Method for screening rabbit new crown neutralizing antibody based on Beacon single cell screening platform
By using the Beacon single-cell screening platform and combining it with a multi-step screening method, the problem of low screening efficiency for rabbit COVID-19 neutralizing antibodies has been solved, enabling efficient and rapid screening of high-affinity antibodies suitable for the treatment and prevention of COVID-19.
Patent Information
- Application Number
- CN202511096537.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-06
- Publication Date
- 2025-11-11
AI Technical Summary
Existing methods for screening rabbit COVID-19 neutralizing antibodies are inefficient and struggle to quickly respond to viral mutations. Traditional hybridoma techniques are time-consuming and cannot efficiently screen for high-quality neutralizing antibodies.
Using the Beacon single-cell screening platform, rabbit SARS-CoV-2 neutralizing antibodies were screened with high throughput and high sensitivity through steps such as antigen preparation, animal immunization, cell isolation, in vitro activation, single-cell sorting, antibody gene amplification and sequencing, antibody expression and purification.
It significantly improves antibody screening efficiency, rapidly screens out high-affinity neutralizing antibodies that can combat the novel coronavirus and its variants, and is simple and easy to standardize.
Smart Images
Figure CN120927972A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, and in particular relates to a method for screening rabbit SARS-CoV-2 neutralizing antibodies based on the Beacon single-cell screening platform. Background Technology
[0002] Neutralizing antibodies can effectively block viral invasion of cells and are an important means of treating and preventing COVID-19. Currently, several neutralizing antibodies have been approved for marketing or have entered clinical trials, but problems such as insufficient neutralizing activity and decreased effectiveness against variant strains still exist. Rabbit-derived antibodies have advantages such as high diversity, strong affinity, and ease of humanization. However, traditional hybridoma technology for screening rabbit monoclonal antibodies is inefficient and time-consuming, making it difficult to meet the needs of rapid response to viral mutations. The Beacon single-cell antibody screening platform can screen antigen-specific memory B cells with high throughput and high sensitivity while preserving the natural pairing of antibodies, providing a new technical means for rapid screening of highly effective neutralizing antibodies.
[0003] Currently, methods for screening rabbit SARS-CoV-2 neutralizing antibodies mainly include hybridoma technology, phage display technology, flow cytometry, and antibody modification. While these methods are widely used, they are inefficient and do not necessarily yield naturally paired antibodies. The Beacon single-cell antibody screening platform, however, integrates culture, cloning, detection, screening, and export. It can load four chips at once, screening 50,000 single cells in one day. From immunization to obtaining positive naturally paired antibodies takes only 35 days. This system preserves the diversity of memory B cells, allowing for the acquisition of high-quality positive cells at an early stage. Therefore, this application provides a highly efficient and rapid method for screening neutralizing antibodies. Summary of the Invention
[0004] This invention provides a method for screening rabbit SARS-CoV-2 neutralizing antibodies based on the Beacon single-cell screening platform, aiming to solve the above-mentioned problems.
[0005] This invention is implemented as follows: a method for screening rabbit SARS-CoV-2 neutralizing antibodies based on the Beacon single-cell screening platform, comprising the following steps: Preparation of screening antigens: The S1 and RBD proteins of the novel coronavirus were conjugated with Assay Beads containing streptavidin; Animal immunization: Rabbits were immunized with SARS-CoV-2 antigen to obtain immune serum; Serum titers and specificity were detected using ELISA, and peripheral blood was collected from rabbits with good titers. Isolation of peripheral blood mononuclear cells (PBMCs): PBMCs were isolated from the peripheral blood of immunized rabbits using density gradient centrifugation; Enrichment of rabbit memory B cells: Antibodies were isolated from biotin-labeled memory B cells, incubated with PBMCs, and then memory B cells were enriched using streptavidin magnetic beads; In vitro activation: The enriched memory B cells were activated in vitro using an activation medium to transform them into plasma cells; Single-cell sorting: Activated plasma cells are loaded onto the Beacon single-cell antibody screening platform for single-cell sorting; Antibody gene amplification and sequencing: The sorted single cells are subjected to antibody gene amplification and sequencing to obtain the variable region sequences of the antibody heavy chain and light chain; Antibody expression and purification: The obtained antibody gene sequence is cloned into an expression vector and transfected into mammalian cells for antibody expression and purification; Activity assay: The purity, activity, and affinity of the obtained antibodies are tested.
