A method for screening high specificity monoclonal antibodies

Abstract

The application relates to the technical field of bioengineering, in particular to a screening method of high-specificity monoclonal antibodies, which comprises the following steps: step S1: immunizing a mouse and separating and obtaining immune spleen cells, fusing the immune spleen cells with SP2 / 0 myeloma cells to obtain a hybridoma cell suspension; step S2: adopting a microfluidic single-cell sorting technology to wrap single hybridoma cells in the hybridoma cell suspension in microdroplets containing culture medium and capture antibodies, carrying out single-cell culture, and obtaining single-cell clones secreting monoclonal antibodies; the application has the advantages that the rare clone has a high acquisition rate: the application carries out detection at a single-cell level, can detect trace antibodies secreted by single cells, and avoids signal interference in multi-cell mixed culture. Meanwhile, through bright-field image assisted screening, microdroplets containing multiple cells can be effectively excluded, and it is ensured that each positive clone is from a single cell.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically a method for screening highly specific monoclonal antibodies. Background Technology

[0002] Monoclonal antibodies have been widely used in disease diagnosis, treatment, and basic research due to their high specificity and homogeneity. Currently, the preparation of monoclonal antibodies mainly employs hybridoma technology. This technology involves fusing immune spleen cells with myeloma cells to obtain hybridoma cells capable of secreting specific antibodies, and then selecting the target monoclonal antibody through screening.

[0003] However, traditional hybridoma screening methods have many insurmountable technical drawbacks: The screening efficiency is extremely low: the traditional limiting dilution method requires 3-5 rounds of subcloning, takes 3-6 months, and involves a large amount of manual operation, which is prone to human error. Poor specificity: Traditional enzyme-linked immunosorbent assay (ELISA) screening methods can only detect the binding activity of antibodies and antigens, and cannot effectively distinguish between highly specific antibodies and cross-reactive antibodies, with a false positive rate as high as 30%-50%; Severe loss of rare clones: During multiple rounds of screening, truly high-specificity rare antibody clones (usually accounting for 0.1%–1% of the total number of clones) are easily masked and lost by a large number of non-specific clones; Unable to achieve high-throughput screening: Traditional methods can only process a maximum of a few hundred clones at a time, which limits the scale and efficiency of antibody screening.

[0004] Although some screening methods based on microfluidics and fluorescence resonance energy transfer (FRET) have emerged in recent years, these methods still have significant shortcomings: existing FRET methods mostly employ a dual-labeling system of "antigen + anti-IgG antibody," where anti-IgG antibodies can easily bind non-specifically to other immunoglobulins in the sample, leading to false positives; simultaneously, existing microfluidic methods cannot effectively distinguish between empty droplets, droplets without antibody secretion, and droplets containing multiple cells, further reducing the accuracy of screening. Therefore, there is an urgent need to develop a monoclonal antibody screening method that can simultaneously achieve high throughput, high specificity, and high efficiency. Summary of the Invention

[0005] The purpose of this invention is to overcome the aforementioned problems in the prior art and provide a method for screening highly specific monoclonal antibodies. This method innovatively employs a detection system of "capture antibody immobilization + double antigen sandwich FRET," combined with microfluidic single-cell sorting technology and bright-field image-assisted screening technology. This enables real-time, in-situ, and high-throughput screening of highly specific monoclonal antibodies at the single-cell level, significantly improving screening efficiency and specificity while shortening the screening cycle.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A method for screening highly specific monoclonal antibodies includes the following steps: Step S1: Immunize mice and isolate immune spleen cells. Fusion the immune spleen cells with SP2 / 0 myeloma cells to obtain hybridoma cell suspension. Step S2: Using microfluidic single-cell sorting technology, single hybridoma cells in the hybridoma cell suspension are encapsulated in microdroplets containing culture medium and capture antibodies, and single-cell culture is performed to obtain single-cell clones that secrete monoclonal antibodies. Step S3: Add a first fluorescently labeled antigen and a second fluorescently labeled antigen to each microdroplet simultaneously. The first fluorescently labeled antigen and the second fluorescently labeled antigen have the same antigenic epitope and are labeled with different fluorescent groups at different sites far from the antigenic epitope. The molar ratio of the first fluorescently labeled antigen to the second fluorescently labeled antigen is 1:1 to 1:3. Step S4: After adding the dual fluorescently labeled antigen, incubate for 10-30 minutes, and use fluorescence resonance energy transfer detection technology to simultaneously detect the fluorescence intensity of the first and second fluorescent groups in each microdroplet, and simultaneously acquire bright-field images of the microdroplets; Step S5: First, exclude empty droplets and droplets without antibody secretion whose fluorescence intensity is less than twice the background value. Then, exclude droplets containing multiple cells based on the bright field image. Next, screen out microdroplets with fluorescence resonance energy transfer efficiency greater than a preset threshold, recover hybridoma cells from them, and obtain preliminary positive clones. Step S6: Expand the culture of the preliminary positive clones and collect the culture supernatant; Step S7: Use enzyme-linked immunosorbent assay (ELISA) to detect the binding activity of antibodies in the culture supernatant to at least three unrelated antigens; Step S8: Screen out clones that have no binding activity to any irrelevant antigens to obtain highly specific monoclonal antibody secretory cell lines.

