Method for high-throughput screening of monoclonal plasma cell secreting antibody

Abstract

The invention discloses a method for high-throughput screening of monoclonal plasma cell secreting antibodies, and relates to the technical field of antibody screening. According to the method, a micro-droplet is used as a reaction container, a large-particle substance carrying a target antigen is used as a matrix, a positive secretion antibody and a fluorescence detection antibody are mediated to generate an aggregation strong fluorescence signal, and high-sensitivity quantitative detection is realized. According to the screening method disclosed by the invention, the cell sample and the report cell sample are sealed by the sealing antibody in advance, so that the possibility of nonspecific binding of the subsequent detection antibody is reduced; and dead cells in the liquid drop system are marked in real time by using a dead cell fluorescent dye, so that false positive signals generated by autofluorescence or non-specific adsorption of the dead cells are eliminated, and the success rate of subsequent cell culture and BCR amplification processes is increased. By adopting the screening method disclosed by the invention, main non-target signals in a current system are basically eliminated, the true positive proportion of a screened product can be greatly improved, and screening of a sample with an extremely low positive rate becomes possible.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody screening, and in particular to a method for high-throughput screening of monoclonal plasma cell secretory antibodies. Background Art

[0002] Monoclonal secretory antibodies are antibodies produced by a single B cell clone that can be continuously secreted in vitro and target specific antigenic epitopes. Screening and obtaining high-purity monoclonal antibodies is of great significance for scientific research, disease diagnosis and treatment.

[0003] Currently, there are many technologies available for screening monoclonal antibodies, with flow cytometry sorting and hybridoma fusion being the most widely used. The former is mainly used to obtain surface antibodies of memory cells, and it is difficult to screen secretory antibodies and antibodies against membrane surface antigens; the latter is only applicable to limited species, and has low throughput, long cycles, and large workloads, and is also difficult to deal with samples with low positive rates.

[0004] Currently, there is also a technology that uses fluorescent image recognition and light manipulation to screen secretory monoclonal antibodies. However, this type of solution has high operating costs, low throughput, and high requirements on the quality of plasma cell samples, and has not yet been promoted on a large scale.

[0005] There are also methods for high-throughput screening based on microfluidics technology, but the system itself is susceptible to interference from various types of false positive signals, making the screening process unstable and limiting the signal sensitivity and binding positivity rate.

[0006] There is an urgent need to develop a high-throughput screening method for monoclonal secretory antibodies that is more comprehensive and accurate. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a high-throughput screening method for monoclonal secretory antibodies with more comprehensive screening and high accuracy based on the defects mentioned in the background technology.

[0008] In order to solve the above problems, the present invention proposes the following technical solutions:

[0009] In a first aspect, the present invention provides a method for high-throughput screening of monoclonal plasma cell secretory antibodies, the screening method comprising:

[0010] (1) Providing a plurality of nanoliter or picoliter droplets, each droplet comprising:

[0011] a. No more than one antibody-secreting cell;

[0012] b. microbeads coated with target antigen, or at least one reporter cell with target antigen on its membrane surface;

[0013] c. Detection antibodies and / or dead cell fluorescent dyes;

[0014] (2) incubating the plurality of nanoliter or picoliter droplets under appropriate conditions to allow the antibody-secreting cells to secrete the target antibody, thereby obtaining incubated droplets;

[0015] (3) passing the incubated droplets through a droplet sorting chip one by one to detect fluorescence signals, and sorting and enriching target droplets according to the fluorescence signal values ​​of the droplets;

[0016] In (1), the antibody-secreting cells are pre-treated with a blocking antibody; the reporter cells carrying the target antigen on the membrane surface are pre-treated with a blocking antibody;

[0017] The blocking antibody can specifically bind to the target antibody; the detection antibody can specifically bind to the target antibody; and the blocking antibody cannot be recognized and bound by the detection antibody; the detection antibody carries a first fluorescent marker, and the fluorescent signal generated by the first fluorescent marker is different from the fluorescent signal generated by the dead cell fluorescent dye.

[0018] A further technical solution is that in said (2), the incubation temperature is 20-37 degrees Celsius and the time is 0.5-8 hours.

[0019] It can be understood that the sorting and enrichment methods include, after identifying the target droplet, applying a dielectrophoretic force or other methods to it, attracting it to the target channel close to one side of the electrode; collecting the liquid flow at the end of the target channel, thereby obtaining the target droplet. The target droplet contains target antibody-secreting cells that can secrete antigen-specific antibodies. The obtained target antibody-secreting cells can be subsequently used for cell culture, hybridoma preparation, BCR sequence capture, sequencing, recloning and antibody preparation, antibody affinity detection and other applications.

