Microfluidic assays and uses thereof

Abstract

The present disclosure relates to a microfluidic method for determining antibody secreting cells (ASCs), comprising the steps of: separating the ASCs in droplets such that each droplet encapsulates only one ASC; incubating the droplet of step a) to accumulate the antibody within the droplet; injecting a virus picoliter into the droplet of step b) to form an immune complex droplet; injecting the host cells into the immune complex droplets in a picoliter manner to form neutralized droplets and infected droplets; and sorting neutralized droplets from the infected droplets based on infection of the host cell by the virus to determine ASC within the neutralized droplets. The disclosure also relates to a microfluidic platform thereof.

Description

Technical Field

[0001] The present invention generally relates to microfluidic assays and uses thereof. Background Art

[0002] Pathogenic viruses are responsible for numerous infectious diseases that plague humanity. As part of our adaptive immune response, neutralizing antibodies (nAbs) play an integral role in conveying protection against viruses. Indeed, in vivo nAb titers are strongly correlated with protection against a variety of viral infections, including those with dengue virus, SARS-CoV-2, and respiratory syncytial virus (RSV). However, the slow onset of natural nAb production following viral infection (ranging from days to weeks) may predispose elderly or immunocompromised individuals to severe health consequences. In such cases, administration of exogenously produced monoclonal nAbs can often prevent infected individuals from progressing to severe stages of the disease.

[0003] Currently, nAb discovery is under tremendous pressure due to the rapid mutation of existing virus strains and the continuous emergence of new viral threats. Although fluorescence activated sorting (FACS) and display systems can be used to screen a large number of antibody secreting cells (ASCs) based on the binding of cells to specific protein antigens, binding affinity often does not reflect the true functional efficacy of nAb candidates. Many high-affinity mAbs bind to non-neutralizing epitopes on viral antigens and are therefore not suitable for therapeutic applications ( Figure 1 a). Worst of all, administration of non-neutralizing antibodies may lead to antibody-dependent enhancement (ADE) effects, increasing the severity of multiple viral infections. Meanwhile, although cells producing potent nAbs can be directly screened through hybridoma generation and single B cell activation / expansion, the cumbersome nature of these procedures results in lengthy workflows. Due to the low throughput of existing virus neutralization assays (10 2 -10 3 Therefore, there remains a need for an integrated system that can rapidly perform functional Ab neutralization assays on large numbers of ASCs, with the ability to not only identify, but also isolate promising candidates.

[0004] The droplet microfluidics platform presents several key advantages that make it an ideal platform for functional screening of ASCs, including: 1) 10-10 41) high throughput of droplets, 2) mature toolkits for droplet operations such as fusion, splitting and sorting, enabling complex multi-step assays, and 3) the ability to accommodate multiple assay reagent types through co-encapsulation, especially reporters or effector cells required for most Ab functional assays. Although droplet-based ASC screening by Ab binding affinity has been well established, more clinically relevant ASC screening by true virus neutralization assays is still lacking due to technical challenges in performing complex multi-step virus neutralization assays. A recent study describes a platform for visualizing the virus neutralization of ASC in microfluidic droplets. However, this method is limited to evaluating the virus neutralization activity of 100-1000 droplets in the field of view and lacks the key ability to sort and retrieving potent nAb secreting cells for downstream analysis or amplification.

[0005] It would be desirable to overcome or ameliorate at least one of the above problems. Summary of the invention

[0006] In order to fill at least one current gap, the present invention relates to a high-throughput droplet microfluidic system that is capable of selecting and recovering ASC based on the neutralizing function of antibodies secreted by single cells. As shown herein, as an example, the platform can be used to enrich cells that secrete nAbs against Chikungunya virus (CHIKV). High-throughput screening of functional ASCs using droplet microfluidics can be a new model for the rapid discovery of effective and functional biologics. In addition, it is also confirmed that the present invention can achieve similar enrichment of low percentages (~2%) of functional nAb-producing cells in the presence of excess cells that secrete irrelevant antibodies, suggesting that the present invention can be used as a potential prospect for the first round of enrichment platform for functional ASCs.

[0007] The present invention provides a microfluidic method for measuring antibody secreting cells (ASC), comprising the following steps:

[0008] a) separating ASCs in droplets so that each droplet encapsulates only one ASC;

[0009] b) incubating the droplets of step a) to accumulate antibodies in the droplets;

[0010] c) injecting the virus into the droplets of step b) to form immune complex droplets;

[0011] d) injecting picoliters of host cells into immune complex droplets to form neutralizing droplets and infectious droplets; and

[0012] e) Based on the infection of the virus to the host cells, neutralizing droplets are sorted out from the infected droplets to measure the ASCs within the neutralizing droplets.

[0013] In some embodiments, neutralizing droplets are sorted from infectious droplets using a dielectrophoretic sorter.

[0014] In some embodiments, the microfluidic method further comprises a step of generating droplets in the presence of lipopolysaccharide prior to step a).

[0015] In some embodiments, the microfluidic method further comprises a step of incubating the immune complex droplets after step c).

[0016] In some embodiments, the droplets of steps a) and b) have one or both of the following characteristics:

[0017] A volume of about 160 pL to about 200 pL;

[0018] The diameter is about 40 μm to about 100 μm, or preferably about 70 μm to about 90 μm.

[0019] In some embodiments, the incubation step (step b) is performed for at least 1 hour.

[0020] In some embodiments, the microfluidic method further comprises a step of incubating the neutralization droplets and the infection droplets after step d).

[0021] In some embodiments, the picoliter injecting step is performed using an electric field of about 0.5 Vpp to about 2 Vpp and a sine wave of about 10 kHz to about 30 kHz.

[0022] In some embodiments, the method is characterized by a rate of about 200 droplets per second to about 500 droplets per second, or preferably about 300 droplets per second.

[0023] In some embodiments, the microfluidic method further comprises a step of recovering the ASC within the neutralized droplet.

[0024] In some embodiments, the recovering step comprises demulsifying the neutralized droplets.

[0025] The present invention provides a microfluidic platform, comprising:

[0026] a) a droplet generator for generating droplets, wherein each droplet encapsulates an antibody secreting cell (ASC);

[0027] b) a first picoliter syringe chip fluidically connected to a droplet generator, the first picoliter syringe chip comprising a first nozzle for delivering viruses into droplets to form immune complex droplets;

[0028] c) a second picoliter syringe chip fluidically connected to the first picoliter syringe chip, the second picoliter syringe chip comprising a second nozzle for delivering host cells into immune complex droplets to form neutralization droplets and infection droplets; and

[0029] d) a droplet sorter fluidically connected to the second picoliter injector chip for sorting droplets based on infection of host cells by the virus, the droplet sorter comprising a first channel and a second channel, the second channel being configured to have a flow resistance greater than that of the first channel.

[0030] In some embodiments, the second channel is configured to have a flow resistance at least twice that of the first channel.

[0031] In some embodiments, the droplet generator includes an aqueous channel for transporting droplets and two oil channels intersecting the aqueous channel, wherein the two oil channels are configured to flow oil to squeeze an aqueous medium in the aqueous channel into droplets.

[0032] In some embodiments, the aqueous channel comprises an outlet configured to extrude the aqueous medium into droplets.

[0033] In some embodiments, the microfluidic platform further comprises a first container fluidically connected to the droplet generator, the first container configured to incubate the droplets.

[0034] In some embodiments, the microfluidic platform further includes a second container fluidically connected to the first picoliter syringe, the second container configured to incubate the immune complex droplets.

[0035] In some embodiments, the microfluidic platform further includes a third container fluidically connected to the second picoliter syringe, the third container configured to incubate the neutralization droplets and the infection droplets.

[0036] In some embodiments, the microfluidic platform further comprises a shielding electrode.

[0037] In some embodiments, the outlet is a constriction.

[0038] In some embodiments, the first picoliter syringe contains a fluid containing the virus at a pressure selected to deliver the virus to the droplets.

[0039] In some embodiments, the second picoliter syringe contains a fluid containing the virus at a pressure selected to deliver the host cells to the immune complex droplets.

[0040] In some embodiments, the first picoliter syringe is configured with a first electric field, wherein the first nozzle and the first electric field are configured to act simultaneously to deliver the virus to the droplet.

[0041] In some embodiments, the second picoliter syringe is configured with a second electric field, wherein the second nozzle and the second electric field are configured to act simultaneously to deliver the host cells to the immune complex droplets.

[0042] In some embodiments, the droplet sorter comprises a channel having a width greater than about 100 μm.

[0043] In some embodiments, the droplets include:

[0044] a) a basal medium comprising Dulbecco's modified Eagle's medium / Nutrient mixture F-12 (DMEM / F-12), about 20 mg / L to about 30 mg / L sodium bicarbonate, and about 50 mg / L to about 60 mg / L sodium pyruvate;

[0045] b) about 10% v / v to about 20% v / v of fetal bovine serum relative to the basal medium; and

[0046] c) about 10% v / v to about 20% v / v of density gradient medium relative to the basal medium.

[0047] In some embodiments, the droplets further include a mixture of penicillin G and streptomycin at about 1% v / v relative to the basal medium.

[0048] In some embodiments, the volume of each of the neutralization droplet and the infection droplet is about 65 pL to about 1000 pL.

[0049] In some embodiments, the volume of the neutralized droplets and the infected droplets is less than 2 times the volume of the droplets of steps a) and / or b).

[0050] In some embodiments, the host cells are delivered at a cell density of about 80 million / mL to about 150 million / mL, or preferably about 100 million / mL.

[0051] In some embodiments, the ASC is a B cell or a transfected cell, or preferably a murine memory B cell.

[0052] In some embodiments, the droplets of step b) are characterized by an antibody concentration of about 0.1 μg / mL to about 20 μg / mL, or preferably about 10 μg / mL.

[0053] In some embodiments, the volume of each immune complex droplet is about 35 pL to about 800 pL.

[0054] In some embodiments, the immune complex droplets are characterized by an increase in volume of about 40% to about 60% relative to the droplet.

[0055] In some embodiments, the immune complex droplets are characterized by a diameter of about 35 μm to about 120 μm, or preferably about 65 μm to about 90 μm.

[0056] In some embodiments, the immune complex droplets are characterized by a viral titer of about 5 kPFU / μL to about 100 kPFU / μL, or preferably about 75 kPFU / μL.

[0057] In some embodiments, the neutralized droplets and infectious droplets are characterized by an increase in volume of about 30% to about 40% relative to the immune complex droplets.

[0058] In some embodiments, the neutralizing droplets and infectious droplets are characterized by a diameter of about 45 μm to about 120 μm, or preferably about 70 μm to about 100 μm.

[0059] In some embodiments, the neutralization droplets and the infection droplets are characterized by a density of about 5 host cells per droplet to about 15 host cells per droplet.

[0060] In some embodiments, the neutralized droplets and the infectious droplets are characterized by a host cell viability of greater than 80% after 30 hours of incubation, or preferably a host cell viability of greater than 90% after 24 hours of incubation.

