CN110879293A - Method for screening hybridoma cell strain secreting pairing monoclonal antibody and application - Google Patents

Abstract

The invention discloses a method and application for screening hybridoma cell lines secreting paired monoclonal antibodies. In the method provided by the invention, Oleyl-PEG4000-NHS is coupled with a capture antibody, the conjugate is added to the hybridoma cell clone to be screened, and cultured; the supernatant is discarded and washed; Wash; add antigen that can be combined with capture antibody, culture; discard supernatant, wash; add fluorescent substance-labeled anti-mouse secondary antibody, culture; discard supernatant, wash; add incomplete medium, observe cells under visible light and fluorescence The cells of the positive clones were isolated and cultured to obtain a hybridoma cell line secreting paired monoclonal antibodies. The method is fast, simple and accurate, and can directly screen positive hybridoma cell lines that secrete antibodies paired with the capture antibody.

Description

A method and application for screening hybridoma cell lines secreting paired monoclonal antibodies

technical field

The invention belongs to the technical field of immunological detection, and particularly relates to a method and application for screening hybridoma cell lines secreting paired monoclonal antibodies.

Background technique

和Milstein，首先报道了应用小鼠骨髓瘤细胞和绵羊红细胞致敏的小鼠脾细胞融合，结果发现一部分融合的细胞既能生长，又能分泌抗羊红细胞抗体。这一发现使制备单一抗原决定簇的单克隆抗体的难题获得了解决，开创了利用细胞融合技术制备单克隆抗体技术先河。杂交瘤技术作为一项突破性的生物技术，为生物科学、临床医学等众多学科的研究提供了广阔应用前景。In 1975, German scholar

and Milstein, who first reported the fusion of mouse spleen cells sensitized with mouse myeloma cells and sheep erythrocytes, and found that some of the fused cells could both grow and secrete anti-sheep erythrocyte antibodies. This discovery solved the difficult problem of preparing monoclonal antibodies with a single antigenic determinant, and created a precedent for the preparation of monoclonal antibodies by cell fusion technology. As a breakthrough biotechnology, hybridoma technology provides broad application prospects for research in many disciplines such as biological sciences and clinical medicine.

Monoclonal antibodies have the characteristics of high purity, strong specificity and high titer, and have been widely used in the development of immunological detection products, the innovation of immunological technology and the improvement of medical treatment and diagnosis methods. It can be said that the application of monoclonal antibodies has become an indispensable and important means in biology and medicine.

At present, the commonly used methods for screening monoclonal antibodies are enzyme-linked immunosorbent assay (ELISA) and other methods. The double-antibody sandwich principle is widely used in the field of basic biological research and the development of immunoassay products. These methods take a long time, can measure only one antibody at a time, or require large sample volumes. Traditional screening of paired antibodies requires first screening a large number of positive hybridoma cell lines that secrete monoclonal antibodies, then preparing a large number of antibodies for purification, and then pairing them by double-antibody sandwich ELISA. The process is complicated, the success rate is low, and it is time-consuming and labor-intensive. Therefore, it is necessary to establish a method that can directly screen cell lines secreting paired monoclonal antibodies to improve the screening efficiency and screening accuracy.

SUMMARY OF THE INVENTION

The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and to establish a method for screening hybridoma cell lines secreting paired monoclonal antibodies.

Another object of the present invention is to provide the application of the above-mentioned method for screening hybridoma cell lines secreting paired monoclonal antibodies.

The object of the present invention is achieved through the following technical solutions:

A method for screening hybridoma cell lines secreting paired monoclonal antibodies, comprising the steps of:

(1) Conjugate Oleyl-PEG4000-NHS with the capture antibody to obtain Oleyl-PEG4000-NHS-Ab1, where Ab1 is the capture antibody;

(2) Washing the hybridoma cell clones to be screened; adding Oleyl-PEG4000-NHS-Ab1, and culturing; discarding the supernatant and washing the hybridoma cell clones;

(3) adding an antibody blocking agent and culturing; discarding the supernatant and washing the hybridoma cell clone;

(4) adding an antigen that can be combined with the capture antibody, and culturing; discarding the supernatant and washing the hybridoma cell clone;

(5) adding a fluorescent substance-labeled anti-mouse secondary antibody, and culturing; discarding the supernatant and washing the hybridoma cell clone;

(6) Add the incomplete medium, observe the cell clusters under visible light and fluorescence, take pictures, mark the cell clusters that produce fluorescence as positive clones, and mark the cell clusters that do not produce fluorescence as negative clones;

(7) The cells of the positive clonal cell mass are isolated and cultured to obtain a hybridoma cell line that secretes a monoclonal antibody capable of pairing with the capture antibody.

The capture antibody described in 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-PEG4000-NHS: capture antibody=molar ratio 5～7:5; more preferably Oleyl-PEG4000-NHS: capture antibody=molar ratio 6: 5.

The coupling method in the coupling described in step (1) is conventional coupling, that is, Oleyl-PEG4000-NHS and the capture antibody can be directly coupled in proportion.

