CN114134163A

CN114134163A - Single antigen specific transgenic hybridoma cell screening method

Info

Publication number: CN114134163A
Application number: CN202111306352.6A
Authority: CN
Inventors: 王战辉; 沈建忠; 温凯; 余文博; 于雪芝; 江海洋; 李园; 李培培
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2021-11-05
Filing date: 2021-11-05
Publication date: 2022-03-04

Abstract

The invention discloses a screening method of a single antigen specific transgenic hybridoma cell, which is characterized in that a transgenic Sp2/0 myeloma cell expressing EGF-R-AVI-tag fusion protein is prepared, a spleen cell fusion technology of a traditional hybridoma technology is combined, the transgenic hybridoma cell capable of displaying secretory specific antibodies on cell membranes is obtained, and a single specific transgenic hybridoma cell is screened by utilizing a single cell sorting system. The invention provides an accurate and high-flux transgenic hybridoma monoclonal antibody production platform based on a transgenic technology. Compared with the traditional hybridoma technology, the preparation time and cost of single antigen specificity hybridoma cell are obviously shortened, the single cell rate and the positive rate are improved, and the titer and the affinity of the prepared monoclonal antibody are also obviously improved. The preparation technology of the transgenic hybridoma antibody provided by the invention can be used for preparing rare monoclonal antibodies with high specificity, high sensitivity and high stability, and has wide application prospect.

Description

Single antigen specific transgenic hybridoma cell screening method

Technical Field

The invention relates to the technical field of biology, in particular to a method for screening single antigen specific transgenic hybridoma cells.

Background

Monoclonal antibodies (mabs) are widely used in the fields of biology, medicine, environmental testing, food science, and the like. In recent years, phage display technology and single B cell technology have provided wide space for the development of monoclonal antibodies, but these methods are still in the initial stage and have certain limitations for the wide production of antibodies. Therefore, the traditional hybridoma technology is still the most important method for preparing monoclonal antibody at present. In the traditional hybridoma technology, the limited dilution method is time-consuming, labor-consuming and low in efficiency for screening the hybridoma cells with antigen specificity, and meanwhile, the secretion amount of the antibody is extremely low in the early stage of the hybridoma cells, so that efficient and accurate detection is difficult to realize. Therefore, efficient screening and selection of antigen-specific hybridoma cells is particularly important.

In recent years, membrane receptor-based flow cytometric sorting (FACS) has been widely used for screening antigen-specific hybridoma cells. The lack of antigen binding receptors on the surface of hybridoma cells results in a higher incidence of false positives in the screening process. In order to realize the display of the antibody on the surface of the hybridoma cell, thereby improving the accuracy of screening the antigen-specific hybridoma cell and reducing false positive. Through a chemical covalent modification method, a fatty chain (Oleyl-PEG4000-NHS) is coupled on the surface of a hybridoma cell membrane, so that the display of the antibody on the surface of the hybridoma cell is realized, and the screening efficiency of the antigen-specific hybridoma cell is obviously improved. However, cell surface-coupled fatty chains are very unstable and are lost as the hybridoma cells divide, and chemical covalent modification can also cause irreversible damage to the normal function and viability of the hybridoma cells. The epidermal growth factor receptor (EGF-R) is a transmembrane glycoprotein, and the transmembrane domain protein of the EGF-R is widely applied to display of foreign proteins on the surface of a cell membrane by a fusion expression technology.

The antigen-specific antibody is displayed on the surface of the transgenic hybridoma, and the rapid identification and accurate analysis of the antibody binding performance and expression level can be realized, so that the accurate identification of the transgenic hybridoma cells with strong secretion capacity and high antibody affinity can be completed.

Therefore, it is important to develop a high-throughput, automatic and precise screening technology for single antigen-specific hybridoma and antibody preparation.

Disclosure of Invention

The invention aims to provide a method for screening single antigen specific transgenic hybridoma cells.