[0061] In some embodiments, the recovering step is characterized by an ASC enrichment ratio of greater than about 1.8.

[0062] In some embodiments, the virus is selected from Chikungunya virus, dengue virus, SARS-CoV-2, respiratory syncytial virus (RSV), Zika virus, EV71, influenza virus, HIV, and norovirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Embodiments of the invention will now be described by way of non-limiting examples and with reference to the accompanying drawings, in which:

[0064] Figure 1 A comparison between bulk and single-cell virus neutralization assays is shown. (a) nAbs must bind in a way that blocks viral infection. Many Abs selected solely based on their ability to bind viruses lack neutralizing function. (b) Top: Typical workflow and timeframe required to generate ASC clonal populations and screen them for virus neutralization activity. Bottom: The ability to perform virus neutralization assays in droplets from single cells has significantly accelerated the discovery of functional Abs. Sorting of droplets with high virus neutralization activity enables the recovery of functional ASCs.

[0065] Figure 2Shown is infection of HEK 293T cells with CHIKV-ZSGreen. (a) Batch infection of HEK 293T cells during 44 hours, wherein the cells are stained by CHIKV (top and bottom rows). As CHIKV replicates in cells, the green fluorescence signal increases over time (bottom row). (b) CHIKV virus-infected cells are counted by flow cytometry analysis. (c) Incubation with CHIKV virus for 22 hours in the presence of different amounts of 8B10 neutralizing mAb, the observed degree of infection of HEK 293T cells.

[0066] Figure 3 Characterization of the picoliter injector is shown. (a) 70 μm diameter droplets generated using a standard flow focusing chip design; (b) droplets after picoliter injection of CHIKV, resulting in droplets of approximately 80 μm diameter; and (c) droplets after subsequent picoliter injection of cells, resulting in droplets of approximately 90 μm diameter. The scale bar represents 100 μm. (d) Population distribution of picoliter injected host HEK293T cells at different cell densities. (e) Changes in cell viability within droplets over time for picoliter injection of 200 million / mL host HEK 293T.

[0067] Figure 4 CHIKV infection and signal readout within droplets are shown. (a) Picoliter injection of 37.5 kPFU / μL CHIKV and host HEK 293T cells in droplets containing 20 μg / mL 8B10 nAb or no nAb. Images show droplets at 0 hours and 20 hours from HEK 293T picoliter injection. Scale bar represents 100 μm. (b) Average CHIKV signal intensity of the droplets at the 20 hour time point quantified by imaging and (c) PMT measurement. (d) Changes in the percentage of infected droplets over time at different picoliter injection CHIKV virus titers and in the presence of 8B10 nAb.

[0068] Figure 5 Characterization of the sorter based on CHIKV infection signal is shown. (a) PMT signal of droplets containing CHIKV-infected HEK 293T cells. In this example, the sorting pulse is triggered when the PMT signal exceeds the 0.4V threshold. (b) Population of droplets before sorting, containing 50% infected droplets and 50% uninfected droplets. Images of droplets recovered at (c) collection outlet and (d) waste outlet after sorting at a 0.48V threshold. At least 95% of infected cells were collected. The scale bar represents 250μm. (e) ROC curve of the sorter obtained by sorting droplets in the signal threshold range of 0.12 to 3.24V.

[0069] Figure 6The complete intra-droplet single-cell neutralization assay workflow is shown. (a) Single-cell neutralization assay workflow. ASC (stained red) and mock-transfected cells (stained blue) were first encapsulated in 70 μm diameter droplets and allowed to accumulate nAbs over a 24-hour period. Cell staining was used only for downstream identification of cell types. CHIKV and host HEK293T cells were then injected into the droplets and incubated to allow infection to occur prior to sorting. (b) When sorting using a 0.4 V threshold, sorted droplets were recovered from the top collection channel and the bottom waste channel, respectively. The scale bar represents 100 μm.

[0070] Figure 7 Droplets recovered from a single-cell neutralization assay using a mixed population of ASCs secreting 8B10 (CHIKV nAb) and 5A6 (unrelated SARS-COV-2 nAb) are shown. The complete single-cell neutralization workflow described in the text was performed on 8B10 ASCs (dyed red) and 5A6 ASCs (dyed blue). (a) Droplets before sorting. CHIKV infected cells show green fluorescence signal. Sorted droplets recovered from (b) the top collection channel and (c) the bottom waste channel, respectively, when sorting using a 0.4V green fluorescence signal threshold. Scale bars represent 100 μm.

[0071] Figure 8 A schematic diagram of the picoliter syringe chip is shown.

[0072] Fig. 9 Shown is (a) a schematic diagram of the sorter chip. The blue electrodes represent ground electrodes, while the red electrodes represent active electrodes. (b) A magnified view of the boxed area where sorting occurs. DETAILED DESCRIPTION

[0073] The present invention is based on the understanding that the main bottleneck of antibody development is to find candidate antibodies with strong functional activity against the target of interest. In order to develop therapeutic monoclonal antibodies, one or several antibody clones with functional activity against the target of interest must be identified and isolated from a large pool of antibody clones. Current high-throughput methods for such identification rely on binding to target-derived antigens (also called hooks) (e.g., flow cytometry-based methods; phage / yeast library methods), or rely on low-throughput functional assays. The inability to screen candidate antibodies with high throughput by function reduces the chance of finding the best candidate antibody from a large number of available clone pools. Recently, methods have been developed to generate picoliter to nanoliter-sized water-in-oil droplets in fluorinated oil media using microfluidic devices. However, most of these platforms use the binding affinity of antibodies to antigens as a surrogate for functional efficacy for selection, rather than using functional-based assays. Enriching functionally relevant ASCs using droplet-based functional assays is inherently difficult because the increased biological complexity of the assays compared to simple binding assays imposes technical limitations that have so far not been compatible with the high throughput required to screen antibodies at a scale comparable to conventional antibody discovery techniques. First, the sequential addition of reagents, which is typically performed by droplet-to-droplet fusion, cannot achieve 100% fusion efficiency due to its high reliance on synchronization of the two droplets. This reduces the specificity of high-throughput assays, a key parameter for identifying rare antibody clones.

[0074] Second, functional assays require the ability to maintain cell viability and functionality within droplets for extended periods of time, which requires large droplet volumes to provide nutrients and waste buffers. However, large droplet volumes result in slower sorting rates due to their larger size.

[0075] Additionally, other microfluidic assay designs employ 1-to-1 droplet matching, which is limited by greater reliance on synchronization of paired droplets, and thus a higher probability of delivery failure of viral / cellular cargo to the original droplet. Other microfluidic assay designs use droplets of smaller size (<70 μm) that are unable to provide adequate nutrients for robust mammalian cell culture.

[0076] The present invention provides a microfluidic method for measuring antibody secreting cells (ASC), comprising the following steps:

[0077] a) separating ASCs in droplets so that each droplet encapsulates only one ASC;

[0078] b) incubating the droplets of step a) to accumulate antibodies in the droplets;

[0079] c) injecting picoliters of virus into the droplets of step b) to form immune complex droplets;

[0080] d) injecting picoliters of host cells into immune complex droplets to form neutralizing droplets and infectious droplets; and

[0081] e) Neutralizing droplets are sorted out from infectious droplets to measure the ASCs within the neutralizing droplets.

[0082] The volume of each of the neutralized droplet and the infected droplet may be about 300 pL to about 400 pL. The volume of the neutralized droplet and the infected droplet may be less than 2 times the initial droplet volume of step a).

[0083] The assay uses picoliter injection to achieve high-throughput delivery of virus and reporter / host cells, resulting in more robust delivery of cargo to all droplets, which is necessary for high-specificity neutralization assays. In addition, the assay can produce droplets of optimal size, where the size is large enough to allow the culture of mammalian cells for the duration of the bioassay, while remaining small enough to allow efficient droplet sorting. The assay also includes a sorting step to enrich for cells secreting nAbs, illustrating its potential application in the discovery of mAbs against viral diseases.

[0084] The microfluidic assays disclosed herein are capable of screening at least 100,000 candidates, and ideally >1 million candidates. In contrast, conventional antibody discovery techniques can only screen no more than about 10,000 candidates by manual manipulation and no more than about 100,000 candidates by robotic manipulation.

[0085] The inventors have also discovered that other factors may also be relevant:

[0086] Time: Since cell viability is lost over time at room temperature, and additional variations introduced in droplet nutrient state and infection time will increase assay variability, the time it takes for all droplets to run through the microfluidic chip is preferably less than 2 hours. Assuming a candidate:droplet ratio of 1:10 (droplet occupancy of 10%, which is required to achieve a single cell per droplet in the Poisson limit), this limits the lower limit of the droplet flux required at each step to at least 140 Hz. For example, at 5% single cell occupancy, the screening time is about 2.8 hours; at 10% single cell occupancy, the screening time is about 1.4 hours; at 20% single cell occupancy, the screening time is about 0.7 hours.

[0087] Droplet fusion throughput: Standard droplet fusion cannot achieve the required throughput, which is typically 10-100 droplets / second. In contrast, the picoliter injection method of the present invention can achieve a throughput of, for example, about 300 droplets / second, thereby achieving a process throughput of 1 million droplets / hr.

[0088] Droplet Sorting Throughput: The droplet fusion process and the nutritional requirements required to implement the bioassays used in the article result in very large droplet volumes (about 5nl droplets). At these volumes, standard droplet sorting cannot achieve the required throughput, and in fact, droplets of this size cannot be sorted using current sorting designs because: due to the increased inertia associated with the larger mass of the droplets, sufficiently large dielectrophoretic forces cannot be achieved without destroying the droplets. In contrast, the picoliter injection procedure of the present invention allows small volumes of fluid to be reliably delivered to each droplet, allowing us to obtain a small final droplet size (200pl) after adding all reagents. In combination with an optimized sorting design to deliver sufficiently large dielectrophoretic forces, while preventing unwanted coalescence of droplets by adding additional electric field shielding elements, we were able to achieve the required sorting throughput of about 200 droplets / second.

[0089] Reliability: Droplet fusion requires precise control of the periodicity of the droplets, and in this regard, desynchronization can easily occur, especially when the droplet frequency is increased (e.g., to increase throughput). This will lead to high false positive rates when the antibody-secreting cells are not properly co-encapsulated with the host cells or infectious agents. In contrast, picoliter injection can very reliably add reagents to all droplets that pass through, and even if there is a malfunction in the process (e.g., too high a pressure), the main effect is the formation of additional small droplets that do not contain antibody-secreting cells, and therefore does not affect sensitivity or specificity.

[0090] In some embodiments, the ASC occupancy of the droplet is about 6% to about 15%, or about 6% to about 20%. Occupancy refers to the volume of the droplet occupied by entities such as cells relative to the total volume of the droplet. In other embodiments, the occupancy is about 7% to about 15%, about 8% to about 15%, about 9% to about 15%, about 10% to about 15%, about 10% to about 14%, about 10% to about 13%, or about 10% to about 12%. In other embodiments, the occupancy is about 10%.

