High-throughput antibody screening method based on single-cell microgel analysis technology

Abstract

This invention discloses a high-throughput antibody screening method based on single-cell microgel analysis technology. The hydrogel in this method is formed by a Michael addition reaction between the thiol groups of thiolated sodium hyaluronate and the double bonds of hyperbranched polyethylene glycol diacrylate. It is self-assembled in a microfluidic device using picoliter injection technology, encapsulating spleen cells obtained from immunized mice. Rod-shaped magnetite beads conjugated with secondary antibodies are used to identify and classify hydrogels with high antibody expression. This method achieves target-free cell analysis, improves selectivity, shortens detection time, reduces operational complexity, and saves costs, providing an effective solution for high-throughput screening of single-cell antibodies.

Description

Technical Field

[0001] This invention relates to a high-throughput antibody screening method, and more particularly to a high-throughput antibody screening method based on single-cell microgel analysis technology. Background Technology

[0002] Antibodies, also known as immunoglobulins, with IgG being the most abundant antibody in the bloodstream. The phenotypic diversity of target-specific IgG components in animals serves as a protective basis during infection or immunity and provides a rich natural antibody library for screening high-performance monoclonal antibodies. Therefore, analyzing the activity and sequence of IgG is of great significance. Currently, emerging single-cell manipulation methods can screen target-specific antibodies at the single-cell level. However, flow cytometry fluorescence sorting (FACS) is limited to the expression of antibodies on the cell surface; traditional hybridoma techniques have limitations such as low B-cell-myeloma cell fusion efficiency and the need for experimental animals; fluorescence-based droplet microfluidic screening requires passing cell-encapsulated droplets one by one through fluorescent activation points in a channel for detection and sorting, demanding high droplet uniformity. Superparamagnetic nanoparticles have been widely used for cell separation; however, using magnetic nanoparticles attached to the cell surface for screening may lead to cell death due to the Fenton reaction or alter cell function through non-specific endocytosis. Therefore, establishing a simple, mild, and more cell-compatible screening method is an urgent problem to be solved. Summary of the Invention

[0003] Purpose of the invention: The present invention aims to provide a simple, mild, and highly cell-compatible high-throughput antibody screening method based on single-cell microgel analysis technology.

[0004] Technical solution: This invention provides a high-throughput antibody screening method based on single-cell microgel analysis technology, comprising the following steps:

[0005] (1) Cells were suspended in a culture medium containing hyperbranched polyethylene glycol diacrylate (HB-PEGDA) as the aqueous phase and fluorinated oil containing surfactants as the oil phase. Hydrogel precursor droplets were prepared in a microfluidic device. Thio-modified sodium hyaluronate (HA-SH) was injected into the incubated droplets using pico-injection technology. After standing, HB-PEGDA and HA-SH underwent a Michael addition reaction, and the droplets were fully gelled to form microspheres.

[0006] (2) Wash away the surfactant on the surface of the microspheres, resuspend them in the culture medium, add rod-shaped Fe3O4 magnetic beads with coupled secondary antibody, incubate, and magnetically separate.

[0007] Preferably, the droplet generating chip used in step (1) to prepare droplets in the microfluidic device is a poly(dimethylsiloxane) (PDMS) chip with a chip size of 40×40×25μm and the diameter of the prepared droplets is 30-45μm.

[0008] Preferably, the culture medium used in the aqueous phase in step (1) is either DMEM or 1640, wherein 5-20% (v / v) fetal bovine serum and 1% (v / v) streptolysin / penicillin mixture are added, and the HB-PEGDA concentration is 6.67% (w / v).

[0009] Preferably, the cells are spleen cells from immunized mice.

[0010] Preferably, the surfactant used in the oil phase is any one of Pico-Surf 1, FS-Kryjeff D900, and EA, and the concentration of the surfactant added to the oil phase is 1-5% (w / w).

[0011] Furthermore, the concentration of HA-SH injected in step (1) using the pico-injection technique is 5.33% (w / v).

[0012] Further, in step (2), pure fluorinated oil is added to wash away the surfactant, centrifuged and the fluorinated oil is discarded. The washing is repeated until the gel microspheres agglomerate into a clump, indicating that the surfactant has been washed away. The gel microspheres are then transferred to the culture medium, centrifuged, and repeated three times. The microspheres are then resuspended in the culture medium for later use.