The addition amount of Oleyl-PEG4000-NHS-Ab1 described in step (2) is preferably calculated by adding 0.02-0.03 mg to each well of a 96-well plate; more preferably, adding 0.025 mg to each well of a 96-well plate.

The culture described in steps (2), (3), (4) and (5) is cultured under cell culture conditions, such as 37° C., 5% CO ₂ .

The culturing time in step (2) is preferably 30-60 min; more preferably 60 min.

The antibody blocking agent described in step (3) is preferably an anti-mouse secondary antibody; more preferably a goat anti-mouse IgGFc secondary antibody.

The added amount of the antibody blocking agent is preferably calculated by adding 0.2 to 0.4 mg to each well of a 96-well plate; more preferably, it is calculated by adding 0.2 mg to each well of a 96-well plate;

The culturing time in step (3) is preferably 45-60 min; more preferably 60 min.

The amount of the antigen that can be combined with the capture antibody described in step (4) is preferably calculated by adding 0.0625 mg to 0.6 mg to each well of a 96-well plate; more preferably, it is calculated by adding 0.3 mg to 0.6 mg to each well of a 96-well plate . The higher the antigen purity, the smaller the amount added per well.

The incubation time described in step (4) is preferably 60 min.

The fluorescent substances described in step (5) include fluorescein isothiocyanate (FITC), tetraethyl rhodamine (RB200), and tetramethyl rhodamine isothiocyanate (TRITC).

The anti-mouse secondary antibody described in step (5) is preferably a goat anti-mouse IgGFc secondary antibody.

The incubation time described in step (5) is preferably 60 min.

The washing described in steps (2), (3), (4) and (5) is preferably phosphate buffered saline (PBS) or incomplete medium.

The phosphate buffered saline (PBS) was 0.015M phosphate buffered saline at pH 7.4.

The preparation method of the incomplete medium is as follows: the mixture of streptomycin and penicillin is mixed with RPMI-1640 medium according to a volume ratio of 1:99, wherein in the mixture of streptomycin and penicillin, the concentration of streptomycin and penicillin All are 1U/ml.

Described in the step (7), the cells of the positive clone cell cluster are separated, and the specific operation is preferably as follows: adding a semi-solid medium to the hybridoma cell clone containing the positive clone cell cluster, picking the cells of the positive clone cell cluster, and moving to New complete medium.

The semi-solid medium is preferably methyl cellulose semi-solid medium.

The preparation method of the methyl cellulose semi-solid medium is as follows: the incomplete medium and the methyl cellulose solution with a mass concentration of 2.7% are uniformly mixed according to a volume ratio of 1:1 to obtain a methyl cellulose semi-solid culture medium. base.

The added amount of the semi-solid medium is suitable to just cover the bottom of the culture plate. Add 100 μl to each well of a 96-well plate.

The above method for directly screening cell lines secreting paired monoclonal antibodies is applied in the field of immunology, and is used to rapidly screen hybridoma cells that can secrete monoclonal antibodies paired with capture antibodies.

Oleyl-PEG4000-NHS is an amphiphilic substance, one end of which is lipophilic and can be inserted into the cell membrane surface, and the other end of the PEG polymer is hydrophilic, and its end is a carboxyl group activated by NHS, which can be combined with the amino group on the antibody . After 5 to 6 days of cell fusion, add the coupled Oleyl-PEG4000-NHS-Ab1, Oleyl-PEG4000-NHS-Ab1 is inserted into the cell membrane surface, and an antibody blocking agent (anti-mouse secondary antibody) is added to the antibody (the Fc segment of Ab1). ) to block, through the principle of specific binding of antibodies and antigens, add antigens that can specifically bind to Ab1, and bind antigens to antibodies specifically, and the antigens will capture the antibodies secreted by hybridoma cells that can be paired with the capture antibody (Ab1) ( Ab2, secreted by hybridoma cells), wash off the unbound antibody (Ab2), then add fluorescent secondary antibody, and use the principle of specific binding between fluorescent secondary antibody and secreted antibody to stain and locate positive hybridoma cell lines. Finally, methylcellulose semi-solid medium was added, and the positive hybridoma cell lines were directly picked out and cultured in new culture wells. In this way, not only positive hybridoma cell lines can be directly screened, but also positive hybridoma cell lines secreting paired antibodies can be directly screened, and the screened cells can be cloned by this method.

