CN110879293A - Method for screening hybridoma cell strain secreting pairing monoclonal antibody and application - Google Patents
Method for screening hybridoma cell strain secreting pairing monoclonal antibody and application Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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Abstract
The invention discloses a method for screening hybridoma cell strains secreting pairing monoclonal antibodies and application. Coupling Oleyl-PEG4000-NHS with a capture antibody, adding the conjugate into a hybridoma clone to be screened, and culturing; discarding the supernatant, and cleaning; adding antibody blocking agent, and culturing; discarding the supernatant, and cleaning; adding an antigen capable of being combined with the capture antibody, and culturing; discarding the supernatant, and cleaning; adding an anti-mouse secondary antibody marked by fluorescent substance, and culturing; discarding the supernatant, and cleaning; adding an incomplete culture medium, observing cell clusters under visible light and fluorescence, taking a picture, and marking the cell clusters generating fluorescence as positive clones; separating the cells of the positive clone cell mass, and culturing to obtain the hybridoma cell strain secreting the paired monoclonal antibody. The method is rapid, simple and convenient, has high accuracy, and can directly screen out positive hybridoma cell strains secreting and capturing antibody paired antibodies.
Description
Technical Field
The invention belongs to the technical field of immunological detection, and particularly relates to a method for screening hybridoma cell strains secreting pairing monoclonal antibodies and application of the hybridoma cell strains.
Background
1975 German scholars
And Milstein, first reported the use of mouse myeloma cells fused with sheep red blood cell sensitized mouse spleen cells, and found that a portion of the fused cells were capable of both growing and secreting anti-sheep red blood cell antibodies. The discovery solves the problem of preparing the monoclonal antibody with a single antigenic determinant, and initiates the technology of preparing the monoclonal antibody by using a cell fusion technology. The hybridoma technology is a breakthrough biotechnology and provides a wide application prospect for the research of numerous disciplines such as bioscience, clinical medicine and the like.
Monoclonal antibodies have the characteristics of high purity, strong specificity, high potency and the like, and are widely used for development of immunological detection products, innovation of immunological technology and improvement of medical treatment and diagnosis methods at present. It can be said that the application of monoclonal antibodies has become an important means essential in biology and medicine.
The currently common methods for screening monoclonal antibodies are enzyme-linked immunosorbent assay (ELISA) and other methods, and the double-antibody sandwich principle is widely applied to the fields of biological basic research and immunoassay product development. These methods require long times and can only measure one antibody at a time, or require large sample volumes. The traditional method for screening the paired antibodies needs to screen a large number of positive hybridoma cell strains secreting monoclonal antibodies, then prepare a large number of antibodies for purification, and then pair the positive hybridoma cell strains through double-antibody sandwich ELISA, so that the process is complicated, the success rate is low, and the time and the labor are consumed. Therefore, it is necessary to establish a method for directly screening a cell strain secreting a monoclonal antibody of a mate pair to improve the screening efficiency and the screening precision.
Disclosure of Invention
The invention aims at overcoming the defects of the prior art and establishing a method for screening hybridoma cell strains secreting pairing monoclonal antibodies.
The invention also aims to provide the application of the method for screening the hybridoma cell strain secreting the pairing monoclonal antibody.
The purpose of the invention is realized by the following technical scheme:
a method for screening hybridoma cell strains secreting pairing monoclonal antibodies comprises the following steps:
(1) coupling Oleyl-PEG4000-NHS with a capture antibody to obtain Oleyl-PEG4000-NHS-Ab1, wherein Ab1 is the capture antibody;
(2) cleaning hybridoma cell clones to be screened; adding Oleyl-PEG4000-NHS-Ab1, and culturing; discarding the supernatant, and cleaning hybridoma cell clones;
(3) adding antibody blocking agent, and culturing; discarding the supernatant, and cleaning hybridoma cell clones;
(4) adding an antigen capable of being combined with the capture antibody, and culturing; discarding the supernatant, and cleaning hybridoma cell clones;
(5) adding an anti-mouse secondary antibody marked by fluorescent substance, and culturing; discarding the supernatant, and cleaning hybridoma cell clones;
(6) adding an incomplete culture medium, observing cell clusters under visible light and fluorescence, taking a picture, marking the cell clusters generating fluorescence as positive clones, and marking the cell clusters not generating fluorescence as negative clones;
(7) separating the cells of the positive clone cell mass, and culturing to obtain the hybridoma cell strain secreting the monoclonal antibody capable of being matched with the capture antibody.
The capture antibody in the step (1) is preferably an H7 subtype avian influenza virus monoclonal antibody.
The coupling ratio in the coupling described in step (1) is preferably Oleyl-PEG 4000-NHS: the capture antibody is 5-7: 5; more preferably, Oleyl-PEG 4000-NHS: capture antibody-to-mole ratio 6: 5.
the coupling mode in the coupling in the step (1) is conventional coupling, namely the Oleyl-PEG4000-NHS is directly coupled with the capture antibody in proportion.
The addition amount of the Oleyl-PEG4000-NHS-Ab1 in the step (2) is preferably calculated according to the addition amount of 0.02-0.03 mg in each hole of a 96-hole plate; more preferably, it is calculated as 0.025mg per well of a 96-well plate.
The culture described in the steps (2), (3), (4) and (5) is a culture under cell culture conditions, e.g., 5% CO at 37 ℃%2。
The culture time in the step (2) is preferably 30-60 min; more preferably 60 min.
The antibody blocking agent in the step (3) is preferably an anti-mouse secondary antibody; more preferably, the secondary antibody is goat anti-mouse IgGFc.
The addition amount of the antibody blocking agent is preferably calculated according to the addition amount of 0.2-0.4 mg in each hole of a 96-hole plate; more preferably 0.2mg per well of a 96-well plate;
the culture time in the step (3) is preferably 45-60 min; more preferably 60 min.