[0091] In some embodiments, the microfluidic method further comprises a step of generating droplets in the presence of lipopolysaccharide before step a). For example, lipopolysaccharide from Escherichia coli O111:B4 can be used.

[0092] In some embodiments, the droplets of steps a) and b) are characterized by a volume of about 30 pL to about 600 pL. In other embodiments, the volume is about 30 pL to about 550 pL, about 30 pL to about 500 pL, about 30 pL to about 450 pL, about 30 pL to about 400 pL, about 30 pL to about 350 pL, about 30 pL to about 300 pL, about 30 pL to about 250 pL, about 30 pL to about 200 pL, about 30 pL to about 150 pL, about 30 pL to about 100 pL, about 30 pL to about 80 pL, about 30 pL to about 70 pL, about 30 pL to about 80 pL, about 30 pL to about 90 pL, about 30 pL to about 100 pL, about 30 pL to about 110 pL, about 30 pL to about 120 pL, about 30 pL to about 130 pL, about 30 pL to about 140 pL, about 30 pL to about 150 pL, about 30 pL to about 100 pL, about 30 pL to about 150 pL, about 30 pL to about 100 pL, about 30 pL to about 120 pL, about 30 pL to about 130 pL, about 30 pL to about 140 pL, about 30 pL to about 150 pL, about 30 pL to about 150 pL, about 30 pL to about 100 pL, about 30 pL to about 150 pL, about 30 pL to about 150 pL, about 30 pL to about pL, about 30 pL to about 60 pL, about 30 pL to about 50 pL, about 50 pL to about 600 pL, about 100 pL to about 600 pL, about 150 pL to about 600 pL, about 200 pL to about 600 pL, about 250 pL to about 600 pL, about 300 pL to about 600 pL, about 350 pL to about 600 pL, about 400 pL to about 600 pL, about 450 pL to about 600 pL, or about 500 pL to about 600 pL. Preferably, the droplet is characterized by a volume of about 160 pL to about 200 pL.

[0093] In some embodiments, the droplets of steps a) and b) are characterized by a diameter of about 40 μm to about 100 μm, or preferably about 70 μm to about 90 μm.

[0094] In some embodiments, ASC is a B cell. In other embodiments, ASC is a murine memory B cell, including VDJ-humanized murine memory B cells or human memory B cells. In other embodiments, ASC is a cell that has been transfected or transduced to secrete antibodies. In other embodiments, ASC is a transfected cell. Transfection generally refers to the introduction of nucleic acid into eukaryotic cells, or more specifically, into animal cells. Traditionally, the term transfection is used to indicate the ingestion of viral nucleic acid from a prokaryotic infection virus or bacteriophage, resulting in infection and the production of mature immune complex particles. However, the term has acquired its current meaning, covering any process of artificially introducing foreign nucleic acid into a cell. In transfection, the introduced nucleic acid may be transiently present in the cell and is therefore only expressed during a limited period of time and is not replicated. Accordingly, transiently transfected genetic material will not be passed from one generation to the next by cell division, and may be lost or diluted during cell division due to environmental factors. However, high-copy transfected genetic material can lead to high levels of protein expression during its presence in the cell. Alternatively, the transfected genetic material can be stably integrated into the genome of the recipient (transduction), and replicated when the host genome replicates.

[0095] The ability to use and analyze B cells (for example) can bypass the lengthy process of generating hybridoma cells and facilitate the screening of a much larger proportion of the immune repertoire, including that of humans as well as that of animals with humanized antibody systems.

[0096] Cell culture media for all biological agents required to support co-culture neutralization may also be optimized to result in good assay quality to obtain sufficient specificity and sensitivity to allow for enrichment and / or isolation of hits.

[0097] In some embodiments, the droplet comprises:

[0098] a) a basal medium comprising Dulbecco's modified Eagle's medium / Nutrient mixture F-12 (DMEM / F-12), about 20 mg / L to about 30 mg / L sodium bicarbonate, and about 50 mg / L to about 60 mg / L sodium pyruvate;

[0099] b) about 10% v / v to about 20% v / v fetal bovine serum relative to the basal medium; and

[0100] c) about 10% v / v to about 20% v / v of density gradient medium relative to the basal medium.

[0101] It has been found that the above media are generally optimal, especially considering the need to co-culture different cell types in this microfluidic system.

[0102] For example, sodium bicarbonate may be about 24.5 mg / L, and sodium pyruvate may be about 55 mg / L. The concentration of the density gradient medium may be about 15% v / v. The density medium may be Optiprep.

[0103] In some embodiments, the droplets further include a mixture of penicillin G and streptomycin at about 1% v / v relative to the basal medium.

[0104] The incubation step (step b) can be performed in an incubation chamber or container. The container can be removed from the chip. For off-chip incubation, parameters such as temperature and oxygen levels can be more easily adjusted when droplets need to be incubated for a long time at various steps of the procedure (e.g., for antibody accumulation, immune complex formation, and infection). Since the droplets can be coated with a surfactant, they will not coalesce.

[0105] In some embodiments, the incubation step (step b) is performed for at least 1 hour, 2 hours, 4 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 36 hours or 48 hours. In some embodiments, the incubation step (step b) is performed at a temperature of about 30°C to about 50°C.

[0106] Once sufficient antibody has accumulated within the droplet, the droplet can proceed to the next step. In some embodiments, the droplet of step b) is characterized by an antibody concentration of about 0.1 μg / mL to about 20 μg / mL. In other embodiments, the concentration is about 0.2 μg / mL to about 20 μg / mL, about 0.4 μg / mL to about 20 μg / mL, about 0.5 μg / mL to about 20 μg / mL, about 0.7 μg / mL to about 20 μg / mL, about 0.8 μg / mL to about 20 μg / mL, about 1 μg / mL to about 20 μg / mL, about 2 μg / mL to about 20 μg / mL, about 4 μg / mL to about 20 μg / mL, about 6 μg / mL to about 20 μg / mL, about 8 μg / mL to about 20 μg / mL, about 8 μg / mL to about 20 μg / mL, about 8 μg / mL to about 18 μg / mL, about 8 μg / mL to about 16 μg / mL, about 8 μg / mL to about 14 μg / mL, about 8 μg / mL to about 12 μg / mL, or about 8 μg / mL to about 10 μg / mL. In other embodiments, the concentration is about 10 μg / mL.

[0107] The average antibody concentration in the droplets can vary depending on the cell type, antibody potency, and assay detection limit. For example, if the antibody has high potency, a lower average antibody concentration can be used, thereby shortening the incubation period.

[0108] Once the droplets have formed, picoliter injections of virus are injected into the droplets to form immune complex droplets. Picoliter injection is a controlled way to add reagents to the droplets. It is insensitive to variations in the droplet cycle and allows for uniform injection even if droplets enter irregularly. The volume of reagent added can be adjusted by varying the injection time or injection pressure. Picoliter injections can be triggered using an electric field that destabilizes the surfactant on the droplet. To stop a picoliter injection, simply turn off the electric field to allow the surfactant already on the droplet to restabilize the interface.

[0109] It has been found that by picoliter injection, the droplet size that occurs when the three droplets (ACS, virus and host cell) are fused together can be avoided, which reduces the throughput and reliability. Picoliter injection can also improve the accuracy of the assay. Specifically, by keeping the volume of the droplets at an appropriate size, the time for antibody accumulation, virus-antibody interaction and host cell infection can be reduced, thereby improving throughput.

[0110] In some embodiments, the picoliter injection step is performed using an electric field of about 0.5 Vpp to about 2 Vpp and a sine wave of about 10 kHz to about 30 kHz. The sine wave can be amplified by 100 times. In other embodiments, the electric field is about 1 Vpp and the sine wave is about 20 kHz.

[0111] In some embodiments, the virus is injected in a picoliter volume of about 20 pL to about 120 pL. In other embodiments, the volume is about 25 pL to about 120 pL, about 30 pL to about 120 pL, about 35 pL to about 120 pL, about 40 pL to about 120 pL, about 40 pL to about 110 pL, about 40 pL to about 100 pL, about 40 pL to about 95 pL, about 40 pL to about 90 pL, about 40 pL to about 85 pL, or about 40 pL to about 80 pL.

[0112] In some embodiments, the volume of each immune complex droplet is about 35 pL to about 800 pL, about 40 pL to about 800 pL, or about 40 pL to about 750 pL. In other embodiments, the volume of each immune complex droplet is about 35 pL to about 700 pL, about 35 pL to about 650 pL, about 35 pL to about 600 pL, about 35 pL to about 500 pL, about 35 pL to about 400 pL, about 35 pL to about 300 pL, about 40 pL to about 300 pL, about 50 pL to about 300 pL, about 60 pL to about 300 pL, about 70 pL to about 300 pL, about 80 pL to about 300 pL, about 90 pL to about 300 pL, about 100 pL to about 300 pL, about 120 pL to about 300 pL, or about 150 pL to about 300 pL. Preferably, in some embodiments, the volume of each immune complex droplet is about 165 pL to about 250 pL.

[0113] In some embodiments, the volume of the immune complex droplet is no more than 1.8 times, no more than 1.7 times, no more than 1.6 times, no more than 1.5 times, or no more than 1.4 times the droplet volume.

[0114] In some embodiments, the immune complex droplets are characterized by an increase in volume of about 40% to about 60% relative to the droplets of steps a) and / or b). In other embodiments, the volume increases by about 45% to about 60%, about 50% to about 60%, or about 50% to about 55%.

[0115] In some embodiments, the immune complex droplets are characterized in that their diameter is about 35 μm to about 120 μm, about 35 μm to about 110 μm, about 35 μm to about 100 μm, about 35 μm to about 90 μm, about 35 μm to about 80 μm, about 35 μm to about 70 μm, about 35 μm to about 60 μm, about 40 μm to about 50 μm; about 95 μm to about 120 μm, or about 100 μm to about 110 μm. In some embodiments, the immune complex droplets are characterized in that their diameter is about 65 μm to about 90 μm, or preferably about 75 μm to about 85 μm.