The invention has the following conception: the invention stably integrates the transmembrane region of EGF-R and biotin receptor peptide AVI-tag into a hybridoma cell genome based on PiggyBac transposase, realizes the stable expression of EGF-R-AVI-tag fusion protein on the surface of a transgenic hybridoma cell, and realizes the capture of a secretory antibody by coupling the streptavidin modified antigen with biotin ligase, thereby achieving the display of an antigen specific antibody on the surface of the hybridoma cell and laying a foundation for the efficient and accurate screening of the antigen specific hybridoma cell.

To achieve the object of the present invention, in a first aspect, the present invention provides a single antigen-specific transgenic hybridoma cell screening method, comprising: preparing transgenic Sp2/0 myeloma cells expressing EGF-R-AVI-tag fusion protein, combining spleen cell fusion technology of traditional hybridoma technology to obtain hybridoma cells capable of displaying secretory specific antibody on cell membranes, and screening single specific transgenic hybridoma cells by using a single cell sorting system (such as a Cellenone full-automatic single cell sorting system).

The EGF-R-AVI-tag fusion protein is as follows:

(a) 1, a protein consisting of an amino acid sequence shown in SEQ ID NO;

(b) 1, protein which is derived from (a) and has the same function by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 1.

In the foregoing method, the nucleic acid sequence encoding the EGF-R-AVI-tag fusion protein is introduced into the Sp2/0 myeloma cell by a plasmid or is integrated into the genome of the Sp2/0 myeloma cell by genetic engineering means.

Preferably, the nucleic acid sequence encoding the EGF-R-AVI-tag fusion protein is integrated into the genome of the SP2/0 myeloma cell by the PiggyBac transposition system.

The method, before fusing with splenocytes, further comprises the following steps: transgenic Sp2/0 myeloma cells were sequentially biotinylated in vitro and PE-streptavidin labeled, then enriched by FACS and single PE-streptavidin labeled transgenic Sp2/0 myeloma cells were prepared for subsequent cell fusion.

In a second aspect, the invention provides single specific transgenic hybridoma cells selected according to the method.

In a third aspect, the invention provides the transgenic hybridoma cell screening method or application of the transgenic hybridoma cell screened according to the method in preparation of monoclonal antibodies.

In a fourth aspect, the invention provides a hybridoma cell, 9a 6.

In a fifth aspect, the invention provides an antibody secreted by hybridoma cell 9a 6.

In a sixth aspect, the invention provides any one of the following uses of the antibody:

1) the method is used for detecting chloramphenicol;

2) is used for preparing a chloramphenicol detection reagent or a chloramphenicol detection kit.

In a seventh aspect, the invention provides a detection reagent or kit comprising said antibody.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

the invention is based on a CellenONE full-automatic single cell sorting system, carries out visual screening on single transgenic hybridoma cells, realizes non-destructive cell screening based on an accurate piezoelectric acoustic distribution technology, and has profound significance on screening and preparing high-affinity rare antibodies.

The invention provides an accurate and high-flux transgenic hybridoma monoclonal antibody production platform based on a transgenic technology. Compared with the traditional hybridoma technology, the preparation time and cost of single antigen specificity hybridoma cell are obviously shortened, the single cell rate and the positive rate are improved, and the average titer and the affinity of the prepared monoclonal antibody are also obviously improved. The preparation technology of the transgenic hybridoma antibody provided by the invention is a method for preparing a rare monoclonal antibody with high specificity, high sensitivity and high stability, and the technology has wide prospects in scientific research and practical application.

Drawings

FIG. 1 shows the design of the carrier for the cell membrane surface fusion protein in the preferred embodiment of the present invention.

FIG. 2 is a schematic representation of flow screening of transgenic Sp2/0 myeloma cells according to a preferred embodiment of the present invention. Wherein, A: negative control, B: and (6) positive screening.

FIG. 3 is a graph showing the identification of the expression level of AVI-tag in the first 10 individual transgenic Sp2/0 myeloma cells that exhibit good growth performance in a preferred embodiment of the invention. Wherein, A: identification of mRNA expression levels, B: and (4) identifying the protein expression level.