[0013] Furthermore, the preparation method of the rod-shaped iron(Fe3O4) magnetic beads for the conjugated secondary antibody is as follows: rod-shaped Fe3O4 magnetic beads are prepared using a polytetrafluoroethylene reactor; sodium dodecyl sulfate and methacrylic acid are added to the suspension to prepare carboxylated rod-shaped Fe3O4 magnetic beads; after activating the carboxylated magnetic beads, streptavidin is added to synthesize streptavidin magnetic beads; and these are co-incubated with biotin-VHH anti-mouse κ light chain to form rod-shaped Fe3O4 magnetic beads for the conjugated secondary antibody.

[0014] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) By encapsulating single cells in HA-SH / HB-PEGDA hydrogel microspheres with high biocompatibility to form a microreactor, direct contact between cells and magnetic nanoparticles can be prevented, improving cell compatibility and system stability; (2) Compared with spherical magnetic beads, rod-shaped magnetic beads can achieve more precise magnetic orientation, have better sedimentation stability, and promote higher packing density of proteins coupled on their relatively "flat" cylindrical surfaces; (3) This method is based on magnetism and uses rod-shaped magnetic beads to separate hydrogel microspheres. Compared with FACS, it realizes target-free analysis and detection of cells, which is non-invasive and low-cost. At the same time, cells with different antibody expression levels can be separated and collected at different times according to the speed of magnetic bead movement, improving selectivity. Compared with hybridoma technology, single-cell culture retains more biological information and can analyze cells from different species. Fluorescence-based droplet microfluidic screening technologies typically use an oil phase as the continuous phase. The single-cell microgel reactor used in this invention transfers single cells to an aqueous phase for analysis. It has good compatibility with classic analytical instruments such as FACS, has low requirements for droplet uniformity, and utilizes magnetism to achieve rapid screening of positive microgels. The system is mild, reduces operational complexity and cost, and shortens detection time, providing an effective solution for high-throughput screening of single-cell antibodies. Attached Figure Description

[0015] Figure 1 A is an experimental flowchart of the hydrogel-based antibody screening method in this invention; B is the experimental flowchart of the traditional hybridoma preparation of monoclonal antibodies; and C is a schematic diagram of the principle of the hydrogel-based antibody screening method.

[0016] Figure 2 In the image, A and B are electron micrographs of rod-shaped Fe3O4 magnetic beads and magnetic orientation verification, respectively.

[0017] Figure 3 The images show the fluorescence microscopy results of the surface antibody on the fluorescent secondary antibody localization antibody microgel; A and B are the bright field and fluorescence field results of the blank microgel after incubation with the fluorescent secondary antibody, respectively; C and D are the bright field and fluorescence field results of the antibody microgel after incubation with the fluorescent secondary antibody, respectively.

[0018] Figure 4 The image shows the fluorescence microscopy results of co-incubation of magnetic beads with antibody microgels and blank microgels.

[0019] Figure 5 Image showing the results of screening antibody microgels using magnetic beads. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0021] Example 1

[0022] Preparation of dual-resistance coupled rod-shaped Fe3O4 magnetic beads:

[0023] (1) Preparation of rod-shaped iron(III) oxide (Fe3O4) magnetic beads

[0024] In a 100 mL polytetrafluoroethylene reactor, 1 mmol FeSO4 7H2O and 2 mmol FeCl3 were dissolved in 40 mL of ultrapure water and sonicated at room temperature. 5 mL of ethylenediamine was added as a template agent, and the mixture was heated at 120 °C for 20 h. After cooling naturally to room temperature, the mixture was filtered, washed, and dried to prepare rod-shaped Fe3O4 magnetic beads with an average length of about 200 nm.

[0025] (2) Preparation of rod-shaped streptavidin magnetic beads

[0026] 1 g of rod-shaped Fe3O4 magnetic beads prepared in (1) and 1.15 g of sodium dodecyl sulfate were dissolved in 45 ml of ultrapure water. 0.96 ml of methacrylic acid was added, and the mixture was heated to 70 °C for 45 min using a magnetic stirrer (300-400 rpm). 1.98 g of potassium persulfate initiator was added, and the reaction was carried out at 70 °C for 2 h. The reaction was cooled to room temperature, and the magnetic beads were separated by a strong magnet. The magnetic beads were washed with ultrapure water at least 5 times and finally dispersed in deionized water to obtain carboxylated rod-shaped Fe3O4 magnetic beads. A 5 mg / mL solution of EDC and NHS was prepared using MES solution, and the carboxylated magnetic beads were activated at 37 °C for 45 min. The activated magnetic beads were coupled with an appropriate amount of streptavidin (10:1) and reacted at 37 °C for 1 h. The magnetic beads were then recovered using a magnetic separator.