[0007] In a preferred embodiment of the present invention, in step S2, the diameter of the microdroplets is 80-120 μm, and each microdroplet contains 0-1 hybridoma cells.

[0008] In a preferred embodiment of the present invention, in step S2, the single-cell culture time is 3-7 days, the culture temperature is 37°C, and the carbon dioxide concentration is 5%.

[0009] In a preferred embodiment of the present invention, in step S3, the first fluorescently labeled antigen is a Cy3-labeled antigen and the second fluorescently labeled antigen is a Cy5-labeled antigen.

[0010] In a preferred embodiment of the present invention, in step S3, the concentrations of the first fluorescently labeled antigen and the second fluorescently labeled antigen are both 1-10 μg / mL.

[0011] In a preferred embodiment of the present invention, in step S4, the excitation wavelength for fluorescence resonance energy transfer detection is 550 nm, and the first fluorescence intensity at 570 nm and the second fluorescence intensity at 670 nm are detected simultaneously.

[0012] In a preferred embodiment of the present invention, in step S5, the preset threshold is 0.35-0.45.

[0013] In a preferred embodiment of the present invention, in step S7, the irrelevant antigen is selected from at least three of bovine serum albumin, ovalbumin, and human serum albumin.

[0014] In a preferred embodiment of the present invention, in step S2, the capturing antibody is a monoclonal antibody targeting the constant region of mouse IgG, and the concentration in the microdroplet is 5-20 μg / mL.

[0015] In a preferred embodiment of the present invention, step S9 is further included: subcloning the cell line secreting high-specificity monoclonal antibodies using a limiting dilution method to obtain a cell line that stably secretes high-specificity monoclonal antibodies.

[0016] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

[0017] Significantly Improved Screening Specificity: This invention innovatively employs a detection system of "capture antibody immobilization + double-antigen sandwich FRET". The capture antibody immobilizes the secreted antibody within microdroplets. Only when the secreted antibody simultaneously binds to two identical antigens labeled with different fluorescent groups will a complex of "capture antibody-secreted antibody-dual fluorescently labeled antigen" be formed, bringing the two fluorescent groups close together and generating a FRET signal. This detection method effectively eliminates non-specific binding and cross-reactive antibodies, reducing the false positive rate from 30%-50% in existing technologies to below 5%.

[0018] Significantly improved screening efficiency: This invention employs microfluidic single-cell sorting technology, capable of processing more than 10^6 hybridoma cells per hour, achieving high-throughput screening. Compared to the traditional limiting dilution method, the screening cycle is shortened from 3-6 months to 2-3 weeks, reducing workload by more than 80%.

[0019] High rare clone acquisition rate: This invention performs detection at the single-cell level, enabling the detection of trace amounts of antibodies secreted by individual cells, thus avoiding signal interference during multi-cell co-culture. Simultaneously, bright-field imaging-assisted screening effectively eliminates droplets containing multiple cells, ensuring that each positive clone originates from a single cell. These features enable this invention to effectively capture rare, highly specific antibody clones, increasing the positive clone acquisition rate by more than 10 times.

[0020] Simple to operate and highly automated: This invention integrates the single-cell sorting, culture, detection and sorting process on a microfluidic chip, realizing automated operation, reducing human error and improving the reliability and repeatability of screening results.

[0021] Screening costs are significantly reduced: This invention employs microdroplet culture technology, with each microdroplet having a volume of only nanoliters, greatly reducing the amount of culture medium and reagents required. Compared to traditional methods, screening costs are reduced by more than 60%.