[0020] A further technical solution is that the target droplet comprises:

[0021] ① In the fluorescence channel of the detection antibody, the droplet has a significant difference between the fluorescence signal value of the droplet and the background fluorescence value of the droplet; ② In the fluorescence channel of the dead cell dye, the droplet has no significant difference between the fluorescence signal value of the droplet and the background fluorescence value of the droplet;

[0022] Alternatively, the target droplet comprises:

[0023] 1) In the fluorescence channel of the detection antibody, the fluorescence signal value of the droplet is 1.2 times or more of the background fluorescence value of the droplet; 2) In the fluorescence channel of the dead cell dye, the fluorescence signal value of the droplet is less than or equal to 1.1 times the background fluorescence value of the droplet.

[0024] A further technical solution is that the blocking antibody carries a second fluorescent marker, and the fluorescent signal generated by the second fluorescent marker is different from the fluorescent signal generated by the first fluorescent marker.

[0025] It can be understood that the blocking antibody can be an antibody without a fluorescent marker, an antibody with a second fluorescent marker, or a combination of an antibody without a fluorescent marker and an antibody with a second fluorescent marker.

[0026] A further technical solution is that the target droplet further includes: ③ a droplet whose fluorescence signal value of the droplet has no significant difference with the background fluorescence value of the droplet in a closed antibody fluorescence channel;

[0027] Alternatively, the target droplet also includes: 3) a droplet whose fluorescence signal value of the droplet has no significant difference from the background fluorescence value of the droplet in a closed antibody fluorescence channel.

[0028] A further technical solution is that the significant difference refers to a statistically significant p value < 0.05; the insignificant difference refers to a statistically significant p value > 0.01.

[0029] A further technical solution is that the method for preparing the droplets comprises the following steps:

[0030] S1. preparing the antibody-secreting cells into a single cell suspension, adding a blocking antibody for blocking treatment, washing after the blocking is completed, removing the excess blocking antibody, and obtaining a single cell suspension of the antibody-secreting cells;

[0031] S2, preparing the reporter cells with the target antigen on the membrane surface into a single cell suspension, adding blocking antibodies for blocking treatment, washing after the blocking is completed, removing the excess blocking antibodies, and obtaining a single cell suspension of the reporter cells with the target antigen on the membrane surface; or coating the target antigen on the surface of microbeads to obtain microbead antigens;

[0032] S3, preparing a buffer solution containing a detection antibody and / or a dead cell fluorescent dye, and removing large particles of fluorescent substances in the droplet buffer solution by filtering or high-speed centrifugation to obtain a droplet buffer solution;

[0033] S4. Resuspend the antibody-secreting cells and the reporter cells with the target antigen on the membrane surface in the droplet buffer respectively, mix the resuspended single-cell suspension of the antibody-secreting cells and the single-cell suspension of the reporter cells with the target antigen on the membrane surface to prepare a plurality of nanoliter or picoliter droplets; or, resuspend the antibody-secreting cells and the microbead antigen in the droplet buffer respectively, mix the resuspended single-cell suspension of the antibody-secreting cells and the microbead antigen to prepare a plurality of nanoliter or picoliter droplets.

[0034] A further technical solution is that in the steps S1 and S2, the temperature of the sealing treatment is 0-37°C, and the sealing time can be 30 seconds to 12 hours. Preferably, the temperature of the sealing treatment is 2-25°C, and the sealing time can be 5 to 20 minutes. More preferably, the temperature of the sealing treatment is 2-8°C, and the sealing time can be 5 to 10 minutes.

[0035] A further technical solution is that in step S3, the microporous filter membrane used for filtration is a bacterial filter membrane of 0.45 μm or less, preferably a bacterial filter membrane of 0.22 μm; and the centrifugal force of high-speed centrifugation is not less than 8000G.

[0036] A further technical solution is that the antibody-secreting cells belong to the B cell lineage, such as plasma cells, hybridoma cells, or other antibody-secreting cells, and their source can be human peripheral blood mononuclear cells (PBMC) or other species or tissue sources.

[0037] A further technical solution is that the blocking antibody and the detection antibody are each independently at least one of a monoclonal antibody and a polyclonal antibody, for example, a sheep anti-human IgG-Fc fragment antibody, a non-human anti-human IgG-Fc fragment antibody, a non-human anti-human IgG (heavy chain + light chain) antibody or an immunoglobulin antibody.

[0038] In the droplet buffer obtained in step S3, the final concentration of the detection antibody can be any concentration between 2 nM and 400 nM, preferably 2-200 nM, and more preferably 10-80 nM.