[0027] (3) Preparation of dual-resistance coupled rod-shaped Fe3O4 magnetic beads

[0028] The rod-shaped streptavidin magnetic beads prepared in step (2) were incubated with 1% bovine serum albumin (BSA) in PBS solution (pH 8.0) for 1 h to block the hydrophobic sites on the surface of the magnetic beads and reduce their non-specific adsorption. Subsequently, the magnetic beads were co-incubated with biotin-VHH anti-mouse κ light chain at room temperature for 1 h to form rod-shaped Fe3O4 magnetic beads with conjugated secondary antibody.

[0029] Example 2

[0030] Preparation of spherical Fe3O4 magnetic beads and their characterization and comparison with rod-shaped Fe3O4 magnetic beads:

[0031] (1) Preparation of spherical Fe3O4 magnetic beads

[0032] In a 100 mL polytetrafluoroethylene reactor, 4.30 g FeCl3·6H2O, 2.35 g trisodium citrate, and 8.75 g sodium acetate (NaAc) were dissolved in 150 mL ethylene glycol. The solution was sonicated at room temperature, heated to 200 °C for 10 h, and then allowed to cool naturally to room temperature. After filtration, washing, and drying, spherical Fe3O4 magnetic beads with an average particle size of approximately 200 nm were obtained.

[0033] (2) Characterization of magnetic orientation of rod-shaped Fe3O4 magnetic beads and spherical Fe3O4 magnetic beads

[0034] 5 mg of rod-shaped Fe3O4 magnetic beads and spherical Fe3O4 magnetic beads were respectively suspended in PBS containing 1% (w / w) PF127 and dissolved by sonication for 5 min to form the microfluidic aqueous phase; the oil phase was Novec HFE-7500 fluorinated oil containing 2% (w / w) surfactant, and uniform droplets were generated in a PDMS chip with a size of 40×40×25μm.

[0035] The droplets were collected in a urine sediment counting chamber and attracted directionally under a microscope using a ring magnet. The results showed (see...). Figure 2 Rod-shaped magnetic beads can form a single line in a droplet due to the attraction of a magnet, while spherical magnetic beads are more dispersed, indicating that rod-shaped magnetic beads have better magnetic orientation.

[0036] Example 3

[0037] Preparation of antibody microgel system from immunized mouse serum: Fluorescent secondary antibody was used to target the surface antibody of the microgel.

[0038] (1) Preparation of HA-SH / HB-PEGDA antibody microgels

[0039] Hydrogel precursor droplets were prepared in a PDMS droplet generation chip with dimensions of 40×40×25μm. The aqueous phase consisted of HB-PEGDA dissolved in a culture medium (containing 10% fetal bovine serum and 1% streptolysin / penicillin mixture) and the oil phase consisted of Novec HFE-7500 fluorinated oil containing 2% (w / w) surfactant. The droplet diameter was approximately 34μm, the volume was 21pL, and the concentration of HB-PEGDA in the aqueous phase was 6.67% (w / v). The generated droplets were collected in sterile syringes and incubated in a saturated humidity incubator at 37°C and 5% CO2 for a period of time. Then, HA-SH at a concentration of 5.33% (w / v) was injected into the droplets using pico-injection technology. The pico-injection chip had dimensions of 40×60×25μm. At this point, the droplet diameter is approximately 43 μm and the volume is 42 pL. After standing at room temperature for 2 hours, the HB-PEGDA and HA-SH in the droplet undergo a Michael addition reaction, resulting in complete gelation and the formation of microgels. The surfactant on the surface of the microgels is washed away with pure fluorinated oil. The microgels are centrifuged at 1000 x g for 1 min, the fluorinated oil is discarded, and the washing process is repeated until the gel microspheres aggregate into a clump, indicating that the surfactant has been completely removed. The gel microspheres are then transferred to a culture medium, centrifuged at 1000 x g for 1 min, and this process is repeated three times. The microspheres are then resuspended in the culture medium for later use. 1% Pluronic F-127 (PF-127) is added to prevent microgel adhesion.