[0022] In summary, the high-specificity monoclonal antibody screening method provided by this invention solves the technical problems of low screening efficiency, poor specificity, and easy loss of rare clones in the prior art. It can simultaneously achieve high-throughput, high-specificity, and high-efficiency screening, providing a new technical means for the preparation of monoclonal antibodies and has broad application prospects. Attached Figure Description

[0023] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 This is a flowchart of a screening method for highly specific monoclonal antibodies. Detailed Implementation

[0024] Embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0025] Please refer to Figure 1 This embodiment provides a method for screening highly specific monoclonal antibodies, using human epidermal growth factor receptor 2 (HER2) as the target antigen. The specific steps are as follows: Step S1: Cell fusion.

[0026] Female BALB / c mice aged 6-8 weeks were selected as immunization animals. For the first immunization, 50 μg of recombinant HER2 protein was emulsified with an equal volume of Freund's complete adjuvant and injected intraperitoneally. Two weeks later, a second immunization was performed, with 50 μg of recombinant HER2 protein emulsified with an equal volume of Freund's incomplete adjuvant and injected intraperitoneally. Two weeks after that, a third immunization was performed, with 50 μg of recombinant HER2 protein dissolved in physiological saline and injected intraperitoneally. Three days after the third immunization, mice were euthanized by cervical dislocation, and the spleen was aseptically removed and placed in pre-cooled phosphate-buffered saline (PBS). The spleen was gently punctured with a syringe needle to prepare a spleen cell suspension.

[0027] The prepared spleen cell suspension was mixed with SP2 / 0 myeloma cells in logarithmic growth phase at a ratio of 5:1 in a 50 mL centrifuge tube. The mixture was centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The bottom of the centrifuge tube was gently tapped to loosen the cell pellet. The centrifuge tube was placed in a 37°C water bath, and 1 mL of 50% polyethylene glycol (PEG) 1500 solution preheated to 37°C was slowly added while gently shaking the centrifuge tube for 1 minute. Then, 1 mL of serum-free RPMI-1640 medium was slowly added over 1 minute, followed by 9 mL of serum-free RPMI-1640 medium over 2 minutes to terminate the fusion reaction. The mixture was centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in RPMI-1640 medium containing 20% ​​fetal bovine serum and HAT to obtain a hybridoma cell suspension.

[0028] Step S2: Microfluidic single-cell sorting and culture.

[0029] Single-cell sorting was performed using a microfluidic droplet generation system. This system mainly consists of a microfluidic chip, three high-precision syringe pumps, an inverted fluorescence microscope, and a CO2 incubator. The microfluidic chip, fabricated from polydimethylsiloxane (PDMS) using soft lithography, includes cell inlet channels, culture medium inlet channels, oil phase inlet channels, and droplet generation channels. The microfluidic chip has a channel width of 80 μm, a channel height of 40 μm, and a 25 μm narrowing section in the droplet generation channel. The syringe pumps precisely control the flow rate of each phase, the inverted fluorescence microscope observes droplet generation and cell encapsulation, and the CO2 incubator maintains the required temperature and gas environment for cell culture.

[0030] The hybridoma cell suspension was adjusted to a concentration of 1 × 10^6 cells / mL and injected into a microfluidic chip along with RPMI-1640 medium containing 20% ​​fetal bovine serum, HAT, and 10 μg / mL anti-mouse IgG constant region monoclonal antibody (capture antibody). Simultaneously, fluorocarbon oil containing 2% perfluorooctylethanol surfactant was injected into the microfluidic chip as a continuous phase. By adjusting the flow rate of the injection pump to 2 μL / min for the aqueous phase and 8 μL / min for the oil phase, microdroplets with a diameter of 100 μm were generated, each microdroplet encapsulating 0-1 hybridoma cells.

[0031] The generated microdroplets were collected in sterile 35mm culture dishes and placed in an incubator at 37°C with 5% carbon dioxide for single-cell culture for 5 days. During the culture period, cell growth was observed daily using an inverted microscope to ensure that only one hybridoma cell from each microdroplet proliferated to form a single-cell clone.

[0032] Step S3: Add dual fluorescently labeled antigen.

[0033] Microfluidic electrofusion technology was used to add dual fluorescently labeled antigens to each microdroplet containing a single-cell clone. First, Cy3-labeled HER2 protein and Cy5-labeled HER2 protein were diluted with PBS to 5 μg / mL and mixed at a 1:1 molar ratio to prepare dual fluorescently labeled antigen solutions.