FIG. 4 is a diagram of a preferred embodiment of the present invention for screening of transgenic hybridoma cells specific for a single chloramphenicol based on CellonONE. Wherein, A: screening schematic for CAP-specific transgenic hybridoma cells, B: brightfield pictures of single chloramphenicol-specific transgenic hybridoma cells in celenone, C: fluorescence field pattern of single chloramphenicol-specific transgenic hybridoma cells in celenone, D-F: schematic of negative controls and pictures in the celenone light and fluorescence fields.

FIG. 5 is an immunofluorescence analysis of chloramphenicol specific transgenic hybridoma cells in a preferred embodiment of the invention. Wherein, A: bright field picture, B: nuclear staining picture, C: fluorescent staining of chloramphenicol-specific transgenic hybridoma cell membrane surface fusion proteins, D: merged images of nuclear staining and cell membrane staining.

FIG. 6 shows a preferred embodiment of the present inventionBased on the comparison of antibody titer and affinity prepared by transgenic technology and traditional hybridoma technology. Wherein, A is the comparison of antibody titer, B is the antibody IC₅₀Comparison of (1).

FIG. 7 is a comparison of the stability of antibodies prepared based on transgenic technology and conventional hybridoma technology in a preferred embodiment of the invention. Wherein, A: analysis of antibody Tm and Tagg values for antibody 2E10 prepared by traditional hybridoma techniques, B: analysis of antibody Tm and Tagg values for antibody 9a6 prepared by transgenic hybridoma technology.

Detailed Description

The invention is based on PiggyBac transposon system, prepares transgenic SP2/0 myeloma cell line with stable expression of EGF-R-AVI-tag fusion protein on the cell membrane surface, combines the traditional cell fusion technology, applies a CellenONE full-automatic single cell sorting system, realizes the high-efficiency and accurate screening of single antigen specific hybridoma cells, establishes a novel single transgenic hybridoma cell antibody preparation technology, and lays a foundation for the preparation of high-affinity rare monoclonal antibodies. Further, the present invention targets Chloramphenicol (CAP) (formula 1) and Balb/c mice as model animals.

One of the technical objects of the present invention is to provide a method for preparing transgenic Sp2/0 myeloma cells.

The second technical object of the present invention is to provide a method for screening individual transgenic Sp2/0 myeloma cells.

The technical purpose of the invention is to provide a method for immunizing BALB/c mice by chloramphenicol immunogen

The fourth technical purpose of the invention is to provide a method for identifying the serum performance of BALB/c mice.

The fifth technical purpose of the invention is to provide a preparation method of a transgenic hybridoma cell and a traditional hybridoma cell.

The sixth technical purpose of the invention is to provide a method for screening single chloramphenicol specific hybridoma cells.

The seventh technical object of the present invention is to provide a method for comparing the transgenic hybridoma technology with the conventional hybridoma technology.

The invention also provides a method for comparing the performances of the monoclonal antibodies prepared by the transgenic hybridoma technology and the traditional hybridoma technology.

The invention adopts the following technical scheme:

transgenic Sp2/0 myeloma cells were prepared as follows: inserting a signal peptide of EGF-R at the N terminal and inserting a transmembrane region of EGF-R at the C terminal of an AVI-tag sequence of a biological receptor peptide, transfecting the N terminal and the C terminal into the genome of Sp2/0 myeloma cells by a PiggyBac transposon system, screening the genome for 21 days by using a cell culture medium containing 10% (v/v) FCS, 2mM glutamine and 2 mu g/mL puromycin, and then carrying out expansion culture on the puromycin-resistant transgenic Sp2/0 myeloma cells by using a conventional complete culture medium.

The screening method for single transgenic Sp2/0 myeloma cells was as follows: transgenic Sp2/0 myeloma cells were first biotinylated in vitro, the biotinylated transgenic Sp2/0 myeloma cells were resuspended in PBS containing 2% BSA and 2mM PE-streptavidin conjugate (PE is fluorescein, i.e., Phycoerythrin (PE)), incubated at 4 ℃ for 20 minutes, washed 3 times with PBS, enriched for PE-streptavidin labeled transgenic Sp2/0 myeloma cells and single cell preparation was performed by FACS. And finally, selecting 10 transgenic Sp2/0 myeloma cells with good growth performance for clonal amplification, and analyzing the expression of mRNA and protein of an exogenous protein core element AVI-tag on the surface of the transgenic Sp2/0 myeloma cells by a real-time fluorescence quantitative PCR (polymerase chain reaction) technology and a western blot method, thereby selecting the transgenic Sp2/0 myeloma cells with the highest AVI-tag expression level and the best growth for a subsequent cell fusion test.

