Methods and systems for screening ligands and receptors

Abstract

The present invention provides methods for identifying compounds having a particular biological function, such as binding affinity to a target or the ability to internalize into a cell. Certain parameters are selected and combined, including the limit of detection of the assay and the concentration of the target, such that the methods can be performed.

Description

Technical Field

[0001] This invention belongs to the field of cell and molecular biology and relates to methods for identifying compounds with specific biological functions. This invention also belongs to the field of microfluidics and relates to microfluidic systems and their use in bioassays. Background Technology

[0002] Molecules exhibit inherent affinity for one another due to electrostatic forces, hydrogen bonds, and dispersion forces. The non-covalent interactions resulting from these affinityes are particularly important in biological processes, including the catalysis of chemical reactions by enzymes, the neutralization of pathogens by antibodies and / or T cells, and the stimulation of cellular activity by interactions between hormones, cytokines, chemokines, and generally any two molecules. The interaction between a biological receptor and its natural ligand can be defined according to various properties, such as specificity, affinity, saturation, binding constant, and physiological response.

[0003] In the context of therapeutic antibodies, affinity is one of the key properties characterizing the potency, and to some extent, mechanism of action, pharmacodynamics, and pharmacokinetics of these macromolecules. Antibody affinity describes the strength of binding of a single antibody to a specific epitope in its antigen. Therefore, antibodies with higher affinity may allow for lower doses or longer dosing intervals during treatment. Furthermore, high affinity can significantly impact the commercial success of therapeutic antibody drugs due to the complex manufacturing systems and therapeutic dosages required for antibodies. In some cases, lower affinity may be desired. For example, a novel approach to developing a vaccine against coronaviruses has recently been developed using a modified spike protein with lower binding affinity (Ratswohl et al., Eur. J. Immunol. 2023, 0: 2350408). Moreover, immunomodulatory antibodies designed to modulate receptor signaling, compared to naturally occurring or directly targeted therapeutic antibodies, provide greater activity through enhanced aggregation due to lower affinity rather than higher affinity. This method delivers enhanced immune cell activation, in vivo T cell expansion, and antitumor activity (Yu et al., Nature, 2023, 614:539-547).

[0004] Another characteristic characterizing therapeutic antibodies is their ability to internalize into cells, rather than simply binding to antigens on the cell surface (Dumontet et al., Nat Rev Drug Discov 2023, 22(8):641-661). Antigen-mediated antibody internalization plays a crucial role in several antibody-based therapies because it allows for drug delivery to cancer cells via antibody-drug conjugates (ADCs), removal or degradation of surface receptors from cancer cells, and antibody-based immunotherapies to identify tumor cells for killing by immune cells (e.g., antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP)).

[0005] Methods known in the art for assessing antibody affinity are based solely on kinetic analysis (Guo et al., RSC Adv. 2016, 6:13837-13845) or in combination with other techniques (Lupu et al., Int. J. Mol. Sci. 2021, 22(23):12832). Droplet-based microfluidic assays also allow for high-throughput characterization of antigen-specific antibody-secreting cells. Gérard et al. described a droplet-based microfluidic platform for antibody screening at the single-cell level, using a dual-fluorescent sandwich ELISA for screening, identification, sorting, and V testing of antigen-specific IgG antibodies produced by antibody-secreting cells from immunized mice. H -V L Paired sequencing (Gérard et al., Nat. Biotechnol. 2020, 38(6):715-721). Other methods for studying antibody-secreting cells in droplets include collecting droplets on a horizontal plane and measuring changes in droplet fluorescence at multiple time points using stationary droplet-based assays (Bucheli et al., Eur. J. Immunol. 2021, 51(6):1334-1347) or DropMap (Eyer et al., Nat. Biotechnol. 2017, 35(10):977-982; Canales-Herrerias et al., J. Clin. Invest. 2022,132(12):e153580). Beacon ® (Berkeley Lights, now part of Bruker) and Cyto-Mine ® The (Sphere Fluidics) commercial platform represents a further approach for researching therapeutic antibodies.

[0006] However, current methods for characterizing antibody affinity and internalization are post-screening processes that require antibody identification before testing to determine its affinity (e.g., by Octet® SPR analysis) or internalization properties (e.g., by flow cytometry analysis).

[0007] Furthermore, the discovery of therapeutically and / or functionally relevant antibodies requires screening antibody-secreting cells (primary or engineered). However, the frequency of cells secreting relevant antibodies is relatively low (less than 1%), mainly due to the vast diversity of antibody libraries and low immune responses.

[0008] In addition, antibodies need to be diluted to working concentrations for different applications. The final working concentration is crucial for the precision and accuracy of each assay. The optimal final concentration is a balance between assay sensitivity, antibody affinity for its epitope, and nonspecific binding (i.e., cross-reactivity) between the antibody and other antigens, which generates background noise. If the working concentration is too low, the target protein or antigen may be undetectable. If the working concentration is too high, the background noise may be high, resulting in a poor signal-to-noise ratio. Therefore, appropriate dilution of the working concentration needs to be optimized for each antibody, application, and sample to obtain the best signal-to-noise ratio. While some antibodies have a wide range of acceptable working concentrations, many antibodies only produce an acceptable signal-to-noise ratio at precise concentrations. Optimizing assay conditions such as protein / reagent concentrations and / or antibody dilution is laborious and time-consuming.

[0009] The method according to the invention aims to address the shortcomings and limitations of current assays by providing a direct screening method for compound-target interactions. In particular, the method disclosed herein allows for the identification of specific biological functions of compounds by selecting the limit of detection (LOD) and target concentration without adjusting or calculating the optimal working concentration of the compound (e.g., dilution). Furthermore, the method allows for the parallel identification and classification of multiple compounds and their ranking based on their specific biological functions, such as their affinity for and / or internalization capacity to the target. Summary of the Invention

[0010] In one aspect, the present invention provides a method for identifying and classifying compounds capable of binding to a target in an assay having a limit of detection (LOD) for the compound, the method comprising the following steps: (a) Provide microreactors containing compounds and targets, (b) Incubate the compound and the target under conditions sufficient to allow the formation of a complex between the compound and the target. (c) The complex is identified by detecting signals above or below a first threshold or a second threshold, wherein: (i) If the signal is below the first threshold and the target is present at a concentration equal to or at most five times the LOD, the compound is classified as having the highest binding affinity for the target; (ii) If the signal is above the first threshold and the target is present at a concentration of at least five times the LOD, the compound is classified as having the lowest binding affinity for the target; (iii) If the signal is above the second threshold and the target is present at a concentration below the LOD, the compound is classified as exhibiting internalization ability, or (iv) If the signal is above the second threshold, and the target and / or detector and / or compound is present at a concentration below the LOD, then the compound is classified as exhibiting internalization ability. The limits of detection (LOD) for the compounds to be identified and classified range from about 0.01 nM to about 10 nM.

[0011] In another aspect, the present invention provides a system for screening compounds in an assay having a predetermined limit of detection (LOD), the system comprising: (a) The first module, which is configured to collect compounds and targets; (b) A second module configured to incubate the compound and the target; (c) A third module configured to detect the interaction between the compound and the target; (d) The fourth module, which is configured to screen the compounds. Attached Figure Description

[0012] Figure 1 shows detectable compound affinity (x-axis) as a function of initial compound concentration (each chart title, IgG0) and captured target (y-axis, Ag) in systems with LODs of 1 nM (A and B) and 0.2 nM (C and D). Solid lines represent assays with an initial target concentration (Ag0) of 30 nM, and dotted lines represent assays with an initial target concentration of 5 nM. For assays with an LOD of 1 nM: using 5 nM antigen (Ag0) for assay readings and using cells secreting 2 nM or 5 nM antibody (IgG0), corresponding to the median and mean primary B cell secretion rates (after 1 hour of secretion in 80 pL droplets), the detected antibody affinity was below 4 nM or 12 nM. Working at higher antigen concentrations (Ag0 = 30 nM), the method was able to screen for antibodies with affinity from 30 nM to 95 nM (see Figure 1A-B). Regarding the determination of LOD of 0.2 nM, the method is capable of detecting antibodies with a wide affinity range (high to low affinity) (see Figures 1C-D).

[0013] Figure 2 The figure shows the detectable compound affinity as a function of initial compound concentration (x-axis) and target concentration (y-axis) in a system with a LOD of 1 nM. Examples with different antibody concentrations (IgG0, nM) and antigen (Ag) concentrations (Ag0, nM) are shown. The curves define the maximum detectable KD (up to 10 nM, 50 nM, 100 nM, 500 nM and above) under different IgG0 and Ag0 conditions in a system with a LOD of 1 nM.

[0014] Figure 3 The results show the detectable compound affinity as a function of initial compound concentration (x-axis) and target concentration (y-axis) in a system with a LOD of 1 nM. By selecting an assay with a LOD of 1 nM and using 5 nM antigen (Ag0) for assay readings, and using cells secreting 2 nM or 5 nM antibody (IgG0), which correspond to approximately the median and mean primary plasma B cell secretion rates (after 1 hour of secretion in an 80 pL droplet), the method enriches positive events for antibodies with affinity below 16 nM.

[0015] Figure 4The results show the detectable compound affinity as a function of initial compound concentration (x-axis) and target concentration (y-axis) in a system with a LOD of 1 nM. By selecting an assay with a LOD of 1 nM and using 10 nM antigen (Ag0) for assay readings, and using cells secreting 2 nM or 5 nM antibody (IgG0), which correspond to approximately the median and mean primary plasma B cell secretion rates (after 1 hour of secretion in an 80 pL droplet), the method will report positive events for antibodies with affinity below 50 nM.