The addition amount of the antigen capable of binding to the capture antibody in the step (4) is preferably calculated by adding 0.0625mg to 0.6mg per well of a 96-well plate; more preferably, the amount of the additive is 0.3mg to 0.6mg per well of a 96-well plate. The higher the purity of the antigen, the smaller the amount added per well.
The incubation time in step (4) is preferably 60 min.
The fluorescent substance in the step (5) comprises Fluorescein Isothiocyanate (FITC), tetraethylrhodamine (RB200) and Tetramethyl Rhodamine Isothiocyanate (TRITC).
The anti-mouse secondary antibody in the step (5) is preferably a goat anti-mouse IgGFc secondary antibody.
The incubation time in step (5) is preferably 60 min.
The washing described in steps (2), (3), (4) and (5) is preferably performed in Phosphate Buffered Saline (PBS) or incomplete medium.
The Phosphate Buffer Solution (PBS) is 0.015M phosphate buffer solution with pH7.4.
The preparation method of the incomplete culture medium comprises the following steps: the mixture of streptomycin and penicillin was mixed with RPMI-1640 medium according to 1: 99 volume ratio, wherein the concentrations of the streptomycin and the penicillin in the mixed solution of the streptomycin and the penicillin are both 1U/ml.
The specific operation of separating the cells of the positive clone cell mass in the step (7) is preferably: adding semi-solid culture medium into hybridoma cell clone containing positive clone cell mass, picking up cell of positive clone cell mass, and transferring to new complete culture medium.
The semisolid culture medium is preferably a methylcellulose semisolid culture medium.
The preparation method of the methyl cellulose semisolid culture medium comprises the following steps: mixing incomplete culture medium and a methylcellulose solution with the mass concentration of 2.7% according to the volume ratio of 1: 1, uniformly mixing to obtain the methyl cellulose semisolid culture medium.
The semi-solid medium is preferably added in an amount to just cover the bottom of the plate. Add 100. mu.l per well of 96-well plate.
The method for directly screening the cell strain secreting the paired monoclonal antibodies is applied to the field of immunology and is used for quickly screening the hybridoma cells capable of secreting the monoclonal antibodies paired with the capture antibodies.
Oleyl-PEG4000-NHS is an amphiphilic substance, one end of which is lipophilic and can be inserted into the surface of a cell membrane, and the other end of which is a PEG polymer which is hydrophilic and the end of which is a carboxyl group activated by NHS and can be combined with an amino group on an antibody. After 5-6 days after cell fusion, adding coupled Oleyl-PEG4000-NHS-Ab1, inserting Oleyl-PEG4000-NHS-Ab1 on the surface of a cell membrane, adding an antibody blocking agent (anti-mouse secondary antibody) to block an antibody (Fc segment of Ab1), adding an antigen capable of being specifically combined with Ab1 according to the principle of specific combination of the antibody and the antigen, specifically combining the antigen and the antibody, capturing the antibody (Ab2, secreted by hybridoma) which is secreted by the hybridoma and can be matched with the captured antibody (Ab1), washing away the unbound antibody (Ab2), adding a fluorescent secondary antibody, and staining and positioning a positive hybridoma cell strain by using the principle of specific combination of the fluorescent secondary antibody and the secreted antibody. And finally, adding a methylcellulose semisolid culture medium, directly picking out the positive hybridoma cell strain, and putting the positive hybridoma cell strain into a new culture hole for culture. Therefore, not only can positive hybridoma cell strains be directly screened, but also positive hybridoma cell strains secreting the paired antibodies can be directly screened, and simultaneously, the screened cells can be cloned by using the method.
Compared with the prior art, the invention has the following advantages and effects:
the conventional monoclonal screening generally requires at least one month to screen positive hybridoma cell strains, and after stable cell strains are established, the monoclonal antibody is quantitatively produced in half a month, purified and quantified, and paired antibodies are screened by using a kit. The invention can rapidly and directly screen hybridoma cells secreting monoclonal antibodies matched with the capture antibodies through an amphiphilic substance (Oleyl-PEG 4000-NHS). Compared with the traditional method for screening the paired antibodies, the method reduces the processes of screening, antibody preparation, antibody purification, antibody sandwich ELISA (enzyme-linked immunosorbent assay) matching and the like in the traditional monoclonal antibody preparation process, greatly reduces the workload, and obviously improves the probability of successful screening. The method can directly screen positive hybridoma cell strains and positive hybridoma cell strains secreting the paired antibodies, and can clone the screened cells by using the method, so that the method greatly reduces the workload compared with a limited dilution method used by the traditional method, and the positive clones are not easy to lose. Hybridoma cell strains secreting pairing antibodies can be screened within 10-15 days by the method, and the screening and pairing identification time is shortened; the antibody secretion condition of each cell in the hole and the variation condition of cell clone can be accurately evaluated, and the combination of the semisolid culture medium is favorable for selecting stable hybridoma cells secreting the paired antibody.
Drawings
FIG. 1 is a SDS-PAGE graph of Oleyl-PEG4000-NHS-Ab1 solution prepared in example 1; wherein, lane 1 is Marker, lane 2 is Oleyl-PEG4000-NHS, lane 3 is Ab1, and lane 4 is Oley-PEG4000-NHS-Ab 1.
FIG. 2 is a chart of UV-Vis spectrophotometer scan results of Oleyl-PEG4000-NHS-Ab1 solution prepared in example 1; wherein, curve A is Oleyl-PEG4000-NHS, curve B is Oleyl-PEG4000-NHS-Ab1, and curve C is Ab 1.
