 20mM pH7.0 PB buffer solution into the collected precipitate, completely dissolving, loading into a dialysis bag, dialyzing with PB buffer solution at 4deg.C, dialyzing for 48 hr, centrifuging at 4000r/min for 20min, and filtering the supernatant with 0.22 μm filter to obtain primary pure antibody;
specific operation of blue gel affinity chromatography purification:
a. taking out the chromatographic column filled with the blue gel filler from the temperature of 4 ℃, balancing to room temperature, flushing the chromatographic column with a balancing buffer solution with the volume of 5 times of the column volume, and balancing the chromatographic column;
the equilibration buffer was 20mM PB, pH7.0;
b. adding the initially pure antibody into a chromatographic column, collecting effluent liquid, controlling the flow rate to be 1.0mL/min, repeatedly loading the sample for 2-3 times, adding an equilibrium buffer solution with the volume of 10 times of the column into the chromatographic column for flushing after the loading is finished, balancing the chromatographic column, and collecting the effluent liquid;
the equilibration buffer was 20mM PB, pH7.0;
c. respectively adding eluent 1, eluent 2, eluent 3 and eluent 4 with the volume of 10 times of the column volume into a chromatographic column, sequentially eluting, collecting all the eluents, detecting the purity of SDS-PAGE, and determining the eluent with the optimal purity of the protein;
eluent 1 was 20mM PB, 20mM NaCl, pH7.0;
eluent 2 was 20mM PB, 50mM NaCl, pH7.0;
eluent 3 was 20mM PB, 200mM NaCl, pH7.0;
Eluent 4 was 20mM PB, 1M NaCl, pH7.0;
d. adding buffer solution with the volume of 5 times of the column volume into the chromatographic column, flushing the chromatographic column, and then flushing and preserving the chromatographic column by using 20% ethanol solution with the volume of 5 times of the column volume;
the buffer was 20mM PB, 2M NaCl, pH7.0;
e. and c, loading the eluent with the optimal purity of the protein in the step c into a dialysis bag, concentrating by using PEG20000, putting the dialysis bag into PBS buffer solution for dialysis for 48 hours, centrifuging for 20 minutes at 4000r/min after the dialysis is finished, and filtering the supernatant by using a 0.22 mu m filter to obtain the purified monoclonal antibody 1 against the human hepatitis B surface antigen.
The purification method of the anti-human hepatitis B surface antigen monoclonal antibody 2 prepared by the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain comprises the following steps:
(1) Preparation of ascites: selecting balb/c male mice of about 12 weeks of age, injecting 0.8mL liquid paraffin into each mouse intraperitoneally, and injecting screened anti-human hepatitis B surface intraperitoneally after 5 daysAntigen monoclonal antibody hybridoma cell strain 2, number of which is 10 6 Closely observing the state of the mice after injection, collecting ascites after the abdomen of the mice swells obviously, centrifuging for 10min at 8000rpm and 4 ℃, removing lipid by adsorption of silicon dioxide, filtering by qualitative filter paper, and collecting supernatant;
(2) Purification of anti-human hepatitis B surface antigen monoclonal antibody 2
Taking out the ascites from the refrigerator at the temperature of minus 20 ℃, centrifuging for 10min at 9000r/min after complete melting, filtering by using a 0.45 mu M filter membrane, purifying by using a protein-G column through an affinity chromatography, purifying by using an ion exchange chromatography, collecting the purified antibody, dialyzing by using a 0.01M PBS buffer solution, and filtering by using a 0.22 mu M filter to obtain the purified anti-human hepatitis B surface antigen monoclonal antibody 2.
Specific procedures for purification by affinity chromatography of the protein-G:
taking 1 part by volume of ascites, adding 1 part by volume of balance buffer solution, fully and uniformly mixing, and filtering by using a microporous filter membrane with the thickness of 0.45 mu m;
taking out the chromatographic column filled with Protein-G filler from 4 ℃, balancing to room temperature, and flushing the balanced chromatographic column with 5 times of column volume of balanced buffer solution;
c. adding the filtered ascites into a chromatographic column, collecting effluent, controlling the flow rate to be 1.5mL/min, repeatedly loading the sample for 2-3 times, adding a balancing buffer solution with the volume of 10 times of the column into the chromatographic column after the loading is finished for washing impurities, balancing the chromatographic column, and collecting the effluent;
d. adding eluent with the volume of 10 times of the column volume into the chromatographic column, collecting the eluent, and regulating the eluent to be neutral by using a neutralization solution;
e. adding 5 times of column volume of balance buffer solution into the chromatographic column, flushing the balance chromatographic column, and then balancing and preserving the chromatographic column by using 20% ethanol solution with 5 times of column volume;
f. Loading the neutral eluate into a dialysis bag, placing the dialysis bag into dialysis buffer solution, dialyzing at 4deg.C for 48 hr, centrifuging at 4000r/min for 20min, and filtering the supernatant with 0.22 μm filter to obtain primary pure antibody;
wherein,,
balanced buffer 20mM Na 2 HPO 4 And 0.15M NaCl mixed solution,pH7.0；
the eluent is 0.1M glycine with pH 3.0;
the neutralization solution is 1M Tris-HCl, and the pH value is 8.5;
the dialysis buffer was 20mM Tris-HCl, pH8.0;
specific operation of purification by ion exchange chromatography:
a. taking out the chromatographic column filled with the Q-Bestarose HP filler from the temperature of 4 ℃, balancing to room temperature, flushing the chromatographic column with 5 times of the volume of the balancing buffer solution, and balancing the chromatographic column;
the equilibration buffer was 20mM Tris-HCl, 5mM MgCl 2 ，pH8.0；
b. Adding the initially pure antibody into a chromatographic column, collecting effluent liquid, controlling the flow rate to be 1.2mL/min, repeating the sample application for 3 times, adding an equilibrium buffer solution with the volume of 10 times of the column into the chromatographic column after the sample application is finished, washing the equilibrium chromatographic column, and collecting the effluent liquid;
the equilibration buffer was 20mM Tris-HCl, pH8.0;
c. respectively adding eluent 1, eluent 2 and eluent 3 with the volume of 10 times of the column volume into the chromatographic column, sequentially eluting, collecting all eluting proteins, detecting the purity of SDS-PAGE, and determining the eluent with the optimal purity of the proteins;
Eluent 1 is 20mM Tris-HCl, 50mM NaCl, pH8.0, eluent 2 is 20mM Tris-HCl, 200mM NaCl, pH8.0, eluent 3 is 20mM Tris-HCl, 1000mM NaCl,pH8.0;
d. adding buffer solution with the volume of 5 times of the column volume into the chromatographic column, flushing the regenerated chromatographic column, and then flushing and preserving the chromatographic column by using 20% ethanol solution with the volume of 5 times of the column volume;
the buffer solution is 20mM Tris-HCl, 2000mM NaCl,pH8.0;
e. and c, loading the eluent with the optimal purity of the protein collected in the step c into a dialysis bag, concentrating by using PEG20000, putting the dialysis bag into PBS buffer solution for dialysis for 48 hours, centrifuging for 20 minutes at 4000r/min after the dialysis is finished, and filtering the supernatant by using a 0.22 mu m filter to obtain the purified anti-human hepatitis B surface antigen monoclonal antibody 2.
The invention adopts the technical proposal and has the following advantages: the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain 1 and the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain 2 and passage cell strains thereof can respectively and stably secrete the anti-human hepatitis B surface antigen monoclonal antibody 1 and the anti-human hepatitis B surface antigen monoclonal antibody 2; the monoclonal antibody secreted by the hybridoma cell strain is subjected to saturated ammonium sulfate salt and blue gel affinity chromatography treatment or protein-G chromatography and ion exchange chromatography treatment respectively, so that impurities can be effectively removed, and the monoclonal antibody with strong specificity, high purity, high titer, good stability, good uniformity and strong repeatability is obtained; the monoclonal antibody obtained after purification is used for the production of detection reagents, and the sensitivity and the specificity of the reagents are greatly improved.
The invention is further described below with reference to the drawings and the detailed description.