[0116] The concentration of the virus can be controlled so that all of the virus can be complexed within the immune complex droplet if the appropriate antibodies are present. In some embodiments, the immune complex droplet is characterized by a virus titer of about 5 kPFU / μL to about 100 kPFU / μL, about 10 kPFU / μL to about 100 kPFU / μL, about 15 kPFU / μL to about 100 kPFU / μL, about 20 kPFU / μL to about 100 kPFU / μL, about 25 kPFU / μL to about 100 kPFU / μL, about 30 kPFU / μL to about 100 kPFU / μL, about 40 kPFU / μL to about 100 kPFU / μL, about 50 kPFU / μL to about 100 kPFU / μL, about 60 kPFU / μL to about 100 kPFU / μL, about 70 kPFU / μL to about 100 kPFU / μL, about 80 kPFU / μL to about 100 kPFU / μL, about 90 kPFU / μL to about 100 kPFU / μL, about 100 kPFU / μL to about 100 kPFU / μL, about 15 ... PFU / μL, about 40kPFU / μL to about 100kPFU / μL, about 50kPFU / μL to about 100kPFU / μL, about 60kPFU / μL to about 100kPFU / μL, about 70kPFU / μL to about 100kPFU / μL, about 70kPFU / μL to about 90kPFU / μL, about 70kPFU / μL to about 80kPFU / μL, or preferably about 75kPFU / μL. A virus titer of about 5kPFU / μL corresponds to an average of 1 virus / 200pl droplets. In some embodiments, the virus titer is at least about 12kPFU / μL.

[0117] In some embodiments, the microfluidic method further comprises a step of incubating the immune complex droplets after step c). The incubation allows the virus to be fully complexed with the antibody.

[0118] In some embodiments, the incubating step is performed for at least 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, or 4 hours.

[0119] In some embodiments, the incubating step is performed in a container.

[0120] As used herein, "neutralizing droplets" refers to droplets comprising ASC, antibodies, viruses, and host cells, and in which a substantial amount of host cells are not infected. For example, as a positive control, when 20 μg / mL 8B10 (mouse antibody) was added, only 9.75% of the cells were infected, thus showing a lower level of infection; ie, neutralized. Therefore, when less than 20% of the cells in a droplet are infected, the droplet can be characterized as a neutralizing droplet. Accordingly, an "infected droplet" refers to a droplet in which a substantial amount of host cells (ie, more than 20%) are infected.

[0121] In some embodiments, host cells are delivered to the immune complex droplets at a rate of about 200 droplets per second to about 500 droplets per second, about 250 droplets per second to about 500 droplets per second, about 300 droplets per second to about 500 droplets per second, about 300 droplets per second to about 450 droplets per second, about 300 droplets per second to about 400 droplets per second, or preferably about 300 droplets per second.

[0122] In some embodiments, the host cells are delivered at a cell density of about 80 million / mL to about 150 million / mL, about 80 million / mL to about 140 million / mL, about 80 million / mL to about 130 million / mL, about 80 million / mL to about 120 million / mL, about 80 million / mL to about 110 million / mL, about 90 million / mL to about 110 million / mL, or preferably about 100 million / mL.

[0123] In some embodiments, host cells are injected in a volume of about 20 pL to about 120 pL. In other embodiments, the volume is about 25 pL to about 120 pL, about 30 pL to about 120 pL, about 35 pL to about 120 pL, about 40 pL to about 120 pL, about 45 pL to about 120 pL, about 50 pL to about 120 pL, about 50 pL to about 115 pL, or about 50 pL to about 110 pL.

[0124] In some embodiments, the volume of each of the neutralization droplet and the infection droplet is about 65 pL to about 1000 pL, about 100 pL to about 1000 pL, or about 200 pL to about 1000 pL. In other embodiments, the volume of each of the neutralization droplet and the infection droplet is about 65 pL to about 85 pL, about 90 pL to about 115 pL, about 115 pL to about 140 pL, about 700 pL to about 800 pL, about 750 pL to about 900 pL, or about 800 pL to about 1000 pL. In some embodiments, the volume of each of the neutralization droplet and the infection droplet is about 250 pL to about 320 pL, about 270 pL to about 380 pL, or about 300 pL to about 400 pL.

[0125] In some embodiments, the volume of the neutralized droplets and the infectious droplets is no more than 1.9 times, no more than 1.8 times, no more than 1.7 times, no more than 1.6 times, or no more than 1.5 times the volume of the droplets.

[0126] In some embodiments, the neutralized droplets and infectious droplets are characterized by an increase in volume of about 30% to about 40% relative to the immune complex droplets.

[0127] In some embodiments, the neutralization droplets and the infection droplets are characterized by a diameter of about 45 μm to about 120 μm, or about 70 μm to about 120 μm. In other embodiments, the neutralization droplets and the infection droplets are characterized by a diameter of about 45 μm to about 65 μm, or preferably about 50 μm to about 60 μm; about 100 μm to about 125 μm, or preferably about 110 μm to about 120 μm. In some embodiments, the neutralization droplets and the infection droplets are characterized by a diameter of about 70 μm to about 100 μm, or preferably about 80 μm to about 90 μm.

[0128] In some embodiments, the neutralization droplets and infection droplets are characterized by a density of about 5 host cells / droplet to about 15 host cells / droplet, about 7 host cells / droplet to about 15 host cells / droplet, or about 7 host cells / droplet to about 10 host cells / droplet.

[0129] In some embodiments, the droplets have about 6% to about 15% occupancy of ASCs and host cells. In other embodiments, the occupancy is about 7% to about 15%, about 8% to about 15%, about 9% to about 15%, about 10% to about 15%, about 10% to about 14%, about 10% to about 13%, or about 10% to about 12%. In other embodiments, the occupancy is about 10%.

[0130] In some embodiments, the microfluidic method further comprises a step of incubating the neutralization droplets and the infection droplets after step d).

[0131] In some embodiments, the incubating step is performed for at least 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 36 hours, or 48 hours.

[0132] In some embodiments, the incubating step is performed in a container.

[0133] In some embodiments, the neutralized droplets and the infectious droplets are characterized by a host cell viability of greater than 80% after 30 hours of incubation, or preferably greater than 90% after 24 hours of incubation.

[0134] In the two picoliter injection steps of forming immune complex droplets and forming neutralization droplets and infection droplets, fluidic resistors (e.g., narrow serpentine channels) can be added to the virus solution and host cell solution to stabilize the injection process.

[0135] The assay also includes a sorting step that provides sufficient piezoelectric force to sort large droplets, thereby allowing high throughput sorting of the larger droplet sizes used in the assay.

[0136] In some embodiments, neutralized droplets are sorted from infected droplets by a dielectrophoretic sorter. The sorter can receive power from about 100 Hz to about 200 Hz, about 100 Hz to about 180 Hz, about 100 Hz to about 160 Hz, about 100 Hz to about 150 Hz, about 110 Hz to about 150 Hz, about 120 Hz to about 150 Hz, about 130 Hz to about 150 Hz, or about 140 Hz to about 150 Hz.

[0137] The sorting step enables enrichment of neutralizing droplets even if they are present at low frequency. The presence of large numbers of cells secreting irrelevant antibodies does not negatively affect the enrichment.

[0138] In some embodiments, the microfluidic method further comprises a step of recovering the ASC within the neutralized droplets. In some embodiments, the recovering step comprises demulsifying the neutralized droplets. The mixture of oil and aqueous medium can then be separated to separate the aqueous medium (e.g., by centrifugation), from which the ASC can be recovered.

[0139] In some embodiments, the recovery step is characterized by an ASC enrichment ratio of about 1.8 to about 3, or preferably about 1.90 to about 2.75. In other embodiments, the ASC enrichment ratio is greater than about 1.8, about 2, about 2.2, about 2.4, about 2.6, about 2.8, or about 3.

[0140] In some embodiments, the virus is selected from Chikungunya virus, dengue virus, SARS-CoV-2, respiratory syncytial virus (RSV), Zika virus, EV71, influenza virus, HIV, and norovirus.

[0141] In some embodiments, the method is characterized in that the rate is about 200 drops / second to about 500 drops / second, or preferably about 300 drops / second. In some embodiments, the method is characterized in that its flux is about 100 drops / second (or Hz) to about 400 drops / second, or preferably about 200 drops / second to about 300 drops / second. In some embodiments, each step independently has the following characteristics: the flux is about 100 drops / second to about 400 drops / second, or preferably about 140 drops / second to about 300 drops / second.

[0142] In some embodiments, the method (from steps a to e) is characterized in that the duration is less than 2 hours.

[0143] The present invention also provides a microfluidic platform, which comprises:

[0144] a) a droplet generator for generating droplets, each droplet encapsulating an antibody secreting cell (ASC);

[0145] b) a first picoliter syringe chip fluidically connected to a droplet generator, the first picoliter syringe chip comprising a first nozzle for delivering viruses into droplets to form immune complex droplets;

[0146] c) a second picoliter syringe chip fluidically connected to the first picoliter syringe chip, the second picoliter syringe chip comprising a second nozzle for delivering host cells into immune complex droplets to form neutralization droplets and infection droplets; and

[0147] d) a droplet sorter fluidically connected to the second picoliter syringe chip for sorting droplets based on infection of host cells by the virus, the droplet sorter comprising a first channel and a second channel, the second channel being configured to have a flow resistance greater than that of the first channel.

[0148] In some embodiments, the microfluidic platform comprises:

[0149] a) a droplet generator comprising at least one aqueous channel having a width of about 40 μm to about 100 μm, the aqueous channel being used to transport droplets, each droplet encapsulating an antibody secreting cell (ASC);

[0150] b) a first picoliter injector chip fluidically connected to a droplet generator, the first picoliter injector chip comprising a first nozzle and a first electric field, the first nozzle and the first electric field configured to act simultaneously to deliver the virus into the droplets to form immune complex droplets;

[0151] c) a second picoliter injector chip fluidically connected to the first picoliter injector chip, the second picoliter injector chip comprising a second nozzle and a second electric field, the second nozzle and the second electric field configured to act simultaneously to deliver host cells into immune complex droplets to form neutralization droplets and infection droplets; and

[0152] d) A droplet sorter fluidically connected to the second picoliter syringe chip, the droplet sorter comprising a top channel and a bottom channel, the bottom channel being configured to have a flow resistance at least twice that of the top channel to sort neutralizing droplets from infected droplets.

[0153] The volume of the neutralization droplet and the infection droplet can each be about 300 pL to about 400 pL. The volume of the neutralization droplet and the infection droplet can be no more than 2 times the droplet volume.

[0154] In some embodiments, the droplet generator further includes two oil channels intersecting the aqueous channel, the two oil channels being configured to flow oil to squeeze the aqueous medium in the aqueous channel into droplets.

[0155] In some embodiments, the aqueous channel comprises an outlet configured to squeeze the aqueous medium into droplets. In some embodiments, the outlet is a constriction.

[0156] In some embodiments, the microfluidic platform further includes a first container fluidically connected to the droplet generator, the first container configured to incubate the droplets.

[0157] In some embodiments, the microfluidic platform further includes a second container fluidically connected to the first picoliter syringe, the second container configured to incubate the immune complex droplets.

[0158] In some embodiments, the microfluidic platform further includes a third container fluidically connected to the second picoliter syringe, the third container configured to incubate the neutralization droplets and the infection droplets.

[0159] In some embodiments, the microfluidic platform further comprises a shielding electrode.

[0160] In some embodiments, the first picoliter syringe contains a fluid containing the virus at a pressure selected to deliver the virus to the droplets. In some embodiments, the first picoliter syringe is configured with a first electric field, and the first nozzle and the first electric field are configured to act simultaneously to deliver the virus to the droplets.