[0006] Preferably, the preparation of the screening antigen specifically includes: The S1 and RBD proteins of the novel coronavirus were biotinylated using a biotinylation kit, and then excess biotin was removed and concentrated using a concentration tube. Biotinylated antigens and Assay Beads containing streptavidin were incubated with shaking at a mass ratio of 1:5, and then the conjugation was detected by immune serum.
[0007] Preferably, the animal immunization specifically includes: Multiple rabbits (e.g., three rabbits) were immunized with the novel coronavirus S1 at a dose of 0.5 mg per rabbit. The immunization schedule consisted of a primary immunization and two booster immunizations. Preferably, the initial primary immunization uses an equal volume of Freund's complete adjuvant emulsified antigen injected subcutaneously at multiple points on the back of the neck of mice. 21 days later, a booster immunization is performed, in which the emulsified antigen is injected intraperitoneally into rabbits using Freund's incomplete adjuvant. The interval between each booster immunization is 14 days. After three immunizations, 1 mL of blood was collected, and the blood was first incubated at 37°C for 0.5 h, then at 4°C for 10 min, and then centrifuged at 7000 r / min for 10 min. The supernatant serum was collected and the antibody titer was detected by indirect ELISA.
[0008] Preferably, the separation of peripheral blood mononuclear cells, enrichment of rabbit memory B cells, and in vitro activation specifically involve: 10 mL of peripheral blood was taken from rabbits with good serum titer and specificity, and centrifuged at 1000 g for 20 min using density gradient centrifugation at 4°C, with the centrifuge brake turned off. Remove and wash the middle layer of PBMCs, add 50uL of normal mouse serum, count the cells, add rabbit memory B cell isolation antibody, incubate at 4℃ for 20 min, then add 100uL of Ati-Biotin Micro Beads Ultrapure, incubate at 4℃ for 15 min, centrifuge, reselect the cells, then load them onto Miltenyi MS columns, and finally squeeze the syringe to elute the cells. Then, using activation medium, at a rate of 5000 cells / well, the cells were seeded into 96-well V-plates and incubated at 37°C in a CO2 incubator for 5 days.
[0009] Preferably, single-cell sorting specifically involves: After reselecting and centrifuging the activated cells, count the number of viable cells at a ratio of 1×10⁻⁶. 7 / mL is loaded into the screening chip, and the Beacon instrument sorts the individual cells into individual chambers, which are then cultured for 30 minutes to induce antibody secretion. Then, rabbit IgG, RBD, and S1 microbeads were loaded onto the chip for screening. Finally, single cells that were positive for rabbit IgG, RBD, and S1 were exported to a 96-well plate.
[0010] Preferably, the antibody gene amplification and sequencing specifically involve: RNA was extracted from the above single cells and reverse transcribed into cDNA. The variable regions of the heavy and light chains of the cells were amplified and sequenced. The correctly sequenced gene is synthesized and cloned into an expression vector for subsequent expression.
[0011] Preferably, the antibody expression and purification specifically involve: The products synthesized from the above genes were transfected into HEK 293 cells at a certain ratio; Five days later, the supernatant was collected by centrifugation and then purified using an AKTA protein purification instrument.
[0012] Preferably, the antibody purity determination specifically involves: Prepare the concentrated gel and separating gel in a certain ratio, and let them solidify at room temperature; The purified antibody was diluted to a certain concentration and loaded at a dose of 3 μg. Add 5X Loading buffer to each sample, then heat at 95℃ for 10 min before loading. Run at 80V for 30 min, then run at 120V to the bottom of the sample. Stain with Coomassie Brilliant Blue for 20 min, then decolorize with 20% ethanol for 3 h. Finally, take a picture for observation.