The method for immunizing BALB/c mice by chloramphenicol immunogen is as follows: the chloramphenicol immunogen was CAP-KLH and was prepared in this laboratory. The immunization method is a neck and back multipoint injection immunization method, the immunization times are 4 times, each time interval is 3 weeks, the immunogen dose is 0.6mg, the first immunization is primary immunization, Freund complete adjuvant is adopted, other immunizations all adopt Freund incomplete adjuvant, and the detection of serum titer and affinity is carried out after 1 week of each immunization.

The method for identifying the serum performance of BALB/c mice is as follows: the serum identification method is based on Enzyme Linked immunosorbent Assay (ELISA) and Indirect competitive Enzyme-Linked immunosorbent Assay (ICELISA).

The transgenic hybridoma and the traditional hybridoma are prepared as follows: after the fourth immunization, mouse antiserum was detected, spleen cells of mice with the highest serum affinity were collected under aseptic conditions, and half of the spleen cells were fused with the transgenic Sp2/0 myeloma cells to prepare transgenic hybridoma cells. The other half of the spleen cells were fused with conventional Sp2/0 myeloma cells to prepare conventional hybridoma cells. Preferably, the fusion method is a chemical induction method of polyethylene glycol.

The screening method of the single chloramphenicol specific hybridoma cell is as follows: first, the transgenic hybridoma cells were biotinylated in vitro. And resuspending the cells with PBS containing 2% BSA and 10. mu.g/mL CAP-streptavidin conjugate, incubating the cells at 4 ℃ for 15min, and washing the cells twice to realize CAP display on the surfaces of the transgenic hybridoma cells. Transgenic hybridoma cells displaying CAP were resuspended in 1mL PBS containing 25mM EDTA, and 5. mu.g/mL PE-Fab secondary antibody at 37 ℃ and 5% CO₂And (3) incubating for 3h, so as to promote the secretion of specific antibodies and the binding of the specific antibodies to the CAP on the cell surface. Labeled transgenic hybridoma cells, PBS washed 3 times, 100-. The procedure was repeated by drawing 20 μ L of cell suspension into a Piezoelectric Dispenser Capillary (PDC), empirically optimizing the PDC jet and sedimentation zones, repositioning the PDC to the camera station via a shaft system, and repeating until each drop was able to stably wrap a single cell. The jet boundary and detection parameters are adjusted and a maximum of 120 fluorescence values is set. Defining a gate that allows entry of cells that meet the parameters of detection ensures efficient sorting of individual chloramphenicol specific transgenic hybridomas. The single chloramphenicol specific transgenes after sortingHybridoma cells were seeded into 96-well plates containing approximately 1000 feeder cells per well. After sorting is finished, the nozzle image of each screened cell is rechecked to ensure the monoclonality, and the fluorescence value of each cell is subjected to preliminary statistical analysis. In the traditional hybridoma technology, screening of single chloramphenicol specific hybridoma cells is carried out by a limiting dilution method.

The method of comparing the transgenic hybridoma technology with the conventional hybridoma technology is as follows: the present invention makes systematic comparisons in terms of single hybridoma cell viability, screening time and reagent consumption, and chloramphenicol-specific antibody production time and reagent consumption.

The method for comparing the performance of monoclonal antibodies based on the transgenic hybridoma technology and the traditional hybridoma technology is as follows: comprehensive comparisons were made in terms of antibody titer, affinity, specificity and stability. Antibody titer is characterized by antibody dilution factor and coating antigen dilution factor, and antibody affinity is characterized by inhibition rate and IC₅₀Value characterisation, antibody specificity was assessed by analysis of the cross-reactivity rates with the three chloramphenicol analogues thiamphenicol, florfenicol and florfenicol amine, and the stability of the monoclonal antibody was characterised by the dissolution temperature Tm value and the aggregation onset temperature Tagg value.