The present invention has the following advantages and effects relative to the prior art:

Conventional monoclonal screening generally takes at least one month to screen positive hybridoma cell lines. After establishing stable cell lines, it takes half a month for the quantitative production of monoclonal antibodies. After purification and quantification, the kits are used to screen for paired antibodies. In the present invention, an amphiphilic substance (Oleyl-PEG4000-NHS) can rapidly and directly screen the hybridoma cells that secrete the monoclonal antibody paired with the capture antibody. Compared with the traditional method of screening paired antibodies, it reduces the screening, antibody preparation, antibody purification, antibody sandwich ELISA pairing and other processes in the traditional monoclonal antibody preparation process, which greatly reduces the workload and significantly improves the probability of successful screening. The method of the invention can not only directly screen positive hybridoma cell lines, but also directly screen positive hybridoma cell lines secreting paired antibodies, and at the same time, the screened cells can be cloned by the method, which is limited compared with the traditional method. The dilution method greatly reduces the workload, and positive clones are not easily lost. The method of the invention can screen hybridoma cell lines that secrete paired antibodies within 10 to 15 days, shortens the time of screening and pairing identification; can accurately evaluate the antibody secretion of each cell in the well and the variation of cell clones, combined with semi-solid The medium facilitates selection of stable hybridoma cells secreting paired antibodies.

Description of drawings

Fig. 1 is the SDS-PAGE detection result of the 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-Ab1.

Fig. 2 is the UV-Vis spectrophotometer scanning result of the Oleyl-PEG4000-NHS-Ab1 solution prepared in Example 1; wherein, curve A is Oleyl-PEG4000-NHS, curve B is Oleyl-PEG4000-NHS-Ab1, curve C is Ab1.

Fig. 3 is a photograph of the direct immunofluorescence identification result of the Oleyl-PEG4000-NHS-Ab1 solution prepared in Example 1; wherein, A1 is SP2/0, first add Oleyl-PEG4000-NHS-Ab1 to incubate for 1 h, and then add 100×FITC Labeled goat anti-mouse IgGFc was incubated for 45 min, and the fluorescence image was 200× under the microscope; A2 was the 400× fluorescence image corresponding to A1; B1 was the 200× visible light image corresponding to A1, B2 was the 400× visible light image corresponding to A2; A3 was SP2/0 The cells were added with diluted 100 × FITC-labeled goat anti-mouse IgGFc and incubated for 45 min. The microscope 200 × fluorescence image, A4 is the 400 × fluorescence image corresponding to A3; B3 is the 200 × visible light image corresponding to A3, and B4 is the 400 × visible light image corresponding to A4.

Figure 4 shows the results of some positive clone cell clusters screened 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 conditions, and B and B are the same. D is observed under visible light conditions.

Figure 5 shows the direct immunofluorescence results of blocking antibodies with different concentrations of goat anti-mouse IgGFc in Example 2; wherein, A is the blocking results of antibodies blocked with 0.125 mg/ml goat anti-mouse IgGFc, and B is the blocking results with 0.25 mg/ml Blocking result of goat anti-mouse IgGFc blocking antibody, C is the blocking result with 0.5 mg/ml goat anti-mouse IgGFc blocking antibody, D is the blocking result with 1 mg/ml goat anti-mouse IgGFc blocking antibody, E is the blocking result with 2 mg/ml goat anti-mouse IgGFc Figure of the blocking result of blocking antibody with 4 mg/ml goat anti-mouse IgGFc, F is the blocking result of blocking antibody with 4 mg/ml goat anti-mouse IgGFc.

Fig. 6 is the direct immunofluorescence results of blocking antibodies with different concentrations of horse serum in Example 2; wherein, A is the blocking results of antibodies blocked with 0.125 mg/ml horse serum, and B is the blocking results of antibodies blocked with 0.25 mg/ml horse serum , C is the blocking result of antibodies blocked with 0.5mg/ml horse serum, D is the blocking result of antibodies blocked with 1 mg/ml horse serum, and E is the blocking result of antibodies blocked with 2 mg/ml horse serum , F is the result of blocking antibody with 4 mg/ml horse serum.

FIG. 7 is a graph showing the direct immunofluorescence results of the antibody without blocking agent in Example 2. FIG.

Detailed ways

The present invention will be described in further detail below with reference to the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field. Unless otherwise specified, the reagents and materials used in the present invention can be obtained commercially, wherein:

The preparation method of phosphate buffered saline (PBS, 0.015mol/L, pH7.4) is as follows: Weigh NaCl 8.0g, Na ₂ HPO ₄ 12H ₂ O 2.9g, KCl 0.2g, KH ₂ PO ₄ 0.2g, add Dissolve in deionized water, adjust pH to 7.4, and dilute to 1000mL.

The preparation method of the incomplete medium or basal medium is as follows: the mixture of streptomycin and penicillin is mixed with RPMI1640 medium according to the volume ratio of 1:99, and the concentration of streptomycin and penicillin in the mixture of streptomycin and penicillin is the same. is 1U/mL.

Complete medium: fetal bovine serum and the above-mentioned incomplete medium are evenly mixed at a volume ratio of 1:9 to obtain a complete medium;

HAT medium: HAT, fetal bovine serum and the above-mentioned incomplete medium are mixed evenly in a volume ratio of 2:10:88 to obtain HAT medium;

HT medium: HT, fetal bovine serum and the above-mentioned incomplete medium are mixed uniformly in a volume ratio of 2:10:88 to obtain HT medium;

The preparation method of the methyl cellulose semi-solid medium is as follows: the above-mentioned incomplete medium and the methyl cellulose solution with a mass concentration of 2.7% are uniformly mixed according to a volume ratio of 1:1 to obtain a methyl cellulose semi-solid medium.