FIG. 3 is a photograph showing the results of direct immunofluorescence assay of Oleyl-PEG4000-NHS-Ab1 solution prepared in example 1; wherein, A1 is SP2/0, firstly adding Oleyl-PEG4000-NHS-Ab1 for incubation for 1h, then adding 100 XFITC labeled goat anti-mouse IgGFc for incubation for 45min, and taking a 200 Xfluorescence image under a microscope; a2 is 400 Xfluorescence image corresponding to A1; b1 is a 200 × visible light image for a1, and B2 is a 400 × visible light image for a 2; a3 is microscope 200 Xfluorescence image when SP2/0 cell is added with diluted 100 XFITC labeled goat anti-mouse IgGFc and incubated for 45min, A4 is 400 Xfluorescence image corresponding to A3; b3 is a 200 × visible light image for A3, and B4 is a 400 × visible light image for a 4.
FIG. 4 is a graph showing the results of the cell masses of some positive clones selected in example 1; wherein, the cell clones in A and B are the same, the cell clones in C and D are the same, A and C are observed under fluorescent condition, B and D are observed under visible condition.
FIG. 5 is a graph showing the direct immunofluorescence results of blocking antibodies with varying concentrations of goat anti-mouse IgGFc in example 2; wherein A is a graph showing the blocking results of the blocking antibody with the goat anti-mouse IgGFc of 0.125mg/ml, B is a graph showing the blocking results of the blocking antibody with the goat anti-mouse IgGFc of 0.25mg/ml, C is a graph showing the blocking results of the blocking antibody with the goat anti-mouse IgGFc of 0.5mg/ml, D is a graph showing the blocking results of the blocking antibody with the goat anti-mouse IgGFc of 1mg/ml, E is a graph showing the blocking results of the blocking antibody with the goat anti-mouse IgGFc of 2mg/ml, and F is a graph showing the blocking results of the blocking antibody with the goat anti-mouse IgGFc of 4 mg/ml.
FIG. 6 is a graph of the direct immunofluorescence results of blocking antibodies with different concentrations of horse serum in example 2; wherein A is a blocking result graph of blocking antibody with 0.125mg/ml horse serum, B is a blocking result graph of blocking antibody with 0.25mg/ml horse serum, C is a blocking result graph of blocking antibody with 0.5mg/ml horse serum, D is a blocking result graph of blocking antibody with 1mg/ml horse serum, E is a blocking result graph of blocking antibody with 2mg/ml horse serum, and F is a blocking result graph of blocking antibody with 4mg/ml horse serum.
FIG. 7 is a graph of the direct immunofluorescence results of example 2 without blocking the antibody with blocking agent.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise indicated, reagents and materials for use in the invention are commercially available, wherein:
the phosphate buffer solution (PBS, 0.015mol/L, pH7.4) was prepared as follows: weighing NaCl 8.0g and Na2HPO4·12H2O 2.9g、KCl 0.2g、KH2PO40.2g, addAdding deionized water for dissolution, adjusting the pH value to 7.4, and fixing the volume to 1000 mL.
The preparation method of the incomplete culture medium or the basic culture medium comprises the following steps: the mixture of streptomycin and penicillin was mixed with RPMI1640 medium according to 1: 99 volume ratio, wherein the concentrations of the streptomycin and the penicillin in the mixed solution of the streptomycin and the penicillin are both 1U/mL.
Complete medium: fetal bovine serum and the incomplete medium were mixed in a ratio of 1: 9 volume ratio to obtain a complete culture medium;
HAT medium: HAT, fetal bovine serum and the above incomplete medium were mixed as follows: 10: 88 volume ratio to obtain HAT culture medium;
HT medium: HT, fetal bovine serum and incomplete medium described above were run at 2: 10: 88 volume ratio to obtain HT culture medium;
the preparation method of the methyl cellulose semisolid culture medium comprises the following steps: mixing the incomplete culture medium with a methylcellulose solution with the mass concentration of 2.7% according to the volume ratio of 1: 1, uniformly mixing to obtain the methyl cellulose semisolid culture medium.
PBST-T Wash: tween-200.5 mL and 0.015mol/L, pH 7.4.4 PBS were added to 1000 mL.
Example 1 screening of hybridoma cell lines secreting paired monoclonal antibodies
(1) Obtaining hybridoma cells
1) Immunization: the immunogen is an avian influenza H7N9 subtype standard detection antigen (Harbin Virgidae biology, Inc.) provided by Shenzhen entry-exit inspection and quarantine bureau, 2ml of PBS (0.015 mol/ml) is added into each bottle of standard antigen according to the instruction to be dissolved, and then the antigen and the concentration of each screening peptide are determined by a BCA method. BALB/C female mice (purchased at southern medical university, same below) at 5 weeks of age were immunized with the treated avian influenza antigen and the immunization strategy was as follows:
TABLE 1 subtype H7 avian influenza Virus immunization strategy
10 days after the four-immunization of the mice, 50 mu l of blood is collected by a tail vein blood sampling mode, the blood is firstly placed in a constant temperature incubator at 37 ℃ for standing for 1h, then the blood is placed in a refrigerator at 4 ℃ for overnight, the serum is naturally separated out, the upper layer of serum is collected by centrifugation at 1500rpm for 15 minutes on the next day, and the separated serum is placed at-20 ℃ for storage. And measuring the serum titer by indirect ELISA, and fusing the mice with the highest titer, namely the best immune effect.
Killing mice by an eyeball picking method before fusion, collecting all blood, placing in a constant temperature incubator at 37 ℃ for standing for 1h, then placing in a refrigerator for overnight at 4 ℃, fully separating out serum for the next day, centrifuging at 1500rpm for 15min, collecting upper layer serum, subpackaging, and placing at-20 ℃ for storage.