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.

EXAMPLE 1 preparation of transgenic Sp2/0 myeloma cells

1. The signal peptide (amino acids 1-24) of EGF-R is inserted into the N-terminal of the biotin receptor peptide (AVI-tag) sequence, and the transmembrane region of EGF-R (amino acids 25-668) is inserted into the C-terminal (FIG. 1). The amino acid sequence of the EGF-R-AVI-tag fusion protein is shown in SEQ ID NO: 1.

2. 1 day before transfection, 0.5X 10⁵-1.0×10⁵Sp2/0 myeloma cells were plated in 6-well plates to achieve an optimal cell density of 70-80%.

3. On the day of transfection, the medium supernatant was discarded, 2mL of complete medium was added, 0.2. mu.g of transposase plasmid (mPB, helper plasmid) and 2. mu.g of AVI-tag-EGF-R plasmid (pPB EF1a, donor plasmid) were mixed and made up to 100. mu.L of Opti-MEM buffer, and added

Reagent, incubation at room temperature.

4. The incubated solution was added dropwise to Sp2/0 myeloma cells, after 18-24h, selection medium containing 2. mu.g/mL puromycin was added, and after 21 days of puromycin prescreening, expansion culture of transgenic Sp2/0 myeloma cells was performed using conventional complete medium.

EXAMPLE 2 selection of Single transgenic Sp2/0 myeloma cells

1. Will be 4X 10⁶Transgenic Sp2/0 myeloma cells were combined with optimized cells containing 5mM MgCl₂10mM ATP, 5. mu.M biotin and 0.85. mu.g of BirA in PBS and mixed at 37 ℃ with 5% CO₂And incubation for 30min to achieve in vitro biotinylation of transgenic Sp2/0 myeloma cells.

2. With a solution containing 5mM MgCl₂The biotinylated transgenic Sp2/0 myeloma cells were washed with PBS containing 2% BSA, the cells were resuspended, 2mM PE-streptavidin conjugate was added, incubated at 4 ℃ for 20 min, and single PE-streptavidin labeled transgenic Sp2/0 myeloma cells were enriched and prepared by FACS (FIG. 2, A and B).

The transgenic Sp2/0 myeloma cells with good growth state in the top ten are selected, and the expression of mRNA and protein of an exogenous protein core element AVI-tag on the surface of the transgenic Sp2/0 myeloma cells is analyzed by a real-time fluorescence quantitative PCR technology and a Western blotting method, so that the transgenic Sp2/0 myeloma cells with the highest AVI-tag expression level and the best growth are selected for a subsequent cell fusion test (FIG. 3, A and B).

EXAMPLE 3 Chloramphenicol immunogen immunization of BALB/c mice

The concentration of the immunogen (CAP-KLH, see CN202011112975.5) was adjusted to 1mg/mL using PBS buffer, and the concentration of the immunogen was determined by Bicinchonic Acid method.

8 female BALB/c mice weighing about 20g are respectively numbered as CAP-KLH-1, CAP-KLH-2, CAP-KLH-3, CAP-KLH-4, CAP-KLH-5, CAP-KLH-6, CAP-KLH-7 and CAP-KLH-8, and are immunized for 4 times in total, wherein the immunization mode is neck and back multi-point injection immunization;

first immunization: 0.6mL of priming preparation (1 mg of CAP-KLH per 1mL of priming preparation) was administered to each immunization with Freund's complete adjuvant;

second to fourth immunizations: the number of days counted from the start of the first immunization was 3 weeks, and 3 boosts were given, each 0.6mg of CAP-KLH.

Example 4 characterization of serum Performance in BALB/c mice

The antisera obtained one week after immunization were subjected to the measurement of titer and sensitivity. The assay method was an iclelisa.