PBST-T washing solution: take 0.5 mL of Tween-20, 0.015 mol/L, pH 7.4 PBS and dilute to 1000 mL.

Example 1 Screening of hybridoma cell lines secreting paired monoclonal antibodies

(1) Acquisition of hybridoma cells

1) Immunization: The immunogen is the avian influenza H7N9 subtype standard detection antigen provided by Shenzhen Entry-Exit Inspection and Quarantine Bureau (Harbin Veken Biological Co., Ltd.), according to the instructions, add 2ml of 0.015mol/ml PBS to each bottle of standard antigen to dissolve, and then use BCA method was used to determine the concentration of antigen and each screening peptide. Immunize 5-week-old BALB/C female mice (purchased from Southern Medical University, the same below) with the treated avian influenza antigen. The immunization strategy is as follows:

Table 1 H7 subtype avian influenza virus immunization strategy

10 days after the fourth immunization of the mice, 50 μl of blood was collected through the tail vein, first placed in a 37 °C constant temperature incubator for 1 h, and then placed in a refrigerator at 4 °C overnight to allow the serum to precipitate naturally, and centrifuge at 1500 rpm the next day. The upper serum was collected after 15 minutes, and stored at -20°C after aliquoting. Serum titer was measured by indirect ELISA, and the mouse with the highest titer, that is, the best immune effect, was used for fusion.

Mice were killed by enucleation before fusion. All blood was collected and placed in a constant temperature incubator at 37 °C for 1 h, then placed in a refrigerator at 4 °C overnight, and the serum was fully analyzed. Store at -20°C.

2) Fusion:

Feeder cells were prepared the day before fusion. The scissors, tweezers, needles and foam boxes were placed on a clean table for ultraviolet irradiation for 30 minutes in advance, and then the mice were killed by enucleating the eyeballs and immersed in a beaker containing 75% alcohol for 5 minutes. Take out the mouse and place it on the foam box, fix its limbs with needles, gently lift its abdominal fur with tweezers, and it can be seen that the fur is separated from the peritoneum. Tear off the skin at the small mouth. A syringe sucked 5 mL of basal medium, injected it into the abdominal cavity of the mouse, and gently rubbed the abdominal cavity with a cotton ball for 1 min, and then aspirated the basal medium and placed it in a 15 ml centrifuge tube. Then use a syringe to draw 5ml of basal medium and repeat the above operation. The collected basal culture was centrifuged at 1000 rpm on a horizontal centrifuge for 7 min. Resuspend the peritoneal cells in HAT medium, count, dilute the cells to 2×10 ⁸ cells/ml, add 100 μl to each well, and place in a 37°C, 5% CO ₂ cell incubator for culture, and prepare the feeder one day before fusion. cell.

The mice with the best immune effect were selected for fusion. Pre-autoclave scissors, 4 tweezers, 2 glass dishes, one 400-mesh iron gauze, and one glass grinding rod. Mice were killed by enucleation, and the blood of mice was collected as a positive control. Try to drain the mouse blood as much as possible to avoid too many blood cells in the mouse spleen and interfere with fusion. The sacrificed mice were placed in 75% alcohol for 5 minutes, then the peritoneum was opened in the same way as feeder cells, and the peritoneum was cut with clean scissors and tweezers. Gently wash the spleen in a dish prefilled with basal culture. Then put the spleen on the iron mesh, add an appropriate amount of basal medium, cut the spleen into pieces with scissors, and then gently grind the spleen with a grinding rod until no large pieces of tissue are seen. Collect the cell suspension in a centrifuge tube, let it stand for 5 minutes, remove the bulk tissue pellet, record the total volume, and count on a hemocytometer.

One week before fusion, myeloma cells SP2/0 (purchased from Shanghai Institute of Cell Biology) were recovered, and several dishes of SP2/0 in logarithmic growth phase were taken and washed twice with basal medium to remove dead cells. Then add 3ml of basal medium to each dish, gently blow down the cells with a 1ml pipette tip, collect the cells in a 50ml centrifuge tube, read the volume of the cell suspension, take an appropriate amount of the cell suspension and count it with a hemocytometer.

According to the counting results, splenocytes were mixed with SP2/0 in a ratio of 5-10:1, placed in a horizontal centrifuge at 1000 rpm, and centrifuged for 7 min. After centrifugation, the supernatant was discarded and the cells were loosened by tapping the centrifuge tube. Place the centrifuge tube on a beaker filled with deionized water at 37°C, slowly add PEG2000 solution (50% concentration, Sigma) pre-incubated to 37°C, 1ml PEG2000 solution (one spleen of a mouse uses 1ml PE) , first slow then fast, add it within 45 seconds, and then let it stand for 90 seconds. Prepare 15ml of incomplete medium in advance, incubate to 37°C, add a centrifuge tube within 2 to 4 minutes after standing, first slow then fast, and gently shake the centrifuge tube while adding. After the termination was completed, centrifuge in a horizontal centrifuge at 1000 rpm for 7 min. After centrifugation, the supernatant was discarded, the cells were resuspended in HAT medium, and evenly spread on the feeder cell plate. Place in a 37°C, 5% _CO2 cell incubator.