2) Fusing:
feeder cells were prepared the day before fusion. Placing scissors, tweezers, needle head and foam box in an ultra-clean bench for ultraviolet irradiation for 30min, then killing the mouse by an eyeball picking method, and soaking in a beaker filled with 75% alcohol for 5 min. The mouse is taken out and placed on the foam box, the four limbs of the mouse are fixed by the needle, the abdominal skin is slightly lifted by the forceps, the skin is separated from the peritoneum, a small opening (the peritoneum cannot be cut off) is cut on the skin by the scissors, and the epidermis is torn along the small opening by the forceps. The syringe sucks 5mL of the basic culture medium, injects the basic culture medium into the abdominal cavity of the mouse, gently softens the abdominal cavity for 1min by using a cotton ball, sucks out the basic culture medium, and places the basic culture medium in a 15mL centrifuge tube. Then 5ml of the basal medium was aspirated by the syringe, and the above operation was repeated. The collected basal medium was centrifuged for 7min at 1000rpm in a horizontal centrifuge. Resuspending peritoneal cells in HAT medium, counting, diluting cells to 2X 108Adding 100 μ l of the extract into each well, and standing at 37 deg.C with 5% CO2Cultured in a cell incubator, and feeder cells are prepared one day before fusion.
The most immune competent mice were selected for fusion. The scissors and the tweezers are sterilized by high pressure in advance, the number of the glass dishes is 2, the number of the 400-mesh gauze is one, and the number of the glass grinding rods is 1. Mice were sacrificed by the eyespot method and blood was collected from the mice as a positive control. The blood of the mouse is discharged to the greatest extent, so that the excessive blood cells in the spleen of the mouse are avoided, and the fusion is prevented from being interfered. The sacrificed mice are placed in 75% alcohol for 5min, then the peritoneum is opened by taking the feeder cells, the peritoneum is cut off by replacing clean scissors and tweezers, then the spleen of the mice is clamped by the tweezers, and the connective tissues are cut off by the scissors. The spleen was gently washed in a dish previously filled with basal medium. Then the spleen was placed on an iron mesh, an appropriate amount of basal medium was added, the spleen was cut up with scissors, and then the spleen was gently ground with a grinding bar until no large tissue was seen. Collecting cell suspension in a centrifuge tube, standing for 5min, removing large tissue precipitate, recording total volume, and counting with a blood counting plate.
Myeloma cells SP2/0 (purchased from Shanghai institute of cell biology) were recovered one week before fusion, cultured dishes of SP2/0 in the logarithmic growth phase were taken, and the cells were gently washed twice with a basal medium to remove dead cells. Then 3ml of basal medium is added into each dish, the cells are gently blown down by a 1ml gun head, the cells are collected in a 50ml centrifuge tube, the volume of cell suspension is read, and a proper amount of cell suspension is taken and counted by a blood counting chamber.
And mixing the splenocytes and SP2/0 according to the counting result in a ratio of 5-10: 1, placing the mixture in a horizontal centrifuge at 1000rpm, and centrifuging the mixture for 7 min. After centrifugation, the supernatant was discarded and the tubes were tapped to loosen the cells. The centrifuge tubes were placed in a beaker containing 37 ℃ deionized water, and 1ml of PEG2000 solution (50% strength, Sigma) incubated at 37 ℃ in advance, 1ml of one spleen PE of one mouse, was added slowly and then rapidly within 45 seconds, and then left to stand for 90 seconds. Preparing 15ml of incomplete culture medium in advance, incubating to 37 ℃, adding the centrifuge tube within 2-4 min after standing, and slowly and quickly adding the centrifuge tube while slightly shaking the centrifuge tube. After completion of termination, the mixture was centrifuged at 1000rpm for 7min in a horizontal centrifuge. After centrifugation, the supernatant was discarded, and the cells were resuspended in HAT medium and plated evenly onto feeder cells plates previously plated. Placing at 37 ℃ and 5% CO2Culturing in a cell culture box.
(2) Coupling and identification of Oleyl-PEG4000-NHS and avian influenza virus
Weighing coupling agent Oleyl-PEG4000-NHS 10mg, dissolving in 1ml PBS buffer solution, placing 100 μ l in a small reagent bottle, placing into a rotor, and slowly adding 2mg avian influenza virus antigen (Harbin Vitaceae biology, Co., Ltd.) on a stirring apparatus. And placing the reagent bottle in the avian influenza virus antigen. The vial was placed at 4 ℃ and stirred overnight.
The purpose of ultrafiltration is to remove unconjugated Oleyl-PEG4000-NHS molecules, which have a molecular weight of about 4KD and AIV above 5000KD, so a 30KD pore size ultrafiltration tube was chosen to remove unconjugated coupling agent, the ultrafiltration procedure was as follows:
1) 5ml of PBS (0.015M, pH7.4) was added to a 30KD ultrafilter tube and centrifuged at 5000rpm for 10 min. The operation was repeated 3 times.
2) Adding the sample into an ultrafiltration tube, centrifuging at 5000rpm for 15min, adding 5ml PBS to slightly blow and wash the membrane in the ultrafiltration tube when the retentate is less than 100 μ l, continuing centrifuging, repeating ultrafiltration for 3 times, and collecting the retentate (controlled within 500 μ l).
3) The ultrafiltration membrane was repeatedly flushed with PBS to remove residual impurities sufficiently, and then centrifuged. The operation was repeated 3 times.
4) The ultrafiltration tube was filled with 20% ethanol treated with a 0.45 μm filter and stored in a refrigerator at 4 ℃.
And scanning and identifying by using an ultraviolet-visible spectrophotometer. The principle is to judge whether the coupling is successful by observing the shift of the absorption peak. Oleyl-PEG4000-NHS, AIV and Oleyl-PEG4000-NHS-AIV all have specific absorption peaks, and whether the coupling is successful or not can be judged by detecting the existence and the position of each characteristic peak.
The BSA standard protein concentration in the kit was diluted with PBS to eight gradients of 1.5mg/mL, 1mg/mL, 0.75mg/mL, 0.5mg/mL, 0.25mg/mL, 0.125mg/mL, 0.0625mg/mL, 0mg/mL, and the samples to be tested were subjected to multiple gradients, and 25. mu.l of each sample was added to wells of an ELISA plate. The color developing solution is prepared in situ, the solution A and the solution B are mixed uniformly according to the volume of 50:1, 200 mu L of the mixture is added into each hole, and the mixture is placed in a constant temperature box at 37 ℃ and reacted for 30min in a dark place. After the reaction is finished, placing the ELISA plate in an ELISA reader to detect the absorption light value at 570nm, drawing a standard curve of the absorption light value of the standard protein curve, and calculating the concentration of the sample to be detected according to a fitting curve formula.