The specific steps of the iclELISA are as follows: 50 μ L of small molecule competitor (CAP or its structural analogue) and diluted antiserum solution were added to the coated ELISA plates, incubated at 37 ℃ for 30min in an incubator, and then washed 3 times with PBST solution and patted dry. The highest inhibitory rate of antisera available from CAP-KLH-7# mice was 85% (Table 1). CAP-KLH-7# was therefore selected for cell fusion assays.

TABLE 1 characterization of serum Performance in immunized mice

EXAMPLE 5 preparation of transgenic hybridoma cells and conventional hybridoma cells

1. CAP-KLH-7# mice were injected intraperitoneally with 0.2mg of immunogen (dissolved in 0.5mL PBS), the mice were sacrificed 3 days later, spleens were harvested under sterile conditions, homogenized, centrifuged at 300g for 10min, diluted with DMEM medium, and filtered through a 70mm filter to obtain single cell suspensions.

2. Half of the spleen lymphocytes were fused with transgenic Sp2/0 myeloma cells, and the other half were fused with normal Sp2/0 myeloma cells.

3. Cells fused with transgenic Sp2/0 myeloma cells were resuspended in 10% HAT medium, dispensed into 1T 75 cell culture flask and 5 96-well plates, and after 10 days of culture, culture supernatants from fused cells were screened by ELISA and ICELISA, and cells were selected for further sorting. Cells fused with conventional Sp2/0 myeloma cells were resuspended in 10% HAT medium and plated evenly into 10 96-well plates.

EXAMPLE 6 screening of Single Chloramphenicol-specific transgenic hybridoma cells

1. Transgenic hybridoma cells and cells containing 5mM MgCl₂10mM ATP, 5. mu.M biotin and 0.85. mu.g of BirA in PBS and mixed at 37 ℃ with 5% CO₂Biotinylated transgenic hybridoma cells were resuspended in PBS containing 2% BSA, incubated at 4 ℃ for 15min, and washed twice.

2. The labeled transgenic hybridomas were resuspended in 1mL PBS containing 25mM EDTA, 10. mu.g/mL CAP-streptavidin conjugate, and 5. mu.g/mL PE-Fab antibody at 37 ℃ with 5% CO₂And incubated for 3 hours.

3. The labeled transgenic hybridoma cells were washed with PBS and resuspended in 1mL degassed PBS containing 25mM EDTA at a concentration of 100-.

4. The procedure was repeated by drawing 20 μ L of cell suspension into a Piezoelectric Dispenser Capillary (PDC), empirically optimizing the PDC jet and sedimentation zones, repositioning the PDC to the camera station via a shaft system, and repeating until each drop was able to stably wrap a single cell.

5. The jet boundary and detection parameters are adjusted and a maximum of 120 fluorescence values is set. A gate was drawn that allowed entry of cells with set detection parameters, ensuring efficient sorting of individual chloramphenicol specific transgenic hybridoma cells.

6. The sorted single chloramphenicol-specific transgenic hybridoma was inoculated into a 96-well plate containing about 1000 feeder cells per well, and 923 antigen-specific transgenic hybridoma cells were co-screened. The survival rate of single cells was as high as 86%. After sorting, the nozzle images of each chloramphenicol specific transgenic hybridoma screened were reviewed to ensure monoclonality, and preliminary statistical analysis of fluorescence values of each cell was performed (fig. 4, a-F).

7. The screened transgenic hybridoma cells are subjected to clone amplification, an antigen-antibody display system on the surface of a cell membrane is identified through a cell immunofluorescence test, and the result shows that the fusion protein EGF-R-AVI-tag can biotinylate the AVI-tag through biotin ligase BirA, is coupled with CAP-streptavidin, is further combined with a chloramphenicol specific antibody secreted by the transgenic hybridoma cells, and realizes the fluorescence labeling on the surface of the cell membrane through the combination of a PE-Fab antibody and the display antibody on the surface of the cell membrane (figure 5, A-D).

Example 7 comparison of transgenic hybridoma technology with conventional hybridoma technology

1. The single cell rate of the transgenic hybridoma technology is up to 92.3 percent and is obviously higher than 15.6 percent of that of the traditional hybridoma technology by analyzing from the single cell rate aspect, which shows that the transgenic hybridoma technology can obviously improve the single cell rate.