[0039] During the blocking treatment, the final concentration of the blocking antibody in the system can be any concentration between 2 nM and 10 μM, preferably 2-1 μM, and more preferably 10-80 nM.

[0040] A further technical solution is that the detection antibody specifically binds to the heavy chain Fc fragment of the target antibody, or specifically binds to any fragment of the complete target antibody containing the heavy chain and / or light chain.

[0041] A further technical solution is that the blocking antibody specifically binds to the heavy chain Fc fragment of the target antibody, or specifically binds to any fragment of the complete target antibody containing the heavy chain and / or light chain.

[0042] It is understandable that, since there are currently engineered antibodies, their structures may be incomplete, or have only one chain, or be chimeric antibodies from different species, or contain deletions or mutations, and such antibodies are also suitable for the present method.

[0043] A further technical solution is that the blocking antibody and the detection antibody are antibodies derived from the same species, or antibodies derived from different species.

[0044] A further technical solution is that the first fluorescent label includes any fluorescent group such as Alexa Fluor 488, Alexa Fluor 647, PE or BV421;

[0045] The second fluorescent label includes any fluorescent group such as Alexa Fluor 488, Alexa Fluor 647, PE or BV421;

[0046] Furthermore, the fluorescent signal generated by the second fluorescent marker is different from the fluorescent signal generated by the first fluorescent marker.

[0047] A further technical solution is that the dead cell fluorescent dye is a dye such as NucGreen, NucRed or EvaGreen that specifically binds to dead cells and has excitation and emission spectrum information. It is understandable that the dead cell fluorescent dye does not non-specifically bind to living cells, microbeads or proteins.

[0048] In the droplet buffer obtained in step S3, the final concentration of the dead cell fluorescent dye can be any concentration between 5 nM and 5 μM, preferably 50-1 μM.

[0049] It can be understood that the microbeads include magnetic microbeads or non-magnetic microbeads.

[0050] A further technical solution is that the particle size of the microbeads is the maximum size of the microbeads (if non-spherical) which is smaller than the diameter of the droplets, for example, the particle size of the microbeads is 20 nm to 50 μm.

[0051] Compared with the prior art, the technical effects achieved by the present invention include:

[0052] The present invention provides a method for high-throughput screening of monoclonal plasma cell secretory antibodies. The method uses microdroplets as reaction containers, uses microbeads carrying target antigens or large granular materials such as reporter cells as a matrix, mediates positive secretory antibodies and fluorescent detection antibodies to produce aggregative strong fluorescent signals, and realizes high-sensitivity quantitative detection. The screening method of the present invention pre-incubates cell samples and reporter cell samples with blocking antibodies in advance, so that memory cell surface antibodies, immune cell surface FcR receptors and other antibody binding sites are blocked, reducing the possibility of non-specific binding of subsequent detection antibodies, and reducing the intensity of related signals. It can effectively exclude false positives and non-specific signals that may appear in the system; further, dead cell fluorescent dyes are used to mark dead cells in the droplet system, including dead cells before encapsulation and dead cells generated during the incubation process, in real time, and then the false positive signals generated by the autofluorescence or non-specific adsorption of dead cells can be excluded by the negative selection method, and the success rate of subsequent cell culture and BCR amplification processes can also be improved. The screening method of the present invention basically excludes the main non-target signals in the current system, greatly improves the true positive ratio of the screening product, and makes it possible to screen samples with extremely low positive rates.

[0053] Furthermore, the screening method of the present invention can also use blocking antibodies and markers to pre-incubate cell samples and reporter cell samples. While achieving the above-mentioned blocking effect, cells or impurities can also be labeled, so that non-specific signals from this source can be excluded through negative selection methods, further improving the true positive rate of the sample.

[0054] Furthermore, in the screening method of the present invention, before preparing the droplets, membrane filtration or high-speed centrifugation is used to remove large particles of fluorescent proteins or dyes that may exist in the droplet system, thereby effectively eliminating false positive signals generated by such substances.

[0055] In summary, the present invention constructs a highly sensitive reaction and detection system, effectively identifies monoclonal secretory antibodies with high affinity to the target antigen, and strictly excludes non-specific signals and non-target signals that may exist in the system, thereby obtaining screening results with extremely high positive ratios and repeatability. The screening method of the present invention has the characteristics of high throughput, high specificity, and high positive ratio. Compared with existing antibody screening techniques and schemes, it has the advantages of simple process, low cost, and short time consumption, and has significant improvements in efficiency and economy. In addition, the method described in the present invention is applicable to plasma cells from multiple species, and there is no strict restriction on the purity of plasma cells; it can be applied to a variety of antigen types such as soluble proteins, membrane surface antigens, small molecule polypeptides, and modified antigens at the same time, and is also applicable to plasma cell samples modified by genetic engineering, especially for difficult samples such as low cell count and low positive rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying any creative work.