[0016] Figure 5 The results show the detectable compound affinity as a function of initial compound concentration (x-axis) and target concentration (y-axis) in a system with a LOD of 1 nM. By selecting an assay with a LOD of 1 nM and using 15 nM antigen (Ag0) for assay readings, and using cells secreting 2 nM or 5 nM antibody (IgG0), corresponding to approximately the median and mean primary plasma B cell secretion rates (1 hour after secretion in an 80 pL droplet), the method will report positive events for antibodies with medium to low affinity.

[0017] Figure 6 The figure shows the performance of antibodies screened according to the method of the invention in terms of their ability to internalize and enter cells. The figure shows the assay of the compound of interest binding to cells / particles. Under “standard” assay conditions (left panel), the internalized compound resulted in an increased peak fluorescence signal compared to internalization (bottom panel). The signal difference between surface binding and internalization was almost undetectable, and despite the use of specific signal detection / computation methods (see, e.g., Figure 7 Enriching internalized compounds using standard methods (maximum peak value and area) by setting thresholds and specificities is still not straightforward. Under the specific conditions of the "internalization assay" (right figure), only the internalization of the compound within the cell will result in a detectable fluorescent signal (bottom figure). Detecting the signal by the maximum peak value or area will greatly enrich the internalized compound. These examples assume: cell diameter 10 µm; cell surface area = 166π; internalization via receptor-mediated endocytosis, ultimately degrading in lysosomes. Endosome diameter 50 nm, lysosome diameter 1 µm. Receptor-Ab internalization will relocate to organelles approximately 3 µm (maximum). Organelle / internalization surface area = 4.5π. Therefore, when the receptor / antibody is internalized, the signal intensity is expected to be approximately 37 times higher / more concentrated. Thus, assays for internalization-specific antibodies can be designed where the concentration of the detectable molecule is just below the LOD concentration.

[0018] Figure 7This figure illustrates the ability of antibodies to internalize into cells using the method according to the invention. The figure shows the assay of the compound of interest binding to cells / particles and different representations of the results, including data reporting and analysis to identify specific compounds for internalization. In the “standard” assay conditions (left figure), the internalized compound, compared to its binding to cells / particles, results in an increased maximum fluorescence signal (Max Target Fluo, bottom figure) while decreasing the “area under the curve” (Area Target Fluo). Therefore, it is difficult to specifically identify the internalized compound. The signal difference between surface binding and internalization is almost undetectable, and specific signal detection / calculation methods (maximum / area) still make it difficult to enrich internalized compounds using standard methods to set thresholds and specifically enrich them. In the specific conditions of the “internalization assay” (right figure), internalization of the compound within the cell produces a detectable fluorescence signal (Max Target Fluo and Area Target Fluo, bottom figure), while cell surface binding is almost undetectable. In this internalization-specific assay, it is easy to set thresholds for detecting and enriching internalization-specific compounds.

[0019] Figure 8 shows the internalization assay formats. Figure 8A represents different assay formats in which a container (1), cells / particles expressing the target (2), a solution present in the container (3), and optionally, cells / particles producing a biologically functional compound (4) are incubated. Figure 8B – Example of compound localization / detection. Figure 8B represents the behavior of LOD-determined assay formats and internalized target / compound of interest / detector. In this example, cells / particles expressing the target (2), compound of interest (8), detector bound to the compound of interest (9), and target bound to the cell membrane (5) are incubated together. In the left image, the compound of interest binds to the target cell expressed on the surface along with the detector. In the middle image, the compound of interest is internalized into the target cell along with the detector. In the right image, the compound of interest has been degraded / recycled to the cell surface, and only the detector remains inside the cell / detectable due to the internalization process. Figure 8C – Example of different internalization localizations. After the compound is internalized, it is likely that it will pass through the cell / granule membrane (6) expressing the target (5) bound to the cell surface. Inside the cell / granule, the compound of interest / the detection agent bound to it can be repositioned to different intracellular compartments (7) with different shapes as shown in the figure. The detection agent can be directly or indirectly coupled to / bound to the compound of interest.

[0020] Figure 9 shows the beadline assay containing compounds of interest bound to their targets. Figure 9A – Example of a form used to screen compounds of interest using the beadline assay. Under certain conditions, the ligand and acceptor are in solution. Detecting interactions requires an assay form compatible with this class of molecules. Paramagnetic beads are used (… Right now The beaded assay can detect the interaction and affinity of a compound with its ligands. In a container (1), biologically functional cells (4) present in a solution (3) and producing the compound of interest (8) are incubated with a trapping agent (10) that interacts directly or indirectly with the compound of interest (8), which has an affinity for binding its target (11). The interaction between the target (11) and the compound of interest (8) is detected by using a directly or indirectly labeled target (11) and directly or indirectly labeled direct or indirect detection agents (9). Figure 9B – Distinguishing between high-affinity and low-affinity compounds with the target. An example of screening in a container (1) containing a fluid (3) and the compound of interest (8) having a binding affinity for a soluble target (11) and produced by biologically functional cells (4) is shown. The assay using the beaded form contains multiple trapping agents (10). The secretion of the compound of interest (8) by biologically functional cells (4) and its binding to the trapping agent (10) are detected by a detector (9) bound to the compound of interest. Depending on the secretion rate of the biologically functional cells (4) (and / or the amount of the compound of interest (8), some of the detectors (9) in solution may be detectable. In cases of low system LOD (e.g., 1 nM) and low target concentration, under condition (a), the compound of interest has a high affinity for the target and binds tightly to the target (high K). on And low Koff), thereby capturing all targets (11') in solution onto the trapping agent (10) that binds to the compound of interest (8). Binding events will be detected under these conditions. In cases of low system LOD (e.g., 1 nM) and low target concentration, the compound of interest has a low affinity for the target and does not bind tightly to it (low Koff). on and high K off Therefore, the target (11') in solution is in excess relative to its binding to the compound of interest (8) in the form of (11). Under these conditions, binding events will not be detected (see, Figure 9B).

[0021] Figure 10 shows the system and setup for determining the LOD. This figure illustrates examples used to determine the LOD. Beaded LOD determination is performed in (a), single-particle LOD determination is performed in (b), and single-particle LOD determination is performed in (c). The LOD determination is performed using similar assay materials: (optionally paramagnetic) particle (14) concentration and preparation method, or particles (14) of simulated cell size and solution (3) in container (1). To minimize biological interference, the particles are functionalized with fluorescent molecules (ideally using the same dye as in the functional assay). Functionalization can be performed by directly binding the detection particle (13) to the particle (14) and / or indirectly binding via a trapping agent (10) and a reporter molecule (12). Examples of trapping agents and / or reporter molecules include antibodies and their derivatives, proteins, lipids, carbohydrates, nucleic acids, and chemicals.

[0022] Figure 11 shows how the measured LOD was determined using a beaded pattern. Measurements were performed according to Example 1. Histograms of the PMT3 signal for each droplet population at frequencies of 200 Hz (A), 1 kHz (B), and 2 kHz (C) are shown. The average LOD calculated from linear regression of signal intensity was approximately 1.6 nM(D) (LOD = average value). 空白 + 3 (SD 空白 ), where the average value 空白 It is the average of the blanks, SD 空白 It is the standard deviation of the blank measurement, also known as σ).

[0023] Figure 12An example system for compartmentalizing target and reagent mixtures, performing assays (reactors), detecting, analyzing, and enriching target-specific compounds is shown. The compartmentalization module combines reagents and compounds in the same compartment where assays can be performed. The reactor module receives input from the compartmentalization module to initiate the assay reaction, e.g., by heating / cooling the compartment. The detector module scans / reads assay readings (e.g., using a fluorescent molecular excitation device and detector device). The assay analyzer calculates the information received from the detector to visualize and interpret the compound function based on the signals emitted by the reading molecules and detector device. Both the detector and analyzer can adjust the system LOD—increasing the LOD by increasing the excitation power and detection sensitivity / gain, and / or decreasing the LOD by decreasing the excitation power and detection sensitivity / gain. Signal processing and computational methods can influence the system LOD, e.g., by removing electronic noise or compensating for crosstalk / stray light. The analyzer detects background signals and assay-specific signals; the analyzer and / or the user can define assay thresholds to define the assay-specific signals. Based on the signal detected by the detector (and optionally displayed on the user interface), and when the measured signal is above a defined measurement threshold, the enrichment module is able to enrich materials with a specific function, defined as having a signal above the threshold. Standard assay selectivity is determined by reaction volume and reaction time; the critical inflection point is determined by the assay LOD and system LOD to enable specific enrichment / detection of target-specific compounds with a specific function. The assay LOD depends on the reporter molecule / measurement reading (both in terms of reporter molecule identity and quantity), while the system LOD depends on the optical setup (both in terms of excitation and detection path definitions) and signal processing.