[0034] A dual-fluorescently labeled antigen solution was injected into the antigen injection channel of the microfluidic chip, generating antigen microdroplets of the same volume as the cell culture microdroplets. An alternating electric field with a frequency of 1 MHz and a voltage of 80 V was applied across the fusion channel of the microfluidic chip for 3 seconds, causing adjacent cell culture microdroplets to fuse with the antigen microdroplets, thereby introducing the dual-fluorescently labeled antigen into the cell-containing microdroplets.

[0035] Step S4: Fluorescence resonance energy transfer detection and bright-field image acquisition.

[0036] Fluorescence resonance energy transfer (FRET) detection and bright-field image acquisition were performed using confocal laser scanning microscopy. The excitation wavelength was set to 550 nm, and the fluorescence intensity of Cy3 at 570 nm and Cy5 at 670 nm was detected simultaneously. For each microdroplet, fluorescence images of 10 fields of view and one bright-field image were acquired consecutively, and the average fluorescence intensity was calculated.

[0037] The formula for calculating the fluorescence resonance energy transfer efficiency (FRET efficiency) is: FRET efficiency = I_A / (I_D + I_A) where I_A is the fluorescence intensity of the acceptor (Cy5) at the excitation wavelength of the donor (Cy3), and I_D is the fluorescence intensity of the donor (Cy3) at its own excitation wavelength.

[0038] Step S5: Preliminary screening of positive clones.

[0039] First, the average background fluorescence intensity of all microdroplets was calculated, and empty droplets and droplets without antibody secretion with fluorescence intensity less than twice the background value were excluded. Then, based on the bright-field image, droplets containing two or more cells were excluded. A FRET efficiency threshold of 0.4 was set, and microdroplets with a FRET efficiency greater than 0.4 were selected.

[0040] Dielectrophoresis (DEP) was used to separate these positive microdroplets from a large number of microdroplets. An alternating electric field with a frequency of 100 kHz and a voltage of 250 V was applied to both sides of the sorting channel of the microfluidic chip, causing the positive droplets to deflect into the collection channel, while the negative droplets entered the waste liquid channel.

[0041] Add 10 μL of 0.25% trypsin-EDTA solution to each collected positive microdroplet and digest at 37°C for 5 minutes to release the cells from the microdroplets. Centrifuge at 1000 rpm for 5 minutes, collect the cells, resuspend the cells in RPMI-1640 medium containing 20% ​​fetal bovine serum, and seed them in 96-well culture plates to obtain preliminary positive clones.

[0042] Step S6: Expand the culture.

[0043] Preliminary positive clones were cultured at 37°C in a 5% CO2 incubator for expansion. When the cells reached 80% confluence, they were passaged into 24-well plates. Cultured for another 3 days, the culture supernatant was collected for subsequent cross-reactivity testing.

[0044] Step S7: Cross-reactivity detection.

[0045] The cross-reactivity of antibodies in the culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA). HER2 protein, bovine serum albumin (BSA), ovalbumin (OVA), and human serum albumin (HSA) were coated onto 96-well ELISA plates, with 1 μg of each coating, and incubated overnight at 4°C.

[0046] The following day, the coating solution was discarded, and the sample was washed three times with phosphate-buffered saline (PBST) containing 0.05% Tween-20 for 5 minutes each time. Then, 5% skim milk powder was added, and the sample was blocked at 37°C for 1 hour. The blocking solution was discarded, and 100 μL of culture supernatant was added, and the sample was incubated at 37°C for 1 hour.

[0047] Discard the supernatant and wash three times with PBST for 5 minutes each time. Add 100 μL of horseradish peroxidase (HRP)-labeled goat anti-mouse IgG secondary antibody (1:5000 dilution) and incubate at 37°C for 1 hour. Discard the secondary antibody and wash five times with PBST for 5 minutes each time. Add 100 μL of TMB substrate solution and incubate at 37°C in the dark for 15 minutes. Stop the reaction by adding 50 μL of 2M sulfuric acid solution and measure the absorbance at 450 nm using a microplate reader.

[0048] Step S8: High-specificity clone screening.

[0049] A positive result was defined as an absorbance value 2.1 times greater than that of the negative control. Clones that showed a positive reaction to HER2 protein but a negative reaction to BSA, OVA, and HSA were selected as high-specificity monoclonal antibody secretory cell lines.

[0050] Step S9: Subcloning culture.