[0057] Figure 1 The schematic diagram of the method for high-throughput screening of monoclonal plasma cell-secreted antibodies provided by the present invention.

[0058] Figure 2 This is the fluorescent image of the droplet in this embodiment 1.

[0059] Figure 3 This is a positive droplet signal diagram of this Example 1.

[0060] Figure 4 The figure shows the droplet signal scatter plot and positive droplet identification result (gate example) of Example 1.

[0061] Figure 5 This is the ELISA verification result of the antibodies obtained in the positive droplets screened in Example 1.

[0062] Figure 6 These are the bright field and fluorescence images of the droplet in Example 2.

[0063] Figure 7 It is the signal scatter diagram of the droplet in the NucGreen channel and the positive droplet identification result of this embodiment 2.

[0064] Figure 8 It is the signal scatter diagram of the droplet in the PE / AF647 channel and the positive droplet identification result of this embodiment 2.

[0065] Fig. 9 This is the screening and verification result of the positive droplets in Example 2 for the membrane protein antigen target.

[0066] Fig.10 These are the droplet fluorescence images obtained for the filtered group and the unfiltered group in Control Example 1, respectively.

[0067] Fig.11 The signal scatter plots obtained for the filtered group and the unfiltered group in Control Example 1 are shown in FIG.

[0068] Fig.12 The images of the droplets obtained in the bright field and PE fluorescence channel of the blocked group and the unblocked group in Control Example 2 are respectively.

[0069] Fig.13 The fluorescence signals of the droplets in the PE / AF647 channel obtained from the blocked group and the unblocked group in Control Example 2, respectively. DETAILED DESCRIPTION

[0070] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments. Obviously, the embodiments described below are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0071] It should be understood that when used in this specification and the appended claims, the terms "include" and "comprises" indicate the presence of described features, integers, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or combinations thereof.

[0072] It should also be understood that the terms used in this description of the embodiments of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the embodiments of the present invention. As used in the description of the embodiments of the present invention and the appended claims, unless the context clearly indicates otherwise, the singular forms of "a", "an" and "the" are intended to include plural forms.

[0073] See also Figure 1 , which is a schematic diagram of the method for high-throughput screening of monoclonal plasma cell secretory antibodies provided by the present invention. The screening method of the present invention uses microdroplets as reaction containers, and uses large particulate materials such as microbeads or reporter cells carrying target antigens as a matrix to mediate the production of aggregated strong fluorescent signals by positive secretory antibodies and fluorescent detection antibodies, thereby achieving highly sensitive quantitative detection. The present invention obtains screening results with extremely high positive ratios and repeatability by strictly excluding non-specific signals and non-target signals that may exist in the droplet system.

[0074] Example 1

[0075] This embodiment provides a method for high-throughput screening of monoclonal plasma cell secretory antibodies, which specifically includes:

[0076] (1) Preparation of antigen-coated magnetic beads:

[0077] Take 25 μL of evenly resuspended Beaver avidin magnetic beads (10 mg / mL, average diameter 300 nm), wash three times with PBST buffer, resuspend in 200 μL PBST, add biotin-labeled antigen to a final concentration of 100 nM, and incubate at room temperature for 30 minutes;

[0078] After incubation, the supernatant was discarded, the cells were washed twice with PBST, resuspended in 200 μL DPBSB (DPBS+0.5% BSA) buffer, and stored at 4°C for later use.

[0079] (2) Preparation of human peripheral plasma B cell suspension:

[0080] PBMCs were isolated from human peripheral blood samples by density gradient centrifugation using Stemcell Lymphoprep reagent;

[0081] CD38 cells and plasma cells were separated from the human PBMC cells using a Miltenyi human plasma cell enrichment kit, resuspended in 1 mL of DPBSB, and placed on ice for later use.

[0082] (3) Pre-incubation of plasma B cells:

[0083] The enriched plasma B cells were concentrated at a concentration of 1×10 7 / mL was resuspended in DPBSB buffer, and then Jackson DyLight-405 GoatAnti-Human IgG(H+L) antibody and Jackson DyLight-405 F(ab')2Fragment RabbitAnti-Human IgG-Fcγ antibody were added at a volume ratio of 1:100, and incubated at 4℃ in the dark for >10min;

[0084] The pre-incubated plasma B cells were filtered through a 20 μm filter, and then centrifuged at 500 g × 5 min to recover the cells, resuspended in DPBSB, and placed on ice and protected from light for later use.