[0013] Preferably, the antibody activity assay specifically involves: The titer and activity of the screened antibodies were determined using ELISA. RBD was used as the coating agent, diluted with CBS to 1 µg / mL, and then added to the microplate. The plate was coated overnight at 4°C. Shake off the coating solution, wash 3 times, pat dry, add 1% BSA blocking solution, 250µL / well, and block at 37°C for 1-2 hours. Wash 3 times, pat dry, add 50µL of PBS to each well, then dilute the antibody with PBS to 2000, 4000, 8000, 16000, 32000 and 64000 times respectively, add 50µL to each well horizontally in the microplate, and incubate at 37°C for 30-40 min. Wash 3 times, pat dry, dilute HRP-labeled goat anti-rabbit secondary antibody to working concentration of 1:6000 with PBS, add 100µL to each well of the microplate, and incubate at 37°C for 30-40 min. Wash 5 times, pat dry, add 100µL of substrate mixture to each well, incubate at 37°C in the dark for 10-15 min, add 50µL of stop solution, and measure the optical density (OD value) at 450nm wavelength using an automated microplate reader. Select the OD value close to 2.0 as the optimal antibody dilution, and then use this concentration for indirect competition.
[0014] Preferably, the antibody affinity assay specifically involves: The antibody was serially diluted, and its affinity was then determined using the Octet molecular interaction instrument.
[0015] Preferably, the novel coronavirus antigen protein is a fusion protein of the RBD receptor binding domain and the NTD domain of S1, which are linked by a flexible linker and directionally coupled to streptavidin microbeads via a histidine tag. Design of dual epitope tandem antigens: The receptor-binding domain (RBM) of RBD is connected to the NTD domain of S1 via a flexible linker (GGGGS×3); Biotinylated and then coupled to streptavidin microbeads; Directed coupling technology: Microbeads pre-coated with streptavidin-histidine tags The antigen C-terminus is fused with 6×His to achieve targeted immobilization (avoiding epitope masking). It can simultaneously capture antibodies targeting RBM (blocking ACE2 binding) and NTD (blocking membrane fusion), thus improving screening sensitivity; Directional coupling has a low shading rate, avoiding epitope loss.
[0016] Preferably, the adjuvant used in the immunization procedure in step (b) is an oil-in-water emulsion comprising: squalene: 3-8% (v / v); monophospholipid A (MPLA): 50-200 μg / mL; CpG-ODN 2006: 20-100 μg / mL; polysorbate 80: 0.5% (v / v); phosphate buffer: balance; By enhancing antibody titer with squalene, activating the dual TLRs of MPLA+CpG-ODN, and inducing TLR4-TLR9 crosstalk, Th1 / humoral immunity synergy is achieved, thereby increasing the proportion of neutralizing antibody-positive B cells.
[0017] The present invention also provides a rabbit SARS-CoV-2 neutralizing antibody, which is obtained by the above-mentioned method of screening rabbit SARS-CoV-2 neutralizing antibodies based on the Beacon single-cell screening platform.
[0018] The above-mentioned rabbit SARS-CoV-2 neutralizing antibody was used in the preparation of drugs for the prevention or treatment of SARS-CoV-2 infection.
[0019] Compared with the prior art, the embodiments of this application have the following main advantages: The method for screening rabbit SARS-CoV-2 neutralizing antibodies based on the Beacon single-cell screening platform provided by this invention utilizes the Beacon single-cell antibody screening platform, which can screen rabbit SARS-CoV-2 neutralizing antibodies with high throughput and high sensitivity, significantly improving antibody screening efficiency.
[0020] The method of this invention can preserve the natural pairing of antibodies and obtain neutralizing antibodies with high affinity.
[0021] The method of this invention is simple to operate, easy to standardize, and can quickly screen for highly efficient neutralizing antibodies against SARS-CoV-2 and its variants.
[0022] This invention has a short development time and broad application prospects. Attached Figure Description
[0023] Figure 1 This is a flowchart of a method for screening rabbit SARS-CoV-2 neutralizing antibodies based on the Beacon single-cell screening platform provided by the present invention.
[0024] Figure 2 This is a diagram of the platform screening process in a method for screening rabbit SARS-CoV-2 neutralizing antibodies based on the Beacon single-cell screening platform provided by the present invention.