Drawings
FIG. 1 shows the comparison of anti-human hepatitis B surface antigen monoclonal antibody 1 with control 1 activity;
FIG. 2 shows the comparison of anti-human hepatitis B surface antigen monoclonal antibody 2 activity with control 2;
FIGS. 3 and 4 show the comparison between the detection ranges of anti-human hepatitis B surface antigen monoclonal antibodies 1 and 2 and the control;
FIG. 5 shows the purity of anti-human hepatitis B surface antigen monoclonal antibody 1;
FIG. 6 shows the purity of anti-human hepatitis B surface antigen monoclonal antibody 2;
FIG. 7 shows the detection result of anti-human HBV surface antigen monoclonal antibody 1 secreted 20 times after passage of hybridoma cell line;
FIG. 8 shows the detection result of anti-human hepatitis B surface antigen monoclonal antibody 2 secreted by the hybridoma cell line for 20 times;
FIG. 9 shows the results of comparison of the test group (right) with the control group (left) of the anti-human HBV-antigen monoclonal antibody 1 and the anti-human HBV-antigen monoclonal antibody 2;
FIG. 10 shows the results of 6 negative serum samples;
fig. 11 shows the detection results of positive and negative samples of different intensities.
Detailed Description
Embodiments of the present invention will be described in detail with reference to examples, in which specific conditions are not noted, according to conventional conditions or manufacturer's recommended conditions. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The described embodiments are intended to be illustrative of only some, but not all embodiments of the invention, and all other embodiments that may be made by one of ordinary skill in the art without inventive faculty are intended to be within the scope of the invention.
Example 1
The preparation method of the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain comprises the following steps:
1. the preparation method of the natural protein of the human hepatitis B surface antigen comprises the following steps:
(1) Low concentration PEG precipitation: serum from asymptomatic human hepatitis B surface antigen carrier was collected, large particles and lipoproteins were removed by precipitation with PEG6000 at a final concentration of 5%, and the supernatant was removed by centrifugation at 4000rpm for 2h at 4℃for 30 minutes, after which the precipitate was discarded.
This step allows selective removal of intact virus (HBV or other viruses or other viral and immune complexes) and removal of most lipoproteins.
(2) High concentration PEG precipitation: precipitating the supernatant with PEG6000 with final concentration of 10%, standing at 4deg.C for 2 hr, centrifuging at 4000rpm for 30min, discarding the precipitate, and collecting supernatant to obtain primary pure natural protein of human hepatitis B surface antigen.
This step may precipitate and remove albumin, transferrin, and some other proteins from the supernatant.
(3) The preparation method of the immunoaffinity chromatographic column comprises the following steps:
(1) coupling: the outsourced finished medium, namely the cyanogen bromide preactivated agarose gel, is taken and placed in a sand core funnel, and is washed by precooled coupling solution A at 0-4 ℃ for at least 30min.
Coupling solution A was 1mM HCl.
(2) A ligand: and (3) placing the outsourced anti-human hepatitis B surface antigen monoclonal antibody into a coupling solution B for washing, wherein the coupling solution B has high salt concentration, can prevent the formation of a polymer, and has the coupling concentration of 5-10mg/mL of filler.
Coupling solution B was 0.1M NaHCO3, 0.5M NaCl, pH8.3;
(3) diluting the medium washed in the step (1) with a coupling solution A (1 mL of the medium is about 0.5mL of the coupling solution A), and mixing the diluted medium with the monoclonal antibody of the anti-human hepatitis B surface antigen washed in the step (2) in an equal volume, and uniformly mixing the mixture at the room temperature of 20-25 ℃ for 2h or overnight at the temperature of 4 ℃ to obtain gel.
(4) The gel was blocked and washed:
closing: pumping out the coupling supernatant and adding a blocking solution, wherein the blocking solution is 0.1M Tris-HCl, the pH value is 8.0, and the room temperature blocking is carried out for 2-4 hours;
cleaning: the immunoaffinity chromatographic column is obtained by washing with 0.1M Tris-HCl and 0.5M NaCl mixed solution with pH8-9, 0.1M acetate buffer solution with pH3-4 and 0.5M NaCl mixed solution alternately for 3-6 times, 5 times of medium volume of liquid for each time, and PBS buffer solution, and can be used or stored in 20% ethanol, and the immunoaffinity chromatographic column can be repeatedly used.
(4) The affinity chromatography purification of the human hepatitis B surface antigen comprises the following steps:
and (3) connecting the immunoaffinity chromatographic column prepared in the step (3) to a computer ultraviolet detector, wherein the immunoaffinity chromatographic column is used for monitoring the real-time change of a sample OD280 in the purification process when the immunoaffinity chromatographic column is used for purifying, and collecting the purified effective sample according to the OD280 monitoring result.
(1) Balance: the immunoaffinity column was washed with binding solution, which was 10mM PBS, 150mM NaCl, until the first peak detected by the computer UV detector OD280 returned to baseline, i.e., equilibrium was reached.
(2) Loading: and (3) collecting effluent liquid of the immunoaffinity chromatographic column obtained by balancing the natural protein of the primary pure human hepatitis B surface antigen obtained in the step (2) on a computer ultraviolet detector OD280 until the baseline of detection is stable.
(3) Eluting: adding eluent which is glycine-hydrochloric acid solution with pH of 3.0, collecting when the baseline detected by the computer ultraviolet detector OD280 rises, regulating the pH of the collected eluent to 7.4 by using a neutralizing solution, desalting by using a Sephadex G25 column, and packaging and storing at-20 ℃, wherein the neutralizing solution is 1M Tris-HCl with pH of 8.5.
2. The preparation method of the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain 1 and the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain 2 comprises the following steps:
(1) Immunization of animals
Taking 3 male BALB/c mice of 6 weeks old, immunizing with the natural protein of the human hepatitis B surface antigen obtained in the step 1 as immunogen, wherein the first immunization dose is 80 mug/mouse, the second immunization is carried out after one week, the immunization dose is 40 mug/mouse, the third immunization is carried out after one week, and the immunization dose is 40 mug/mouse; mixing immunogen with Freund's complete adjuvant with equal volume, emulsifying thoroughly, and performing subcutaneous multipoint immunization on dorsal inguinal region; performing the fourth immunization after one week, wherein the immunization dose is 50 mug/dose, mixing with an equal volume of sterile physiological saline, and performing intraperitoneal injection immunization; after 5 days, the mice were subjected to tail-breaking blood sampling, serum titers were detected to be more than 10 ten thousand, and cell fusion was performed.
(2) Cell fusion
The method comprises the steps of killing mice, immersing the mice in 75% alcohol for 5min, picking spleens of immunized mice in a sterile environment, grinding dispersed cells, washing and centrifuging to obtain B lymphocytes, mixing and centrifuging the B lymphocytes and myeloma cells SP2/0 in a logarithmic growth phase and in a good growth state according to the ratio of 5:1 of cell number, washing the cells with serum-free DMEM culture medium to ensure no serum in the mixed cells, centrifuging and discarding supernatant, slowly adding 1mL of 50% PEG solution with the mass concentration within 50s in a water bath at 37 ℃, dropwise adding and mixing, slowly adding 40mL of serum-free DMEM culture medium preheated at 37 ℃ after the reaction of the supernatant for 90s, stopping fusion after the slow adding speed, ensuring complete dilution of PEG, centrifuging at 1000rpm for 10min, discarding supernatant, re-suspending cell sediment with the HAT culture medium preheated at 37 ℃, fully mixing uniformly, placing the cell sediment in a 96-hole cell culture plate, and placing the cell culture plate into a culture box with 5% carbon dioxide for culturing.
(3) Hybridoma cell screening and subcloning
And replacing 1/2 of culture solution in the 96-well cell culture plate with HAT culture medium on the third day and the eighth day after fusion, and sucking the cell supernatant on the tenth day, namely the cell supernatant to be detected.
The method for screening the cells to be detected by using an indirect ELISA method comprises the following specific steps:
(1) coating: diluting the natural protein of the hepatitis B surface antigen into 1 mug/mL by using a coating liquid, coating 100 mug/hole into an ELISA plate, coating at 37 ℃ for 2 hours or 4 ℃ overnight, washing the plate for 5 times by using a PBST buffer solution, removing unbound antigen and impurities, and drying the ELISA plate by beating;
the coating solution was a carbonate buffer at pH 9.6.