(3) Screening positive AIV hybridoma cells by a cell surface immunofluorescence method:
and (4) screening positive hybridoma cells by using a cell surface fluorescence adsorption method after 5-7 days of fusion. Gently aspirating supernatant from wells of the cell culture plate, adding 100. mu.l of incomplete medium, gently washing, and thenThe medium was aspirated off, the operation was repeated and the wash was performed twice. 100 μ l of Oleyl-PEG4000-NHS-AIV diluted to 0.1mg/ml was added to each well, and the mixture was incubated at 37 ℃ with 5% CO2Culturing in a cell culture box for 1 h. The vial was placed at 4 ℃ and stirred overnight. And repeating the cleaning process in the first step and washing twice. A200-fold dilution of FITC-labeled goat anti-mouse IgG antibody was added at 100. mu.l per well. Placing at 37 ℃ and 5% CO2Culturing in a cell culture box for 1 h. The washing process in the first step was repeated, washed twice, 100. mu.l of incomplete medium was added, and the plate was then placed under a fluorescence microscope and observed in an excited state. The green fluorophore was observed and labeled at the bottom of the plate.
The semi-solid medium method picks up positive AIV hybridoma cell mass. After marking the positive hybridoma cell mass, moving the cell culture plate to a clean bench, removing the culture medium, adding 100 mul of prepared cellulose semisolid culture medium, then slightly sucking the cell mass at the marked position by using a 10 mul gun head, moving the cell mass to the cell culture plate with the HT culture medium added in advance, and recording. And culturing the cells for about 3 days, performing fluorescent staining operation again, and cloning the cells by combining a semisolid culture medium method until a stable cell strain 16 is obtained. The result of selection was detected by indirect ELISA (the specific procedure is as follows), and a positive cell line 11A11 with good specificity was obtained.
Indirect ELISA:
1) coating antigen: coating solution (0.05M carbonate buffer solution of pH 9.6: Na)2CO31.5g、NaHCO32.9g plus ddH2Dissolving in 800mL of O water, adjusting pH to 9.6, adding water to 1000mL), diluting H5, H7N9 and H9N2 (Harbin Vitaceae biology, Ltd.) to 0.002mg/mL, adding into wells of an enzyme-labeled plate respectively, keeping the wells at 100 μ L, standing overnight (about 12H) at 4 ℃ in a refrigerator, discarding the coating solution, washing the plate with PBS-T for 3 times, 3min each time;
2) and (3) sealing: preparing 5% (v/w) skimmed milk powder, sealing, placing in a thermostat at 37 deg.C for incubation for 1h, discarding the sealing solution, washing the plate with PBS-T for 3 times, each time for 3 min;
3) primary antibody incubation: adding hybridoma cell supernatant, using SP2/0 cell supernatant as negative control, adding 100 μ L into each well, incubating in 37 deg.C incubator for 1h, discarding cell supernatant, washing plate with PBS-T for 3 times, each time for 3 min;
4) and (3) secondary antibody incubation: diluting goat anti-mouse IgG secondary antibody 8000 times with PBS-T, adding 100 μ L per well, incubating in 37 deg.C incubator for 45min, discarding the secondary antibody solution, washing the plate with PBS-T for 5 times, each time for 3 min;
5) color development: adding a color development liquid: solution A (solution A, dissolving TMB powder in DMSO to a final concentration of 11mg/ml, adding 1/10 volumes of glycerol (all operating in dark place, then placing into a black bottle and storing in dark place at 4 deg.C)) at 50 μ L/well; solution B (0.2 moL/L disodium hydrogen phosphate and 0.1moL/L citric acid buffer solution prepared into a solution with 0.74mg/mL of urea peroxide concentration and with pH of 5.5) 50 μ L/well. Reacting at room temperature in dark for 10 min;
6) and (4) terminating: stop with the addition of ELISA stop solution (2% v/v sulfuric acid), 50. mu.L per well;
7) and (3) detection: enzyme-linked immunosorbent assay (ELISA), and the absorption value at the wavelength of 450 nm.
The cell lines had low affinity (small OD value) for the antigens H5 and H9N2 and high affinity (large OD value) for H7N9, and were highly specific.
(4) Preparation of capture antibody:
1) BALB/C mice over 8 weeks old are selected, and according to the dose of 500 mu L/mouse, the mice are injected with Freund incomplete adjuvant one week in advance, and after completion, the abdomen of the mice is gently softened to be uniformly dispersed. Hybridoma cells 11A11 with mellow luster and good state and positive indirect ELISA detection are selected. RPMI-1640 was washed once to remove most of the dead cells; adding a certain volume of RPMI-1640, gently blowing up cells, taking quantitative suspension to calculate total cell number, placing in a centrifuge tube, centrifuging at 1000rpm for 7min, discarding supernatant, and resuspending cells to 1 × 10 with RPMI-16406one/mL. Mice were also injected intraperitoneally at a dose of 500 μ L/mouse, gently abdominally to distribute the cells evenly and marked.
2) The abdomen begins to swell and enlarge 7-10 days after the mice are inoculated with the cells. When the abdomen of the mouse is obviously swollen, 75% alcohol is used for disinfecting the periphery of the abdomen of the mouse, and a No. 12 medical needle is used for drainage and ascites of a mobile phone are used. Continuously raising the mice, observing the state of the mice every other day, and collecting ascites again if the abdomens of the mice swell again. The ascites fluid which is just collected is put into a centrifuge and centrifuged for 30min at 4000rpm and 4 ℃, and the supernatant fluid is taken and packed to-20 ℃ for storage.