[0024] Figure 13 shows typical results of cell-based assay LOD determination using different reading systems (droplet fluorescence detector and image analyzer). (A) Gating strategy used during droplet scanning to detect droplets containing live cells with specific signals on the cell surface. From left to right: Droplets selected based on size (y-axis) over time (x-axis), then droplet demultiplexing based on different Dy754 droplet encoding concentrations detected by the PMT5 channel, while having the same width (x-axis), from which droplets containing live cells (unincorporated NucGreen, therefore PMT2 negative and CellTrace Violet labeled, therefore PMT1 positive) were selected for further analysis of antibody-cell binding. Right now (A) PMT4 signal above the background. (B) Following a similar gating strategy to (A), examples of antibody binding to cells are shown for different primary antibody concentrations (0 to 10 nM) (histogram below). The figure above shows cells without cells (…). Right nowHistogram of antibody signal (PMT4) in droplets (PMT1 negative). Under these conditions, for this type of cell-based assay, the LOD was 1.09 × 10e6 fluorescent units (reported by PMT) (i.e., blank mean + 3 standard deviations). )。 Using the same amount of reporter molecule, this corresponds to an assay LOD capable of detecting primary antibodies down to approximately 0.7 nM (estimated by plotting linear regression of 1 nM and 10 nM values). (C) Bright-field, Dy754 (indicating droplet coding / droplet conditions), AF647 (indicating antibody signal), CTV (indicating cell presence), and NucGreen (indicating cell viability) images of droplets used to assess the LOD of cell-based assays using reporter cells. (D) Quantification of LOD assessment in still images. From left to right: Identification of droplet conditions using DY754 (increased Dy754 reflects increased primary antibody concentration from 0 to 10 nM, as shown), assessment of droplet diameter uniformity, identification of droplets containing cells (CTV positive), identification of droplets containing viable cells (NucGreen negative), and finally, staining of the cell membrane (CM) with AF647-labeled detection molecular weight primary antibody binding. Under these conditions, the LOD for this type of cell-based assay is 7151 fluorescent units (reported by image acquisition). ( That is, blank mean + 3 standard deviation). Using the same amount of reporter molecules, this corresponds to the assay LOD that can detect down to about 1.0 nM primary antibody (estimated by plotting linear regression of 1 nM and 10 nM values).

[0025] Figure 14 shows the enrichment of compounds with specific functions based on LOD and target concentration. (A) The left panel shows the distribution of IgG secretion frequency (in nM) and the cell count distribution of antibody affinity (estimated for screening 10) based on data published in Eyer et al., Nature Biotechnology 2017, 35(10): 977-982 and Molari et al., eLife 2020, 9: e55678. 6 (a) The recovery distribution of function-specific antibodies, considering cell secretion and antibody affinity (both independent of each other, as shown by Eyer et al., Nature Biotechnology 2017, 35(10): 977-982), is shown in Figure A in an assay with a LOD of 1 nM and a target of 20 nM. Most antibodies (>99.999%) have medium to low affinity. At constant antibody concentration / using a cell secretion threshold selection ( Right now Under the condition of approximately 5 nM and a relatively deep histogram, the critical KD (KDc) is 76 nM. Right nowUnder these conditions, the maximum detectable Kd was 100 nM. Under these conditions, the assay retained the vast majority of medium / low affinity antibodies. (C) The recovery distribution of function-specific antibodies considering cell secretion and antibody affinity (which are independent of each other, as shown by Eyer et al., Nature Biotechnology 2017, 35(10): 977-982) in an assay with a LOD of 1 nM and a target of 5 nM is shown in Figure A. Under these conditions, the maximum detectable Kd was 100 nM, and the enrichment of high affinity antibodies was significant. Under constant antibody concentration / using cell secretion threshold selection ( Right now Under conditions of approximately 5 nM and a relatively deep histogram, the critical KD is 16 nM. Under these conditions, the vast majority of high-affinity antibodies were retained.

[0026] Figure 15 The statistical data shown in Figure 14 is based on the histogram. This is according to the set critical K. D (KDc), based on LOD and target concentration (Ag0), efficiency ( Right now Based on the total cell pool with KD < KDc, the percentage of cells recovered with KD < KDc) and purity ( Right now The percentage of cells with specific functions among the total number of recovered cells is affected. Therefore, an assay can be designed to highly enrich specific functions based on the determination of LOD and antigen concentration, regardless of compound concentration.

[0027] Figure 16 shows the antibody affinity and concentration distribution calculated based on a set critical KD (KDc). KDc is a user-defined KD used to screen for compounds with affinity below (or above) KDc. Since KDc depends on the target concentration (Ag0), the measured LOD, and the compound concentration (IgG0), molecules of interest can be screened by varying Ag0 when IgG0 is similar. IgG0 can be estimated using reporter molecules. At the average / median compound concentration (IgG0), the reporter molecule signal will cover most of the signal population (…). Right now The compound concentrations follow a Gaussian distribution – see [link to relevant documentation] Figure 14AThe horizontal line represents the KDc threshold, the vertical dashed line (2 nM) is the average compound concentration obtained under the assay conditions, and the vertical solid line (5 nM) is the average compound concentration obtained under the assay conditions. (A) By performing assays with a LOD of 1 nM and Ag0 of 5 nM, and simultaneously screening for compound molecules secreted in average or median populations, the KDc was set to 16 nM. The target population of compounds of interest was as low as 0.2% (dark gray). (B) By performing assays with a LOD of 1 nM and Ag0 of 10 nM, and simultaneously screening for compound molecules secreted in average or median populations, the KDc was set to 36 nM. The target population of compounds of interest was as low as 1.2% (dark gray). (C) By performing assays with a LOD of 1 nM and Ag0 of 15 nM, and simultaneously screening for compound molecules secreted in average or median populations, the KDc was set to 76 nM. The target population of compounds of interest was as low as 2.5% (dark gray). Detailed Implementation

[0028] The interaction between two binding molecules (e.g., a receptor and its ligand) is characterized by two important parameters: affinity and concentration. In the context of antibodies, antibody affinity can be described as the strength of the reversible binding between the antibody (Ab) and the antigen (Ag), expressed by the following formula:

[0029] The strength of antibody-antigen binding increases with a rapid binding rate and a slow dissociation rate, and the binding rate is related to the binding rate constant (k). on or k a The dissociation rate is directly proportional to the dissociation rate constant (k). off or k d It is directly proportional to the equilibrium dissociation constant (K). D To describe it.

[0030] Determining antibody affinity using current methods such as surface plasmon resonance (SPR) or biolayer interferometry (BLI) is relatively challenging because these methods may still include the detection of some unrelated antibodies. Furthermore, current methods have several limitations. In terms of cost, many of these techniques require specialized equipment and consumables, making their setup and maintenance relatively expensive. In this context, some affinity measurements may require relatively large quantities of purified antibodies and / or antigens for the experiment. This can be a limitation when dealing with limited or precious samples, especially when antibodies or antigens are difficult to produce or purify. In terms of time consumption, some affinity measurements, such as SPR or BLI, involve multiple steps and can take several hours to complete a single experiment. This long timeframe may not be suitable for situations requiring rapid screening or evaluation of large antibody assemblies. Additionally, some methods, such as SPR and BLI, may face challenges when handling complex biological samples (such as serum or crude cell lysates) due to potential interference from other components present in the sample. Special considerations or additional steps may be needed to address these challenges.

[0031] The method disclosed herein provides a direct and reliable approach for identifying compounds of interest with specific biological functions without requiring any pretreatment, such as adjusting or calculating optimal working concentrations (e.g., dilutions). This finding offers several advantages because the method allows for affinity-based differentiation of compounds in solution using complex biological samples (e.g., cellular composition). This method can be applied to screen antibody-producing immune cells in patients with cancer and / or immunized animals, monitor antibody responses over time in patients following administration of therapeutic antibodies, determine the presence of autoantibodies in autoimmune diseases, assess the potency of monoclonal antibodies after their generation, or evaluate the clonality of antibody-producing cells.

[0032] In particular, the inventors have discovered that selecting an assay with a predetermined limit of detection (LOD) and selecting a target concentration allows for the screening of compounds of interest that can bind to the target by means of the biophysical properties of the compounds (e.g., affinity and / or internalization).

[0033] LOD stands for an important parameter characterizing the analytical performance of a bioassay. In many clinical laboratories and diagnostic applications, LOD is used interchangeably with "sensitivity," "analytical sensitivity," or "limit of detection." However, this can be confusing because the term "sensitivity" is also used in other ways. For example, in some applications, "sensitivity" refers to the slope of the calibration curve, which is the definition used by the International Union of Pure and Applied Chemistry (IUPAC).

[0034] LOD is the lowest level of analyte signal that can be statistically distinguished from a blank sample. Under certain conditions, especially to minimize false positive event detection (<0.27%), a higher LOD value can be selected and defined as follows: LOD = Average 空白 + 3 (SD 空白 ) Among them, average 空白 It is the average of the blanks, SD 空白 It is the standard deviation of the blank measurement.

[0035] The mean of the blank was determined using a zero calibrator because it is difficult to find samples completely lacking the analyte of interest. The zero calibrator was tested at least 20 times in the same run, and the mean and standard deviation of the results were calculated. LOD was considered to be above the blank mean by 3 standard deviations. Using this formula, the probability of a positive event misclassified as a compound of interest is 0.27%. Example 1 and Figure 11 show how to calculate the LOD of a droplet microfluidic assay using a standard curve. In this example, the fluorescence signal (in V, y-axis) is plotted as a function of the compound concentration (in nM, x-axis).

[0036] The linear equation f(x) = ax + b can be identified based on the following calibration curve, where f(x) corresponds to the measured signal (e.g., voltage, light emission, energy, etc.), "b" is the value of the intersection of the line and the vertical axis, "a" is the sensitivity of the system (i.e., the slope of the line, or a function relating the measured signal to the quantity to be measured), and "x" is the value of the quantity to be determined from the signal f(x) (e.g., temperature, concentration, pH, etc.). The LOD of "x" is calculated as the value of "x" when f(x) equals the blank mean "y" plus "t" times its standard deviation "s" (or, if zero, the standard deviation corresponding to the lowest measured value), where "t" is the selected confidence value (e.g., for a 99.3% confidence level, t = 3, determined by the blank limit). Therefore, the LOD of x = [f(x) - b] / a = (y + 3 s – b) / a.