[0085] (4) Preparation of droplet buffer:

[0086] Take 816 μL of DPBio droplet basal buffer, add 10 μL of Jackson PE GoatAnti-Human IgG-Fcγ antibody and 4.1 μL of NucGreen fluorescent dye, mix well, filter through a 0.22 μm filter membrane, and place on ice to avoid light for later use.

[0087] (5) Preparation, incubation and sorting of microdroplets:

[0088] Take 100 μL of the antigen-coated magnetic bead suspension from step (1), discard the supernatant, evenly resuspend the magnetic beads in 120 μL of the droplet buffer from step (4), and then add it to the No. 1 injection hole of the DPBio droplet generation chip;

[0089] Take 0.6×10 6 The pre-incubated plasma B cells in step (3) are uniformly resuspended in 120 μL of the droplet buffer in step (4), and then added to injection well No. 2;

[0090] Take 500 μL of droplet generation oil and add it to injection hole No. 3;

[0091] The three gas pressures on the DPBio droplet generator were set to 2.7 psi, 2.7 psi, and 5.4 psi, respectively, so that the oil in injection hole No. 3 co-encapsulated the magnetic beads in injection hole No. 1 and the plasma cells in injection hole No. 2 to form microdroplets;

[0092] The microdroplets were incubated at 37°C for 2 h;

[0093] After the incubation is completed, the microdroplets are introduced into the droplet sorting chip and sequentially pass through the fluorescence detection window of the chip at a frequency of 200 Hz under the conditions that the sample flow, the interval flow, and the support flow gas pressure are 3.8 psi-3.5 psi-10.0 psi respectively;

[0094] Set the 405nm laser power to 10mW and the 488nm laser power to 10mW; set the NucGreen, PE, and DyLight-405 channel gain parameters to 620 respectively; collect the signals generated in each fluorescence channel when each droplet passes through the detection window, and analyze the maximum intensity value of each droplet signal;

[0095] In this embodiment, the fluorescence image of the droplet is shown in Figure 2 According to the droplet sorting criteria, droplets with NucGreen signal value <400, PE signal value >1900, and DyLight-405 signal value <400 were selected as positive droplets; the results are shown in Figure 3 As can be seen from the figure, there are positive droplets with PE fluorescence on the surface of the magnetic beads, and there are negative droplets with DL405 fluorescence (no PE fluorescence) on the surface of the cells. The positive droplet image is consistent with the signal curve, with a peak signal in the PE channel and only background signals in the DL405 and NucGreen channels.

[0096] See also Figure 4 , which is a droplet signal scatter plot and positive droplet identification result of this embodiment. It can be seen from the figure that a large number of droplets have spike signals in the DL405 channel, and some of the droplets also have spike signals in the PE channel. Through the above sorting criteria, this embodiment can select droplets whose signal intensity has significant differences only in the PE channel but no significant differences in the DL405 channel as positive droplets of this embodiment, thereby improving the positive rate of sorting.

[0097] (6) Enrichment and verification of positive droplets:

[0098] The sorting voltage was set to 1600 V and the voltage duration was 1000 μs. The positive droplets were driven to the positive collection channel using dielectrophoretic force and fell into the microwells containing cell lysate and RNase inhibitor.

[0099] After sorting, the DPBio Human Single Cell BCR Amplification and Library Construction Kit was used to amplify the BCR of each positive cell, and the light and heavy chain paired DNA sequences were obtained by second-generation sequencing.

[0100] Figure 5 This is the ELISA verification result (against soluble antigen targets) of the antibodies obtained in the positive droplets screened in this example. It can be seen that 90% of the antibodies have affinity for the target antigen.

[0101] Example 2

[0102] This embodiment provides a method for high-throughput screening of monoclonal plasma cell secretory antibodies, which specifically includes:

[0103] (1) Preparation of mouse spleen plasma B cell suspension:

[0104] Fresh mouse spleen was obtained, placed in a 40 μm cell strainer and 1640 culture medium, and spleen cell suspension was obtained by grinding;

[0105] CD138 cells and plasma B cells were separated from the spleen cells of the mice using the Stemcell Mouse Plasma Cell Enrichment / Release Kit, resuspended in 1 mL of DPBSB, and placed on ice for later use.

[0106] (2) Pre-incubation of reporter cells:

[0107] CHO reporter cells were prepared into a single cell suspension at a concentration of 1×10 7 / mL was resuspended in DPBSB buffer, and then Jackson Goat Anti-Mouse IgG (H+L) antibody was added at a volume ratio of 1:200, and incubated at 4℃ in the dark for 10min, and then Jackson Alexa-Fluor-647 F(ab')2 Fragment Rabbit Anti-Mouse IgG-Fcγ antibody was added at a volume ratio of 1:200, and incubated at 4℃ in the dark for >10min;

[0108] The pre-incubated reporter cells were filtered through a 20 μm filter, and then centrifuged at 500 g × 5 min to recover the cells, resuspended in DPBSB, and placed on ice and protected from light for later use.