(2) Conjugation and identification of Oleyl-PEG4000-NHS and avian influenza virus

Weigh 10 mg of the coupling agent Oleyl-PEG4000-NHS, dissolve it in 1 ml of PBS buffer, take 100 μl into a small reagent bottle, put it in a rotor and add 2 mg of avian influenza virus antigen (Harbin Veken Biological Co., Ltd.) slowly on the stirrer. Place the reagent bottle on the avian influenza virus antigen. Place the vial at 4°C and stir overnight.

The purpose of ultrafiltration is to remove the unconjugated Oleyl-PEG4000-NHS molecule, whose molecular weight is about 4KD, while the molecular weight of AIV is above 5000KD, so choosing a 30KD pore size ultrafiltration tube can remove the uncoupled coupling agent, ultrafiltration The process is as follows:

1) Take 5ml of PBS (0.015M, pH7.4) into a 30KD ultrafiltration tube, and centrifuge at 5000rpm for 10min. Repeat the operation 3 times.

2) Add the sample to the ultrafiltration tube and centrifuge at 5000rpm for 15min. When the remaining retentate is less than 100μl, add 5ml PBS to lightly rinse the membrane in the ultrafiltration tube, continue to centrifuge, repeat the ultrafiltration 3 times, and collect the retentate (control within 500μl) .

3) The ultrafiltration tube membrane was repeatedly rinsed with PBS to fully remove residual impurities, and then centrifuged. Repeat the operation 3 times.

4) Fill the ultrafiltration tube with 20% ethanol treated with a 0.45 μm filter membrane, and store it in a refrigerator at 4°C.

Use UV-Vis spectrophotometer to scan and identify. 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 their own specific absorption peaks, and the success of the coupling can be determined by detecting the presence or absence of each characteristic peak and its out position.

The BSA standard protein concentration in the kit was diluted with PBS to 1.5mg/mL, 1mg/mL, 0.75mg/mL, 0.5mg/mL, 0.25mg/mL, 0.125mg/mL, 0.0625mg/mL, 0mg/mL Make multiple gradients for the sample to be tested, and then add 25 μl of each sample to the wells of the microtiter plate. The color developing solution is prepared and used now. Mix A and B by volume 50:1, add 200 μL to each well, place in a 37°C incubator, and react in the dark for 30 minutes. After the reaction, place the microplate in a microplate reader to detect the absorption light value at 570 nm, draw a standard curve of the absorption light value of the standard protein curve, and calculate the concentration of the sample to be tested according to the fitting curve formula.

(3) Screening of positive AIV hybridoma cells by cell surface immunofluorescence method:

After 5-7 days of fusion, positive hybridoma cells were screened by cell surface fluorescence adsorption method. Gently aspirate the supernatant from the wells of the cell culture plate, add 100 μl of incomplete medium to gently wash, then aspirate the medium, repeat the operation, and wash twice. 100 μl of Oleyl-PEG4000-NHS-AIV diluted to 0.1 mg/ml was added to each well, and cultured in a 37° C., 5% CO ₂ cell incubator for 1 h. Place the vial at 4°C and stir overnight. Repeat the cleaning process in step 1, two times. Add 200-fold diluted FITC-labeled goat anti-mouse IgG antibody, 100 μl per well. Place in a 37°C, 5% _CO2 cell incubator for 1 h. Repeat the washing process in the first step, wash twice, add 100 μl of incomplete medium, and then place the plate under a fluorescence microscope and observe it in the excited state. Observe and label the position of the green fluorophore at the bottom of the plate.

The positive AIV hybridoma cell clusters were picked by semi-solid medium method. After labeling the positive hybridoma cell clusters, move the cell culture plate to the ultra-clean workbench, remove the medium, add 100 μl of the prepared cellulose semi-solid medium, and then use a 10 μl pipette tip to gently aspirate the cell clusters at the marked position. , moved to the cell culture plate pre-added with HT medium, and recorded. After culturing the cells for about 3 days, the fluorescent staining operation was performed again, and the cells were cloned in combination with the semi-solid medium method until 16 stable cell lines were obtained. The selection results were detected by indirect ELISA (the specific process is as follows), and the positive cell line 11A11 with good specificity was obtained.