[0037] In the context of this invention, the term "limit of detection" or "LOD" refers to the lowest concentration of an analyte that can be reliably measured by an analytical procedure for a given assay. Therefore, as used herein, the term "limit of detection" or "LOD" refers to the "method LOD" or "assay LOD," which does not correspond to the "instrument LOD." The instrument LOD can be obtained by analyzing the analyte in pure solvent. Therefore, the "instrument LOD" only indicates the instrument's ability to detect the analyte and can only be used to compare different instruments. As used herein, the term "threshold" refers to the critical KD "KDc" in the context of measuring the binding affinity of a compound of interest and to the "assay LOD" in the context of measuring the internalization ability of a compound of interest. To further distinguish these thresholds, the term "first threshold" refers to the critical KD "KDc" herein, and the term "second threshold" refers to the "assay LOD" herein. Therefore, "method LOD" or "assay LOD" is used to determine the internalization ability of a compound capable of binding a target, while "KDc" is used to determine the affinity of a compound capable of binding a target. The “Method LOD” also takes into account the impact of sample preparation and measurement procedures on the analytical results. The LOD can be determined using a matched matrix calibration curve method, as illustrated in Examples 1, 2, and Figure 11.

[0038] LOD may be affected by several parameters, such as Figure 12 As shown. While the assay reaction may be affected by the assay volume and reaction time, the determination of LOD is influenced by the quality, properties, and quantity / concentration of the instruments and reagents used to read the assay reaction. For example, fluorescence-based determination of LOD may be affected by the brightness and / or extinction coefficient and / or fluorescence quantum yield of the fluorescent dye. Fluorescence energy transfer determination of LOD will depend on the molar extinction coefficient, quantum yield, and / or fluorescence half-life.

[0039] It should be understood that, in some cases, the measured LOD may vary due to system performance and calibration issues / randomness. Assessing system performance and calibration reliability may be preferable by testing the system LOD using several methods and comparing the measured LOD before and after calibration.

[0040] Since compound, target, and / or reporter molecule signaling is central to the determination and notification of compound / target responses, the determination of LOD can be influenced by the quantity of such molecules. In some cases, these molecules can be expressed by cells, in which case very high and / or very low promoter expression of genes can affect the determination of LOD. The determination of LOD depends on the reporter molecule / assay reading (in terms of both reporter molecule identity and quantity).

[0041] The method of measurement can also affect the determination of LOD. Using a solid support (including but not limited to particles, beads, and slides), the amount of support, cell support, or reacting in solution can alter the LOD for the same reading. In the context of this invention, LOD is determined using a beaded measurement (see Examples 1 and 2). For exemplary phenotyping assays, see, for example, Gérard et al., Nat. Biotechnol. 2020, 38(6): 715-721; Eyer, K., Doineau, R., Castrillon, C. et al. Single-cell deep phenotyping of IgG-secreting cells for high-resolution immune monitoring. Nat Biotechnol 35, 977–982 (2017); Bounab, Y., Eyer, K., Dixneuf, S. et al. Dynamic single-cell phenotyping of immune cells using the microfluidic platform DropMap. NatProtoc 15, 2920–2955 (2020); and PCT / EP2015 / 073942, disclosed as WO2016059182. However, when it comes to the measurement of internalization capacity, the determination of LOD is made by cell-based assays (see, for example, Example 3). For example, see, for example, Gérard et al., Nat. Biotechnol. 2020, 38(6): 715-721.

[0042] In some embodiments, it may be combined with any aspect or embodiment of this disclosure, wherein the assay is a beading assay. Exemplary beading assays that can be used with the methods and systems of this disclosure are described, for example, in Gérard et al., Nat. Biotechnol. 2020, 38(6): 715-721; Eyer, K., Doineau, R., Castrillon, C. et al. Single-cell deep phenotyping of IgG-secreting cells for high-resolution immune monitoring. Nat Biotechnol 35, 977–982 (2017); Bounab, Y., Eyer, K., Dixneuf, S. et al. Dynamic single-cell phenotyping of immune cells using the microfluidic platform DropMap. Nat Protoc 15, 2920–2955 (2020); and PCT / EP2015 / 073942, disclosed as WO2016059182. In some embodiments, it may be combined with any aspect or implementation of this disclosure, wherein the assay is a cell-based assay. Exemplary cell-based assays that can be used with the methods and systems of this disclosure are described, for example, in Gérard et al., Nat. Biotechnol. 2020, 38(6): 715-721.

[0043] In the context of this invention, another parameter used for screening compounds is the critical KD (KDc). KDc depends on the target concentration (Ag0), the measured LOD, and the compound concentration (IgG0), and is defined by the following formula:

[0044] Compound concentrations can be estimated using reporter molecules. At the average / median concentration of the compound, the reporter molecule signal will cover most of the signal population. Right now The compound concentrations follow a Gaussian distribution – see [link to relevant documentation] Figure 14A )

[0045] The inventors have surprisingly discovered that by adjusting the measurement of LOD and target concentration (Ag0), specific functionalities can be enriched. Right now (Affinity), and select compounds whose "signal" is either higher or lower than KDc for the vast majority of cells.

[0046] Furthermore, for a given assay with a fixed LOD and target concentration (Ag0), compounds identified and / or classified can be ranked based on their specific functions (e.g., affinity or internalization capacity). This ranking is performed by calculating an index called “affinity magnitude” for each compound, which can be the ratio of the target signal to the compound signal (see, for example, equation (a) below). Alternatively, affinity magnitude can be calculated as the ratio of target relocation to compound relocation. In this context, for a given analyte (target or compound), “relocation” refers to the ratio of the signal at the reading location (e.g., beaded line or cell) to the background signal (see, for example, equation (b) below).

[0047] These affinity forces can therefore be calculated as follows: (a) ,or (b)

[0048] Wherein “target signal (peak value)” and “compound signal (peak value)” are the peak signals of the target or compound detected in the microreactor, respectively, and “target signal (average value outside the peak value)” and “compound signal (average value outside the peak value)” are the average signals of the target or compound measured in the microreactor (excluding the peak value).

[0049] Therefore, in some embodiments, the methods and systems disclosed herein include sorting different compounds (e.g., compounds analyzed in parallel microreactors and optionally classified in the same category) based on their binding affinity to a target. Alternatively, in some embodiments, the methods and systems disclosed herein include sorting different compounds (e.g., compounds analyzed in parallel microreactors and optionally classified in the same category) based on their internalization ability.

[0050] According to one aspect, the present invention relates to a method for identifying and classifying compounds capable of binding to a target in an assay having a limit of detection (LOD) for said compound, the method comprising the following steps: (a) Provide microreactors containing compounds and targets, (b) Incubate the compound and the target under conditions sufficient to allow the formation of a complex between the compound and the target. (c) The complex is identified by detecting signals above or below a first threshold or a second threshold, wherein: (i) If the signal is below the first threshold and the target is present at a concentration equal to or at most five times the LOD, the compound is classified as having the highest binding affinity for the target; (ii) If the signal is above the first threshold and the target is present at a concentration of at least five times the LOD, the compound is classified as having the lowest binding affinity for the target; (iii) If the signal is above the second threshold and the target is present at a concentration below the LOD, the compound is classified as exhibiting internalization ability, or (iv) If the signal is above the second threshold, and the target and / or detector and / or compound is present at a concentration below the LOD, then the compound is classified as exhibiting internalization ability. The limits of detection (LOD) for the compounds to be identified and classified range from about 0.01 nM to about 10 nM.

[0051] As used in this article, the indefinite article "a / an" can also refer to a plural number of / types. Right now "One or more" or "at least one". For example, the term "a compound" includes "one or more compounds".

[0052] As used herein, the term "compound" refers to a molecule capable of binding to a specific target. Conversely, the term "target" refers to a molecule capable of being bound by a specific compound. Thus, since both a compound and its target can be defined as binding partners, a molecule that acts as a compound can also act as a target, and vice versa.

[0053] In one implementation, the detector may be directly or indirectly coupled to / bound to the compound of interest (see Figure 8).

[0054] The compound of interest can be any receptor, and the target can be any ligand, and vice versa.

[0055] In one embodiment, the compound is selected from antibodies, ligands, and receptors.

[0056] In one implementation, the compound or target is an antibody.

[0057] As used herein, the term "antibody" refers to an immunoglobulin molecule that is immunoreactive with a specific antigen, or a protein molecule that acts as a receptor for a specific antigen (e.g., a T-cell receptor (TCR)-like antibody). Therefore, the term "antibody" includes monoclonal antibodies, bispecific antibodies, antibody-drug conjugates (ADCs), engineered antibodies, nanobodies, polyclonal antibodies, recombinant antibodies, intact antibodies (antibodies consisting of at least two heavy chains and two light chains linked by disulfide bonds), and antibody fragments, such as single-chain variable fragments (ScFv), antigen-binding fragments (Fab), and F(ab')2 and Fc fusion proteins. Intact antibodies include IgA, IgD, IgE, IgM, and IgG, the latter including subclasses IgG1, IgG2, IgG3, and IgG4. Non-restrictive examples of IgG subclasses include: human IgG subclasses: IgG1, IgG2, IgG3, and IgG4—some of which (if not all) are retained in transgenic animals; mouse IgG subclasses: IgG1, IgG2a, IgG2b, and IgG3; rat IgG subclasses: IgG1, IgG2a, IgG2b, and IgG2c; and rabbit and goat IgG.

[0058] Although the concentration information of the compounds to be identified and classified may be known, for example, from the supplier, the methods disclosed herein do not require determining the ideal concentration at which the compound binds to its target.

[0059] In one embodiment, the compound is provided in a microreactor at an undetermined concentration.

[0060] Those skilled in the art will understand that concentration depends on the amount of substance (mass or number of molecules) and the volume in which the measurement is performed. A larger volume will result in a lower concentration for a fixed number of molecules. In this embodiment, the concentration is provided in nM. In this embodiment, the reaction is measured in a volume of 80 pL.