[0109] (3) Pre-incubation of plasma B cells:

[0110] The enriched plasma B cells were concentrated at a concentration of 1×10 7 / mL was resuspended in DPBSB buffer, and then Jackson Goat Anti-Mouse IgG (H+L) antibody and Jackson Alexa-Fluor-647 F(ab')2Fragment Rabbit Anti-Mouse IgG-Fcγ antibody were added at a volume ratio of 1:100, and incubated at 4℃ in the dark for >10min;

[0111] The pre-incubated plasma B cells were filtered through a 20 μm filter, and then centrifuged at 500 g × 5 min to recover the cells, resuspended in DPBSB, and placed on ice and protected from light for later use.

[0112] (4) Preparation of droplet buffer:

[0113] Take 816 μL of DPBio droplet basal buffer, add 10 μL of Jackson PE GoatAnti-Mouse IgG-Fcγ antibody and 4.1 μL of NucGreen fluorescent dye, mix well, filter through a 0.22 μm filter membrane, and place on ice to avoid light for later use.

[0114] (5) Preparation, incubation and sorting of microdroplets:

[0115] Take 0.6×10 6 The plasma B cells pre-incubated in step (3) are recovered by centrifugation and uniformly resuspended in 120 μL of the droplet buffer in step (4), and then added to the No. 1 injection hole of the DPBio droplet generation chip;

[0116] Take 4.5×10 6 The pre-incubated reporter cells in step (2) are recovered by centrifugation and uniformly resuspended in 120 μL of the droplet buffer in step (4), and then added to injection well No. 2;

[0117] Take 500 μL of droplet generation oil and add it to injection hole No. 3;

[0118] The three gas pressures on the DPBio droplet generator were set to 2.7 psi, 2.7 psi, and 5.4 psi, respectively, so that the oil in injection hole No. 3 co-encapsulated the plasma cells in injection hole No. 1 and the reporter cells in injection hole No. 2 to form microdroplets;

[0119] The microdroplets were incubated at 37°C for 2 h;

[0120] After the incubation is completed, the micro-droplets are introduced into the droplet sorting chip, and pass through the fluorescence detection window of the chip in sequence at a frequency of 200 Hz under the conditions that the sample flow, the interval flow, and the support flow pressure are 3.8 psi-3.5 psi-10.0 psi respectively;

[0121] Set the 488nm laser power to 10mW and the 635nm laser power to 30mW; set the gain parameters of NucGreen, PE, and AF647 channels to 620 respectively; collect the maximum signal of each droplet in each fluorescence channel;

[0122] According to the droplet sorting criteria, droplets with NucGreen signal value <400, PE signal value >1800, and AF647 signal value with no significant difference from the background signal are selected as positive droplets; in this embodiment, the droplet bright field image and PE / AF647 fluorescence image are shown in Figure 6 As can be seen from the figure, a single plasma cell and several reporter cells are co-encapsulated in some droplets, and obvious PE channel fluorescence signals are present on the surface of some co-encapsulated reporter cells, which are binding signals of positive secreted antibodies; at the same time, AF647 signals from blocking antibodies are also present on the cell surfaces in some droplets.

[0123] Further, Figure 7 and Figure 8 They are the signal scatter diagrams of the droplets in the NucGreen channel and the PE / AF647 channel and the positive droplet identification results of this embodiment, respectively. Figure 7 It shows that there are a large number of droplets containing dead cells and showing strong NucGreen fluorescence signals, and the proportion of droplets without dead cells is >97%. Figure 8 It showed that there were a large number of AF647-positive droplets, some of which were also PE-positive, which might come from multi-packed droplets of negative and positive plasma cells, or cells that did not fully achieve the blocking effect, or false positive signals that gradually accumulated during the incubation process.

[0124] According to the screening criteria for positive droplets in this embodiment, that is, droplets with significant differences from the droplet background signal in the PE channel but no significant differences in the AF647 channel are selected as positive droplets, non-target droplets containing false positive signals on the cell surface or negative and positive plasma cell multi-pack droplets can be excluded, which has the advantage of high accuracy. In this embodiment, due to the presence of fluorescence crosstalk, PE positive droplets have a weak signal drift in the AF647 channel, which does not affect the identification of positive droplets.