[0025] Figure 3 This is an SDS-PAGE image of an antibody obtained by a method for screening rabbit SARS-CoV-2 neutralizing antibodies based on the Beacon single-cell screening platform provided by the present invention.
[0026] Figure 4This invention provides a method for screening rabbit SARS-CoV-2 neutralizing antibodies based on the Beacon single-cell screening platform, and the resulting antibody titer detection graph is shown.
[0027] Figure 5 This invention provides a method for screening rabbit SARS-CoV-2 neutralizing antibodies based on the Beacon single-cell screening platform, which yields an antibody affinity detection map. Detailed Implementation
[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terminology used herein in the specification of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application; the terms "comprising" and "having," and any variations thereof, in the specification, claims, and foregoing drawings of this application are intended to cover non-exclusive inclusion. The terms "first," "second," etc., in the specification, claims, or foregoing drawings of this application are used to distinguish different objects, not to describe a particular order.
[0029] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.
[0030] Example 1 This invention provides a method for screening rabbit SARS-CoV-2 neutralizing antibodies based on the Beacon single-cell screening platform, such as... Figure 1 As shown, it includes the following steps: Preparation of screening antigens: The S1 and RBD proteins of the novel coronavirus were conjugated with Assay Beads containing streptavidin; Animal immunization: Rabbits were immunized with SARS-CoV-2 antigen to obtain immune serum; Serum titers and specificity were detected using ELISA, and peripheral blood was collected from rabbits with good titers. Isolation of peripheral blood mononuclear cells (PBMCs): PBMCs were isolated from the peripheral blood of immunized rabbits using density gradient centrifugation; Enrichment of rabbit memory B cells: Antibodies were isolated from biotin-labeled memory B cells, incubated with PBMCs, and then memory B cells were enriched using streptavidin magnetic beads; In vitro activation: The enriched memory B cells were activated in vitro using an activation medium to transform them into plasma cells; Single-cell sorting: Activated plasma cells are loaded onto the Beacon single-cell antibody screening platform for single-cell sorting. The screening process is as follows: Figure 2 As shown; Antibody gene amplification and sequencing: The sorted single cells are subjected to antibody gene amplification and sequencing to obtain the variable region sequences of the antibody heavy chain and light chain; Antibody expression and purification: The obtained antibody gene sequence is cloned into an expression vector and transfected into mammalian cells for antibody expression and purification; Activity assay: The purity, activity, and affinity of the obtained antibodies are tested.
[0031] The preparation of the screening antigen specifically involves: The S1 and RBD proteins of the novel coronavirus were biotinylated using a biotinylation kit, and then excess biotin was removed and concentrated using a concentration tube. Biotinylated antigens and Assay Beads containing streptavidin were incubated with shaking at a mass ratio of 1:5, and then the conjugation was detected by immune serum.
[0032] Furthermore, the animal immunization specifically refers to: Multiple rabbits (e.g., three rabbits) were immunized with the novel coronavirus S1 at a dose of 0.5 mg per rabbit. The immunization schedule consisted of a primary immunization and two booster immunizations. Furthermore, for the first primary immunization, an equal volume of Freund's complete adjuvant emulsified antigen was injected subcutaneously at multiple points on the back of the neck of mice. 21 days later, a booster immunization was performed. For the booster immunization, an emulsified antigen was injected intraperitoneally into rabbits using Freund's incomplete adjuvant. The interval between each booster immunization was 14 days. After three immunizations, 1 mL of blood was collected, and the blood was first incubated at 37°C for 0.5 h, then at 4°C for 10 min, and then centrifuged at 7000 r / min for 10 min. The supernatant serum was collected and the antibody titer was detected by indirect ELISA.
[0033] In this embodiment, the separation of peripheral blood mononuclear cells, enrichment of rabbit memory B cells, and in vitro activation specifically involve: 10 mL of peripheral blood was taken from rabbits with good serum titer and specificity, and centrifuged at 1000 g for 20 min using density gradient centrifugation at 4°C, with the centrifuge brake turned off. Remove and wash the middle layer of PBMCs, add 50uL of normal mouse serum, count the cells, add rabbit memory B cell isolation antibody, incubate at 4℃ for 20 min, then add 100uL of Ati-Biotin Micro Beads Ultrapure, incubate at 4℃ for 15 min, centrifuge, reselect the cells, then load them onto Miltenyi MS columns, and finally squeeze the syringe to elute the cells. Then, using activation medium (existing conventional activation medium), cells were seeded into 96-well V-plates at a rate of 5000 cells / well and incubated at 37°C in a CO2 incubator for 5 days.