[0061] In the context of this invention, the ligand may be, for example, a T-cell antigen (from a TCR / T-cell antigen recognition pair), a B-cell antigen (from a B-cell receptor / B-cell antigen recognition pair), a stimulatory immune checkpoint molecule (e.g., OX40 from an OX40L / OX40 pair), an inhibitory immune checkpoint molecule (e.g., PD-1 from a PD-L1 / PD-1 pair), a cytokine (from a cytokine / cytokine receptor pair), a carbohydrate (from a selectin / carbohydrate pair), an immunoglobulin superfamily member (from a pair containing two members of an immunoglobulin superfamily), a selectin (from an immunoglobulin superfamily member / selectin pair), a chemokine (from a chemokine / chemokine receptor pair), a hormone (from a hormone / hormone receptor pair), a growth factor (from a growth factor / growth factor receptor pair), a GPCR ligand (from a GPCR / corresponding ligand pair), or a substrate (from an enzyme / corresponding substrate pair). In some embodiments, the ligand set is a group of T-cell antigens (peptides, glycolipids, or small metabolites such as 5-A-RU derivatives), preferably bound to major histocompatibility complexes (MHC molecules, which may be class I, II, or MR1) or to CD1a, b, c, or d molecules.

[0062] In one embodiment, the ligand is selected from T-cell antigens, B-cell antigens, stimulatory immune checkpoint molecules, inhibitory immune checkpoint molecules, cytokines, carbohydrates, immunoglobulin superfamily members, selectins, chemokines, hormones, growth factors, GPCR ligands, or substrates.

[0063] In one implementation, the compound or target is a cell receptor.

[0064] In another implementation, the compound or target is a T-cell receptor.

[0065] In the context of this invention, the receptor may be, for example, a T-cell receptor (TCR, from a TCR / T-cell antigen recognition pair), a B-cell receptor (from a B-cell receptor / B-cell antigen recognition pair), a stimulatory immune checkpoint molecular receptor (e.g., OX40L from an OX40L / OX40 pair), an inhibitory immune checkpoint molecular receptor (e.g., PD-L1 from a PD-L1 / PD-1 pair), a cytokine receptor (from a cytokine / cytokine receptor pair), a selectin (from a selectin / carbohydrate pair), or an integrin (from an integrin / immune... Immunoglobulin superfamily member pairs), immunoglobulin superfamily members (from immunoglobulin superfamily member / selectin pairs, or from pairs containing two members of the immunoglobulin superfamily), cadherins (from pairs containing two cadherins), chemokine receptors (from chemokine / chemokine receptor pairs), hormone receptors (from hormone / hormone receptor pairs), growth factor receptors (from growth factor / growth factor receptor pairs), G protein-coupled receptors (GPCRs, from GPCR / corresponding ligand pairs), chimeric antigen receptors or enzymes (from enzyme / corresponding substrate pairs).

[0066] In one embodiment, the receptor is selected from T cell receptors, B cell receptors, stimulatory immune checkpoint molecule receptors, inhibitory immune checkpoint molecule receptors, cytokine receptors, selectins, integrins, immunoglobulin superfamily members, cadherins, chemokine receptors, hormone receptors, growth factor receptors, G protein-coupled receptors, or enzymes.

[0067] In the context of this invention, compounds and / or targets may be provided directly or indirectly to a microreactor. Direct provision of compounds and / or targets refers to the event that the compounds and / or targets are present in the microreactor at a predefined concentration. Indirect provision of compounds and / or targets refers to the situation where the compounds and / or targets are generated in the microreactor following a chemical reaction or incubation. A non-limiting example of indirect provision of compounds and / or targets is providing antibody-secreting cells that, after incubation, produce antibodies within a certain concentration range. Another example of indirect provision of compounds and / or targets is providing the compounds and / or targets in precursor form, which, after a chemical reaction, produces compounds and / or targets within a certain concentration range.

[0068] In one embodiment, the compound is provided in a microreactor at a concentration of about 0.1 nM to about 100 nM, preferably about 0.5 nM to about 50 nM.

[0069] In another embodiment, the target is provided in the microreactor at a concentration of about 0.1 nM to about 75 nM, preferably about 1 nM to about 30 nM.

[0070] In the context of this invention, the term “about” is used herein to mean “close to” or “near”. When the term “about” is used in conjunction with a particular value or range of values, it modifies that value or range by extending the boundaries above and below the stated value. As used herein, the term “about” modifies a value to a variance that is above or below 10% of the stated value, up or down (higher or lower).

[0071] As used herein, the term "assay" refers to an assay for the formation and / or detection of a compound-target complex. Such assays include, but are not limited to, Western blotting, immunoprecipitation, immunofluorescence, immunocytochemistry, immunohistochemistry, fluorescence-activated cell sorting (FACS), fluorescence in situ hybridization (FISH), immunomagnetic assays, ELISA, ELISPOT, agglutination assays, aggregation assays, solubility assays, sandwich immunoassays, cell-based assays, cell-cell interaction assays, flocculation assays, cell panning, etc. Suitable assays for carrying out the methods according to the claimed invention are described below.

[0072] As used herein, the term “cell” refers to eukaryotic and / or prokaryotic cells, primary cells, engineered cells, and / or synthetic cells.

[0073] LOD can vary depending on the assay chosen, due to factors such as the selected fluorophore, particle / cell size, and detection method. Therefore, the LOD for an assay used to detect targets bound to a solid support may not correspond to the LOD for an assay used to detect the target exposed on a single cell surface.

[0074] Typically, the Level of Detail (LOD) for many assays is determined using a series of blank measurements to describe measurement error. All assays involve numerous possible sources of reproducibility and / or measurement error. For antibody-based assays, pipetting errors, incomplete mixing of samples and / or reagents, contamination, nonspecific binding, and randomized instrumentation processes can lead to inconsistencies and inaccuracies. Possible sources of test variability and / or measurement error include sample preparation, contamination, and randomized instrumentation processes. "Instrument LOD" includes contamination from reagents but not from sample preparation. "Method LOD" includes contamination from both reagent and sample preparation processes.

[0075] The LOD range for the determination used to carry out the method according to the invention is 0.01 nM to 10 nM. Preferably, it is 0.1 nM to 8 nM. More preferably, it is 0.1 nM to 5 nM. Ideally, the determination has an LOD of 1 nM.

[0076] The KDc range used for the determination according to the method of the invention is from 0.001 nM to 1000 nM. Preferably, it is from 0.01 nM to 100 nM. More preferably, it is from 0.1 nM to 10 nM. Ideally, the determination has a KDc of 5 nM.

[0077] The binding affinity of ligands to GPCRs typically falls in the nanomolar (nM) to micromolar (µM) range. Some small molecule drugs may have binding affinities in the low picomolar (pM) range. Enzyme-linked receptors (including receptor tyrosine kinases) typically have binding affinities for their ligands in the nanomolar (nM) to picomolar (pM) range. Nuclear receptors, responding to small lipophilic hormones, typically have binding affinities in the nanomolar (nM) range. The binding affinity of ligands (including neurotransmitters and drugs) to ion channels varies considerably, ranging from micromolar (µM) to picomolar (pM) concentrations.

[0078] In the context of this invention, when the compound is an antibody, the expression "high affinity" refers to having K... D Antibodies with a molecular weight of < 10 nM, preferably < 1 nM, are preferred. When the compound is a TCR, the expression "high affinity" refers to having K+. D < 10 µM, ideally below 1 µM TCR-pMCH. When the compound is a ligand-GPCR, the expression "high affinity" refers to having K0... D Ligand-GPCR with a molecular weight of < 5 nM, ideally < 1 nM.

[0079] In the context of this invention, when the compound is an antibody, the expression "moderate affinity" means having 50 nM > K. D Antibodies with >10 nM. When the compound is a TCR, "intermediate affinity" means having >100 µM K. D >10 µM TCR-pMCH. When the compound is a ligand-GPCR, "moderate affinity" means having 50 nM > K D >5 nM ligand-GPCR.

[0080] In the context of this invention, when the compound is an antibody, the expression "low affinity" refers to having K0... D Antibodies with a molecular weight of >50 nM. When the compound is a TCR, the expression "low affinity" refers to having K+. D >100 µM TCR-pMCH. When the compound is a ligand-GPCR, the expression "low affinity" refers to having K D >50 nM ligand-GPCR.

[0081] In the context of this invention, the target may be expressed or displayed on the surface of cells (or multiple cells), synthetic cells, beads and / or engineered APC-like beads (as disclosed by Neal et al., J. Immunol. Res. Ther. 2017, 2: 68-79), or encoded in vitro as disclosed by Grubaugh et al. (Grubaugh et al., Vaccine 2013, 31: 3805-3810).

[0082] In one implementation, the target is either bound to a solid support or exposed on the surface of a single cell.

[0083] As used herein, the term "solid support" refers to any non-biological matrix (e.g., magnetic beads, gel matrix / beads, or affinity matrix / beads) that has a given specificity for target molecules, such that the target molecules can be immobilized on the support, which allows for the separation of target molecules from the contents contained in the microreactor.

[0084] In another embodiment, the target is a macromolecule selected from proteins, peptides, carbohydrates, lipids, and nucleic acids.

[0085] In the context of this invention, the term "microreactor" refers to a container for carrying out chemical or physical processes within a series of fluidly connected modules. A microreactor is characterized by at least one cross-sectional dimension, such as depth, width, length, diameter, etc., ranging from about 0.001 nanometers to about 15 x 10⁻⁶ nanometers. 6 Nanoscale. Examples of fluid-connected modules include chambers, channels, reservoirs, etc. A non-limiting example of a microreactor is a microfluidic droplet within a microfluidic device.