(2) Closing: each well was blocked by adding 1w/v% BSA in PBST buffer, 150. Mu.L/well, incubated at 37℃for 2h, plates were washed 5 times with PBST buffer, and plates were dried.
(3) Adding cell supernatant to be detected: the cell supernatant to be detected is added into an ELISA plate, 100 mu L/hole is added, meanwhile, mouse immune serum is used as positive control, HAT culture medium is used as blank control, water bath is carried out for 30min at 37 ℃, PBST buffer solution is used for washing the plate for 5 times, and the ELISA plate is dried by beating.
(4) Adding enzyme-labeled secondary antibodies: HRP-labeled goat anti-mouse antibody was diluted 1000-fold with PBS buffer, added to the elisa plate, 50 μl/well, and water-bath at 37 ℃ for 30min. The plates were washed 5 times with PBST buffer and the ELISA plates were dried.
(5) Color development: adding TMB color development liquid to develop color, and incubating for 10min at 37 ℃ with 100 mu L/hole; stopping with 2mol/L sulfuric acid, detecting 50 mu L/hole at 450nm wavelength of enzyme label instrument, and detecting sample OD value/negative control OD value not less than 2.1 to obtain positive result, thus completing one-time screening.
After the first screening, the HAT culture medium is used for completely changing culture solution, indirect ELISA screening is carried out every other day, screening is repeatedly carried out for three times, the cells with strong positive results (OD 450 is stronger than that of positive control) are selected for three times for subcloning, the screened positive holes are transferred to a new 96-hole cell culture plate, subcloning is carried out by adopting a multiple dilution method, and after seven days, the culture medium is changed into a DMEM complete culture medium containing 20v/v% FBS, and the culture is continued; detecting culture supernatant by indirect ELISA method after 8-12 days, continuously screening and subcloning twice, selecting single positive hole of cell mass, continuing subcloning, and simultaneously enlarging culture and freezing the rest cells cloned each time; the subcloning is carried out for 2-3 times until the positive rate of the 96-well plate reaches 100%, and two anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strains, namely an anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain 1 and an anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain 2, are obtained after the subcloning is finished.
(4) Preservation of anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain
Performing expansion culture on the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain obtained in the step (3), collecting cells when the cell growth state is good and the cell density reaches 80%, freezing the cells by using a cell freezing solution, placing the cells in a liquid nitrogen gas state for 1h according to the gradient freezing method of the chamber, and preserving the liquid nitrogen in a liquid nitrogen liquid state for a long time.
The cell cryopreservation solution consisted of 10v/v% DMSO, 30% DMEM high glucose incomplete broth and 60v/v% FBS.
(5) Cell subtype detection
The mouse monoclonal antibody subtype (IgG 1, igG2a, igG2b, igG3, igA and IgM) of two anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strains are detected by using the mouse monoclonal antibody subtype rapid detection kit, the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain 1 is the IgA subtype, and the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain 2 is the IgG1 subtype.
The anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain 1 and the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain 2 are respectively preserved in China center for type culture collection, and the preservation number of the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain 1 is CCTCC NO: c202361, the preservation code of the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain 2 is CCTCC NO: C202362.
example 2
The purification method of the anti-human hepatitis B surface antigen monoclonal antibody 1 comprises the following steps:
(1) Preparation of ascites: selecting balb/c male mice of about 12 weeks of age, injecting 0.8mL liquid paraffin into each mouse, and injecting screened anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain 1 into the abdominal cavity after 5 days, wherein the number of the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strains is 10 6 After injection, closely observing the state of the mice, collecting ascites after the abdomen of the mice swells obviously, centrifuging for 10min at 8000rpm and 4 ℃, removing lipid by adsorption of silicon dioxide, filtering by qualitative filter paper, collecting supernatant, detecting antibody titer in the ascites by ELISA method, and storing in a refrigerator at-20 ℃ for standby after passing.
(2) Purification of anti-human hepatitis B surface antigen monoclonal antibody 1
Taking out the ascites from the refrigerator at the temperature of minus 20 ℃, centrifuging for 10min at 9000r/min after complete melting, filtering with a 0.45 mu M filter membrane, pretreating the ascites by an ammonium sulfate salting-out method, purifying by a blue gel affinity chromatography column, collecting the purified antibody, dialyzing with a 0.01M PBS buffer solution, filtering with a 0.22 mu M filter, and then obtaining the purified monoclonal antibody 1 of the anti-human hepatitis B surface antigen, packaging and storing in the refrigerator at the temperature of minus 20 ℃.
Specific operation of purification by ammonium sulfate salting-out method:
a. taking 1 part by volume of ascites, adding 1 part by volume of saturated ammonium sulfate solution to ensure that the saturation degree is 50 percent, stirring the solution while fully and uniformly mixing the solution, stirring the solution for 30min, centrifuging the solution for 20min at 9000r/min, discarding the supernatant, and collecting the precipitate.
b. Adding 0.8 volume part of 20mM pH7.0 PB buffer solution into the collected precipitate, completely dissolving, loading into a dialysis bag, dialyzing with PB buffer solution at 4deg.C, dialyzing for 48 hr, centrifuging at 4000r/min for 20min, and filtering the supernatant with 0.22 μm filter to obtain primary pure antibody.
Specific operation of blue gel affinity chromatography purification:
a. taking out the chromatographic column filled with the blue gel filler from the temperature of 4 ℃, balancing to room temperature, flushing the chromatographic column with a balancing buffer solution with the volume of 5 times of the column volume, and balancing the chromatographic column;
the equilibration buffer was 20mM PB, pH7.0;
b. adding the initially pure antibody into a chromatographic column, collecting effluent liquid, controlling the flow rate to be 1.0mL/min, repeatedly loading the sample for 2-3 times, adding an equilibrium buffer solution with the volume of 10 times of the column into the chromatographic column for flushing after the loading is finished, balancing the chromatographic column, and collecting the effluent liquid;
the equilibration buffer was 20mM PB, pH7.0;
c. respectively adding eluent 1, eluent 2, eluent 3 and eluent 4 with the volume of 10 times of the column volume into a chromatographic column, sequentially eluting, collecting all the eluents, detecting the purity of SDS-PAGE, and determining the eluent with the optimal purity of the protein;
eluent 1 was 20mM PB, 20mM NaCl, pH7.0;
eluent 2 was 20mM PB, 50mM NaCl, pH7.0;
eluent 3 was 20mM PB, 200mM NaCl, pH7.0;
eluent 4 was 20mM PB, 1M NaCl, pH7.0;
d. adding buffer solution with the volume of 5 times of the column volume into the chromatographic column, flushing the chromatographic column, and then flushing and preserving the chromatographic column by using 20% ethanol solution with the volume of 5 times of the column volume;
the buffer was 20mM PB, 2M NaCl, pH7.0.
e. And c, loading the eluent with the optimal purity of the protein in the step c into a dialysis bag, concentrating by using PEG20000, putting the dialysis bag into PBS buffer solution for dialysis for 48 hours, centrifuging for 20 minutes at 4000r/min after the dialysis is finished, filtering the supernatant by using a 0.22 mu m filter to obtain the purified monoclonal antibody 1 against the human hepatitis B surface antigen, subpackaging, and storing in a refrigerator at the temperature of minus 20 ℃.
Example 3
The purification method of the anti-human hepatitis B surface antigen monoclonal antibody 2 comprises the following steps:
(1) Preparation of ascites: selecting balb/c male mice of about 12 weeks of age, injecting 0.8mL liquid paraffin into each mouse, and injecting screened anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain 2 into the abdominal cavity after 5 days, wherein the number of the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strains is 10 6 Closely observing the state of mice after injection, collecting ascites after the abdominal distention is obvious, centrifuging at 8000rpm for 10min at 4 ℃, removing lipid by adsorption of silicon dioxide, filtering with qualitative filter paper, collecting supernatant, detecting antibody titer in ascites by ELISA method, and preserving in a refrigerator at-20deg.C after passing the standardAnd storing for standby.