[0125] (6) Enrichment and verification of positive droplets:

[0126] The sorting voltage was set to 1600 V and the voltage duration was 1000 μs. The single positive droplet was sorted into the positive collection channel using dielectrophoresis force and fell into the microwell containing cell lysate and RNase inhibitor;

[0127] After sorting, the DPBio Mouse Single Cell BCR Amplification and Library Construction Kit was used to amplify the BCR of each positive cell, and the light and heavy chain paired DNA sequences were obtained by Sanger sequencing or second-generation sequencing.

[0128] Fig. 9 This is the FACS verification result of the antibodies obtained from the positive droplets screened in this example (targeting membrane protein antigens). It can be seen that more than 80% of the antibodies have affinity for the reporter cell surface membrane antigens (RL1 and BL1 double positive samples).

[0129] Comparative Example 1

[0130] This control example is based on Example 1, except that the control example 1 does not include step (2), that is, droplets without plasma cells are prepared, so there is no true positive signal in the droplets; a filtered group and an unfiltered group are set at the same time: the filtered group is that the preparation method of step (4) is the same as that of Example 1; the unfiltered group is that in step (4), the prepared droplet buffer is not filtered by a 0.22 μm filter membrane. The rest of the operations are the same as those in Example 1.

[0131] By observing the droplet fluorescence images of the filtered and unfiltered groups and detecting the droplet fluorescence signals, it was shown that filtering the droplet buffer containing fluorescent antibodies has the effect of reducing nonspecific signals.

[0132] Results Fig.10 and Fig.11 . Fig.10 It is the fluorescence image of the droplets obtained from the filtered group and the unfiltered group in the PE channel, showing that the unfiltered droplets contain fluorescent bright spots of varying sizes and intensities, while the filtered droplets basically contain no fluorescent bright spots.

[0133] Fig.11 It is the fluorescence signal of the droplets obtained from the filtered group and the unfiltered group in the PE channel. It can be seen that there is basically no non-target fluorescence signal in the droplet samples after filtering, while there are a large number of non-target fluorescence signals in the droplet samples without filtering.

[0134] Comparative Example 2

[0135] This control example is based on Example 2, except that in step (2) of Example 2, negative CHO reporter cells that do not express the target antigen are used instead of positive reporter cells that express the target antigen, so there is no true positive signal in the obtained droplets; and a closed group and an unclosed group are set at the same time: the closed group is that in steps (2) and (3), the pre-incubation treatment of the reporter cells and plasma B cells is the same as in Example 2; the unclosed group is that in steps (2) and (3), the reporter cells and plasma B cells are not pre-incubated. The rest of the operations are the same as in Example 2.

[0136] By observing the droplet fluorescence images of the blocked and unblocked groups and detecting the droplet fluorescence signals, it was shown that blocking treatment of plasma cell samples had the effect of reducing and marking nonspecific cell signals.

[0137] Results Fig.12 and Fig.13 . Fig.12 These are images of droplets in the blocked and unblocked groups in the bright field and PE fluorescence channels, showing that droplets that have not been blocked contain cell surface PE fluorescence, while droplets that have been blocked contain significantly reduced cell surface PE fluorescence signals.

[0138] Fig.13 It is the fluorescence signal of the droplets in the blocked group and the unblocked group in the PE / AF647 channel. It can be seen that there is basically no strong PE signal in the droplet samples that have been blocked, but there are a large number of AF647 signals, while there are a large number of non-target PE fluorescence signals in the droplet samples that have not been blocked.

[0139] From the results of Control Example 1 and Control Example 2, it can be seen that the cells are pre-blocked (pre-incubated) and the droplet buffer containing the fluorescent antibody is filtered, which can reduce the interference of non-specific signals and non-target signals in the system, thereby improving the recognition rate of positive droplets and the positive rate.

[0140] In the above embodiments, the description of each embodiment has its own emphasis. For parts that are not described in detail in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0141] The above is a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can easily think of various equivalent modifications or replacements within the technical scope disclosed by the present invention, and these modifications or replacements should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A method for high-throughput screening of monoclonal plasma cell secretory antibodies, characterized in that: The screening method comprises: (1) Providing a plurality of nanoliter or picoliter droplets, each droplet comprising: a. No more than one antibody-secreting cell; b. microbeads coated with target antigen, or at least one reporter cell with target antigen on its membrane surface; c. Detection antibodies and / or dead cell fluorescent dyes; (2) incubating the plurality of nanoliter or picoliter droplets under appropriate conditions to allow the antibody-secreting cells to secrete the target antibody, thereby obtaining incubated droplets; (3) passing the incubated droplets through a droplet sorting chip one by one to detect fluorescence signals, and sorting and enriching target droplets according to the fluorescence signal values ​​of the droplets; In (1), the antibody-secreting cells are pre-treated with a blocking antibody; the reporter cells carrying the target antigen on the membrane surface are pre-treated with a blocking antibody; The blocking antibody can specifically bind to the target antibody; the detection antibody can specifically bind to the target antibody; and the blocking antibody cannot be recognized and bound by the detection antibody; the detection antibody carries a first fluorescent marker, and the fluorescent signal generated by the first fluorescent marker is different from the fluorescent signal generated by the dead cell fluorescent dye.