[0161] In some embodiments, the second picoliter syringe contains a fluid containing host cells at a pressure selected to deliver the host cells to the immune complex droplets. In some embodiments, the second picoliter syringe is configured with a second electric field, and the second nozzle and the second electric field are configured to act simultaneously to deliver the host cells to the immune complex droplets.

[0162] In some embodiments, the droplet sorter includes channels having a width greater than about 100 μm. Thus, each channel may be greater than about 100 μm in width.

[0163] Working principle of microfluidic platform

[0164] Figure 1 b (top) shows a conventional workflow for screening and isolating ASCs with virus neutralizing ability. Because patient-derived antibody-secreting B cells have limited proliferation capacity, they must first be immortalized or stimulated to proliferate (e.g., by hybridoma formation or addition of cytokines), a process that is both time-consuming and low-yielding. Subsequently, single B cells are isolated and clonally expanded in well plates (e.g., 96-well plates or 384-well plates). This amplification step can take many weeks to many months and is essential for obtaining sufficient antibody concentrations in large-volume (10-100 μL) well plates. Subsequently, the secreted antibodies are recovered from each well, mixed with a predetermined amount of target virus particles for binding, and finally incubated with susceptible host cells to assess the extent of viral infection. A very important last step is to specifically recover ASCs based on their neutralizing activity for downstream sequencing or amplification. This conventional workflow is not sufficient to meet the needs of rapid discovery of nAbs against novel viruses and their variants because it requires multiple months of processing time and is limited to screening a limited number of ASCs.

[0165] It is reasonable to infer that if antibodies secreted by single ASCs can approach the concentration levels required for effective virus neutralization, the most time-consuming steps in the current workflow (i.e., cell immortalization and clonal expansion) can be eliminated. Confining a single ASC to a small volume will allow for rapid accumulation of antibodies because their diffusion is prevented. Therefore, in this platform, single ASCs are encapsulated in picoliter droplets to promote rapid accumulation of antibodies ( Figure 1 b, bottom). Live virus is then delivered to each droplet to allow binding, and finally susceptible host cells are delivered to the same droplet for viral infection. Most importantly, this platform allows high-throughput detection (at a rate of 700,000 droplets / hour) of viral infection, as well as integrated sorting and recovery of droplets that have been neutralized and have low viral infection.

[0166] The working principle of the present invention will be described in detail below. In the following embodiments, for consistency, the present invention will be described in conjunction with some conditions to demonstrate the present invention. However, those skilled in the art will appreciate that the present invention is not limited thereto.

[0167] As an example, a Chikungunya virus (CHIKV) infection model was used to validate the principle and performance of the platform. Chikungunya virus is a re-emerging pathogen that is endemic in many parts of Africa and Asia, with large outbreaks in recent decades and millions of cases. There are currently no clinically licensed vaccines or therapies available for Chikungunya infection, but monoclonal nAb treatment has shown positive results in Phase I clinical trials. The ability to rapidly screen monoclonal nAbs will help develop better treatment options for Chikungunya infection.

[0168] 1.1 Bulk infection and neutralization assay

[0169] We first characterized the bulk infection behavior of CHIKV. The CHIKV-ZSGreen strain encodes the fluorescent ZSGreen protein under the control of a subgenomic promoter. Infection and replication of CHIKV-ZSGreen in host cells produce a green fluorescence signal. As expected, infection of HEK 293T cells in 96-well plates with CHIKV-ZSGreen resulted in a time-dependent increase in cells expressing green fluorescence, reaching a plateau at 22 h, with 73% of infected cells at an MOI of 10. In addition, to investigate the infection behavior of CHIKV in the presence of nAbs, the previously reported anti-CHIKV monoclonal nAb 8B10 was allowed to bind to CHIKV, and then susceptible cells were added. Addition of 8B10 nAb resulted in a dose-dependent reduction in infection in HEK 293T cells ( Figure 2 b). When 20 μg / mL 8B10 was added, only 9.75% of the cells were infected ( Figure 2 c).

[0170] 1.2 Phased approach to implement the intra-droplet virus neutralization assay

[0171] Conventional batch virus neutralization assays typically include four steps: 1. Single-cell isolation of ASCs followed by clonal expansion to allow antibodies to accumulate in the supernatant over time. 2. The antibody-containing supernatant is mixed with viruses to allow them to bind. 3. The antibody-virus mixture is added to host cells to allow infection. 4. Viral infection is measured and ASC candidates with virus neutralization activity are recovered. Similarly, in order to adapt this assay to a droplet-based scenario, the following multiple microfluidic operations are required; 1. Single-cell isolation of ASCs by droplet encapsulation (without clonal expansion) and accumulation of antibodies in droplets. 2. Virus delivery into droplets by picoliter injection. 3. Host cells are delivered into droplets by picoliter injection. 4. Virus neutralization within droplets is measured and ASC separation is performed by droplet sorting.

[0172] 1.2.1 ASC encapsulation

[0173] ASCs were first encapsulated individually into 70 μm diameter droplets using a standard flow focusing chip and then incubated for 24 hours to accumulate nAbs. The average concentration of nAbs in each ASC-containing droplet at 24 hours was quantified by ELISA to be approximately 10 μg / mL, which is well within the range of nAb concentrations required for batch production of effective virus neutralization ( Figure 2 c).

[0174] 1.2.2 Virus and host cell delivery by picoliter injection

[0175] The workflow is a multi-step process that requires the addition of viruses and host cells in sequence. Therefore, implementing a robust method to perform these operations is essential to ensure the quality of the final assay. Previous reports on virus neutralization assays in droplets relied on a droplet-to-droplet fusion system to combine ASC, virus, and host cells, with a final droplet volume of about 5 nL. However, droplet-to-droplet fusion is highly dependent on the synchronicity of droplet pairs, and any disruption to this synchronicity (e.g., caused by some coalesced droplets) can result in poor fusion efficiency. In addition, although large droplet sizes are acceptable for visualization, they are incompatible with high-throughput droplet sorting because larger droplets tend to break when sorting at high speeds if ASCs are to be recovered.

[0176] A strategy was developed to deliver viruses and host cells into droplets containing ASCs using picoliter injection. Because the volume delivered by picoliter injection is only a fraction of the volume entering the droplet, the contents can be successfully delivered without significantly increasing the droplet size. The self-triggering mechanism of picoliter injection enables robust performance even when the droplet cycle may vary during the experiment. This ensures that the reagents are delivered to all droplets to maximize the proportion of droplets in which CHIKV infection can occur. After each round of picoliter injection, an increase in droplet diameter of approximately 10 μm, or a corresponding increase in volume of 50.9% and 35.8%, respectively, was observed.

[0177] Figure 8 An example schematic of a picoliter injector chip is shown. In the workflow, a picoliter injector chip is first used to deliver CHIKV into a fully formed 70 μm diameter droplet containing a single ASC. Subsequently, a second picoliter injector chip delivers host cells into the droplet at a rate of 300 droplets per second. High cell densities ranging from 50 million to 200 million cells / mL are used to ensure that multiple host cells can be delivered into each droplet. Figure 3 d shows that the number of host cells delivered into each droplet can be controlled by varying the cell density. Finally, we investigated the survival of host cells delivered by picoliter injection. At 24 h, the cell survival rate was still as high as 94.6% ( Figure 3 e), indicating that the process is suitable for intra-droplet infection. Likewise, the excellent survival rate of multiple cells during 41 hours of incubation in droplets (88.4%) clearly indicates that sufficient nutrients are available to maintain both ASCs and host cells throughout the workflow. The greatly enhanced cell survival rate in droplets may be partly attributed to the optimized nutrient-rich medium in the droplets and the improved gas exchange of the droplets during incubation in 12-well plates (see the "Methods" section).

[0178] 1.3 Intra-droplet infection and purified nAb neutralization

[0179] Intra-droplet CHIKV infection was performed by adding 37.5 kPFU / μL CHIKV to 70 μm diameter droplets containing cell culture medium, followed by the addition of host cells by picoliter injection. For all intra-droplet infection experiments, an average of 8 host cells were delivered to each droplet during picoliter injection (100 million cells / mL at the injection port). 20 hours after the addition of host cells, detectable green fluorescence signals were observed in most droplets, indicating successful intra-droplet viral infection ( Figure 4 a).

[0180] To investigate the effect of nAbs on CHIKV infection, droplets containing 20 μg / mL purified 8810 nAb were injected with 37.5 kPFU / μL CHIKV, and intra-droplet infection was compared with a virus-only control ( Figure 4 a). Analysis of droplet signal intensity by imaging (average of 2D images) ( Figure 4 b) and compared with a continuous-flow PMT detection system (peak fluorescence per droplet) ( Figure 4 c) A significant reduction in the average CHIKV infection signal was observed in most nAb-containing droplets.

[0181] To determine the optimal viral titer for use in subsequent single-cell neutralization experiments, two conditions need to be considered. First, the CHIKV viral titer to be used should reach a high infection rate required to reduce the number of false positives in the downstream droplet sorting step. Second, in order to reduce the probability of false negatives due to infection even in the presence of nAb, the viral titer should be as low as possible. Therefore, to define the specificity window, there is a delicate balance between infection duration and viral titer. During the 48 hours after host cell injection, the infection rate of droplets was studied for different viral titers ( Figure 4 d). In general, the higher the viral titer of picoliter injection, the greater the percentage of infection droplets at all time points. In addition, no matter how much the viral titer used is, the infection rate seems to tend to a plateau after 24 hours. According to the results, a viral titer of 75kPFU / μL was selected for the single cell neutralization assay because it can provide a high droplet infection rate of 93.2% after 20 hours. As a positive control for the presence of nAb, droplets containing 9μg / mL 8B10 nAb (to simulate the average concentration of Ab accumulated in the droplets after 24 hours) were injected with 75kPFU / μL CHIKV virus, and then host cells were injected for infection. We observed that when the droplets contained nAb, the percentage of droplets with infected cells was consistently lower at all times, indicating that they have a protective effect against CHIKV infection in this droplet-based assay. In this way, it is expected that nAb can be enriched by collecting droplets containing cells that exhibit lower infection levels.

[0182] 1.4 Droplet sorter characterization

[0183] The previously reported droplet-based virus neutralization assay does not allow downstream recovery of nAb-secreting ASCs because it lacks a critical step that enables the separation of nAb-containing droplets. To address this issue, the workflow integrates a 100 μm-height droplet dielectrophoresis sorter that is able to separate droplets based on CHIKV infection signals ( Fig. 9To characterize the performance of the sorter, a mixed pool containing a 1:1 ratio of droplets with CHIKV-infected host cells and droplets with uninfected host cells was sorted based on the detected CHIKV fluorescence signal. The high fluorescence of droplets containing infected cells triggered a series of sorting pulses that redirected them to a waste reservoir ( Figure 5 a). We defined positive droplets as those containing uninfected cells (indicating the presence of nAb). We then evaluated the false positive and true positive rates of the sorted droplets over a sorting threshold range of 0.12 to 3.24 V ( Figure 5 e). Image of droplets before sorting when the sorting threshold is 0.48V ( Figure 5 b) Collect the droplet images in the reservoir ( Figure 5 c) and images of droplets in the waste reservoir ( Figure 5 d) shows the highly accurate performance of the integrated sorter in the microfluidic platform.