[0086] In one embodiment, the microreactor is selected from droplets, pores, chambers, and tubes.

[0087] As used herein, the term "droplet" refers to an isolated portion of a fluid that is immiscible with its surrounding environment. In the context of this invention, a "droplet" can be spherical, substantially spherical, or non-spherical. The shape may depend on various parameters, such as the external environment.

[0088] As used herein, the terms “orifice,” “chamber,” and “tube” refer to any structure capable of containing a volume of fluid. These structures can have different volumetric dimensions (picoli, nanoliters, microliters, milliliters).

[0089] In another embodiment, the volume of the microreactor ranges from approximately 10. -6 From µL to about 2000 µL, preferably from about 0.000001 µL to about 1 µL, more preferably from about 0.000001 µL to about 0.01 µL.

[0090] In the context of this invention, the incubation step is intended to allow the compound to be produced in a detectable amount and to allow the compound to bind to its target, thereby forming a complex.

[0091] Incubation conditions can vary depending on the assay being performed or the concentration of the compound and / or target being studied.

[0092] When assaying antibodies, the incubation step is an important aspect because it allows the antibody to bind to its antigen. Appropriate incubation conditions must be carefully determined for each assay. Excessively high antibody concentrations and excessively long incubation times may result in nonspecific background signals. Conversely, excessively low concentrations and excessively short incubation times may result in very weak or absent signals. Increasing the incubation time can increase the time it takes for the antibody to find and bind to its antigen, which is important when using low-affinity antibodies or when antibody accessibility to the antigen is limited.

[0093] In the context of this invention, the incubation parameters are suitable for the efficient detection of proteins of interest on the surface and inside the cell (e.g., intracellular cytokines, transcription factors).

[0094] Typical incubation temperatures for eukaryotic cells and compounds range from 4°C to 37°C ± 2°C, while those for prokaryotic / viral / archaic cells and compounds range from 4°C to > 95°C. Typical incubation times range from milliseconds (for kinetic assays) to over 24 hours (for assays of cell-cell interaction-mediated regulation of compound production).

[0095] In one embodiment, the incubation step is carried out in at least one microreactor for about 1 to about 24 hours.

[0096] In the context of this invention, the identification step allows for the detection of the formation of a complex between a compound and its target.

[0097] In the context of this invention, a detection signal above a threshold may originate from the direct detection of a complex between at least one compound and at least one target, or as a result of indirect detection of the complex (e.g., secondary antibody detection agent, indirect coupling, or secretion of inducing molecules, intracellular signal transduction, etc.).

[0098] In one implementation, the identification step is performed via fluorescence reading, luminescence reading, or phosphorescence reading. The signal is emitted as a result of a chemical or enzymatic reaction.

[0099] As used herein, the term “fluorescence reading” includes, but is not limited to, direct fluorescence and indirect fluorescence (e.g., FRAP, FLIP, FRET, BRET, FLIM, PRIM).

[0100] In one embodiment, the method disclosed herein further includes ranking the compounds based on their classification and comparison with one or more additional compounds, and / or based on their binding affinity and / or internalization ability to the target compared with the binding affinity and / or internalization ability of one or more additional compounds to the target.

[0101] In one embodiment, the binding affinity and / or internalization ability of the one or more additional compounds to the target are analyzed in a parallel microreactor according to the methods disclosed herein, optionally wherein the one or more additional compounds are classified in the same category as the compound.

[0102] In one embodiment, the method disclosed herein further includes calculating an affinity magnitude, based on

[0103] (a) ,or

[0104] (b)

[0105] Wherein “target signal (peak)” and “compound signal (peak)” are the peak signals of the target and compound detected in the microreactor, respectively, and “target signal (average outside peak)” and “compound signal (average outside peak)” are the average signals of the target and compound measured in the microreactor, respectively, with the corresponding peak signals optionally excluded.

[0106] As used in this article, the term "mean" refers to any mathematical measure of central tendency. Means include the arithmetic mean, median, or mode.

[0107] In one embodiment, a higher affinity level corresponds to a higher affinity and / or a lower KD; the affinity level is related to the compound's affinity for the target and is negatively correlated with KD; the target signal is obtained by direct or indirect target signal detection; and / or the compound signal is obtained by direct or indirect compound signal detection.

[0108] In one implementation, the sorting is performed using an affinity magnitude calculated according to either equation (a) or equation (b): (a) ,or (b)

[0109] Wherein, “target signal (peak)” and “compound signal (peak)” are the peak signals of the target and compound detected in the microreactor, respectively, and “target signal (off-peak average)” and “compound signal (off-peak average)” are the average signals of the target and compound measured in the microreactor, respectively, with the corresponding peak signals optionally excluded. In one embodiment, a higher affinity level corresponds to a higher affinity and / or a lower KD; the affinity level is related to the compound’s affinity for the target and is negatively correlated with KD; the target signal is obtained by direct or indirect target signal detection; and / or the compound signal is obtained by direct or indirect compound signal detection.

[0110] In another embodiment, the sorting is performed using an affinity factor calculated as follows: - Higher affinity corresponds to higher affinity and / or lower KD; - The affinity level is related to the compound's affinity for the target and is negatively correlated with KD; - The target signal is obtained through direct or indirect target signal detection; and / or - The compound signal is obtained through direct or indirect compound signal detection.

[0111] In one embodiment, the detection agent is a reagent that directly or indirectly binds to the compound.

[0112] In one implementation, the detection agent is coupled to the reporter molecule.

[0113] In one implementation, when the target concentration is at most 5 nM and the signal is below a first threshold, the compound with the highest binding affinity to the target is enriched.

[0114] In another embodiment, when the target concentration is as high as 15 nM and the signal is above a first threshold, the compound with the lowest binding affinity to the target is enriched.

[0115] In the context of this invention, the selection of a determination having 1 nM LOD, and the provision of targets with concentrations ranging from about 5 nM to about 15 nM and compounds with concentrations ranging from about 1 nM to about 10 nM, enable the methods disclosed herein to enrich compounds having specific biological functions.

[0116] In one embodiment, if the measured LOD is about 1 nM, and the compound is present at a concentration of about 1 nM to about 5 nM, and

[0117] i. If the target is present at a concentration of about 5 nM, the detected complex is enriched with the highest binding affinity for the target; ii. If the target is present at a concentration of approximately 15 nM, the detected complex is enriched with the lowest binding affinity for the target.

[0118] In one embodiment, if the measured LOD is about 1 nM, the signaling molecule is present at a concentration below the LOD, and the target is present at any concentration, then the compound is classified as exhibiting internalization ability when a signal above a second threshold is detected.

[0119] In one embodiment, if the measured LOD is about 1 nM, the compound is present at a concentration below the LOD, and the target is present at any concentration, then the compound is classified as exhibiting internalization ability when a signal above a second threshold is detected.

[0120] According to another aspect, the present invention relates to a system for screening compounds in an assay having a predetermined limit of detection (LOD), the system comprising: (a) The first module, which is configured to collect compounds and targets; (b) A second module configured to incubate the compound and the target; (c) A third module configured to detect the interaction between the compound and the target; (d) The fourth module, which is configured to screen the compounds.

[0121] In the context of this invention, the first module is directly or indirectly fluidly connected to the second module, the second module is directly or indirectly fluidly connected to the third module, and the third module is directly or indirectly fluidly connected to the fourth module. The LOD associated with the first and second modules refers to "method LOD." The LOD associated with the third and fourth modules refers to "instrument LOD." The third module is configured to detect signals above or below a threshold. Right now The threshold refers to the formation of a complex between at least one compound and at least one target, or the absence of said complex. The threshold may refer to a first threshold or a second threshold. Depending on the nature of the signal to be detected, other suitable modules may be selected accordingly. For example, a fluorometer may be used to measure spectral fluorescence parameters (300 – 1000 nm).

[0122] The implementation methods and definitions disclosed herein also apply to the systems disclosed herein.

[0123] Example

[0124] Example 1

[0125] LOD was determined using a beaded thread method.

[0126] To determine the LOD of a given system, measurements are performed under conditions simulating the actual measurement. In the case of beadwork measurements, the same reagents should be used. Right now The setup includes paramagnetic beads, assay buffer, and reporter molecules, similar to the conditions used for the corresponding screening assays. An example of a beaded assay is shown here, where the molecule of interest is detected using the DL550 dye. Other assay methods and dyes can be used. The paramagnetic beads are coupled to streptavidin, and the reporter molecule (DL550) is indirectly attached to the beads via biotinylated oligonucleotides, such that a single DL550 molecule binds to a single oligonucleotide. The number of biotinylate sites per bead per container exceeds the number of biotinylated oligonucleotides incubated / used for LOD.

[0127] To determine the minimum sensitivity of the assay, different molecular / dye conditions were tested for each bead: 1 – 0 nM Oligonucleotide DL550 2 – 1 nM oligonucleotide DL550 3-5 nM oligonucleotide DL550 4 – 25 nM Oligonucleotide DL550 5 – 125 nM Oligonucleotide DL550 The assay buffer solution was prepared as follows (final concentration):

[0128] The paramagnetic bead mixture was prepared as follows:

[0129] The beads were pretreated as described by Gérard et al., Nat. Biotechnol. 2020, 38(6): 715-721. In short: Take 33 µL of beads for every 100 µL of Coflow 1 solution.

[0130] Place the beads on a magnet (Thermo MagJet rack 12x1.5) for 1 minute, then resuspend them in PBS at 33%.

[0131] Wash with PBS and place on a magnet for 1 minute.

[0132] Discard the supernatant and repeat the PBS wash.

[0133] The beads were resuspended in 10% pluronic F127 (20% beads).