(2) Purification of anti-human hepatitis B surface antigen monoclonal antibody 2
Taking out ascites from a refrigerator at-20 ℃, centrifuging for 10min at 9000r/min after complete thawing, filtering with a 0.45 mu M filter membrane, purifying by affinity chromatography through a protein-G column, purifying by ion exchange chromatography, collecting purified antibodies, dialyzing with a 0.01M PBS buffer, filtering with a 0.22 mu M filter, and then obtaining the purified anti-human hepatitis B surface antigen monoclonal antibody 2, split charging and storing in the refrigerator at-20 ℃.
Specific procedure for protein-G affinity chromatography purification:
taking 1 part by volume of ascites, adding 1 part by volume of balance buffer solution, fully and uniformly mixing, and filtering by using a microporous filter membrane with the thickness of 0.45 mu m;
taking out the chromatographic column filled with Protein-G filler from 4 ℃, balancing to room temperature, and flushing the balanced chromatographic column with 5 times of column volume of balanced buffer solution;
c. adding the filtered ascites into a chromatographic column, collecting effluent, controlling the flow rate to be 1.5mL/min, repeatedly loading the sample for 2-3 times, adding a balancing buffer solution with the volume of 10 times of the column into the chromatographic column after the loading is finished for washing impurities, balancing the chromatographic column, and collecting the effluent;
d. adding eluent with the volume of 10 times of the column volume into the chromatographic column, collecting the eluent, and regulating the eluent to be neutral by using a neutralization solution;
e. adding 5 times of column volume of balance buffer solution into the chromatographic column, flushing the balance chromatographic column, and then balancing and preserving the chromatographic column by using 20% ethanol solution with 5 times of column volume.
f. Loading the neutral eluate into dialysis bag, placing the dialysis bag into dialysis buffer solution, dialyzing at 4deg.C for 48 hr, centrifuging at 4000r/min for 20min, and filtering the supernatant with 0.22 μm filter to obtain primary pure antibody.
Wherein,,
balanced buffer 20mM Na 2 HPO 4 And 0.15M NaCl mixed solution, pH7.0;
The eluent is 0.1M glycine with pH 3.0;
the neutralization solution is 1M Tris-HCl, and the pH value is 8.5;
the dialysis buffer was 20mM Tris-HCl, pH8.0.
Specific operation of purification by ion exchange chromatography:
a. taking out the chromatographic column filled with the Q-Bestarose HP filler from the temperature of 4 ℃, balancing to room temperature, flushing the chromatographic column with 5 times of the volume of the balancing buffer solution, and balancing the chromatographic column;
the equilibration buffer was 20mM Tris-HCl, 5mM MgCl 2 ，pH8.0；
b. Adding the initially pure antibody into a chromatographic column, collecting effluent liquid, controlling the flow rate to be 1.2mL/min, repeating the sample application for 3 times, adding an equilibrium buffer solution with the volume of 10 times of the column into the chromatographic column after the sample application is finished, washing the equilibrium chromatographic column, and collecting the effluent liquid;
the equilibration buffer was 20mM Tris-HCl, pH8.0;
c. respectively adding eluent 1, eluent 2 and eluent 3 with the volume of 10 times of the column volume into the chromatographic column, sequentially eluting, collecting all eluting proteins, detecting the purity of SDS-PAGE, and determining the eluent with the optimal purity of the proteins;
eluent 1 is 20mM Tris-HCl, 50mM NaCl, pH8.0, eluent 2 is 20mM Tris-HCl, 200mM NaCl, pH8.0, eluent 3 is 20mM Tris-HCl, 1000mM NaCl,pH8.0;
d. adding buffer solution with the volume of 5 times of the column volume into the chromatographic column, flushing the regenerated chromatographic column, and then flushing and preserving the chromatographic column by using 20% ethanol solution with the volume of 5 times of the column volume;
The buffer solution is 20mM Tris-HCl, 2000mM NaCl,pH8.0;
e. loading the eluent with the optimal purity of the protein collected in the step c into a dialysis bag, concentrating by using PEG20000, putting the dialysis bag into PBS buffer solution for dialysis for 48 hours, centrifuging for 20 minutes at 4000r/min after the dialysis is finished, filtering the supernatant by using a 0.22 mu m filter to obtain the purified anti-human hepatitis B surface antigen monoclonal antibody 2, sub-packaging, and storing in a refrigerator at the temperature of minus 20 ℃.
Example 4
Monoclonal antibody identification
1. Detection of activity of anti-human hepatitis B surface antigen monoclonal antibody 1 and anti-human hepatitis B surface antigen monoclonal antibody 2
The activity of the anti-human hepatitis B surface antigen monoclonal antibody 1 and the anti-human hepatitis B surface antigen monoclonal antibody 2 prepared by the invention is detected by adopting an indirect ELISA method, and the specific detection method is as follows:
(1) Coating: the anti-human hepatitis B surface antigen monoclonal antibody 1 and the anti-human hepatitis B surface antigen monoclonal antibody 2 prepared by the invention are respectively diluted into 1 mug/mL by coating liquid, 100 mug/hole is coated into an ELISA plate, the coating is carried out at 4 ℃ overnight, the plate is washed 5 times by PBST buffer solution, unbound antigen and impurities are removed, and the ELISA plate is patted dry;
the coating liquid is a carbonate buffer solution with the pH value of 9.6, and the formula is as follows: sodium carbonate 0.85g, sodium bicarbonate 1.4g, constant volume to 500mL.
(2) Closing: each well was blocked by adding 1w/v% BSA in PBST buffer, 150. Mu.L/well, incubated at 37℃for 2h, plates were washed 5 times with PBST buffer, and plates were dried.
(3) Adding a detection object: the anti-human hepatitis B surface antigen monoclonal antibody 1 prepared by the invention, the outsourced anti-human hepatitis B surface antigen monoclonal antibody 1 (short for comparison 1), the anti-human hepatitis B surface antigen monoclonal antibody 2 prepared by the invention and the outsourced anti-human hepatitis B surface antigen monoclonal antibody 2 (short for comparison 2) are respectively added into an ELISA plate, 100 mu L/hole is washed for 30min in a water bath at 37 ℃, and the ELISA plate is dried by beating with PBST buffer solution for 5 times.
(4) Adding enzyme-labeled secondary antibodies: HRP-labeled goat anti-mouse IgG was diluted 5000-fold with PBS buffer, added to the ELISA plate, 50. Mu.L/well, washed 5 times with PBST buffer in a water bath at 37℃for 30min, and the ELISA plate was dried.
(5) Color development: adding TMB color development liquid to develop color, and incubating for 10min at 37 ℃ with 100 mu L/hole; the reaction was stopped with 2M sulfuric acid stop solution, 50. Mu.L/well, and the activity was measured at a wavelength of 450 nm.
The detection results are shown in table 1, fig. 1 and fig. 2:
TABLE 1
The anti-human hepatitis B surface antigen monoclonal antibody 1 and the anti-human hepatitis B surface antigen monoclonal antibody 2 prepared by the invention can be found to have higher titers than the control 1 and the control 2 through the table 1, which shows that the anti-human hepatitis B surface antigen monoclonal antibody 1 and the anti-human hepatitis B surface antigen monoclonal antibody 2 prepared by the invention have good reactivity with the natural human hepatitis B surface antigen, high titers and are superior to outsourcing antibodies.
2. Anti-human hepatitis B surface antigen monoclonal antibody 1, and comparison of detection range and sensitivity of anti-human hepatitis B surface antigen monoclonal antibody 2
The ELISA sandwich method is adopted to detect the detection ranges of the anti-human hepatitis B surface antigen monoclonal antibody 1 and the anti-human hepatitis B surface antigen monoclonal antibody 2, and the specific detection method is as follows:
(1) Coating: the anti-human hepatitis B surface antigen monoclonal antibody 1 and the outsourcing anti-human hepatitis B surface antigen monoclonal antibody 1 (abbreviated as control 1) prepared by the invention are respectively diluted into 1 mug/mL by coating liquid, are coated into an ELISA plate according to 100 mug/hole, are coated overnight at 4 ℃, are washed for 5 times by using PBST buffer solution, remove unbound antigen and impurities, and are patted dry;
the coating liquid is a carbonate buffer solution with the pH value of 9.6, and the formula is as follows: sodium carbonate 0.85g, sodium bicarbonate 1.4g, constant volume to 500mL.