[0184] 1.5 Enrichment of cells secreting nAbs against CHIKV infection using a microfluidic platform

[0185] Finally, we validated the platform’s ability to recover cells that secrete nAbs against CHIKV infection. We prepared an initial ASC population consisting of a 1:2 mixture of cells that secrete 8B10 nAbs (stained red) and cells that do not secrete nAbs (stained blue). Note that cell staining is not used for cell sorting but rather for downstream identification of cell types. Single cells from this mixed population were encapsulated in 70 μm diameter droplets and incubated for 24 h to allow nAb accumulation in the droplets. Subsequently, CHIKV virus and host cells were sequentially added to the droplets and allowed to incubate for 18-22 h for infection to occur ( Figure 6 a). Then, according to the previously established ROC curve, the droplet sorting was performed using the sorting thresholds of 0.4V and 0.6V ( Figure 6 b). The sorted droplets were then demulsified and the recovered cells were analyzed using flow cytometry to determine the percentage of ASC (dyed red) and non-secreting cells (dyed blue) (Table 1). Although flow cytometry showed that the percentage of nAb-secreting cells in the initial population was 26-33%, in experiments performed on three different occasions, the percentage of nAb-secreting cells was higher in the collected droplet collection (42-57%). To reflect the efficiency of enriching nAb-secreting cells, we used an "enrichment ratio" measurement, which is defined as the ratio of the percentage of ASC to non-secreting cells after and before sorting. ASC enrichment ratios of 1.90-2.75 were obtained using this platform, which proves the concept of selecting ASCs that secrete anti-CHIKV nAbs.

[0186] Compared to the antibody affinity assay, the relatively low enrichment ratios for the virus neutralization assay are entirely expected. Unlike the antigen binding assay, which involves simple molecular interactions between antigen and antibody, the virus neutralization assay is much more complex, involving the binding of the antibody to the correct epitope and requiring complex cellular machinery to determine the infectivity of the virus and the permissiveness of the host cell. The incomplete permissiveness of the host cells (also seen in the bulk neutralization assay, where approximately 30% of the cells are uninfected even in the absence of neutralizing antibodies) Figure 2 C)) may lead to the selection of false positive droplets, thus reducing the enrichment ratio. Nevertheless, this is the first demonstration of high-throughput enrichment of virus neutralizing antibodies in a semi-automated microfluidic platform, and it can already be used as a first enrichment step to roughly select nAb secreting cells. We believe that this demonstration lays the foundation for future use in a variety of viral diseases.

[0187] Table 1: Distribution of ASCs and mock-transfected cells before and after sorting using 0.4V and 0.6V thresholds in 3 independent experiments

[0188]

[0189] In addition, in addition to the results of enriching ASCs from non-secreting cells using the present invention, similar enrichment of specific neutralizing antibody-secreting cells can be achieved in more complex scenarios that are more similar to physiological samples. Two important aspects were focused on: 1) the proportion of neutralizing antibody-secreting cells in convalescent / immunized patients is low (0.1-2%), and 2) there is an excess of B cells secreting irrelevant antibodies in patient samples. In order to simulate these conditions, an experiment was performed in which two cells secreting different monoclonal antibodies were prepared (8B10 is a CHIKV neutralizing antibody and 5A6 is an unrelated SARS-COV-2 neutralizing antibody). ASCs secreting 8B10 (marked in red) and ASCs secreting 5A6 (marked in blue) were mixed in a ratio of 1:50 and sorted as described above. Figure 7ac shows representative images of droplets before sorting and sorting droplets from the top collection channel and the bottom waste channel. As before, unsorted droplets are mainly composed of a mixture of virus-infected cells and specific antibody-secreting cells (red) and irrelevant antibody-secreting cells (blue). Droplets recovered from the top collection channel mainly contain uninfected cells, as well as the enrichment of observable specific CHIKV neutralizing antibodies (red). After FACS analysis of these recovered cells, we observed that the sorted cells included 6.27% of CHIKV neutralizing antibody-secreting cells, while CHIKV neutralizing antibody-secreting cells were 2.69% (Table 2) in the unsorted population. At a sorting threshold of 0.4V, the enrichment ratio was 2.42, similar to the enrichment ratio we obtained in earlier experiments. The results show that even when neutralizing antibody-secreting cells are present at low frequencies, the present invention still retains the ability to enrich them, and the presence of cells that secrete irrelevant antibodies in large quantities does not negatively affect the enrichment.

[0190] The successful application of the present invention was demonstrated in a scenario that better simulates physiological conditions, which is an important milestone for the platform to enter clinical application.

[0191] Table 2: Distribution of CHIKV-specific ASC (8B10) and ASC secreting unrelated antibodies (5A6) before and after sorting on the platform of the present invention

[0192]

[0193] discuss

[0194] The emergence of novel viral threats and the rapid mutation of viral strains poses a huge challenge to conventional screening methods in terms of rapid discovery of effective nAbs for the treatment of infected individuals. Here we demonstrate a rapid (<3 days), high-throughput (150,000 cells per hour) integrated microfluidic platform that can detect and sort ASCs based on their virus neutralization activity. Sorted live ASCs are suitable for downstream amplification for large-scale production of nAbs, or can be characterized using techniques such as single-cell RNA sequencing to identify the genes encoding the Abs. This platform can become a new paradigm for significantly shortening the workflow for virus neutralization Ab discovery and production.

[0195] This is the first time that an intra-droplet virus neutralization assay has been demonstrated in the context of an infectious human disease for which there is clinical evidence that exogenously administered nAbs have therapeutic benefits. Previous reports on intra-droplet virus neutralization assays have been limited to characterizing the functional activity of ASCs due to their lack of droplet sorting capabilities. In this article, to realize its true potential to accelerate high-throughput discovery of viral nAbs, we successfully developed and optimized a microfluidic platform with the ultimate goal of functional ASC recovery. Cells secreting monoclonal antibodies were used to validate the platform of the present invention to statistically evaluate the virus neutralization activity within droplets. Since the virus neutralization activity is performed at the level of single antibody-secreting cells, it is compatible with methods for rapidly generating antibody-secreting cells (e.g., transient transfection) without the need for long-term cell immortalization. On the other hand, the application of this method also has great potential for enriching functional populations from polyclonal antibody-secreting cells, such as those typically present in convalescent patients.

[0196] In this example, we demonstrate that a single-round workflow can enrich functional ASCs 1.90-2.75-fold relative to non-Ab secreting cells. Similar enrichment of specific ASCs was achieved when they were present at a low proportion (~2%) in cells that secreted unrelated antibodies in large excess, indicating that the application of this method to enrich functional antibodies from the original sample has promising prospects. The relatively modest enrichment factor is consistent with the partially permissive host cells we used in this proof of concept. Multiple literatures have shown that the selection of host cells and reporter viruses may have a significant impact on the sensitivity of virus neutralization assays. We believe that there are obvious opportunities to improve their permissiveness and infectivity in droplet-based virus neutralization assays through host cell and reporter virus engineering. For example, knocking out antiviral genes in host cells (such as STAT1) can greatly increase viral infection rates, as shown for dengue virus. Overexpression of viral receptors in host cells (such as ACE2 and TMPRSS2 for SARS-CoV-2, MXRA8 for CHIKV) can also significantly increase the efficiency of viral entry into host cells. Engineering approaches to increase viral capsid stability and improved viral manufacturing processes to reduce empty capsids will also increase viral infectivity. These approaches are expected to significantly improve the specificity of the platform for selecting virus-neutralizing ASCs by reducing false positives.

[0197] Although additional specificity can be obtained from host cell and reporter virus engineering, the current enrichment of functional ASCs from convalescent individuals or immunized animals represents a significant advance in terms of generating ASC subpopulations that secrete more potent polyclonal nAbs for immediate application. We note that despite our best efforts to follow the steps of conventional virus neutralization assays in the single-cell droplet workflow, there are still inevitable differences. Among them, the variability of the number of target cells and the heterogeneity of antibody-secreting cells and viruses are exacerbated when present in small numbers in droplets. Therefore, we consider the present invention as a first enrichment step to provide a rough step selection of neutralizing antibody-producing cells. If needed, this enriched population can be used to generate polyclonal antibodies that are more functional than unsorted populations. A second round of workflow can also be performed on the collected cells to improve the enrichment of ASCs. In addition, we consider that the workflow can be performed using ASCs enriched from the previously reported Ab binding affinity assay to obtain nAbs with high affinity. Finally, RNA sequencing of antibody-encoding genes can be performed on cells enriched using the workflow. Antibody sequences enriched relative to the initial population can be identified. This approach has been very successful in identifying therapeutic antibodies even when specific ASCs make up only a small fraction of circulating cells (e.g., from convalescent patients). Combining enrichment and RNA sequencing via the workflow of the present invention is expected to enable rapid identification of functional antibodies from polyclonal mixtures.

[0198] In summary, we provide a complete platform that can be used to rapidly discover and recover functional ASCs. We show an example of a workflow for enriching cells secreting nAbs against the CHIKV virus. Indeed, this platform is versatile and can be used to rapidly screen biologics for the treatment of various viral infections. We envision that this will be of great interest to the scientific community seeking to characterize nAbs against specific viruses and to biotech companies focused on rapidly discovering functional biologics using new paradigms.

[0199] method

[0200] Equipment fabrication and operation

[0201] The polydimethylsiloxane (PDMS) microfluidic chips used in this study were fabricated using well-established soft lithography fabrication techniques. Three types of microfluidic chips, namely, droplet generator, picoliter syringe, and droplet sorter, were used to establish the intra-droplet virus neutralization assay. Flow control of the aqueous and oil phases in all processes was performed by a syringe pump (Pump 11 Elite, Harvard Apparatus, Holliston, MA) operating in infusion mode. The oil phase used in this study was composed of fluorocarbon oil Novec TMHFE-7500 (3M, Singapore), which contains 1% (w / w) Picosurf-1 TM Surfactants (Sphere Fluidics, Cambridge, UK) were used to stabilize the droplets.