[0034] In practice, single-cell sorting is specifically as follows: After reselecting and centrifuging the activated cells, count the number of viable cells at a ratio of 1×10⁻⁶. 7 / mL is loaded into the screening chip, and the Beacon instrument sorts the individual cells into individual chambers, which are then cultured for 30 minutes to induce antibody secretion. Then, rabbit IgG, RBD, and S1 microbeads were loaded onto the chip for screening. Finally, single cells that were positive for rabbit IgG, RBD, and S1 were exported to a 96-well plate.
[0035] Preferably, the antibody gene amplification and sequencing specifically involve: RNA was extracted from the above single cells and reverse transcribed into cDNA. The variable regions of the heavy and light chains of the cells were amplified and sequenced. The correctly sequenced gene is synthesized and cloned into an expression vector for subsequent expression.
[0036] In this embodiment, the antibody expression and purification are specifically as follows: The products synthesized from the above genes were transfected into HEK 293 cells at a certain ratio; Five days later, the supernatant was collected by centrifugation and then purified using an AKTA protein purification instrument.
[0037] The purity determination of antibodies is specifically as follows: Prepare the concentrated gel and separating gel in a certain ratio, and let them solidify at room temperature; The purified antibody was diluted to a certain concentration and loaded at a dose of 3 μg. Add 5X loading buffer to each sample, heat at 95℃ for 10 min, then load the sample. Run at 80V for 30 min, then run at 120V until the sample reaches the bottom. Stain with Coomassie Brilliant Blue for 20 min, then destain with 20% ethanol for 3 h. Finally, take pictures and observe the results. See below. Figure 3 .
[0038] The specific steps for antibody activity assay are as follows: The titer and activity of the screened antibodies were determined using ELISA. RBD was used as the coating agent, diluted with CBS to 1 µg / mL, and then added to the microplate. The plate was coated overnight at 4°C. Shake off the coating solution, wash 3 times, pat dry, add 1% BSA blocking solution, 250µL / well, and block at 37°C for 1-2 hours, preferably 1.5 hours in this embodiment; Wash 3 times, pat dry, add 50µL of PBS to each well, then dilute the antibody with PBS to 2000, 4000, 8000, 16000, 32000 and 64000 times respectively, add 50µL to each well horizontally in the ELISA plate, and incubate at 37°C for 30-40 min, preferably 35 min in this example; Wash 3 times, pat dry, dilute HRP-labeled goat anti-rabbit secondary antibody to working concentration of 1:6000 with PBS, add 100µL to each well of the microplate, and incubate at 37°C for 30-40 min, preferably 35 min in this example; Wash 5 times, pat dry, add 100µL of substrate mixture to each well, and incubate at 37°C in the dark for 10-15 min (preferably 12.5 min in this example). Add 50µL of stop solution and measure the optical density (OD value) at 450nm using an automated microplate reader. Select an OD value close to 2.0 as the optimal antibody dilution, and then use this concentration for indirect competition. The antibody titer results of ELISA are shown below. Figure 4 .
[0039] In practice, the antibody affinity assay is performed as follows: The antibody was serially diluted, and its affinity was then determined using an Octet molecular interaction analyzer. The results are shown in [Figure number missing]. Figure 5 .