3) Ascites was thawed at 4 ℃ and centrifuged at 10000rpm at 4 ℃ for 15min, and the supernatant was collected and its volume was recorded. Placing the collected supernatant into a magnetic stirrer, stirring in ice water bath, slowly adding saturated ammonium sulfate solution with the same volume dropwise, continuously stirring for 15min, and placing into a refrigerator for standing overnight at 4 ℃. The next day, centrifugation was carried out at 10000rpm and 4 ℃ for 30min, the supernatant was discarded, the precipitate was dissolved in 2-fold of PBS, and the precipitate was filtered through a 0.2 μm filter and subjected to the next step. Desalting with desalting column, separating Protein G affinity chromatography column to obtain hybrid Protein, collecting and eluting target antibody with too large volume, and concentrating antibody with 30KD ultrafiltering tube. The antibody 11a11 was collected after ultrafiltration. The prepared 11A11 ascitic monoclonal antibody is used as a capture antibody for standby, and double distilled water is used for preparing an 11A11 strain antibody solution with the concentration of 5 mg/ml.
(5) Coupling and identification of Oleyl-PEG4000-NHS with Capture antibody (Ab1)
1) Oleyl-PEG4000-NHS coupled to Ab1
2mg of Oleyl-PEG4000-NHS (available from NF in Japan) was weighed out and dissolved in 200. mu.l of PBS to obtain an Oleyl-PEG4000-NHS solution.
Adding 150 μ l of Oleyl-PEG4000-NHS into a bottle, placing on an ice box, and stirring; 250. mu.l of a 5mg/ml 11A11 strain antibody solution was slowly added to the above solution, and the mixture was stirred overnight at 4 ℃. Ultrafiltering with 30KD ultrafiltering tube to obtain Oleyl-PEG4000-NHS-11A11 solution. Subsequently, the cells were sterilized by filtration through a 0.22 μm filter and placed at 4 ℃.
2) Identification of Oleyl-PEG4000-NHS-Ab1
The resulting working solution (i.e., Oleyl-PEG4000-NHS-11A11 solution) was identified by SDS-PAGE, UV-Vis spectroscopy. SDS-PAGE identification As shown in FIG. 1, lane 4 is Oley-PEG4000-NHS-Ab1, and the band lags behind lane 3, demonstrating successful coupling; the ultraviolet-visible spectroscopic scan identification is shown in fig. 2.
Diluting the obtained working solution with incomplete culture medium to concentration of 0.98mg/ml, adding SP2/0 cell 100 μ L, 37 deg.C, and 5% CO2Incubate for 1h, wash twice in incomplete medium, add 100 XFITC labeled goat anti-mouse IgGFc (Solarbiolot No. 2018052)2) Incubating for 45 min; in the control group, 100. mu.L of SP2/0 cells was added with an equal volume of incomplete medium. Adding 100 XFITC labeled goat anti-mouse IgGFc and incubating for 45 min; after the incubation, the supernatant was aspirated, 100. mu.L of incomplete medium was added and washed for 2 times, and finally, after 100. mu.L of incomplete medium was added, photographs were taken under a fluorescent microscope. The direct immunofluorescence results are shown in figure 3. The results showed that Oleyl-PEG4000-NHS-11A11 was indeed obtained.
(6) Screening of hybridoma cell masses secreting monoclonal antibodies that mate with the antibody of strain 11A11
Before actual selection, working concentration of Oleyl-PEG4000-NHS-11A11, working concentration of goat anti-mouse IgG (Fc) antibody, blocking time, antigen using concentration and fluorescent secondary antibody using concentration are optimized, and the optimized conditions are adopted in the following operations.
1) BALB/C mice of 6 weeks of age were selected for immunization, and avian influenza virus subtype H7 (avian influenza standard test antigen purchased from Haerbin Vitaceae, Inc.) was immunized for the first time: 800 μ g, multiple injection; boosting, 600 μ g, multiple injection; and (3) sprint immunization: 300 ug, tail vein injection, with 14 days between immunizations. The spleen was removed from the mice and the splenocytes were combined with prepared SP2/0 cells in log phase growth (purchased from shanghai institute of cell biology) according to 6: 1 cell, adding polyethylene glycol (PEG-2000) to fuse the cells, uniformly spreading the cells on 6 96-hole cell culture plates for culture, and starting detection 5-6 days after fusion.
2) Adding 100 μ l of 0.25mg/ml Oleyl-PEG4000-NHS-11A11, placing in a cell culture box at 37 deg.C and 5% CO2Incubate for 60min, discard the supernatant and wash twice with incomplete medium.
3) Mu.l of goat anti-mouse IgG FC (KPL, cat #: 5220-2Incubate for 60min, discard the supernatant and wash twice with incomplete medium.
4) Adding 100 μ l of 6mg/ml avian influenza H7 subtype antigen (Harbin Vitaceae Biotechnology Co., Ltd.), placing in a cell culture box at 37 deg.C and 5% CO2Incubate for 60min, discard the supernatant, wash twice with incomplete medium (used in this experiment)The avian influenza H7 subtype antigen is an avian influenza standard detection antigen, has few effective components and higher use concentration; when the antigen is used, the OD value of the coated and purified antigen is equivalent to 50 ng/well OD value when the amount of the coated plate is 400 ng/well, so that the use amount of the antigen with better purification can be reduced as required, and the addition amount of the fluorescent secondary antibody can be referred to).
5) 100 μ L of FITC-labeled goat anti-mouse IgGFc diluted 100-fold (lebao company, cat #: SF131), placing in a cell culture box at 37 ℃ and 5% CO2Incubate for 60min, discard the supernatant and wash 1 time with incomplete medium.