(2) Closing: each well was blocked by adding 1w/v% BSA in PBST buffer, 150. Mu.L/well, incubated at 37℃for 2h, plates were washed 5 times with PBST buffer, and plates were dried.
(3) Adding natural antigen on the surface of human hepatitis B: diluting the natural antigen on the surface of human hepatitis B to a certain concentration, adding the diluted natural antigen into a corresponding hole of an ELISA plate, washing the plate with PBST buffer solution for 5 times in a water bath at 37 ℃ for 30min at 100 mu L/hole, and drying the ELISA plate.
(4) Adding enzyme-labeled antibody: the HRP is used for marking the anti-human hepatitis B surface antigen monoclonal antibody 2 and outsourcing anti-human hepatitis B surface antigen monoclonal antibody 2 (short for reference 2) prepared by the invention by PBS buffer solution, respectively diluting 5000 times, adding into an ELISA plate, 50 mu L/hole, washing the plate for 30min by PBST buffer solution for 5 times in a water bath at 37 ℃, and drying the ELISA plate by beating.
(5) Color development: adding TMB color development liquid to develop color, and incubating for 10min at 37 ℃ with 100 mu L/hole; the reaction was stopped with 2M sulfuric acid stop solution, 50. Mu.L/well, and the reaction was detected at a wavelength of 450 nm.
The detection results are shown in fig. 3 and 4, and the sensitivity and linearity of the monoclonal antibody against the human hepatitis B surface antigen prepared by the invention are superior to those of a control, which indicates that the purified antibody has high titer, good specificity and wide detection range; the detection range is consistent with the control, and the sensitivity is better than the control.
After repeated experiments, the results prove that: the detection range of the coated anti-human hepatitis B surface antigen monoclonal antibody 1 and the marked anti-human hepatitis B surface antigen monoclonal antibody 2 is better than that of the marked anti-human hepatitis B surface antigen monoclonal antibody 1 and the coated anti-human hepatitis B surface antigen monoclonal antibody 2.
3. Detection of purity of anti-human hepatitis B surface antigen monoclonal antibody
10 mug of anti-human hepatitis B surface antigen monoclonal antibody 1 and anti-human hepatitis B surface antigen monoclonal antibody 2 are respectively taken, and an SDS-PAGE method is adopted, so that the results are shown in fig. 5 and 6, wherein the molecular weight markers of the proteins are as follows from top to bottom: 116KD, 66.4KD, 45KD, 35KD, 25KD, 18.4KD, 14.4KD.
FIGS. 5 and 6 show that the purity of the anti-human hepatitis B surface antigen monoclonal antibody 1 is not less than 95% and the purity of the anti-human hepatitis B surface antigen monoclonal antibody 2 is not less than 95%.
4. Antibody secretion stability detection of anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain
Subculturing the screened anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strains, collecting culture supernatants at 1-20 generations respectively, detecting by ELISA method, and analyzing the stability of cell secretion anti-human hepatitis B surface antigen monoclonal antibody, wherein the specific detection method is as follows:
(1) Coating: the natural antigen on the surface of the human hepatitis B is diluted to 1 mug/mL by using coating liquid, 100 mug/hole is coated into an ELISA plate, the coating is carried out at 4 ℃ for over night, the plate is washed by using PBST buffer solution for 5 times, unbound antigen and impurities are removed, and the ELISA plate is dried by beating.
(2) Closing: each well was blocked by adding 1% BSA in PBST buffer, 150. Mu.L/well, incubated at 37℃for 2h, plates were washed 5 times with PBST buffer, and plates were dried.
(3) And (3) detection: the cell supernatant to be detected was added to the ELISA plate, 100. Mu.L/well, water-bath was performed at 37℃for 30min, the plate was washed 5 times with PBST buffer, and the ELISA plate was dried by pipetting.
(4) Adding enzyme-labeled secondary antibodies: HRP-labeled goat anti-mouse antibody was 2000-fold diluted with PBS buffer and added to the elisa plate at 50 μl/well in a 37 ℃ water bath for 30min. The plates were washed 5 times with PBST buffer (1000 mLPBS and 0.2mL Tween 20 configuration) and the ELISA plates were blotted dry.
(5) Color development: adding TMB color development liquid to develop color, and incubating for 10min at 37 ℃ with 100 mu L/hole; stopping with 2M sulfuric acid stopping solution, 50 μl/well, and detecting at 450nm wavelength; the detection results are shown in fig. 7 and 8.
As can be seen from fig. 7 and 8: repeated experiments prove that the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain is passaged for 20 times to secrete the anti-human hepatitis B surface antigen monoclonal antibody, the titer of the anti-human hepatitis B surface antigen monoclonal antibody is basically unchanged, the consistency is higher, and the stability of the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain is better.
Example 5
Application of monoclonal antibody against human hepatitis B surface antigen
1. Detection of anti-human hepatitis B surface antigen monoclonal antibody 1, 2 titer
The potency of the purified anti-human hepatitis B surface antigen monoclonal antibody is detected by adopting a colloidal gold chromatography method: two groups of experiments are set, the left side is a control group, and the coating and the marking are outsourcing anti-human hepatitis B surface antigen monoclonal antibodies; the right side is coated with the anti-human hepatitis B surface antigen monoclonal antibody 1 and labeled with the anti-human hepatitis B surface antigen monoclonal antibody 2, and the specific detection method is as follows:
(1) Preparation of colloidal gold-anti-human hepatitis B surface antigen monoclonal antibody 2 conjugate: dissolving chloroauric acid in ultrapure water to obtain final concentration of 0.01wt%, adding 1.5mL of 1wt% trisodium citrate aqueous solution per 100mL after boiling, continuously boiling for 15min, cooling, and adding 0.2 mol/L K 2 CO 3 Adjusting pH to 8.2, adding purified anti-human hepatitis B surface antigen monoclonal antibody 2mg under rapid stirring, stirring for 15min, adding bovine serum albumin 240mg, stirring for 5min, and adding 10wt% NaCl aqueous solutionMaking the final concentration of NaCl be 1wt%, uniformly mixing, centrifuging for 10min at 2000r/min, removing precipitate, centrifuging supernatant at 10000rpm for 60min, absorbing supernatant, and dissolving precipitate which is the primary purified colloidal gold-anti-human hepatitis B surface antigen monoclonal antibody conjugate in storage liquor at-20deg.C, storing the storage liquor at 0.02 mol/L, pH7.4 Tris-HCl, polyethylene glycol with molecular weight 20000 of 40mg/100mL, 50v/v% glycerol and 0.02w/v% NaN 3 。
(2) Preparation of immunochromatography test strips: the test strip consists of three parts, namely water-absorbing fiber, nitrocellulose membrane and water-absorbing filter paper. The water-absorbing fiber is partially spotted with a colloidal gold-anti-human hepatitis B surface antigen monoclonal antibody 2 conjugate; the nitrocellulose membrane was coated with 100. Mu.g/mL of anti-human hepatitis B surface antigen monoclonal antibody 1 (detection line) and goat anti-mouse IgG (control line) diluted with 0.02 mol/L of PBS at pH7.4, and the membrane was air-dried and blocked with PBS containing 10v/v% of neonatal bovine serum. Then sequentially sticking on white plastic sheet (support), cutting into strips of 0.8 cm ×8 cm, adding desiccant, and sealing for storage.
(3) Detection of human hepatitis B surface antigen: the end of the test strip with the gold-labeled antibody is immersed in serum, and the serum level does not exceed the mark line. And (3) result judgment: taking two red lines on a cellulose membrane of a test strip as positive, and prompting that serum contains human hepatitis B surface antigen; only one red line (near the end of the bibulous filter paper, control line) appears as negative. Reagent failure is considered if no red line appears. The result determination time: after about 3 seconds, the sample was removed, left to stand, observed for 3-5 minutes, and not effective after 10 minutes.