[0134] Ultrasound treatment on ice for 15 minutes (Ultrasound machine VWR USC-TH).

[0135] Place on a magnet for 4 minutes and resuspend in PBS.

[0136] Dispense into five 400 µL tubes.

[0137] The oligonucleotide solution was prepared as a 100X solution, with 4 µL of labeled oligonucleotide added to each 400 µL bead. Then, 5-fold serial dilutions were performed to achieve the desired final concentration.

[0138] Add the oligonucleotides to the beads and mix immediately.

[0139] Incubate at room temperature for 30 minutes.

[0140] Place on a magnet for 1 minute and discard the supernatant. Resuspend each 100 µL mixture in 200 µL of PBS.

[0141] Incubate at room temperature for 5 minutes

[0142] Repeated PBS washing

[0143] The beads were resuspended in the assay buffer at 33%.

[0144] 80 pL droplets were generated using an oil-in-water emulsion droplet generator known in the art. The five different conditions were distinguished from each other by using different concentrations of another fluorescent dye (Dy754 in this case).

[0145] The prepared emulsions were analyzed on a droplet reader, as described by Gérard et al., Nat. Biotechnol. 2020, 38(6): 715-721. Gating was defined for all five distinct populations, and data for each individual frequency were recorded.

[0146] Figure 11 shows the signal intensity and LOD calculation for each concentration of the reporter dye.

[0147] Example 2

[0148] LOD was determined using a beaded thread method.

[0149] Another possibility for determining the LOD in a given system is to further perform the determination under conditions simulating the actual determination. In the case of bead determination, the same reagents should be used ( Right nowThe assay comprises paramagnetic beads, an assay buffer, the compound of interest, a trapping agent, and an assay reagent coupled to a reporter molecule, under conditions similar to those used for the corresponding screening assay. An example of a bead assay is shown here, where the compound of interest is detected using the DL550 dye. Other assay methods and dyes can be used. The paramagnetic beads are coupled to streptavidin, the trapping agent is indirectly coupled to the paramagnetic beads, and the compound of interest is indirectly detected by an assay reagent bound to it and coupled to the reporter molecule (DL550).

[0150] To determine the minimum sensitivity of the assay in the presence of the compound of interest in the simulated container, different compound of interest conditions were tested for each bead: 1 – 0 nM Compounds of interest 2 – 0.2 nM Compounds of interest 3 – 1 nM compounds of interest Compounds of interest at 4–5 nM The compound of interest, in this embodiment, is a human antibody (IgG), diluted at a specified concentration in an assay buffer containing 25 nM of a DL550-goat anti-human IgG assay agent conjugated with DL550.

[0151] The assay buffer solution was prepared as follows (final concentration):

[0152] The paramagnetic bead mixture was prepared as follows:

[0153] The beads were pretreated as described by Gérard et al., Nat. Biotechnol. 2020, 38(6): 715-721. In short: Take 33 µL of beads for every 100 µL of Coflow 1 solution.

[0154] Place the beads on a magnet (Thermo MagJet rack 12x1.5) for 1 minute, then resuspend them in PBS at 33%.

[0155] Wash with PBS and place on a magnet for 1 minute.

[0156] Discard the supernatant and repeat the PBS wash.

[0157] The beads were resuspended in 10% pluronic F127 (20% beads).

[0158] Ultrasound treatment on ice for 15 minutes (Ultrasound machine VWR USC-TH).

[0159] Place on a magnet for 4 minutes and resuspend in PBS.

[0160] Dispense into five 400 µL tubes.

[0161] Dilute the beads with PBS

[0162] Add the capture antibody to the bead tube.

[0163] Incubate at room temperature for 30 minutes.

[0164] Place on the magnet for 1 minute and discard the supernatant. Wash with assay buffer.

[0165] Incubate at room temperature for 5 minutes

[0166] Repeated PBS washing

[0167] The beads were resuspended in the assay buffer at 33%.

[0168] Four different concentrations of fluorescent dye (Dy754 in this case) were added, each concentration corresponding to a concentration condition of a compound of interest.

[0169] Streptavidin beads resuspended in assay buffer and a primary antibody (i.e., the compound of interest) / detection antibody solution in assay buffer were co-fluidized on a microfluidic chip, and 80 pL droplets were generated using an oil-in-water emulsion droplet generator well-known in the art. The four different conditions were distinguished from each other by using different concentrations of another fluorescent dye (Dy754 in this case).

[0170] The prepared emulsions were analyzed on a droplet reader, as described by Gérard et al., Nat. Biotechnol. 2020, 38(6): 715-721. Gating was defined for all four distinct populations, and all data were recorded at different re-injection frequencies.

[0171] Similar results to those in Figure 11 were obtained and can be used for further determination of LOD calculation.

[0172] Example 3

[0173] In cell-based assays, LOD is defined using particles or cells.

[0174] To determine the LOD of a given system, the assay is performed under conditions simulating the real assay. In the case of cell-based assays, the same reagents should be used. Right nowThe assay (including reporter cells, assay buffer, and reporter molecule) is similar to the conditions used for the corresponding screening assay. An example of a cell-based assay is shown here, where the molecule of interest is detected using the DL550 dye. Other assay methods and dyes can be used. Reporter cells expressing the target of interest bind the reporter molecule (DL550) to compounds of interest that indirectly bind the target. The amount of the target expressed on the surface of the reporter cell may vary from one reporter cell-based assay to another. In the latter example, the expression variability is preferably no more than 10-fold.

[0175] To evaluate the determination of LOD while ensuring specificity, different conditions were run: Condition 1: Reporter cells are present in the presence of the detection molecule but not in the presence of the compound of interest. Condition 2: The presence of a reporter cell with the detection molecule and 1 nM of the compound of interest. Condition 3: The presence of a reporter cell with the detection molecule and 10 nM of the compound of interest. The assay was performed in the following assay buffer.

[0176] The assay buffer was filtered using a syringe and a 0.45µm hydrophobic filter (Cil, ref SF13PT45C).

[0177] The cells were prepared according to the following protocol: Counting reporter cells, Centrifuge for 5 minutes at 400g, then resuspend the cells at a density of 1 million cells per milliliter in warm PBS (phosphate-buffered saline) + CTV (Cell Trace Violet, 1:4000). Incubate at 37°C for 20 minutes. Add 5 times the volume of warm RPMIc (complete RPMI medium) and incubate at 37°C for 5 minutes. Wash twice in cold PBS Cells were filtered through a cell filter (70 µm). Count the cells again. Preparation of reagent solution #1: assay buffer + detection molecule at a final concentration of 12.5 nM (in droplets). Centrifuge and resuspend the cells in reagent solution #1 at 7.5 million cells per milliliter.

[0178] Preparation of primary antibodies containing reporter cells ( Right now A solution of the compound of interest containing 0 nM, 1 nM or 10 nM.

[0179] To distinguish the different conditions used, the reporter dye called 'Dropcode DY754' (Dyomics-754-00, reference number E10-10032) was added together with the primary antibody solution at a final concentration of 0.2 µM, 0.6 µM, or 1.8 µM in the droplet.

[0180] According to Gérard et al., Nat. Biotechnol. 2020, 38(6):715-721, 80 pL droplets containing cells and reagents were generated.

[0181] Collect droplets and incubate at 4°C for 1 hour to allow the primary antibody and detection molecule complex to bind to the reporter cells.

[0182] The specific binding of the primary antibody to the target cell was monitored and detected by detecting the translocation of the molecule to the target cell when the primary antibody was present. Droplet fluorescence analysis was performed on a droplet scanner, as described by Gérard et al., Nat. Biotechnol. 2020, 38(6): 715-721, and on a static droplet analyzer, DropMap, as described by Eyer et al., Nat. Biotech 2017, 35(10): 977-982. More than 10,000 droplets were analyzed under each condition.

[0183] The LOD is determined by the concentration of the primary antibody at which the translocation of the primary antibody to the reporter cell is equivalent to the signal observed in the absence of the primary antibody.

[0184] Typical results are shown in Figure 13. The results indicate that the LOD of measurements using imaging readouts is approximately 1 nM, while the LOD of measurements using a droplet analyzer is approximately 0.7 nM.

[0185] Example 4

[0186] Running high affinity antibody assay

[0187] 1 - PBMC Thawing

[0188] Thaw the PBMC vials in 50 mL of pre-warmed complete culture medium, then centrifuge the cells at 400 g for 5 minutes at 4°C. Resuspend the cells in 10 mL of warm PBS and count the cells.

[0189] 2 - PBMC CTV staining

[0190] Resuspend cells at 1 million cells / mL in warm PBS + CTV (1:4000) and incubate at 37°C for 20 min. Add 5 volumes of warm RPMIc (minimum) and incubate at 37°C for 5 min. Centrifuge cells and wash with warm RPMIc, then wash twice with ice-cold PBS at 4°C and filter through a cell filter (70 µm). Count the cells.

[0191] 3 - Optionally, PBMCs can be further purified into B cells, memory B cells, and memory B cells can be further activated.

[0192] 4 - Perform bead thread measurement

[0193] 4-1 - Preparation of assay buffer

[0194] The assay buffer was filtered using a syringe and a 0.45 µm hydrophobic filter (Cil, ref SF13PT45C).

[0195] 4-2 - Preparation of magnetic beads (=coflow#1)

[0196] - Vortex bead storage liquid 10 seconds.

[0197] - Take 33 µL of beads for every 100 µL of coflow1 solution.

[0198] - Place on a magnet (Thermo MagJet rack 12x1.5) for 1 minute, then resuspend the beads in PBS at 33% (100 µL per 100 µL of Coflow #1 solution).

[0199] Wash with PBS (without incubation) and place on a magnet for 1 minute. Discard the supernatant and repeat the PBS wash.