6) Adding 150 μ l of incomplete culture medium, observing cell clusters under visible light and fluorescence by using a fluorescence microscope, photographing to make negative and positive marks, marking the cell clusters generating fluorescence as positive clones, and marking the cell clusters not generating fluorescence as negative clones. The results of photographing a part of the positive clone cell masses are shown in FIG. 4.
7) The supernatant from the wells containing the positive cell clones was removed, 100. mu.l of methylcellulose semisolid medium was added, and the positive-labeled cells were pipetted into a new cell culture plate for culture, depending on the previous labeling.
As a result: 5 hybridoma cell strains are screened, the cell masses can be observed to be dyed with fluorescence under a fluorescence microscope, and the result shows that the antibody secreted by the cell strains can react with the avian influenza H7 subtype antigen through ELISA detection; ascites is prepared from the 5 hybridoma cells (the preparation process of the ascites of the hybridoma cell strain is referred to in the specific operation), the 5 hybridoma cells are purified, then the 5 hybridoma cells are paired with a secreted antibody of a capture antibody 11A11 strain by a double antibody sandwich ELISA method, and the 5 hybridoma cells are verified to be paired with a secreted antibody of a 11A11 strain.
EXAMPLE 2 determination of suitable antibody blocking Agents and concentrations
(1) Oleyl-PEG4000-NHS-Ab1(11A11), and hybridoma cell clones were obtained in the same manner as in example 1.
(2) Mu.l of 0.25mg/ml Oleyl-PEG4000-NHS-Ab1(11A11) was added to hybridoma clones and placed in a cell culture chamber at 37 ℃ with 5% CO2Incubating for 60min, discarding the supernatant, and washing twice with incomplete culture medium; a. theGroup (2): mu.l of 0.125mg/ml, 0.25mg/ml, 0.5mg/ml, 1mg/ml, 2mg/ml, 4mg/ml goat anti-mouse IgG FC blocking antibody, group B: adding 100 μ l of horse serum blocking antibody 0.125mg/ml, 0.25mg/ml, 0.5mg/ml, 1mg/ml, 2mg/ml and 4mg/ml, placing hybridoma cell clone without blocking agent as positive control group in cell culture box at 37 deg.C and 5% CO2Incubate for 60min, discard the supernatant and wash twice with incomplete medium. The antibody 11A11 strain was used for direct immunofluorescence, and the direct immunofluorescence results of group A, group B, and control group are shown in FIGS. 5, 6, and 7, respectively.
Therefore, the goat anti-mouse IgG FC is used as a blocking agent, the blocking effect can be achieved when the concentration is more than 1mg/ml, and the blocking effect is better when the concentration is more than 2 mg/ml; horse serum failed to achieve a blocking effect.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A method for screening hybridoma cell strains secreting pairing monoclonal antibodies is characterized by comprising the following steps: the method comprises the following steps:
(1) coupling Oleyl-PEG4000-NHS with a capture antibody to obtain Oleyl-PEG4000-NHS-Ab1, wherein Ab1 is the capture antibody;
(2) cleaning hybridoma cell clones to be screened; adding Oleyl-PEG4000-NHS-Ab1, and culturing; discarding the supernatant, and cleaning hybridoma cell clones;
(3) adding antibody blocking agent, and culturing; discarding the supernatant, and cleaning hybridoma cell clones;
(4) adding an antigen capable of being combined with the capture antibody, and culturing; discarding the supernatant, and cleaning hybridoma cell clones;
(5) adding an anti-mouse secondary antibody marked by fluorescent substance, and culturing; discarding the supernatant, and cleaning hybridoma cell clones;
(6) adding an incomplete culture medium, observing cell clusters under visible light and fluorescence, taking a picture, marking the cell clusters generating fluorescence as positive clones, and marking the cell clusters not generating fluorescence as negative clones;
(7) separating the cells of the positive clone cell mass, and culturing to obtain the hybridoma cell strain secreting the monoclonal antibody capable of being matched with the capture antibody.
2. The method for screening hybridoma cell lines secreting paired monoclonal antibodies according to claim 1, wherein:
the capture antibody in the step (1) is an H7 subtype avian influenza virus monoclonal antibody.
3. The method for screening hybridoma cell lines secreting paired monoclonal antibodies according to claim 1, wherein:
the antibody blocking agent in the step (3) is a goat anti-mouse secondary antibody;
and (5) the anti-mouse secondary antibody is a goat anti-mouse secondary antibody.
4. The method for screening hybridoma cell lines secreting paired monoclonal antibodies according to claim 1, wherein:
the coupling ratio of coupling described in step (1) was Oleyl-PEG 4000-NHS: the capture antibody is 5-7: 5;
the addition amount of the Oleyl-PEG4000-NHS-Ab1 in the step (2) is calculated according to the addition amount of 0.02-0.03 mg in each hole of a 96-hole plate;
the adding amount of the antibody blocking agent in the step (3) is calculated according to the addition of 0.2mg in each hole of a 96-hole plate;
the adding amount of the antigen in the step (4) is calculated according to the addition of 0.0625 mg-0.6 mg of the antigen in each hole of a 96-hole plate.
5. The method for screening hybridoma cell lines secreting paired monoclonal antibodies according to claim 1, wherein:
the culture time in the step (2) is 30-60 min;
the culture time in the step (3) is 45-60 min;
the culture time in the step (4) is 60 min;
the culture time in the step (5) is 60 min.
6. The method for screening hybridoma cell lines secreting paired monoclonal antibodies according to claim 1, wherein:
the fluorescent substance in the step (5) is fluorescein isothiocyanate, tetraethyl rhodamine or tetramethyl rhodamine isothiocyanate;
the washing in the steps (2), (3), (4) and (5) is performed by selecting a phosphate buffer solution or an incomplete culture medium.
7. The method for screening hybridoma cell lines secreting paired monoclonal antibodies according to claim 1, wherein:
the step (7) of isolating the cells of the positive clonal cell mass is specifically performed by: adding a semi-solid culture medium into hybridoma cell clones containing positive clone cell masses, selecting cells of the positive clone cell masses, and transferring the cells to a new complete culture medium;
the semisolid culture medium is a methylcellulose semisolid culture medium.