As shown in FIG. 9, each two test strips correspond to the same test sample, and the test groups (right) of the anti-human hepatitis B surface antigen monoclonal antibody 1 and the anti-human hepatitis B surface antigen monoclonal antibody 2 have slightly higher titers than the control groups (left), so that the test samples meet the requirements.
Anti-human hepatitis B surface antigen monoclonal antibody 1 and detection of specificity of anti-human hepatitis B surface antigen monoclonal antibody 2
6 negative serum samples were tested as shown in FIG. 10.
Positive and negative samples of different intensities were tested as shown in fig. 11.
The specific detection method comprises the following steps:
(1) Preparation of colloidal gold-anti-human hepatitis B surface antigen monoclonal antibody 2 conjugate: dissolving chloroauric acid with ultrapure water to make the final concentration of chloroauric acid be 0.01wt%, adding 1wt% trisodium citrate aqueous solution 1.5 mL every 100. 100 mL after boiling, continuously boiling for 15 minutes, adjusting the pH to 8.2 with 0.2 mol/L K CO3 after cooling, adding purified anti-human hepatitis B surface antigen monoclonal antibody 2-2 mg under rapid stirring, continuously stirring for 15 minutes, adding bovine serum albumin 240 mg, stirring for 5 minutes, and finally adding 10wt% NaCl aqueous solution to make the final concentration of NaCl in the system be 1wt%, and uniformly mixing. Centrifuging at 2000 r/min for 10 min, discarding the precipitate, centrifuging the supernatant at 10000 r/min for 60 min, and sucking the supernatant, wherein the precipitate is the primary purified colloidal gold-anti-human hepatitis B surface antigen monoclonal antibody conjugate. Dissolving in storage solution (0.02 mol/L, pH 7.4 Tris-HCl, polyethylene glycol with molecular weight of 20000 of 40 mg/100 mL, 50v/v% glycerol, 0.02wt% NaN 3), and storing at 20deg.C.
(2) Preparation of immunochromatography test strips: the test strip consists of three parts, namely water-absorbing fiber, nitrocellulose membrane and water-absorbing filter paper. The water-absorbing fiber part is coated with gold-anti-human hepatitis B surface antigen monoclonal antibody conjugate; the nitrocellulose membrane was coated with 100. Mu.g/mL of anti-human hepatitis B surface antigen monoclonal antibody 1 (detection line) and goat anti-mouse IgG (control line) diluted with 0.02 mol/L of PBS at pH7.4, and the membrane was air-dried and blocked with PBS containing 10% of newborn calf serum. Then sequentially sticking on white plastic sheet (support), cutting into strips of 0.8 cm ×8 cm, adding desiccant, and sealing for storage.
(3) Human hepatitis B surface antigen detection: the end of the test strip with gold-labeled antibody (i.e., MAX end) is immersed in serum, and the serum level does not exceed the marker line. And (3) result judgment: taking two red lines on a cellulose membrane of a test strip as positive, and prompting that serum contains human hepatitis B surface antigen; only one red line (near the end of the bibulous filter paper, control line) appears as negative. Reagent failure is considered if no red line appears. The result determination time: after about 3 seconds, the sample was removed, left to stand, observed for 3-5 minutes, and not effective after 10 minutes.
6 negative serum samples were tested, as shown in FIG. 10, and the test results: 6 negative serum samples are detected, the film surface is clean, and the requirements are met.
3 cases of strong cations, 3 cases of cations, 1 case of weak cations, 3 cases of negative samples were detected, and the results are shown in fig. 11: the positive gradient is obvious, the negative is free from background, no false positive reaction is caused, no nonspecific adsorption is caused, and the result is accurate.
3. The invention relates to the comparison of the accuracy of the clinical serum detection result of the anti-human hepatitis B surface antigen monoclonal antibody
The pair of anti-human hepatitis B surface antigen monoclonal antibodies of the invention is applied to the comparison of a colloidal gold chromatography method and an outsourcing pair of anti-human hepatitis B surface antigen monoclonal antibodies, and the positive verification result is shown in the following table 2:
TABLE 2
The specificity verification results are shown in table 3 below:
TABLE 3 Table 3
The result shows that the monoclonal antibody of the anti-human hepatitis B surface antigen is applied to the detection of the colloidal gold chromatography, the detection rate is consistent with that of the monoclonal antibody of the anti-human hepatitis B surface antigen purchased externally, and the specificity is superior to that of the monoclonal antibody purchased externally.
In order to better prove that the recovery rate of the antibody can be improved by adopting the operation method of the invention when the anti-human hepatitis B surface antigen monoclonal antibody is purified, the following 3 comparative examples are provided.
Comparative example 1
The difference from example 2 is that: the specific procedure for the purification by blue gel affinity chromatography was 50mM PB, pH7.0 for the equilibration buffer and the specific results are shown in Table 4.
TABLE 4 Table 4
From table 4, it can be found that: when 20mM PB pH7.0 is used as an equilibrium buffer solution, the binding effect of the sample and the affinity column is best, and the recovery rate of the antibody is greatly improved.
Comparative example 2
The difference from example 2 is that: the equilibration buffer was 100mM PB, pH7.0 during the specific procedure for blue gel affinity chromatography purification, and the specific results are shown in Table 5.
TABLE 5
As can be seen from Table 5, when 20mM PB pH7.0 was used as the equilibration buffer, the binding effect of the sample to the affinity column was best, and the recovery rate of the antibody was greatly improved.
Comparative example 3
The difference from example 3 is that: the specific procedure for ion exchange chromatography was followed with an equilibration buffer of 20mM Tris-HCl, pH8.0, with the specific structure shown in Table 6.
TABLE 6
As can be seen from Table 6, 20mM Tris-HCl 5mM MgCl was used 2 The pH value of 8.0 is used as an equilibrium buffer solution, so that the binding effect of a sample and an affinity column can be improved, and the recovery rate of the antibody is improved.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the invention, but is merely illustrative of the embodiments, and all the details not described herein are common knowledge of a person skilled in the art, so that it is possible for a person skilled in the art to modify the technical solutions described in the foregoing embodiments or to make equivalent substitutions for some technical features thereof. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The preparation method of the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain is characterized by comprising the following steps: the preparation method of the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain comprises the following steps of:
(1) Immunization of animals
Taking 3 male BALB/c mice of 6 weeks old, immunizing with natural protein of human hepatitis B surface antigen as immunogen, performing tail-breaking blood collection on the mice, detecting serum titer to more than 10 ten thousand, and performing cell fusion;
(2) Cell fusion
Killing mice, soaking the mice in 75% alcohol for 5min, picking spleen of immunized mice in a sterile environment, grinding dispersed cells, washing and centrifuging to obtain B lymphocytes, mixing B lymphocytes and myeloma cells SP2/0 according to the ratio of cell number to 5:1, centrifuging, washing cells with serum-free DMEM culture medium to ensure that the mixed cells are serum-free, centrifuging to discard supernatant, slowly adding 1mL of 50% PEG solution with mass concentration in a water bath at 37 ℃ at a constant speed, dropwise adding and mixing the supernatant, slowly adding 40mL of serum-free DMEM culture medium at a pre-temperature of 37 ℃ after the reaction of the supernatant for 90s, ensuring that PEG is thoroughly diluted to terminate fusion, centrifuging at 1000rpm for 10min, discarding the supernatant, re-suspending cell sediment with HAT culture medium at the pre-temperature of 37 ℃, fully mixing the supernatant, placing the supernatant into a 96-hole cell culture plate, and culturing in a culture box with 5% carbon dioxide;
(3) Hybridoma cell screening and subcloning
The third day and the eighth day after fusion, 1/2 of culture solution in the 96-hole cell culture plate is replaced by HAT culture medium respectively, and the tenth day absorbs cell supernatant, namely cell supernatant to be detected;
the method comprises the steps of screening cells to be detected by an indirect ELISA method, after the first screening, completely replacing culture solution with HAT culture medium, repeatedly screening for three times, selecting cells with strong positive results from the three times, subcloning the cells for 2-3 times generally until the positive rate of a 96-well plate reaches 100%, and ending subcloning to obtain two anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strains, namely an anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain 1 and an anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain 2.