[0040] This invention provides a method for screening rabbit SARS-CoV-2 neutralizing antibodies based on the Beacon single-cell screening platform. This method can screen for highly efficient neutralizing antibodies with high throughput and high sensitivity, providing a new strategy for the treatment and prevention of COVID-19. Ultimately, two antibodies, R6 and R36, with good activity and high affinity were screened. Sequencing of R6 and R36 antibodies revealed the following: the heavy chain amino acid sequence of R6 is shown in SEQ ID NO. 1, the light chain amino acid sequence of R6 is shown in SEQ ID NO. 2, the heavy chain amino acid sequence of R36 is shown in SEQ ID NO. 3, and the light chain amino acid sequence of R36 is shown in SEQ ID NO. 4. R6 heavy chain amino acid sequence (SEQ ID NO. 1): QSVEESGGRLVTPGTPLTLPCTVSGIDLSTWSMSWVRQAPGKGLEWIGITYSASDGSTYYANWAKGRFTISRASTTVDLKMTSLTTEDTAIYFCCRINYVSSTWGADNLWGQGTLVTVSSWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTANOANAALRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK。
[0041] R6 light chain amino acid sequence (SEQ ID NO. 2): DVVMTQTPSPVSEPVGGTVTIKCQASQSISSYLAWYQQKPGQPPKLLIYDASDLASGVPSRFKGSGSGTQFTLTISDLECADAATYYCQYSNYGSNYVASTFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC。
[0042] R36 heavy chain amino acid sequence (SEQ ID NO. 3): QSVKESEGGLFKPAETLTLTCTVSGAIDLSSDAISWVRQAPGNGLEWIGVIGNGANGYKFYASWAKSRSTITRNTNLNTVDTLKMTSLTAADTATYFCARDLGTTSYIDRLDLWGQGTLVSSWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVS VTSSSAEAANNSRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTIPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLE PKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK.
[0043] R36 heavy chain amino acid sequence (SEQ ID NO. 4): DPVMTQTPSPVSAAVGSTVTISQSVGDNNWLGSCQSWYQQKPGQPPKLLIYAASTLASGVPSRFKGSGSGTQFTLTISGVQCDDAGTYYCAGRGYSGSDLYTFGGGTE VVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC.
[0044] Example 2 The difference between this embodiment and Embodiment 1 is that: 1) The novel coronavirus antigen protein is a fusion protein of the RBD receptor binding domain and the NTD domain of S1. The two are linked by a flexible linker and are directionally coupled to streptavidin microbeads via a histidine tag. 2) The immunization procedure does not use Freund's adjuvant; instead, it uses an oil-in-water emulsion containing: squalene: 3-8% (v / v); monophospholipid A (MPLA): 50-200 μg / mL; CpG-ODN 2006: 20-100 μg / mL; polysorbate 80: 0.5% (v / v); phosphate buffer: balance. Comparative Example 1: The effect of the adjuvant was verified by using only the compound adjuvant (retaining the antigen and B cell activation method of Example 1).
[0045] Comparative Example 2: Using only dual epitope antigen-directed conjugation (retaining the adjuvant and B cell activation method of Example 1), the antigen design effect was verified alone.
[0046] 1. Comparison of immune effects, the results are shown in Table 1 below: Table 1 * indicates p < 0.01 compared to Example 1; The results above show that the combined adjuvant (MPLA+CpG) increases the proportion of memory B cells.
[0047] 2. Comparison of antigen screening efficiencies, the results are shown in Table 2 below: Table 2 * indicates p < 0.01 compared to Example 1; The results above show that dual epitope antigens improve the detection rate of broad-spectrum and high-efficiency antibodies, and targeted conjugation avoids epitope masking.
[0048] It should be noted that, for the sake of simplicity, the foregoing embodiments are all described as a series of actions. However, those skilled in the art should understand that the present invention is not limited to the described order of actions, as some steps may be performed in other orders or simultaneously according to the present invention. Furthermore, those skilled in the art should also understand that the embodiments described in the specification are preferred embodiments, and the actions and modules involved are not necessarily essential to the present invention.
[0049] It should be understood that the disclosed apparatus can be implemented in other ways, given the several embodiments provided in this application. For example, the apparatus embodiments described above are merely illustrative; the division of units described above is only a logical functional division, and in actual implementation, there may be other division methods. For example, multiple units or components may be combined or integrated into another system, or some features may be ignored or not executed. Furthermore, the coupling or communication connections shown or discussed may be through some interfaces; the indirect coupling or communication connections between devices or units may be telecommunications or other forms.