[0040] (2) Preparation of HA-SH / HB-PEGDA blank microgels

[0041] Hydrogel precursor droplets were prepared in a PDMS droplet generation chip with dimensions of 40×40×25μm. The aqueous phase consisted of HB-PEGDA dissolved in a culture medium (containing 10% fetal bovine serum and 1% strepto-penicillin mixture) and an PBS suspension of an equal volume of serum antibody (1). The oil phase consisted of Novec HFE-7500 fluorinated oil containing 2% (w / w) surfactant. The droplet diameter was approximately 34μm, the volume was 21pL, and the concentration of HB-PEGDA in the aqueous phase was 6.67% (w / v). The generated droplets were collected in a sterile syringe and incubated in a saturated humidity incubator at 37°C and 5% CO2 for the same time as in (1). HA-SH at a concentration of 5.33% (w / v) was injected into the droplets using pico-injection technology. The pico-injection chip had dimensions of 40×60×25μm. At this point, the droplet diameter was approximately 43 μm and the volume was 42 pL. After standing at room temperature for 2 hours, the HB-PEGDA and HA-SH in the droplet underwent a Michael addition reaction, resulting in complete gelation and the formation of microgels. The surfactant on the surface of the microgels was washed away with pure fluorinated oil. The microgels were centrifuged at 1000 x g for 1 min, the fluorinated oil was discarded, and the washing process was repeated until the gel microspheres agglomerated into a cluster, indicating that the surfactant had been completely removed. The gel microspheres were then transferred to a culture medium, centrifuged at 1000 x g for 1 min, and this process was repeated three times. The microspheres were then resuspended in the culture medium for later use, and 1% PF-127 was added to prevent microgel adhesion.

[0042] (3) Fluorescent secondary antibody localization microgel surface antibody

[0043] F(ab′)2 anti-mouse IgG Fc-FITC was added to the microgel suspensions of (1) and (2) respectively, and the mixtures were placed on a vertical rotary mixer at room temperature and incubated for 30 min. The results were then examined under a fluorescence microscope.

[0044] Experimental results (see) Figure 3 Under a fluorescence microscope, the HA-SH / HB-PEGDA antibody microgel showed obvious FITC fluorescence on its surface, while the HA-SH / HB-PEGDA blank microgel showed no fluorescence. This indicates that the prepared microgel has a porous structure and that antibodies are exposed on the surface of the microgel and can be identified.

[0045] Example 4

[0046] Preparation of antibody microgel systems from immunized mouse serum: Microgels were screened using rod-shaped Fe3O4 magnetic beads conjugated with secondary antibodies.

[0047] (1) Preparation of HA-SH / HB-PEGDA antibody-fluorescent secondary antibody microgel

[0048] Hydrogel precursor droplets were prepared in a PDMS droplet generation chip with dimensions of 40×40×25μm. HB-PEGDA dissolved in a culture medium (containing 10% fetal bovine serum and 1% streptolysin / penicillin mixture) served as the aqueous phase. Antibodies from immunized mouse serum were co-incubated with F(ab′)2 anti-mouse IgG Fc-FITC to locate the serum antibodies in real time, and then added to the aqueous phase. The oil phase consisted of Novec HFE-7500 fluorinated oil containing 2% (w / w) surfactant. The droplet diameter was approximately 34μm, the volume was 21pL, and the concentration of HB-PEGDA in the aqueous phase was 6.67% (w / v). The generated droplets were collected in sterile syringes and incubated in a saturated humidity incubator at 37°C and 5% CO2 for a period of time. Then, HA-SH at a concentration of 5.33% (w / v) was injected into the droplets using pico-injection technology. The pico-injection chip had dimensions of 40×60×25μm. At this point, the droplet diameter was approximately 43 μm and the volume was 42 pL. After standing at room temperature for 2 hours, the HB-PEGDA and HA-SH in the droplet underwent a Michael addition reaction, resulting in complete gelation and the formation of microgels. The surfactant on the surface of the microgels was washed away with pure fluorinated oil. The microgels were centrifuged at 1000 x g for 1 min, the fluorinated oil was discarded, and the washing process was repeated until the gel microspheres agglomerated into a cluster, indicating that the surfactant had been completely removed. The gel microspheres were then transferred to a culture medium, centrifuged at 1000 x g for 1 min, and this process was repeated three times. The microspheres were then resuspended in the culture medium for later use, and 1% PF-127 was added to prevent microgel adhesion.