2. The method for preparing the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain according to claim 1, wherein the method comprises the following steps: the preparation method of the natural protein of the human hepatitis B surface antigen comprises the following steps:
(1) Low concentration PEG precipitation: collecting serum of asymptomatic human hepatitis B surface antigen carrier, precipitating with 5% final concentration PEG6000 to remove large particles and lipoprotein, standing at 4deg.C for 2 hr, centrifuging at 4000rpm for 30 min, discarding precipitate, and collecting supernatant;
(2) High concentration PEG precipitation: precipitating the supernatant with PEG6000 with final concentration of 10%, standing at 4deg.C for 2h, centrifuging at 4000rpm for 30 min, discarding the precipitate, and collecting supernatant to obtain primary pure natural protein of human hepatitis B surface antigen;
(3) Preparing an immunoaffinity chromatographic column;
(4) The affinity chromatography purification of the human hepatitis B surface antigen comprises the following steps:
connecting the immunoaffinity chromatography column prepared in the step (3) to a computer ultraviolet detector, and when the immunoaffinity chromatography column is used for purifying, monitoring the real-time change of a sample OD280 in the purifying process, and collecting a purified effective sample according to an OD280 monitoring result;
(1) balance: washing the immunoaffinity chromatographic column with a binding solution until the first peak detected by the computer ultraviolet detector OD280 returns to the baseline, namely equilibrium is reached, wherein the binding solution is 10mM PBS and 150mM NaCl;
(2) loading: the immune affinity chromatographic column after balancing the natural protein of the primary pure human hepatitis B surface antigen obtained in the step (2) is used for collecting effluent liquid until the baseline of detection of the computer ultraviolet detector OD280 is stable;
(3) eluting: adding eluent which is glycine-hydrochloric acid solution with pH of 3.0, collecting when the baseline detected by the computer ultraviolet detector OD280 rises, regulating the pH of the collected eluent to 7.4 by using a neutralizing solution, desalting by using a Sephadex G25 column, and packaging and storing at-20 ℃, wherein the neutralizing solution is 1M Tris-HCl with pH of 8.5.
3. The method for preparing the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain according to claim 2, wherein the method comprises the following steps: the preparation method of the immunoaffinity chromatographic column comprises the following steps:
(1) coupling: placing the purchased finished medium-cyanogen bromide preactivated agarose gel in a sand core funnel, washing with a precooled coupling solution A at 0-4 ℃ for at least 30min;
coupling solution A was 1mM HCl;
(2) a ligand: placing outsourced anti-human hepatitis B surface antigen monoclonal antibody into a coupling solution B for washing, wherein the coupling concentration of the anti-human hepatitis B surface antigen monoclonal antibody is 5-10mg/mL of filler;
coupling solution B was 0.1M NaHCO3, 0.5M NaCl, pH8.3;
(3) diluting the medium washed in the step (1) by using a coupling solution A, mixing the diluted medium with the monoclonal antibody of the anti-human hepatitis B surface antigen washed in the step (2) in an equal volume, and uniformly mixing the mixture at the room temperature of 20-25 ℃ for 2h or overnight at the temperature of 4 ℃ to obtain gel;
(4) the gel was blocked and washed:
closing: pumping out the coupling supernatant and adding a blocking solution, wherein the blocking solution is 0.1M Tris-HCl, the pH value is 8.0, and the room temperature blocking is carried out for 2-4 hours;
cleaning: the immunoaffinity chromatography column is obtained by alternately washing with 0.1M Tris-HCl and 0.5M NaCl mixed solution with pH8-9, 0.1M acetate buffer solution with pH3-4 and 0.5M NaCl mixed solution for 3-6 times, washing with 5 times of medium volume liquid each time, and washing with PBS buffer solution.
4. The method for preparing the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain according to claim 1, wherein the method comprises the following steps: the subcloning operation method comprises the following steps: transferring the screened positive holes to a new 96-hole cell culture plate, subcloning by adopting a double-ratio dilution method, and after seven days, changing the culture medium into a DMEM complete culture medium containing 20v/v% FBS, and continuing to culture; after 8-12 days, detecting culture supernatant by using an indirect ELISA method, continuously screening and subcloning twice, selecting single positive holes of cell clusters, continuing subcloning, and simultaneously expanding culture and freezing the rest cells cloned each time.
5. The method for preparing the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain according to claim 1, wherein the method comprises the following steps: the method for screening the cells to be detected by using an indirect ELISA method comprises the following specific steps:
(1) coating: diluting the natural protein of the hepatitis B surface antigen into 1 mug/mL by using a coating liquid, coating 100 mug/hole into an ELISA plate, coating at 37 ℃ for 2 hours or 4 ℃ overnight, washing the plate for 5 times by using a PBST buffer solution, removing unbound antigen and impurities, and drying the ELISA plate by beating;
the coating liquid is carbonate buffer solution with pH of 9.6;
(2) Closing: adding a PBST buffer solution containing 1w/v% BSA into each hole for sealing, incubating for 2 hours at 37 ℃ with 150 mu L/hole, washing the plate with the PBST buffer solution for 5 times, and drying the ELISA plate;
(3) adding cell supernatant to be detected: adding the cell supernatant to be detected into an ELISA plate, using mouse immune serum as positive control, using HAT culture medium as blank control, washing the plate with PBST buffer solution for 5 times in water bath at 37 ℃ for 30min, and drying the ELISA plate;
(4) adding enzyme-labeled secondary antibodies: diluting the HRP-labeled goat anti-mouse antibody 1000 times by using PBS buffer solution, adding the diluted goat anti-mouse antibody into an ELISA plate, 50 mu L/hole, washing the plate for 5 times by using PBST buffer solution in water bath at 37 ℃ for 30min, and drying the ELISA plate by beating;
(5) color development: adding TMB color development liquid to develop color, and incubating for 10min at 37 ℃ with 100 mu L/hole; stopping with 2mol/L sulfuric acid, detecting 50 mu L/hole at 450nm wavelength of enzyme label instrument, and detecting sample OD value/negative control OD value not less than 2.1 to obtain positive result, thus completing one-time screening.
6. The method for preparing the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain according to claim 1, wherein the method comprises the following steps: the method for immunizing by using the natural protein of the human hepatitis B surface antigen as immunogen comprises the following steps: the first immunization dose is 80 mug/one, the second immunization is carried out after one week, the immunization dose is 40 mug/one, the third immunization is carried out after one week, and the immunization dose is 40 mug/one; mixing immunogen with Freund's complete adjuvant with equal volume, emulsifying thoroughly, and performing subcutaneous multipoint immunization on dorsal inguinal region; performing the fourth immunization after one week, wherein the immunization dose is 50 mug/dose, mixing with an equal volume of sterile physiological saline, and performing intraperitoneal injection immunization; after 5 days, mice were bled for tail-breaking.
7. The method for purifying anti-human hepatitis B surface antigen monoclonal antibody 1 prepared by the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain according to claim 1, wherein the method comprises the following steps: the method comprises the following steps:
(1) Preparation of ascites: selecting balb/c male mice of about 12 weeks of age, injecting 0.8mL liquid paraffin into each mouse, and injecting screened anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain 1 into the abdominal cavity after 5 days, wherein the number of the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strains is 10 6 Closely observing the state of the mice after injection, collecting ascites after the abdomen of the mice swells obviously, centrifuging for 10min at 8000rpm and 4 ℃, removing lipid by adsorption of silicon dioxide, filtering by qualitative filter paper, and collecting supernatant;
(2) Purification of anti-human hepatitis B surface antigen monoclonal antibody 1
Taking out the ascites from the refrigerator at the temperature of minus 20 ℃, centrifuging for 10min at 9000r/min after complete melting, filtering with a 0.45 mu M filter membrane, pretreating the ascites by an ammonium sulfate salting-out method, purifying by a blue gel affinity chromatography column, collecting the purified antibody, dialyzing with a 0.01M PBS buffer, and filtering with a 0.22 mu M filter to obtain the purified anti-human hepatitis B surface antigen monoclonal antibody 1.