[0202] A typical 60 μm high, 3-inlet flow focusing channel design is used for droplet generation and single cell encapsulation process. The width of the aqueous channel and the oil channel is designed to be 50 μm, and then narrowed to 40 μm at the outlet to promote the breakup of the droplets. Droplets of 70 μm diameter are produced by infusing aqueous substances and oils. Next, in order to deliver CHIKV and host cells into droplets, a 45 μm high / wide picoliter syringe is used, wherein the picoliter syringe nozzle width is 40 μm. In order to generate a stable interface that destroys the droplets of the re-injection to achieve the electric field required for picoliter injection, a 1Vpp 20kHz sine wave is amplified 100 times and passed to the electrode of the picoliter syringe. Finally, in order to sort the droplets after CHIKV infection, a 100 μm high droplet sorter is used. In order to ensure that the droplets preferentially enter the top outlet channel in the absence of an electric field, the bottom outlet channel is lengthened so that its resistance is about twice that of the top channel. Shielding electrodes are also included around the device to prevent the sorting electrical pulses from causing unwanted coalescence of droplets.

[0203] In order to manufacture the master mould required for producing microfluidic devices, SU-8 2050 negative photoresist (Kayaku Advanced Materials, Westborough, MA) was first spin-coated onto silicon wafers. Subsequently, ultraviolet exposure was performed by a mask aligner (MJB4, SUSS MicroTec, Germany), and development was performed according to the recommended settings of the SU-8 product manual. The recovered mold was then surface treated overnight with trichloro-(1H, 1H, 2H, 2H-perfluorooctyl) silane (Sigma-Aldrich, St.Louis, MO) in a desiccator. Then PDMS (Sylgard 184TM, DowCorning Inc, Midland, MI) was added to the mold, degassed and cured. The cured PDMS microchannel was then removed from the mold using a scalpel, and then an inlet and outlet were created using a 1mm biopsy puncture. The microchannels were then cleaned ultrasonically for 10 min, dried, and irreversibly bonded to the substrate using a plasma cleaner (PDC-32G, Harrick Plasma, Ithaca, NY). Glass slides spin-coated with PDMS were used as substrates for the droplet generator, while uncoated glass slides were used as substrates for the picoliter syringe and sorter. After plasma bonding, to fabricate the electrodes required for the picoliter syringe and sorter, low-melting-point indium alloy wires (WIREBN-52189, Indium Corporation, Clinton, NY) were melted into the electrode inlet channels and then connected to the metal wires and bonded with UV-curable glue (Uni-Seal TM 6322, Incure, Asheville, SC).

[0204] CHIKV-ZSGreen virus production

[0205] Virus stock was prepared using CHIKV (LR2006 OPY1)-ZsGreen infectious molecular clones and then passaged twice in VeroE6 cells to amplify the virus. Virus stock was concentrated by ultracentrifugation at 28,000 g for 4 hours using a sucrose cushion. Virus stock titer was quantified by serial dilution on Vero E6 cells by plaque assay.

[0206] Bulk infection and neutralization assay

[0207] For infection assays under batch conditions, HEK 293T cells were seeded in 96-well plates at 30,000 cells per well and incubated for 5 hours to allow cell adhesion. At the same time, the antibody stock solution was mixed with CHIKV-ZSgreen virus stock solution (6,000pfu / μL) at 37°C for 2 hours to allow immune complex formation. Then 100 μL of virus+antibody mixture was added to the cells and incubated at 5% CO2, 37°C for 22 hours. Then trypsinized cells and fixed to read by flow cytometry. Flow cytometry was performed on a BD LSR II flow cytometer.

[0208] Generation of monoclonal antibody-secreting cells

[0209] A single plasmid (encoding 8B10 or 5A6) was constructed to express the heavy and light chain genes of the specific antibodies using the human ferritin heavy chain promoter and light chain promoter, respectively. HEK 293T cells were seeded in 6-well plates overnight and then transfected with 8B10 plasmid at approximately 80% confluence using Lipofectamine 3000 according to the manufacturer's protocol. Cells were harvested 24 hours after transfection by trypsinization for assay.

[0210] Intra-droplet CHIKV infection and purified nAb neutralization assay

[0211] Intra-droplet infection assays were performed with three different CHIKV virus titers (18.75 / 37.5 / 75 kPFU / μL) to determine the optimal titer for use in subsequent neutralization experiments. Droplets of 70 μm diameter were first generated using two aqueous phases—the first aqueous phase included only growth medium (DMEM / F12 with 20% FBS and 1% Penstrep), while the second aqueous phase included growth medium containing 15.5% (v / v) Optiprep (Sigma-Aldrich, St. Louis, MO). The droplets were collected into syringes and incubated in a 37°C 5% CO2 incubator for 24 hours. The droplets were then injected with CHIKV picoliters, collected into another syringe and placed back in the incubator for 3 hours, followed by a round of host cell picoliter injections. For the host cell picoliter injection process, host cells were resuspended with 15.5% (v / v) Optiprep to reach a cell density of 100 million / mL and loaded into a syringe containing a magnetic stir bar. During the picoliter injection, the syringe was placed on ice and the stirring bar was constantly stirred to ensure that the outflowing cell suspension was homogenous. After the second round of picoliter injection, the droplets were transferred to a 12-well plate to improve gas exchange ( TC-treated 12-well flat-bottom plates (Sigma-Aldrich, St. Louis, MO) containing 2 mL HFE-7500 supplemented with 1% (wt / wt) Picosurf-I TM , sealed with parafilm, and incubated in a 37°C 5% CO2 incubator. At different time points, the droplets were sampled and loaded into a disposable blood cell counter (EVETM cell counting slides, NanoEntek, Seoul), and then imaged by fluorescence microscopy. ImageJ was then used to quantify the infection rate based on the CHIKV green fluorescence signal. Infected droplets were defined as droplets containing at least one host cell with a fluorescence intensity exceeding the background.

[0212] In the in-droplet neutralization experiment of the purified nAb, the purified 8B10 nAb was diluted with growth medium to obtain a concentration of 18 μg / mL, which was used as the first aqueous phase. After dilution with the second aqueous phase, a final in-droplet nAb concentration of 9 μg / mL was obtained. The droplets were then subjected to the in-droplet infection workflow described above to determine the infection rate of nAb-containing droplets over time.

[0213] Optical and electronic devices for droplet sorting

[0214] The optical setup used in droplet sorting included a fluorescence light source (SPECTRA III, Lumencor Inc., USA) and a photomultiplier tube (MMT) detection system (H9306-03, Hamamatsu Photonics KK, Japan). Only the FITC channel was used in all droplet sorting experiments.

[0215] The voltage signal obtained from the PMT is parallelized into two outputs for sorting and signal recording. For signal recording, the PMT analog signal is converted to a digital signal using a data acquisition card (USB-6002, National Instruments, USA) and recorded using the built-in Analog Input Recorder application in MATLAB (R2019b, MathWorks, USA). For sorting, the PMT signal is processed in real time using an Arduino DUE microprocessor (Arduino, USA) to determine whether a specific droplet signal exceeds a predefined threshold. The DUE microprocessor is used to control an Arduino UNO microprocessor (Arduino, USA), which is responsible for generating 8Vpp, 10kHz square waves, which are amplified 100 times by a high-voltage amplifier and then passed to the electrodes of the sorter. After detecting a PMT signal exceeding the threshold, the sorting wave is turned on for 1750μs to guide the droplets to the sorting channel at the bottom.

[0216] For ROC curve characterization, a 50:50 mixed pool of infected and non-infected droplets was sorted at 11 different thresholds ranging from 0.12 to 3.24 V. 0.5 μL of droplets were then sampled from each sorting condition and subjected to fluorescence imaging to determine the number of true and false positives.

[0217] Table 3 shows the PMT signal distribution of virus-free droplets containing 293T cells.

[0218] Table 3: PMT signal distribution

[0219]

[0220] Single-cell droplet neutralization assay

[0221] A single-cell droplet neutralization assay was performed to validate the ability of this workflow to enrich ASCs secreting functional nAbs against CHIKV. A cell suspension containing a 1:2 mixture of ASCs secreting CHIKV nAbs and non-secreting cells was first resuspended in 15.5% (v / v) Optiprep. To later identify these two cell populations, ASCs were pre-stained red (CellTracker TM Red CMTPX Dye, Invitrogen, Waltham, MA), while non-secreting cells were stained blue (Cell Proliferation Dye eFluor TM 450, Invitrogen, Waltham, MA). Droplets of 70 μm diameter were then generated using the cell suspension as the first aqueous phase and the growth medium as the second aqueous phase. A single cell encapsulation efficiency of 20% was used. The droplets were collected into a syringe and placed in an incubator at 37°C with 5% CO2 for 24 hours to allow the nAb to accumulate within the droplets, and then the intra-droplet infection assay workflow was performed. When the total droplet infection rate reached >90%, droplet sorting was performed between 18-22 hours after picoliter injection of host cells. The droplets obtained under each sorting condition and the pre-sorting baseline were then fluorescently imaged, demulsified using 20% ​​(v / v) 1H,1H,2H,2H-perfluoro-1-octanol (Sigma-Aldrich, St. Louis, MO) in HFE7500, and the aqueous phase was recovered. The cells recovered from the droplets were trypsinized and fixed, and the percentage of ASCs and non-secreting cells was analyzed by flow cytometry.

[0222] In order to evaluate the performance of the present invention in enriching specific functional ASCs in the presence of a large number of cells secreting unrelated antibodies, the workflow described above was performed for 8B10 ASC (secreting CHIKV nAb) and 5A6 ASC (secreting unrelated SARS-COV-2nAb). The initial cell population containing 8B10 ASC and 5A6 ASC at a ratio of 1:50 was first encapsulated into droplets of 70 μm diameter to achieve a single cell occupancy of 20%. The droplets were then incubated to allow the antibodies to accumulate in the droplets for 24 hours. The droplets were then injected with 75 kPFU / uL of CHIKV picoliters, incubated for 3 hours to neutralize CHIKV with antibodies, and then a second picoliter injection step was performed to deliver 293T host cells at a cell density of 100 million / mL. The droplets were then incubated for 20 hours to allow infection to occur, followed by dielectrophoresis sorting at a 0.4 / 0.6V signal threshold.

[0223] Droplet culture medium composition

[0224] Table 4 shows the average concentration of nAbs recovered from the supernatant of droplets containing ASCs. 70 μm diameter droplets containing 10% single encapsulated ASCs were incubated for 24 hours to accumulate nAbs, followed by demulsification and quantification by ELISA. DMEM / F12 + 20% FBS (highlighted in blue) was used for all in-droplet processes described in the main text as it showed the best performance.

[0225] Table 4: Average concentration of nAbs in droplets

[0226]

[0227] Droplet size

[0228] Table 5 shows the change in droplet diameter after two rounds of picoliter injection (virus followed by host cells).

[0229] Table 5: Changes in droplet diameter

[0230] Droplet diameter (μm) Estimated droplet volume (pL) Estimated volume change (%) Original droplet size 70.62±0.36 184.4 - After the first round of pi-liter injections 81.00±0.92 278.3 +50.9 After the second round of pi-liter injections 89.70±1.12 377.9 +35.8

[0231] It will be understood that the various aspects of the described embodiments are susceptible to numerous further variations and permutations. Accordingly, the described aspects are intended to embrace all such modifications, changes and variations that fall within the spirit and scope of the appended claims.