[0050] The units described above as separate components may or may not be physically separate. The components shown as units may or may not be physical units; that is, they may be located in one place or distributed across multiple network units. Some or all of the units can be selected to achieve the purpose of this embodiment according to actual needs.
[0051] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on these embodiments, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still combine, add, delete, or otherwise adjust the features of the various embodiments of the present invention according to the circumstances without conflict or creative effort, thereby obtaining different technical solutions that do not fundamentally depart from the concept of the present invention. These technical solutions also fall within the scope of protection of the present invention.
Claims
1. A method for screening rabbit SARS-CoV-2 neutralizing antibodies based on the Beacon single-cell screening platform, characterized in that, Includes the following steps: (a) Conjugating novel coronavirus antigen protein to detection microbeads; (b) Immunize rabbits with the antigen protein and isolate peripheral blood mononuclear cells from rabbits that have reached the titer level; (c) Enrichment of memory B cells from PBMCs and in vitro activation into plasma cells; (d) The activated cells were loaded into the Beacon single-cell antibody screening platform for single-cell sorting and multiple rounds of antigen-specific screening; (e) Export single cells that have been positive in multiple rounds of screening and perform antibody gene amplification; (f) Recombinant expression and purification of the antibody, followed by activity testing.
2. The method for screening rabbit SARS-CoV-2 neutralizing antibodies based on the Beacon single-cell screening platform as described in claim 1, characterized in that, In step (a), the novel coronavirus antigen protein is selected from S1 and RBD proteins, and the detection microbeads are streptavidin-conjugated microbeads.
3. The method for screening rabbit SARS-CoV-2 neutralizing antibodies based on the Beacon single-cell screening platform as described in claim 1, characterized in that, The immunization procedure in step (b) includes basic immunization and at least two booster immunizations, with adjuvants selected from Freund's complete adjuvant and incomplete adjuvant.
4. The method for screening rabbit SARS-CoV-2 neutralizing antibodies based on the Beacon single-cell screening platform as described in claim 1, characterized in that, In step (c), biotin-labeled antibodies and streptavidin magnetic beads were used to enrich memory B cells, and the culture time in the activation medium was 3-7 days.
5. The method for screening rabbit SARS-CoV-2 neutralizing antibodies based on the Beacon single-cell screening platform as described in claim 4, characterized in that, The multi-round antigen-specific screening includes at least: First round: Rabbit IgG positive test; Second round: RBD specific detection; Third round: S1 specificity detection.
6. The method for screening rabbit SARS-CoV-2 neutralizing antibodies based on the Beacon single-cell screening platform as described in claim 1, characterized in that, The antibody gene amplification in step (e) includes: extracting single-cell RNA, reverse transcribing it into cDNA, and amplifying the variable region sequences of the heavy and light chains.
7. The method for screening rabbit SARS-CoV-2 neutralizing antibodies based on the Beacon single-cell screening platform as described in claim 1, characterized in that, The recombinant expression in step (f) involves cloning the heavy chain variable region sequence and the light chain variable region sequence gene into an expression vector and transfecting HEK 293 cells for expression; the activity detection includes detecting the purity, activity, and affinity of the obtained antibody.
8. The method for screening rabbit SARS-CoV-2 neutralizing antibodies based on the Beacon single-cell screening platform as described in claim 2, characterized in that, In step (a), the novel coronavirus antigen protein is a fusion protein of the RBD receptor binding domain and the NTD domain of S1. The two are linked by a flexible linker and are directionally coupled to streptavidin microbeads via a histidine tag.
9. The method for screening rabbit SARS-CoV-2 neutralizing antibodies based on the Beacon single-cell screening platform as described in claim 1, characterized in that, The adjuvant used in the immunization procedure in step (b) is an oil-in-water emulsion containing: squalene: 3-8%; monophospholipid A: 50-200 μg / mL; CpG-ODN 2006: 20-100 μg / mL; polysorbate 80: 0.5%; phosphate buffer: balance.
10. A rabbit SARS-CoV-2 neutralizing antibody, characterized in that, The method described in any one of claims 1-9 for screening rabbit SARS-CoV-2 neutralizing antibodies using the Beacon single-cell screening platform was employed.