[0049] (2) Preparation of HA-SH / HB-PEGDA blank microgels

[0050] Hydrogel precursor droplets were prepared in a PDMS droplet generation chip with dimensions of 40×40×25μm. HB-PEGDA dissolved in culture medium (containing 10% fetal bovine serum and 1% streptolysin / penicillin mixture) was used as the aqueous phase. An equal volume of PBS containing the antibody-fluorescent secondary antibody from (1) was added to the aqueous phase. The oil phase was Novec HFE-7500 fluorinated oil containing 2% (w / w) surfactant. The droplet diameter was approximately 34μm, the volume was 21pL, and the concentration of HB-PEGDA in the aqueous phase was 6.67% (w / v). The generated droplets were collected in a sterile syringe and incubated in a saturated humidity incubator at 37°C and 5% CO2 for the same time as in (1). HA-SH at a concentration of 5.33% (w / v) was injected into the droplets using pico-injection technology. The pico-injection chip had dimensions of 40×60×25μm. At this point, the droplet diameter was approximately 43 μm and the volume was 42 pL. After standing at room temperature for 2 hours, the HB-PEGDA and HA-SH in the droplet underwent a Michael addition reaction, resulting in complete gelation and the formation of microgels. The surfactant on the surface of the microgels was washed away with pure fluorinated oil. The microgels were centrifuged at 1000 x g for 1 min, the fluorinated oil was discarded, and the washing process was repeated until the gel microspheres agglomerated into a cluster, indicating that the surfactant had been completely removed. The gel microspheres were then transferred to a culture medium, centrifuged at 1000 x g for 1 min, and this process was repeated three times. The microspheres were then resuspended in the culture medium for later use, and 1% PF-127 was added to prevent microgel adhesion.

[0051] (3) Screening microgels using rod-shaped Fe3O4 magnetic beads coupled with secondary antibodies

[0052] Mix the microgels prepared in (1) and (2), add the rod-shaped Fe3O4 magnetic beads with coupled secondary antibody prepared in Example 1 to the suspension, place it on a vertical rotating mixer at room temperature, incubate for 30 min, and perform magnetic screening.

[0053] Experimental results: Under a fluorescence microscope, the HA-SH / HB-PEGDA antibody microgel emitted green fluorescence and had rod-shaped magnetic beads coupled to its surface. The blank HA-SH / HB-PEGDA microgel showed no fluorescence and had virtually no rod-shaped magnetic beads adhering to its surface. Under the influence of a magnet, the magnetic beads could carry the HA-SH / HB-PEGDA antibody microgel, indicating that the screening method established in this invention is valid.

[0054] Example 5

[0055] Preparation of a microgel system for immunized mouse spleen cells: Fluorescent secondary antibody was used to target the surface antibody of the microgel.

[0056] (1) Preparation of HA-SH / HB-PEGDA single-cell microgels

[0057] Hydrogel precursor droplets were prepared in a PDMS droplet generation chip with dimensions of 40×40×25μm. The aqueous phase consisted of HB-PEGDA dissolved in a culture medium (containing 10% fetal bovine serum and 1% strepto-penicillin mixture), while the oil phase consisted of Novec HFE-7500 fluorinated oil containing 2% (w / w) surfactant. The droplets had a diameter of approximately 34μm and a volume of 21pL, with an HB-PEGDA concentration of 6.67% (w / v) in the aqueous phase. The generated droplets were collected in sterile syringes and incubated in a humidified incubator at 37°C and 5% CO2 for a period of time to induce antibody secretion from the cells. HA-SH at a concentration of 5.33% (w / v) was injected into the droplets using pico-injection technology. The pico-injection chip had dimensions of 40×60×25μm. At this point, the droplet diameter was approximately 43 μm and the volume was 42 pL. After standing at room temperature for 2 hours, the HB-PEGDA and HA-SH in the droplet underwent a Michael addition reaction, resulting in complete gelation and the formation of microgels. The surfactant on the surface of the microgels was washed away with pure fluorinated oil. The microgels were centrifuged at 1000 x g for 1 min, the fluorinated oil was discarded, and the washing process was repeated until the gel microspheres agglomerated into a cluster, indicating that the surfactant had been completely removed. The gel microspheres were then transferred to a culture medium, centrifuged at 1000 x g for 1 min, and this process was repeated three times. The microspheres were then resuspended in the culture medium for later use, and 1% PF-127 was added to prevent microgel adhesion.