Indirect ELISA:

1) Coating antigen: Dissolve with coating solution (pH9.6, 0.05M carbonate buffer: Na ₂ CO ₃ 1.5 g, NaHCO ₃ 2.9 g and ddH ₂ O water 800 mL, adjust pH 9.6 and add water to 1000mL) Dilute H5, H7N9, H9N2 (Harbin Veken Bio Co., Ltd.) to 0.002mg/ml, add 100μL to each well of the ELISA plate, and store in the refrigerator at 4°C overnight (about 12h), discard the coating solution , wash the plate 3 times with PBS-T, 3 min each time;

2) Blocking: prepare 5% (v/w) nonfat milk powder for blocking, 300 μL per well, incubate in a 37°C incubator for 1 h, discard the blocking solution, and wash the plate 3 times with PBS-T, 3 min each time;

3) Primary antibody incubation: add hybridoma supernatant, SP2/0 cell supernatant as a negative control, add 100 μL to each well, incubate at 37°C for 1 h, discard the cell supernatant, and wash the plate with PBS-T 3 times, 3min each time;

4) Secondary antibody incubation: Dilute the goat anti-mouse IgG secondary antibody by 8000 times with PBS-T, add 100 μL to each well, incubate in a 37°C incubator for 45 min, discard the secondary antibody solution, and wash the plate 5 times with PBS-T. 3min each time;

5) Color development: add color developing solution: A solution (A solution: dissolve TMB powder in DMSO to make the final concentration 11mg/ml, and then add 1/10 volume of glycerol (all are protected from light, and then put into a black bottle) Store in the dark at 4 degrees)) 50μL/well; B solution (pH 5.5, 0.2mol/L disodium hydrogen phosphate and 0.1moL/L citric acid buffer to prepare a solution with a concentration of urea peroxide of 0.74mg/mL) 50μL /hole. React at room temperature for 10 min in the dark;

6) Termination: stop by adding ELISA stop solution (2% v/v sulfuric acid), 50 μL per well;

7) Detection: microplate reader, absorption value at wavelength 450nm.

The cell lines with low affinity for antigen H5 and H9N2 (small OD value) and strong affinity for H7N9 (large OD value) are cell lines with good specificity.

(4) Preparation of capture antibody:

1) Select BALB/C mice over 8 weeks old, and inject the mice with incomplete Freund's adjuvant by intraperitoneal injection at a dose of 500 μL/mice one week in advance. Select the hybridoma cell 11A11 which is round and shiny, in good condition and positive by indirect ELISA. RPMI-1640 was washed once to remove most of the dead cells; then a certain volume of RPMI-1640 was added to gently blow up the cells, the quantitative suspension was taken to calculate the total number of cells, placed in a centrifuge tube and centrifuged at 1000 rpm for 7 min, the supernatant was discarded, and the RPMI- 1640 to resuspend cells to ¹ x 106 cells/mL. The mice were also injected intraperitoneally at a dose of 500 μL/mouse, and the abdomen was gently distributed to make the cells evenly distributed and labeled.

2) 7-10 days after the mice were inoculated with cells, the abdomen began to expand and become larger. When the abdomen of the mice was obviously distended, 75% alcohol was used to disinfect the abdomen of the mice, and a 12-gauge medical needle was used to drain the ascites. Continue to raise the mice, observe the state of the mice on the next day, and collect ascites again if the abdomen expands again. The ascites fluid just collected was placed in a centrifuge, centrifuged at 4000 rpm for 30 min at 4°C, and the supernatant was aliquoted and stored at -20°C.

3) The ascites was thawed at 4°C and centrifuged at 10,000rpm for 15min at 4°C, the supernatant was collected, and the volume was recorded. The collected supernatant was placed on a magnetic stirrer and stirred in an ice-water bath, and an equal volume of saturated ammonium sulfate solution was slowly added dropwise. The next day, centrifuge at 10,000 rpm at 4°C for 30 min, discard the supernatant, dissolve the precipitate with 2 times the initial volume of PBS, and filter through a 0.2 μm filter before proceeding to the next step. After desalting with a desalting column and separating impurity proteins with a Protein G affinity chromatography column, the target antibody collected and eluted is too large to be stored, so the antibody needs to be concentrated by a 30KD ultrafiltration tube. The ultrafiltered antibody 11A11 was collected. The 11A11 ascites-type monoclonal antibody was prepared as a capture antibody for use, and double-distilled water was used to prepare a 11A11 strain antibody solution with a concentration of 5 mg/ml.

(5) Conjugation and identification of Oleyl-PEG4000-NHS and capture antibody (Ab1)

1) Oleyl-PEG4000-NHS coupled with Ab1

2 mg of Oleyl-PEG4000-NHS (purchased from Japan NF Company) was weighed and dissolved in 200 μl of PBS to obtain an Oleyl-PEG4000-NHS solution.

150 μl of Oleyl-PEG4000-NHS was added to the bottle and placed on an ice box with stirring; 250 μl of 5 mg/ml 11A11 strain antibody solution was slowly added to the above solution, and stirred at 4°C overnight. 30KD ultrafiltration tube ultrafiltration to obtain Oleyl-PEG4000-NHS-11A11 solution. Then, filter sterilization was carried out using a 0.22 μm filter membrane, and the mixture was placed at 4°C.

2) Identification of Oleyl-PEG4000-NHS-Ab1

The obtained working solution (ie Oleyl-PEG4000-NHS-11A11 solution) was identified by SDS-PAGE, UV-Vis spectroscopic scanning and identification. SDS-PAGE electrophoresis identification is shown in Figure 1, lane 4 is Oley-PEG4000-NHS-Ab1, and the band lags behind lane 3, which proves that the coupling is successful; UV-Vis spectroscopic scanning identification is shown in Figure 2.