2. The method for high-throughput screening of monoclonal plasma cell secretory antibodies according to claim 1, characterized in that: The target droplet comprises: ① In the fluorescence channel of the detection antibody, the droplet has a significant difference between the fluorescence signal value of the droplet and the background fluorescence value of the droplet; ② In the fluorescence channel of the dead cell dye, the droplet has no significant difference between the fluorescence signal value of the droplet and the background fluorescence value of the droplet; Alternatively, the target droplet comprises: 1) In the fluorescence channel of the detection antibody, the fluorescence signal value of the droplet is 1.2 times or more of the background fluorescence value of the droplet; 2) In the fluorescence channel of the dead cell dye, the fluorescence signal value of the droplet is less than or equal to 1.1 times the background fluorescence value of the droplet.

3. The method for high-throughput screening of monoclonal plasma cell secretory antibodies according to claim 1, characterized in that: The blocking antibody carries a second fluorescent label, and the fluorescent signal generated by the second fluorescent label is different from the fluorescent signal generated by the first fluorescent label.

4. The method for high-throughput screening of monoclonal plasma cell secretory antibodies according to claim 3, characterized in that: The target droplets also include: ③ droplets whose fluorescence signal value of the droplets has no significant difference with the background fluorescence value of the droplets in the closed antibody fluorescence channel; Alternatively, the target droplet also includes: 3) a droplet whose fluorescence signal value of the droplet has no significant difference from the background fluorescence value of the droplet in a closed antibody fluorescence channel.

5. The method for high-throughput screening of monoclonal plasma cell secretory antibodies according to claim 2 or 4, characterized in that: The significant difference refers to a statistically significant p value < 0.05; the no significant difference refers to a statistically significant p value > 0.

01.

6. The method for high-throughput screening of monoclonal plasma cell secretory antibodies according to claim 1, characterized in that: The method for preparing the droplets comprises the following steps: S1. preparing the antibody-secreting cells into a single cell suspension, adding a blocking antibody for blocking treatment, washing after the blocking is completed, removing the excess blocking antibody, and obtaining a single cell suspension of the antibody-secreting cells; S2, preparing the reporter cells with the target antigen on the membrane surface into a single cell suspension, adding blocking antibodies for blocking treatment, washing after the blocking is completed, removing the excess blocking antibodies, and obtaining a single cell suspension of the reporter cells with the target antigen on the membrane surface; or coating the target antigen on the surface of microbeads to obtain microbead antigens; S3, preparing a buffer solution containing a detection antibody and / or a dead cell fluorescent dye, and removing large particles of fluorescent substances in the droplet buffer solution by filtering or high-speed centrifugation to obtain a droplet buffer solution; S4. Resuspend the antibody-secreting cells and the reporter cells with the target antigen on the membrane surface in the droplet buffer respectively, mix the resuspended single-cell suspension of the antibody-secreting cells and the single-cell suspension of the reporter cells with the target antigen on the membrane surface to prepare a plurality of nanoliter or picoliter droplets; or, resuspend the antibody-secreting cells and the microbead antigen in the droplet buffer respectively, mix the resuspended single-cell suspension of the antibody-secreting cells and the microbead antigen to prepare a plurality of nanoliter or picoliter droplets.

7. The method for high-throughput screening of monoclonal plasma cell secretory antibodies according to claim 6, characterized in that: In the step S3, the microporous filter membrane used for filtration is a bacterial filter membrane of 0.45 μm or less; and the centrifugal force of high-speed centrifugation is not less than 8000G.

8. The method for high-throughput screening of monoclonal plasma cell secretory antibodies according to claim 1, characterized in that: The blocking antibody and the detection antibody are each independently at least one of a monoclonal antibody and a polyclonal antibody.

9. The method for high-throughput screening of monoclonal plasma cell secretory antibodies according to claim 3, characterized in that: The first fluorescent label includes any one of Alexa Fluor488, Alexa Fluor 647, PE or BV421 fluorescent groups; The second fluorescent label includes any one of Alexa Fluor 488, Alexa Fluor 647, PE or BV421 fluorescent groups; Furthermore, the fluorescent signal generated by the second fluorescent marker is different from the fluorescent signal generated by the first fluorescent marker.

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