[0058] (2) Preparation of HA-SH / HB-PEGDA blank microgels

[0059] Hydrogel precursor droplets were prepared in a PDMS droplet generation chip with dimensions of 40×40×25μm. HB-PEGDA dissolved in culture medium (containing 10% fetal bovine serum and 1% strepto-penicillin mixture) was used as the aqueous phase. An equal volume of PBS and the cell suspension from (1) were added to the aqueous phase. The oil phase was Novec HFE-7500 fluorinated oil containing 2% (w / w) surfactant. The droplet diameter was approximately 34μm, the volume was 21pL, and the concentration of HB-PEGDA in the aqueous phase was 6.67% (w / v). The generated droplets were collected in a sterile syringe and incubated in a saturated humidity incubator at 37°C and 5% CO2 for the same time as in (1). HA-SH at a concentration of 5.33% (w / v) was injected into the droplets using pico-injection technology. The pico-injection chip had dimensions of 40×60×25μm. At this point, the droplet diameter was approximately 43 μm and the volume was 42 pL. After standing at room temperature for 2 hours, the HB-PEGDA and HA-SH in the droplet underwent a Michael addition reaction, resulting in complete gelation and the formation of microgels. The surfactant on the surface of the microgels was washed away with pure fluorinated oil. The microgels were centrifuged at 1000 x g for 1 min, the fluorinated oil was discarded, and the washing process was repeated until the gel microspheres agglomerated into a cluster, indicating that the surfactant had been completely removed. The gel microspheres were then transferred to a culture medium, centrifuged at 1000 x g for 1 min, and this process was repeated three times. The microspheres were then resuspended in the culture medium for later use, and 1% PF-127 was added to prevent microgel adhesion.

[0060] (3) Fluorescent secondary antibody localization microgel surface antibody

[0061] Add F(ab′)2 anti-mouse IgG Fc-FITC to the microgel suspension in (1), place it on a vertical rotating mixer at room temperature, incubate for 30 min, and observe the results under a fluorescence microscope.

[0062] Experimental results (see) Figure 4 Under a fluorescence microscope, the HA-SH / HB-PEGDA single-cell microgel showed obvious FITC fluorescence on its surface, while the HA-SH / HB-PEGDA blank microgel showed no fluorescence. This indicates that the prepared microgel has a porous structure and that the antibodies secreted by the cells and exposed on the surface of the microgel can be identified.

[0063] Example 6

[0064] Preparation of a microgel system for immunized mouse spleen cells: Microgels were screened using rod-shaped Fe3O4 magnetic beads conjugated with secondary antibodies.

[0065] (1) Preparation of HA-SH / HB-PEGDA single-cell microgels

[0066] Hydrogel precursor droplets were prepared in a PDMS droplet generation chip with dimensions of 40×40×25μm. Immunized mouse spleen cells were suspended in a culture medium (containing 10% fetal bovine serum and 1% strepto-penicillin mixture) with HB-PEGDA dissolved in it as the aqueous phase. The oil phase consisted of Novec HFE-7500 fluorinated oil containing 2% (w / w) surfactant. The droplet diameter was approximately 34μm, the volume was 21pL, and the concentration of HB-PEGDA in the aqueous phase was 6.67% (w / v). The generated droplets were collected in sterile syringes and incubated in a saturated humidity incubator at 37°C and 5% CO2 for a period of time to induce cell secretion of a certain amount of antibody. HA-SH at a concentration of 5.33% (w / v) was injected into the droplets using pico-injection technology. The pico-injection chip had dimensions of 40×60×25μm. At this point, the droplet diameter was approximately 43 μm and the volume was 42 pL. After standing at room temperature for 2 hours, the HB-PEGDA and HA-SH in the droplet underwent a Michael addition reaction, resulting in complete gelation and the formation of microgels. The surfactant on the surface of the microgels was washed away with pure fluorinated oil. The microgels were centrifuged at 1000 x g for 1 min, the fluorinated oil was discarded, and the washing process was repeated until the gel microspheres agglomerated into a cluster, indicating that the surfactant had been completely removed. The gel microspheres were then transferred to a culture medium, centrifuged at 1000 x g for 1 min, and this process was repeated three times. The microspheres were then resuspended in the culture medium for later use, and 1% PF-127 was added to prevent microgel adhesion.