[0232] In this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to mean the inclusion of a stated integer or step, or group of integers or steps, but not the exclusion of any other integer or step, or group of integers or steps.

[0233] In this specification and the claims that follow, unless the context requires otherwise, the phrase "consisting essentially of" and variations thereof (e.g., "consists essentially of") are understood to indicate that the elements described are essential, i.e., essential elements of the invention. The phrase allows for the presence of other undescribed elements that do not materially affect the characteristics of the invention, but excludes other unspecified elements that affect the basic and novel characteristics of the defined method.

[0234] Reference in this specification to any prior publication (or information obtained therefrom) or any known matter is not and should not be regarded as an admission or recognition or any form of implication that the prior publication (or information obtained therefrom) or known matter constitutes part of the common general knowledge in the field to which this specification relates.

Claims

1. A microfluidic method for measuring antibody secreting cells (ASC), comprising the following steps: a) separating ASCs in droplets so that each droplet encapsulates only one ASC; b) incubating the droplets of step a) to accumulate antibodies in the droplets; c) injecting picoliters of virus into the droplets of step b) to form immune complex droplets; d) injecting picoliters of host cells into immune complex droplets to form neutralizing droplets and infectious droplets; and e) Based on the infection of the virus to the host cells, neutralizing droplets are sorted out from the infected droplets to measure the ASCs within the neutralizing droplets.

2. The microfluidic method according to claim 1, wherein a dielectrophoresis sorter is used to sort the neutralized droplets from the infected droplets.

3. The microfluidic method according to claim 1 or 2, further comprising a step of generating droplets in the presence of lipopolysaccharide before step a). 4 . The microfluidic method according to claim 1 , further comprising a step of incubating the immune complex droplets after step c).

5. The microfluidic method according to any one of claims 1 to 4, wherein the droplets of steps a) and b) have one or both of the following characteristics: A volume of about 160 pL to about 200 pL; and The diameter is about 40 μm to about 100 μm, or preferably about 70 μm to about 90 μm.

6. The microfluidic method according to any one of claims 1 to 5, wherein the incubation step (step b) is performed for at least 1 hour.

7. The microfluidic method according to any one of claims 1 to 6, further comprising a step of incubating the neutralization droplets and the infection droplets after step d).

8. The microfluidic method according to any one of claims 1 to 7, wherein the picoliter injection step is performed using an electric field of about 0.5 Vpp to about 2 Vpp and a sine wave of about 10 kHz to about 30 kHz.

9. The microfluidic method according to any one of claims 1 to 8, wherein the method is characterized by a rate of about 200 droplets per second to about 500 droplets per second, or preferably about 300 droplets per second.

10. The microfluidic method according to any one of claims 1 to 9, further comprising a step of recovering the ASC in the neutralized droplet. The microfluidic method of claim 10 , wherein the recovery step comprises demulsifying the neutralized droplets.

12. A microfluidic platform comprising: a) a droplet generator for generating droplets, each droplet encapsulating an antibody secreting cell (ASC); b) a first picoliter syringe chip fluidically connected to a droplet generator, the first picoliter syringe chip comprising a first nozzle for delivering viruses into droplets to form immune complex droplets; c) a second picoliter syringe chip fluidically connected to the first picoliter syringe chip, the second picoliter syringe chip comprising a second nozzle for delivering host cells into immune complex droplets to form neutralization droplets and infection droplets; as well as d) a droplet sorter fluidically connected to the second picoliter syringe chip for sorting droplets based on infection of host cells by the virus, the droplet sorter comprising a first channel and a second channel, the second channel being configured to have a flow resistance greater than that of the first channel.

13. The microfluidic platform of claim 12, wherein the second channel is configured to have a flow resistance at least twice that of the first channel.

14. The microfluidic platform according to claim 12 or 13, wherein the droplet generator comprises an aqueous channel for transporting droplets and two oil channels intersecting the aqueous channel, the two oil channels being configured to allow oil to flow to squeeze the aqueous medium in the aqueous channel into droplets.

15. The microfluidic platform of claim 14, wherein the aqueous channel comprises an outlet configured to squeeze the aqueous medium into droplets.

16. The microfluidic platform according to any one of claims 12 to 15, further comprising a first container fluidically connected to the droplet generator, the first container being configured to incubate the droplets. 17 . The microfluidic platform according to any one of claims 12 to 16 , further comprising a second container fluidically connected to the first picoliter syringe, the second container being configured to incubate immune complex droplets.

18. The microfluidic platform of any one of claims 12 to 17, further comprising a third container fluidically connected to the second picoliter syringe, the third container being configured to incubate the neutralization droplets and the infection droplets.

19. The microfluidic platform according to any one of claims 12 to 18, further comprising a shielding electrode.

20. The microfluidic platform of claim 15, wherein the outlet is a constriction.

21. The microfluidic platform of any one of claims 12 to 20, wherein the first picoliter syringe contains a fluid containing a virus at a pressure selected to deliver the virus to the droplet.

22. The microfluidic platform of any one of claims 12 to 21, wherein the second picoliter syringe contains a fluid containing host cells at a pressure selected to deliver the host cells to the immune complex droplets.

23. The microfluidic platform of any one of claims 12 to 22, wherein the first picoliter syringe is configured with a first electric field, and the first nozzle and the first electric field are configured to act simultaneously to deliver the virus to the droplet.

24. The microfluidic platform of any one of claims 12 to 23, wherein the second picoliter syringe is configured with a second electric field, and the second nozzle and the second electric field are configured to act simultaneously to deliver the host cells to the immune complex droplets.

25. The microfluidic platform of any one of claims 12 to 24, wherein the droplet sorter comprises a channel having a width greater than about 100 μm.

26. The microfluidic method according to any one of claims 1 to 11 or the microfluidic platform according to any one of claims 12 to 15, wherein the droplet comprises: a) a basal medium comprising Dulbecco's modified Eagle's medium / Nutrient mixture F-12 (DMEM / F-12), about 20 mg / L to about 30 mg / L sodium bicarbonate, and about 50 mg / L to about 60 mg / L sodium pyruvate; b) about 10% v / v to about 20% v / v of fetal bovine serum relative to the basal medium; and c) about 10% v / v to about 20% v / v of density gradient medium relative to the basal medium.

27. The microfluidic method or microfluidic platform of claim 26, wherein the droplets further comprise a mixture of penicillin G and streptomycin at about 1% v / v relative to the basal medium.

28. The microfluidic method of any one of claims 1 to 11 or the microfluidic platform of any one of claims 12 to 15, or the microfluidic method or microfluidic platform of claims 16 or 27, wherein the volume of each of the neutralization droplet and the infection droplet is about 65 pL to about 1000 pL.

29. The microfluidic method according to any one of claims 1 to 11 or the microfluidic platform according to any one of claims 12 to 25, or the microfluidic method or microfluidic platform according to any one of claims 26 to 28, wherein the volume of the neutralization droplets and the infection droplets does not exceed 2 times the volume of the droplets.

30. The microfluidic method of any one of claims 1 to 11 or the microfluidic platform of any one of claims 12 to 15, or the microfluidic method or microfluidic platform of any one of claims 26 to 29, wherein the host cells are delivered at a cell density of about 80 million / mL to about 150 million / mL, or preferably about 100 million / mL.

31. The microfluidic method according to any one of claims 1 to 11 or the microfluidic platform according to any one of claims 12 to 25, or the microfluidic method or microfluidic platform according to any one of claims 26 to 30, wherein the ASC is a B cell or a transfected cell, or preferably a murine memory B cell.

32. The microfluidic method according to any one of claims 1 to 11 or the microfluidic platform according to any one of claims 12 to 25, or the microfluidic method or microfluidic platform according to any one of claims 26 to 31, wherein the droplets of step b) are characterized in that the antibody concentration is about 0.1 μg / mL to about 20 μg / mL, or preferably about 10 μg / mL.

33. The microfluidic method of any one of claims 1 to 11 or the microfluidic platform of any one of claims 12 to 25, or the microfluidic method or microfluidic platform of any one of claims 26 to 32, wherein the volume of each immune complex droplet is about 35 pL to about 800 pL.

34. The microfluidic method of any one of claims 1 to 11 or the microfluidic platform of any one of claims 12 to 25, or the microfluidic method or microfluidic platform of any one of claims 26 to 33, wherein the immune complex droplet is characterized by an increase in volume of about 40% to about 60% relative to the droplet.

35. The microfluidic method according to any one of claims 1 to 11 or the microfluidic platform according to any one of claims 12 to 25, or the microfluidic method or microfluidic platform according to any one of claims 26 to 34, wherein the immune complex droplets are characterized by a diameter of about 35 μm to about 120 μm, or preferably about 65 μm to about 90 μm.

36. The microfluidic method according to any one of claims 1 to 11 or the microfluidic platform according to any one of claims 12 to 25, or the microfluidic method or microfluidic platform according to any one of claims 26 to 35, wherein the immune complex droplets are characterized by a viral titer of about 5 kPFU / μL to about 100 kPFU / μL, or preferably about 75 kPFU / μL.

37. The microfluidic method of any one of claims 1 to 11 or the microfluidic platform of any one of claims 12 to 25, or the microfluidic method or microfluidic platform of any one of claims 26 to 36, wherein the neutralization droplets and infection droplets are characterized by an increase in volume of about 30% to about 40% relative to the immune complex droplets.

38. The microfluidic method according to any one of claims 1 to 11 or the microfluidic platform according to any one of claims 12 to 25, or the microfluidic method or microfluidic platform according to any one of claims 26 to 37, wherein the neutralization droplets and infection droplets are characterized by a diameter of about 45 μm to about 120 μm, or preferably about 70 μm to about 100 μm.

39. The microfluidic method of any one of claims 1 to 11 or the microfluidic platform of any one of claims 12 to 25, or the microfluidic method or microfluidic platform of any one of claims 26 to 38, wherein the neutralization droplets and infection droplets are characterized by a density of about 5 host cells per droplet to about 15 host cells per droplet.

40. The microfluidic method according to any one of claims 1 to 11 or the microfluidic platform according to any one of claims 12 to 25, or the microfluidic method or microfluidic platform according to any one of claims 26 to 39, wherein the neutralization droplets and infection droplets are characterized in that the host cell survival rate is greater than 80% after 30 hours of incubation, or preferably the host cell survival rate is greater than 90% after 24 hours of incubation.

41. The microfluidic method of claim 10 or 11, wherein the recovery step is characterized by an ASC enrichment ratio of greater than about 1.

8.

42. The microfluidic method according to any one of claims 1 to 11 or the microfluidic platform according to any one of claims 12 to 25, or the microfluidic method or microfluidic platform according to any one of claims 26 to 41, wherein the virus is selected from Chikungunya virus, dengue virus, SARS-CoV-2, respiratory syncytial virus (RSV), Zika virus, EV71, influenza virus, HIV and norovirus.

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