Dilute the obtained working solution with incomplete medium to a concentration of 0.98 mg/ml, add 100 μL of SP2/0 cells, incubate for 1 h at 37 °C, 5% CO ₂ , wash twice with incomplete medium, add 100 × FITC-labeled Goat anti-mouse IgGFc (Solarbiolot No. 20180522) was incubated for 45 min; the control group was SP2/0 cells with an equal volume of incomplete medium added to 100 μL. Add 100×FITC-labeled goat anti-mouse IgGFc and incubate for 45 min; after the incubation, remove the supernatant, add 100 μL of incomplete medium to wash twice, and finally add 100 μL of incomplete medium, observe and take pictures under a fluorescence 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 clusters secreting monoclonal antibodies paired with 11A11 strain antibodies

Before the actual selection, we have optimized the working concentration of Oleyl-PEG4000-NHS-11A11, goat anti-mouse IgG (Fc) antibody working concentration, blocking time, antigen concentration, and fluorescent secondary antibody concentration. optimized conditions.

1) Select 6-week-old BALB/C mice for immunization, and immunize H7 subtype avian influenza virus (purchased from Harbin Veken Biotech Co., Ltd. for avian influenza standard detection antigen) for the first immunization: 800 μg, multi-point injection; booster immunization, 600 μg, multi-point injection; sprint immunization: 300 μg, tail vein injection, 14 days between each immunization. The spleen was taken from the mouse, and the spleen cells were mixed with the prepared SP2/0 cells in logarithmic growth phase (purchased from Shanghai Institute of Cell Biology) according to the cell ratio of 6:1, and polyethylene glycol (PEG-2000) was added. It was fused, and evenly spread to 6 96-well cell culture plates for culture, and the detection was started on the 5th to 6th day after fusion.

2) Add 100 μl of Oleyl-PEG4000-NHS-11A11 with a concentration of 0.25 mg/ml, incubate in a cell incubator at 37° C., 5% CO ₂ for 60 min, discard the supernatant, and wash twice with incomplete medium.

3) Add 100 μl of goat anti-mouse IgG FC (KPL, product number: 5220-0341) blocking antibody at a concentration of 2 mg/ml, incubate in a cell incubator at 37°C, 5% CO ₂ for 60 min, discard the supernatant, use incomplete The medium was washed twice.

4) Add 100 μl of 6mg/ml avian influenza H7 subtype antigen (Harbin Veken Biotechnology Co., Ltd.), incubate in a cell incubator at 37°C, 5% CO ₂ for 60 min, discard the supernatant, and wash twice with incomplete medium (The avian influenza H7 subtype antigen used in this experiment is the standard avian influenza detection antigen, with few active ingredients, so the concentration used is higher; when using this antigen, the amount of the coated plate is 400 ng/well and the coated and purified antigen is 50 ng/ The OD values of the wells are equivalent, so the use of better-purified antigens can be reduced as appropriate, and the amount of fluorescent secondary antibody added can be referred to).

5) Add 100 μL of FITC-labeled goat anti-mouse IgGFc (Laybold Company, Cat. No.: SF131) diluted 100 times, incubate in a cell incubator at 37°C, 5% CO ₂ for 60 min, discard the supernatant, and wash with incomplete medium 1 time.

6) Add 150 μl of incomplete medium, observe the cell clusters under visible light and fluorescence with a fluorescence microscope, and take pictures to mark the negative and positive ones. The cell clusters that produce fluorescence are marked as positive clones, and the cell clusters that do not produce fluorescence are marked as negative clone. The photographing results of some positive clonal cell clusters are shown in Figure 4 .

7) Remove the supernatant from the culture wells containing positive cell clones, add 100 μl of methylcellulose semi-solid medium, and pipette the positively labeled cells into a new cell culture plate according to the previous labeling.

RESULTS: Five hybridoma cell lines were screened. The cell clusters were stained with fluorescence under a fluorescence microscope. The ELISA test showed that the secreted antibodies could react with avian influenza H7 subtype antigens. The tumor cells were used to prepare ascites (refer to the preparation process of ascites of the hybridoma cell line mentioned above) and purify them. Then, the 5 strains of antibodies were paired with the secreted antibodies of the capture antibody 11A11 strain by double-antibody sandwich ELISA method. After verification, these 5 strains of antibodies were All are paired with 11A11 strain secreted antibodies.

Example 2 Determination of suitable antibody blocking agents and concentrations

(1) By the same method as in Example 1, Oleyl-PEG4000-NHS-Ab1 (11A11), and a hybridoma cell clone were obtained.