[0067] (2) Screening microgels using rod-shaped Fe3O4 magnetic beads coupled with secondary antibodies

[0068] Add the rod-shaped Fe3O4 magnetic beads with coupled secondary antibody prepared in Example 1 to the microgel prepared in (1), place it on a vertical rotating mixer at room temperature, incubate for 30 min, and perform magnetic screening.

[0069] The experiment shows (see Figure 5 The material constructed in this invention enables high-throughput screening of single-cell antibodies, while shortening detection time, reducing operational complexity, and saving costs. See the experimental flowchart and schematic diagram of the screening method described in this invention. Figure 1 .

Claims

1. A high-throughput antibody screening method based on single-cell microgel analysis technology, characterized in that: The method includes the following steps: (1) Cells were suspended in HB-PEGDA dissolved in culture medium as the aqueous phase and fluorinated oil containing surfactant as the oil phase. Hydrogel precursor droplets were prepared in a microfluidic device. HA-SH was injected into the incubated droplets and allowed to stand to allow HB-PEGDA and HA-SH to undergo Michael addition reaction, and the droplets were fully gelled to form microspheres. (2) Wash away the surfactant on the surface of the microspheres, resuspend them in the culture medium, add rod-shaped Fe3O4 magnetic beads with coupled secondary antibody, incubate, and magnetically separate.

2. The antibody high-throughput screening method based on single-cell microgel analysis technology according to claim 1, characterized in that: In step (1), the droplet generation chip used by the microfluidic device to prepare droplets is a poly(dimethylsiloxane) chip.

3. The antibody high-throughput screening method based on single-cell microgel analysis technology according to claim 1, characterized in that: The diameter of the droplet in step (1) is 30-45 μm.

4. The antibody high-throughput screening method based on single-cell microgel analysis technology according to claim 1, characterized in that: The culture medium used in the aqueous phase in step (1) is either DMEM or 1640, and the culture medium contains 5-20% (v / v) fetal bovine serum and 1% (v / v) streptolysin / penicillin mixture, with an HB-PEGDA concentration of 6.67% (w / v).

5. The antibody high-throughput screening method based on single-cell microgel analysis technology according to claim 1, characterized in that: The cells mentioned in step (1) are spleen cells from immunized mice.

6. The antibody high-throughput screening method based on single-cell microgel analysis technology according to claim 1, characterized in that: In step (1), the surfactant used in the oil phase is any one of Pico-Surf 1, FS-Kryjeff D900, or EA, and the concentration of the surfactant added in the oil phase is 1-5% (w / w).

7. The antibody high-throughput screening method based on single-cell microgel analysis technology according to claim 1, characterized in that: In step (1), HA-SH was injected using pico-injection technology, and the concentration of HA-SH was 5.33% (w / v).

8. The antibody high-throughput screening method based on single-cell microgel analysis technology according to claim 1, characterized in that: In step (2), pure fluorinated oil is added to wash away the surfactant. After centrifugation, the fluorinated oil is discarded. The washing is repeated until the gel microspheres agglomerate into a clump, indicating that the surfactant has been completely washed away. The gel microspheres are then transferred to the culture medium, centrifuged, and resuspended in the culture medium for later use.

9. The antibody high-throughput screening method based on single-cell microgel analysis technology according to claim 1, characterized in that: The preparation method of the rod-shaped Fe3O4 magnetic beads used in step (2) is as follows: add rod-shaped Fe3O4 magnetic beads, sodium dodecyl sulfate and methacrylic acid to a solvent, and react to prepare carboxylated rod-shaped Fe3O4 magnetic beads. After activating the carboxylated rod-shaped Fe3O4 magnetic beads, add streptavidin to synthesize streptavidin magnetic beads. Co-incubate them with biotin-VHH anti-mouse κ light chain to form rod-shaped Fe3O4 magnetic beads for the conjugated secondary antibody.

10. The antibody high-throughput screening method based on single-cell microgel analysis technology according to claim 9, characterized in that: The preparation method of the rod-shaped Fe3O4 magnetic beads is as follows: FeSO4·7H2O and FeCl3 are dissolved in a solvent, ethylenediamine is added as a template agent, and rod-shaped Fe3O4 magnetic beads are obtained after the reaction.

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