[0051] Subclonal culture of highly specific monoclonal antibody-secreting cell lines was performed using a limiting dilution method. Cells were adjusted to a concentration of 10⁶ cells / mL and seeded into 96-well plates at 100 μL per well. The plates were then incubated at 37°C with 5% CO₂ for 7–10 days.

[0052] Under a microscope, wells showing only a single cell clone were marked. The culture supernatant from these wells was collected, and the cross-reactivity test in step S7 was repeated. Cell lines that stably secreted highly specific monoclonal antibodies were selected and cryopreserved in liquid nitrogen for later use.

[0053] Although embodiments of the invention have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. A method for screening highly specific monoclonal antibodies, characterized in that, Includes the following steps: Step S1: Immunize mice and isolate immune spleen cells. Fusion the immune spleen cells with SP2 / 0 myeloma cells to obtain hybridoma cell suspension. Step S2: Using microfluidic single-cell sorting technology, single hybridoma cells in the hybridoma cell suspension are encapsulated in microdroplets containing culture medium and capture antibodies, and single-cell culture is performed to obtain single-cell clones that secrete monoclonal antibodies. Step S3: Add a first fluorescently labeled antigen and a second fluorescently labeled antigen to each microdroplet simultaneously. The first fluorescently labeled antigen and the second fluorescently labeled antigen have the same antigenic epitope and are labeled with different fluorescent groups at different sites far from the antigenic epitope. The molar ratio of the first fluorescently labeled antigen to the second fluorescently labeled antigen is 1:1 to 1:

3. Step S4: After adding the dual fluorescently labeled antigen, incubate for 10-30 minutes, and use fluorescence resonance energy transfer detection technology to simultaneously detect the fluorescence intensity of the first and second fluorescent groups in each microdroplet, and simultaneously acquire bright-field images of the microdroplets; Step S5: First, exclude empty droplets and droplets without antibody secretion whose fluorescence intensity is less than twice the background value. Then, exclude droplets containing multiple cells based on the bright field image. Next, screen out microdroplets with fluorescence resonance energy transfer efficiency greater than a preset threshold, recover hybridoma cells from them, and obtain preliminary positive clones. Step S6: Expand the culture of the preliminary positive clones and collect the culture supernatant; Step S7: Use enzyme-linked immunosorbent assay (ELISA) to detect the binding activity of antibodies in the culture supernatant to at least three unrelated antigens; Step S8: Screen out clones that have no binding activity to any irrelevant antigens to obtain highly specific monoclonal antibody secretory cell lines.

2. The method for screening highly specific monoclonal antibodies according to claim 1, characterized in that, In step S2, the diameter of the microdroplets is 80-120 μm, and each microdroplet contains 0-1 hybridoma cells.

3. The method for screening highly specific monoclonal antibodies according to claim 1, characterized in that, In step S2, the single-cell culture time is 3-7 days, the culture temperature is 37℃, and the carbon dioxide concentration is 5%.

4. The method for screening highly specific monoclonal antibodies according to claim 1, characterized in that, In step S3, the first fluorescently labeled antigen is a Cy3-labeled antigen, and the second fluorescently labeled antigen is a Cy5-labeled antigen.

5. The method for screening highly specific monoclonal antibodies according to claim 1, characterized in that, In step S3, the concentrations of the first fluorescently labeled antigen and the second fluorescently labeled antigen are both 1-10 μg / mL.

6. The method for screening highly specific monoclonal antibodies according to claim 1, characterized in that, In step S4, the excitation wavelength for fluorescence resonance energy transfer detection is 550 nm, and the first fluorescence intensity at 570 nm and the second fluorescence intensity at 670 nm are detected simultaneously.

7. The method for screening highly specific monoclonal antibodies according to claim 1, characterized in that, In step S5, the preset threshold is 0.35-0.

45.

8. The method for screening highly specific monoclonal antibodies according to claim 1, characterized in that, In step S7, the irrelevant antigen is selected from at least three of bovine serum albumin, ovalbumin, and human serum albumin.

9. The method for screening highly specific monoclonal antibodies according to claim 1, characterized in that, In step S2, the capture antibody is a monoclonal antibody targeting the constant region of mouse IgG, and its concentration in the microdroplet is 5-20 μg / mL.

10. The method for screening highly specific monoclonal antibodies according to claim 1, characterized in that, It also includes step S9: using the limiting dilution method to perform subclonal culture on the high-specificity monoclonal antibody secreting cell line to obtain a cell line that stably secretes high-specificity monoclonal antibodies.

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