8. The method for purifying anti-human hepatitis B surface antigen monoclonal antibody 1 prepared by the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain according to claim 7, wherein the method comprises the steps of: the specific operation of the ammonium sulfate salting-out method purification is as follows:
a. Taking 1 part by volume of ascites, adding 1 part by volume of saturated ammonium sulfate solution to ensure that the saturation of the ascites is 50 percent, stirring the ascites while fully mixing the ascites, centrifuging the ascites for 20 minutes at 9000r/min after stirring the ascites for 30 minutes, discarding the supernatant, and collecting the precipitate;
b. adding 0.8 volume part of 20mM pH7.0 PB buffer solution into the collected precipitate, completely dissolving, loading into a dialysis bag, dialyzing with PB buffer solution at 4deg.C, dialyzing for 48 hr, centrifuging at 4000r/min for 20min, and filtering the supernatant with 0.22 μm filter to obtain primary pure antibody;
specific operation of blue gel affinity chromatography purification:
a. taking out the chromatographic column filled with the blue gel filler from the temperature of 4 ℃, balancing to room temperature, flushing the chromatographic column with a balancing buffer solution with the volume of 5 times of the column volume, and balancing the chromatographic column;
the equilibration buffer was 20mM PB, pH7.0;
b. adding the initially pure antibody into a chromatographic column, collecting effluent liquid, controlling the flow rate to be 1.0mL/min, repeatedly loading the sample for 2-3 times, adding an equilibrium buffer solution with the volume of 10 times of the column into the chromatographic column for flushing after the loading is finished, balancing the chromatographic column, and collecting the effluent liquid;
the equilibration buffer was 20mM PB, pH7.0;
c. respectively adding eluent 1, eluent 2, eluent 3 and eluent 4 with the volume of 10 times of the column volume into a chromatographic column, sequentially eluting, collecting all the eluents, detecting the purity of SDS-PAGE, and determining the eluent with the optimal purity of the protein;
Eluent 1 was 20mM PB, 20mM NaCl, pH7.0;
eluent 2 was 20mM PB, 50mM NaCl, pH7.0;
eluent 3 was 20mM PB, 200mM NaCl, pH7.0;
eluent 4 was 20mM PB, 1M NaCl, pH7.0;
d. adding buffer solution with the volume of 5 times of the column volume into the chromatographic column, flushing the chromatographic column, and then flushing and preserving the chromatographic column by using 20% ethanol solution with the volume of 5 times of the column volume;
the buffer was 20mM PB, 2M NaCl, pH7.0;
e. and c, loading the eluent with the optimal purity of the protein in the step c into a dialysis bag, concentrating by using PEG20000, putting the dialysis bag into PBS buffer solution for dialysis for 48 hours, centrifuging for 20 minutes at 4000r/min after the dialysis is finished, and filtering the supernatant by using a 0.22 mu m filter to obtain the purified monoclonal antibody 1 against the human hepatitis B surface antigen.
9. The method for purifying anti-human hepatitis B surface antigen monoclonal antibody 2 prepared by the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain according to claim 1, wherein the method comprises the following steps: the method comprises the following steps:
(1) Preparation of ascites: selecting balb/c male mice of about 12 weeks of age, injecting 0.8mL liquid paraffin into each mouse, and injecting screened anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain 2 into the abdominal cavity after 5 days, wherein the number of the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strains is 10 6 Closely observing the state of the mice after injection, collecting ascites after the abdomen of the mice swells obviously, centrifuging for 10min at 8000rpm and 4 ℃, removing lipid by adsorption of silicon dioxide, filtering by qualitative filter paper, and collecting supernatant;
(2) Purification of anti-human hepatitis B surface antigen monoclonal antibody 2
Taking out the ascites from the refrigerator at the temperature of minus 20 ℃, centrifuging for 10min at 9000r/min after complete melting, filtering by using a 0.45 mu M filter membrane, purifying by using a protein-G column through an affinity chromatography, purifying by using an ion exchange chromatography, collecting the purified antibody, dialyzing by using a 0.01M PBS buffer solution, and filtering by using a 0.22 mu M filter to obtain the purified anti-human hepatitis B surface antigen monoclonal antibody 2.
10. The method for purifying the anti-human hepatitis B surface antigen monoclonal antibody 2 prepared by the anti-human hepatitis B surface antigen monoclonal antibody hybridoma cell strain according to claim 9, wherein the method comprises the following steps of: specific procedures for purification by affinity chromatography of the protein-G:
taking 1 part by volume of ascites, adding 1 part by volume of balance buffer solution, fully and uniformly mixing, and filtering by using a microporous filter membrane with the thickness of 0.45 mu m;
taking out the chromatographic column filled with Protein-G filler from 4 ℃, balancing to room temperature, and flushing the balanced chromatographic column with 5 times of column volume of balanced buffer solution;
c. Adding the filtered ascites into a chromatographic column, collecting effluent, controlling the flow rate to be 1.5mL/min, repeatedly loading the sample for 2-3 times, adding a balancing buffer solution with the volume of 10 times of the column into the chromatographic column after the loading is finished for washing impurities, balancing the chromatographic column, and collecting the effluent;
d. adding eluent with the volume of 10 times of the column volume into the chromatographic column, collecting the eluent, and regulating the eluent to be neutral by using a neutralization solution;
e. adding 5 times of column volume of balance buffer solution into the chromatographic column, flushing the balance chromatographic column, and then balancing and preserving the chromatographic column by using 20% ethanol solution with 5 times of column volume;
f. loading the neutral eluate into a dialysis bag, placing the dialysis bag into dialysis buffer solution, dialyzing at 4deg.C for 48 hr, centrifuging at 4000r/min for 20min, and filtering the supernatant with 0.22 μm filter to obtain primary pure antibody;
wherein,,
balanced buffer 20mM Na 2 HPO 4 And 0.15M NaCl mixed solution, pH7.0;
the eluent is 0.1M glycine with pH 3.0;
the neutralization solution is 1M Tris-HCl, and the pH value is 8.5;
the dialysis buffer was 20mM Tris-HCl, pH8.0;
specific operation of purification by ion exchange chromatography:
a. taking out the chromatographic column filled with the Q-Bestarose HP filler from the temperature of 4 ℃, balancing to room temperature, flushing the chromatographic column with 5 times of the volume of the balancing buffer solution, and balancing the chromatographic column;
The equilibration buffer was 20mM Tris-HCl, 5mM MgCl 2 ，pH8.0；
b. Adding the initially pure antibody into a chromatographic column, collecting effluent liquid, controlling the flow rate to be 1.2mL/min, repeating the sample application for 3 times, adding an equilibrium buffer solution with the volume of 10 times of the column into the chromatographic column after the sample application is finished, washing the equilibrium chromatographic column, and collecting the effluent liquid;
the equilibration buffer was 20mM Tris-HCl, pH8.0;
c. respectively adding eluent 1, eluent 2 and eluent 3 with the volume of 10 times of the column volume into the chromatographic column, sequentially eluting, collecting all eluting proteins, detecting the purity of SDS-PAGE, and determining the eluent with the optimal purity of the proteins;
eluent 1 is 20mM Tris-HCl, 50mM NaCl, pH8.0, eluent 2 is 20mM Tris-HCl, 200mM NaCl, pH8.0, eluent 3 is 20mM Tris-HCl, 1000mM NaCl,pH8.0;
d. adding buffer solution with the volume of 5 times of the column volume into the chromatographic column, flushing the regenerated chromatographic column, and then flushing and preserving the chromatographic column by using 20% ethanol solution with the volume of 5 times of the column volume;
the buffer solution is 20mM Tris-HCl, 2000mM NaCl,pH8.0;
e. and c, loading the eluent with the optimal purity of the protein collected in the step c into a dialysis bag, concentrating by using PEG20000, putting the dialysis bag into PBS buffer solution for dialysis for 48 hours, centrifuging for 20 minutes at 4000r/min after the dialysis is finished, and filtering the supernatant by using a 0.22 mu m filter to obtain the purified anti-human hepatitis B surface antigen monoclonal antibody 2.
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