(2) Add 100 μl of 0.25mg/ml Oleyl-PEG4000-NHS-Ab1 (11A11) to the hybridoma cell clone, incubate it in a cell incubator at 37°C, 5% CO ₂ for 60 min, discard the supernatant, and use the incomplete medium Wash twice; group A: add 100μl 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: add 100μl respectively 0.125mg/ml, 0.25mg/ml, 0.5mg/ml, 1mg/ml, 2mg/ml, 4mg/ml horse serum blocking antibody, the hybridoma cell clone without blocking agent was used as a positive control group, and placed in cell culture Incubate at 37°C, 5% CO ₂ for 60 min, discard the supernatant, and wash twice with incomplete medium. The 11A11 strain antibody was used for direct fluorescence immunization, and the direct immunofluorescence results of group A, group B, and control group were shown in Figures 5, 6, and 7, respectively.

It can be seen that when goat anti-mouse IgG FC is used as the blocking agent, the blocking effect can be achieved when the concentration is above 1 mg/ml, and the blocking effect is better when the concentration is above 2 mg/ml; horse serum cannot achieve the blocking effect.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

Claims (10)

1. a method for screening the hybridoma cell line of secreting paired monoclonal antibodies, is characterized in that: comprise the steps: (1) Conjugate Oleyl-PEG4000-NHS with the capture antibody to obtain Oleyl-PEG4000-NHS-Ab1, where Ab1 is the capture antibody; (2) Washing the hybridoma cell clones to be screened; adding Oleyl-PEG4000-NHS-Ab1, and culturing; discarding the supernatant and washing the hybridoma cell clones; (3) adding an antibody blocking agent and culturing; discarding the supernatant and washing the hybridoma cell clone; (4) adding an antigen that can be combined with the capture antibody, and culturing; discarding the supernatant and washing the hybridoma cell clone; (5) adding a fluorescent substance-labeled anti-mouse secondary antibody, and culturing; discarding the supernatant and washing the hybridoma cell clone; (6) Add the incomplete medium, observe the cell clusters under visible light and fluorescence, take pictures, mark the cell clusters that produce fluorescence as positive clones, and mark the cell clusters that do not produce fluorescence as negative clones; (7) The cells of the positive clonal cell mass are isolated and cultured to obtain a hybridoma cell line that secretes a monoclonal antibody capable of pairing with the capture antibody. 2. the method for screening the hybridoma cell line that secretes paired monoclonal antibodies according to claim 1, is characterized in that: The capture antibody described in step (1) is the H7 subtype avian influenza virus monoclonal antibody. 3. the method for screening the hybridoma cell line that secretes paired monoclonal antibodies according to claim 1, is characterized in that: The antibody blocking agent described in step (3) is goat anti-mouse secondary antibody; The anti-mouse secondary antibody described in step (5) is a goat anti-mouse secondary antibody. 4. the method for screening the hybridoma cell line that secretes paired monoclonal antibodies according to claim 1, is characterized in that: The coupling ratio of the coupling described in step (1) is Oleyl-PEG4000-NHS: capture antibody=molar ratio 5-7:5; The amount of Oleyl-PEG4000-NHS-Ab1 described in step (2) is calculated by adding 0.02 to 0.03 mg to each well of a 96-well plate; The addition amount of the antibody blocking agent described in step (3) is calculated by adding 0.2 mg to each well of the 96-well plate; The amount of antigen added in the step (4) is calculated by adding 0.0625 mg to 0.6 mg to each well of the 96-well plate. 5. the method for screening the hybridoma cell line that secretes paired monoclonal antibodies according to claim 1, is characterized in that: The time of culturing described in step (2) is 30～60min; The time of culturing described in step (3) is 45～60min; The incubation time described in step (4) is 60min; The incubation time described in step (5) was 60 min. 6. The method for screening hybridoma cell lines secreting paired monoclonal antibodies according to claim 1, wherein: The fluorescent substance described in step (5) is fluorescein isothiocyanate, tetraethyl rhodamine or tetramethyl isothiocyanate rhodamine; The washing described in steps (2), (3), (4) and (5) is to select phosphate buffered saline or incomplete medium for washing. 7. The method for screening hybridoma cell lines secreting paired monoclonal antibodies according to claim 1, wherein: The specific operation of separating the cells of the positive clonal cell cluster described in step (7) is as follows: adding a semi-solid medium to the hybridoma cell clone containing the positive clonal cell cluster, picking out the cells of the positive clonal cell cluster, and moving it to a new cell. the complete medium; The semi-solid medium is methylcellulose semi-solid medium. 8. the method for screening hybridoma cell lines secreting paired monoclonal antibodies according to claim 7, is characterized in that: The preparation method of the methyl cellulose semi-solid medium is as follows: the incomplete medium and the methyl cellulose solution with a mass concentration of 2.7% are uniformly mixed according to a volume ratio of 1:1 to obtain a methyl cellulose semi-solid culture medium. base. 9 . The application of the method for directly screening cell lines secreting paired monoclonal antibodies according to any one of claims 1 to 8 in the field of immunology. 10 . 10. The application according to claim 9, wherein the method is used to rapidly screen hybridoma cells that secrete a monoclonal antibody paired with the capture antibody.

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