8. The method for screening hybridoma cell lines secreting paired monoclonal antibodies according to claim 7, wherein:
the preparation method of the methyl cellulose semisolid culture medium comprises the following steps: mixing incomplete culture medium and a methylcellulose solution with the mass concentration of 2.7% according to the volume ratio of 1: 1, uniformly mixing to obtain the methyl cellulose semisolid culture medium.
9. The use of the method of any one of claims 1 to 8 for directly screening cell lines secreting paired monoclonal antibodies in the field of immunology.
10. Use according to claim 9, characterized in that: the method is used for rapidly screening hybridoma cells secreting monoclonal antibodies paired with capture antibodies.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112129952A (en) * | 2020-09-21 | 2020-12-25 | 普健生物（武汉）科技有限公司 | Chemiluminescence kit for detecting human soluble CD14 |
| CN115125251A (en) * | 2022-08-22 | 2022-09-30 | 广东忠信生物科技有限公司 | Method for efficiently obtaining specific fully human monoclonal antibody gene and application |
| CN115166241A (en) * | 2022-08-22 | 2022-10-11 | 广东忠信生物科技有限公司 | Efficient screening technology for simultaneously screening memory B cells and plasma cells and application |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0566205A1 (en) * | 1992-04-17 | 1993-10-20 | Akzo Nobel N.V. | Method for the elimination of non-specific reactions in immuno-assays |
| US20060035389A1 (en) * | 2002-05-15 | 2006-02-16 | Paul Peluso | Immobilization of glycorproteins |
| CN101389754A (en) * | 2005-12-29 | 2009-03-18 | 人类起源公司 | Co-culture of placental stem cells and stem cells from a second source |
| CN103266088A (en) * | 2013-04-15 | 2013-08-28 | 北京世纪元亨动物防疫技术有限公司 | H7 subtype avian influenza virus monoclonal antibody of and kit |
| CN104312979A (en) * | 2014-09-30 | 2015-01-28 | 江苏省农业科学院 | Anti-H7 subtype avian influenza virus monoclonal antibody and application thereof |
| CN104977408A (en) * | 2015-06-15 | 2015-10-14 | 暨南大学 | Method for screening hybridoma cells secreting specific monoclonal antibodies, and application thereof |
| CN105264358A (en) * | 2013-02-18 | 2016-01-20 | 赛拉诺斯股份有限公司 | Image analysis and measurement of biological samples |
| CN107475203A (en) * | 2017-07-19 | 2017-12-15 | 武汉科前生物股份有限公司 | A kind of H7 avian influenza virus monoclonal antibody and application |
| CN107904209A (en) * | 2017-11-03 | 2018-04-13 | 武汉科前生物股份有限公司 | A kind of H7 subtype avian influenza virus monoclonal antibody and application |
-
2019
- 2019-11-05 CN CN201911068973.8A patent/CN110879293A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0566205A1 (en) * | 1992-04-17 | 1993-10-20 | Akzo Nobel N.V. | Method for the elimination of non-specific reactions in immuno-assays |
| US20060035389A1 (en) * | 2002-05-15 | 2006-02-16 | Paul Peluso | Immobilization of glycorproteins |
| CN101389754A (en) * | 2005-12-29 | 2009-03-18 | 人类起源公司 | Co-culture of placental stem cells and stem cells from a second source |
| CN105264358A (en) * | 2013-02-18 | 2016-01-20 | 赛拉诺斯股份有限公司 | Image analysis and measurement of biological samples |
| CN103266088A (en) * | 2013-04-15 | 2013-08-28 | 北京世纪元亨动物防疫技术有限公司 | H7 subtype avian influenza virus monoclonal antibody of and kit |
| CN104312979A (en) * | 2014-09-30 | 2015-01-28 | 江苏省农业科学院 | Anti-H7 subtype avian influenza virus monoclonal antibody and application thereof |
| CN104977408A (en) * | 2015-06-15 | 2015-10-14 | 暨南大学 | Method for screening hybridoma cells secreting specific monoclonal antibodies, and application thereof |
| CN107475203A (en) * | 2017-07-19 | 2017-12-15 | 武汉科前生物股份有限公司 | A kind of H7 avian influenza virus monoclonal antibody and application |
| CN107904209A (en) * | 2017-11-03 | 2018-04-13 | 武汉科前生物股份有限公司 | A kind of H7 subtype avian influenza virus monoclonal antibody and application |
Non-Patent Citations (1)
| Title |
|---|
| 孙黎飞主编: "《细胞免疫学实验研究方法》", 甘肃科学技术出版社, pages: 77 - 100 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112129952A (en) * | 2020-09-21 | 2020-12-25 | 普健生物（武汉）科技有限公司 | Chemiluminescence kit for detecting human soluble CD14 |
| CN112129952B (en) * | 2020-09-21 | 2023-06-06 | 普健生物（武汉）科技有限公司 | Chemiluminescent kit for detecting human soluble CD14 |
| CN115125251A (en) * | 2022-08-22 | 2022-09-30 | 广东忠信生物科技有限公司 | Method for efficiently obtaining specific fully human monoclonal antibody gene and application |
| CN115166241A (en) * | 2022-08-22 | 2022-10-11 | 广东忠信生物科技有限公司 | Efficient screening technology for simultaneously screening memory B cells and plasma cells and application |
| CN115166241B (en) * | 2022-08-22 | 2023-03-24 | 广东忠信生物科技有限公司 | Efficient screening technology for simultaneously screening memory B cells and plasma cells and application |
| CN115125251B (en) * | 2022-08-22 | 2023-11-03 | 广东忠信生物科技有限公司 | Method for efficiently obtaining specific fully human monoclonal antibody gene and application |
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