2. The analysis from the aspect of the positive rate of the cells shows that the ratio of the chloramphenicol specific transgenic hybridoma cells screened by the transgenic hybridoma technology is 81.2%, and in the traditional hybridoma technology, the chloramphenicol specific hybridoma cells only account for 18.7%, which indicates that the transgenic hybridoma technology can significantly improve the positive rate of the cells;

3. according to the analysis in the aspect of preparation time, the screening time of a single chloramphenicol specific transgenic hybridoma is 2h, compared with the traditional limited dilution method which requires 28 days to screen a single chloramphenicol specific hybridoma, the preparation efficiency is greatly improved, and the time and the cost of related consumables are saved.

Example 8 comparison of the Performance of monoclonal antibodies prepared based on transgenic and traditional hybridoma technology

1. The antibody titer of mAb 9A6 prepared by the transgenic hybridoma technology is 41754, which is 3.6 times higher than that of mAb 2E10 prepared by the traditional hybridoma technology, and IC is higher than that of mAb 2E10 prepared by the traditional hybridoma technology₅₀Is 0.28ng/mL, is improved by 3 times compared with the antibody prepared by the traditional hybridoma technology (figure 6, A and B), and shows that the titer and the sensitivity of the antibody prepared by the transgenic hybridoma are higher than those of the antibody prepared by the traditional hybridoma technologyAntibodies prepared by tumor technology.

2. Antibody specificity was assessed by analyzing cross-reactivity with the three chloramphenicol analogs thiamphenicol, florfenicol and florfenicol amine, indicating that neither method of producing antibodies crossed the three analogs (table 2). The formula for CR is as follows:

CR＝IC₅₀(CAP)/IC₅₀(CAP structural analogs)

Characterization of the stability of the monoclonal antibody by determining the Tm value of the dissolution temperature of the antibody and the Tagg value of the aggregation initiation temperature of the antibody, Tm of mAb 9A6₁Is 71.1 ℃ Tm₂Is Tm of mAb 2E10 at 87.4 ℃₁Is 67.4 ℃ Tm₂It was 84.5 ℃ and 76.9 ℃ for Tagg of mAb 9A6 and 71.4 ℃ for Tagg of mAb 2E10, indicating that the antibodies produced by the transgenic hybridoma technique were more stable (FIGS. 7, A and B).

TABLE 2 Cross-reactivity analysis of antibodies prepared based on transgenic and traditional hybridoma technology

The experimental results show that the transgenic hybridoma technology can prepare the antibody with better performance.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

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Claims

1. A method for screening individual antigen-specific transgenic hybridoma cells, said method comprising: preparing transgenic Sp2/0 myeloma cells expressing EGF-R-AVI-tag fusion protein, combining spleen cell fusion technology of traditional hybridoma technology to obtain hybridoma cells capable of displaying secretory specific antibody on cell membranes, and screening single specific transgenic hybridoma cells by using a single cell sorting system;

the EGF-R-AVI-tag fusion protein is as follows:

(a) 1, a protein consisting of an amino acid sequence shown in SEQ ID NO;

2. The method of claim 1, wherein the nucleic acid sequence encoding the EGF-R-AVI-tag fusion protein is introduced into the Sp2/0 myeloma cell by a plasmid, or is integrated into the genome of the Sp2/0 myeloma cell by genetic engineering means.

3. The method of claim 2, wherein the nucleic acid sequence encoding the EGF-R-AVI-tag fusion protein is integrated into the genome of the Sp2/0 myeloma cell via the PiggyBac transposition system.

4. The method of claim 1, further comprising, prior to fusion with the splenocytes, the steps of: transgenic Sp2/0 myeloma cells were sequentially biotinylated in vitro and PE-streptavidin labeled, then enriched by FACS and single PE-streptavidin labeled transgenic Sp2/0 myeloma cells were prepared for subsequent cell fusion.

5. Single specific transgenic hybridoma cells selected according to the method of any one of claims 1 to 4.

6. Use of the method of any one of claims 1 to 4 or the transgenic hybridoma cell of claim 5 for the preparation of a monoclonal antibody.