[0200] - Resuspend the beads in 10% pluronic F127 (20% beads).

[0201] - Ultrasonic treatment on ice for 15 minutes (Ultrasonic device VWR USC-TH).

[0202] Place on a magnet for 3-4 minutes and resuspend in PBS (16.5% beads, 200µL per 100µL Coflow #1 solution).

[0203] Add the capture antibody to the bead and mix immediately. Incubate at room temperature for 30 minutes.

[0204] - Place on the magnet for 1 minute, then discard the supernatant.

[0205] - Wash with assay buffer for 5 minutes (use 200 µL per 100 µL of Coflow #1 solution). Place on a magnet for 2 minutes. Discard the supernatant. Repeat the assay buffer wash.

[0206] - Resuspend the beads at 33% in reagent solution #1 (assay buffer + detection antibody + antigen).

[0207] 4-3 - Preparation of cells / antibodies (=Coflow#2)

[0208] - Resuspend the CTV-stained cells in assay buffer at 7.5 million cells per milliliter.

[0209] - Add DY754 to handle dropcode.

[0210] 4-4 - Droplet Formation

[0211] - Generate 80 pL droplets, similar to Gérard et al., Nature Biotechnology 2020, 38(6): 715-721.

[0212] - The collection tube (2 mL tube) is kept on the circular magnet and placed on ice throughout the droplet generation, incubation and droplet re-injection process.

[0213] 4-5 - Cellular secretion

[0214] To allow cells to secrete antibodies to the desired concentration, and given that they are expected to secrete an average of 5 nM (median 2 nM) of antibody per hour in an 80 pL droplet, the collection tube was kept at 37°C for 90 minutes.

[0215] 4-6 - Signal Analysis

[0216] Inject droplets into the droplet analyzer (at 200 Hz, 1 kHz, and 2 kHz) and / or into the imaging chamber.

[0217] For at least the antigen fluorescent molecule, high-affinity antibodies have a detectable signal above the threshold.

[0218] Example 5

[0219] In-process / low affinity antibody assay

[0220] Similar to Example 4, except that the mixing of the assay buffer solution (step 4-1) is as follows:

[0221] For at least the antigen fluorescent molecule, medium / low affinity antibodies have a detectable signal above the threshold.

[0222] Example 6

[0223] Run internalized antibody assay

[0224] Similar to Example 1, where LOD determination is performed, two primary antibodies (one with the property of internalization in reporter cells and the other without) are used at different concentrations (ranging from LOD to above LOD). To identify the internalizing antibody, either the concentration of the detection molecule is calibrated to show an undetectable signal when using the non-internalizing antibody and a detectable signal when using the internalizing antibody, or the target expressed by the reporter cells is below the determination LOD, which is only detectable when the antibody is internalized.

[0225] Example 7

[0226] Screening ligand and receptor libraries

[0227] Similar to Example 3, two cell types are encapsulated in 80 pL droplets: one generates a ligand library, and the other generates a receptor library. Alternatively, a ligand can be screened for the receptor library. Conversely, a receptor type can be screened for the ligand library.

[0228] Receptors, receptor libraries, ligands and / or ligand libraries can also exist as cell-free molecules, for example, in solution, on particles, on particle aggregates, etc.

[0229] The interaction between ligands and receptors can be monitored directly or indirectly by measuring signal transduction molecules as readouts (secreted molecules, fluorescent molecules, expression, internalization, and activation of downstream signal transduction pathways).

[0230] The detection of interactions can be based on determining the LOD, which can be modulated to detect only desired events with selected binding affinity. In this embodiment, the LOD determination is a detection LOD for secreted molecules, fluorescent molecule expression, internalization, or activation of downstream signaling pathways, which can be performed directly or indirectly.

[0231] To select the desired LOD for measurement, one can consider (1) brighter fluorescent dyes, (2) very high and / or very low promoter expression genes, and / or (3) high or low abundance of signal transduction fluorescent molecules.

[0232] Example 8

[0233] Select the assay method based on the desired antibody function.

[0234] Use and Figure 15Under similar experimental conditions set, antibodies with specific functions can be selectively enriched. It is estimated that when screening one million enriched B cells, with similar secretion rates and assay conditions to those of Eyer et al., Nature 2017 ( Right now , primary B cells from spleen or bone marrow), when the critical KD (KDc) is about 10 nM (upper part of the table) and about 20 nM (lower part of the table), antibodies with KD < KDc can be specifically enriched by selecting the antigen concentration (Ag0) and the assay with a selected LOD.

[0235] In short, the assay conditions are as described in Example 4, in particular the composition of the assay buffer, bead preparation, droplet generation and incubation, and reading of the droplet fluorescence signal.

[0236] Example 9

[0237] Selection of Antibodies with Specific Functions

[0238] Using similar experimental conditions set forth in Figure 16, antibodies with specific functions can be selectively enriched. In this example, the secretion rate of primary B cells isolated from immunized animals is about 5 nM (after 1 hour of incubation in 80 pL droplets). When cells are prepared as described in Eyer et al., Nature Biotech 2017 or Gérard et al., Nature Biotech 2020, and droplets are prepared as described in Example 4, it is conceivable to simultaneously detect the antigen bound by secreted antibodies and the amount of secreted molecules by using antibody detection molecules. Under average secretion, the vast majority of cells will show similar antibody detection molecule signal intensities. Based on this information, and working under assay conditions with the required LOD (1 nM herein) and antigen concentration (Ag0), the critical KD (KDc) detected under average secretion is identified (as shown in Figure 16). Under these conditions, antibodies with affinity lower than KDc can be specifically enriched based on the assay LOD, antigen concentration Ag0, and a secretion signal at or below the average cell secretion (without knowing its exact value).

[0239] Example 10

[0240] Ranking of Antibodies Based on Specific Functions

[0241] Using similar experimental conditions as set in Figure 16, and after selecting antibodies with affinity below KDc as described in Example 9, the selected antibodies can be ranked based on their respective affinities. The affinity measure is calculated as the ratio of the target signal to the compound signal. Alternatively, the affinity measure can be calculated as the ratio of target translocation to compound translocation, where "translocation" here refers to the ratio of the peak signal (at the reading position, such as a bead or cell) to the average signal over the remainder of the 80 pL droplet. Affinity scores are used to rank the antibodies, with higher metrics corresponding to better affinity for the target, equivalent to a lower KD. This metric does not provide information on the exact KD value for each antibody, but based on Example 9, it is expected that all selected antibodies have an affinity below KDc, and the lowest-ranked antibody among the selected antibodies likely has an affinity closest to KDc. In conjunction with the selection of antibodies with lower affinity than KDc as described in Example 9, affinity levels are used to further enrich antibodies that have better binding to the target (higher measure), lower binding to the target (lower measure, affinity close to KDc), or intermediate binding to the target (intermediate measure), depending on the desired antibody function.

[0242] With further calibration of the system using calibration curves and standards (e.g., primary antibodies with known affinity for the target), the KD of the individual selected antibody can be estimated using the method described above.

Claims

1. A method for identifying and classifying compounds capable of binding to a target in an assay, said assay having a limit of detection (LOD) for said compound, said method comprising the steps of: (a) Provide microreactors containing compounds and targets, (b) Incubate the compound and the target under conditions sufficient to allow the formation of a complex between the compound and the target. (c) The complex is identified by detecting signals above or below a first threshold or a second threshold, wherein: (i) If the signal is below the first threshold and the target is present at a concentration equal to or at most five times the LOD, the compound is classified as having the highest binding affinity for the target; (ii) If the signal is above the first threshold and the target is present at a concentration of at least five times the LOD, the compound is classified as having the lowest binding affinity for the target; (iii) If the signal is above the second threshold and the target is present at a concentration below the LOD, the compound is classified as exhibiting internalization ability, or (iv) If the signal is above the second threshold, and the target and / or detector and / or compound is present at a concentration below the LOD, then the compound is classified as exhibiting internalization ability. The limits of detection (LOD) for the compounds to be identified and classified range from about 0.01 nM to about 10 nM.

2. The method of claim 1, wherein the compound is provided at an undetermined concentration.

3. The method according to any one of claims 1 and 2, wherein the target is provided in the microreactor at a concentration of about 0.1 nM to about 75 nM.

4. The method according to any one of claims 1 to 3, wherein the compound is provided in the microreactor at a concentration of about 0.1 nM to about 100 nM.

5. The method according to any one of claims 1 to 4, wherein the target is bound to a solid support or exposed on a single cell surface.

6. The method according to any one of claims 1 to 5, wherein the target is a macromolecule selected from the group consisting of proteins, peptides, carbohydrates, lipids and nucleic acids.

7. The method according to any one of claims 1 to 6, wherein the compound is selected from antibodies, ligands, and receptors.

8. The method of claim 7, wherein the ligand is selected from T cell antigens, B cell antigens, stimulatory immune checkpoint molecules, inhibitory immune checkpoint molecules, cytokines, carbohydrates, members of the immunoglobulin superfamily, selectins, chemokines, hormones, growth factors, GPCR ligands or substrates.

9. The method according to claim 7, wherein the receptor is selected from T cell receptors, B cell receptors, receptors for stimulatory immune checkpoint molecules, receptors for inhibitory immune checkpoint molecules, cytokine receptors, selectins, integrins, members of the immunoglobulin superfamily, cadherins, chemokine receptors, hormone receptors, growth factor receptors, G protein-coupled receptors, or enzymes.

10. The method according to any one of claims 1 to 9, wherein the microreactor is selected from droplets, pores, chambers, and tubes.

11. The method according to any one of claims 1 to 10, wherein the volume of the microreactor is in the range of 10. -6 µL to 2000 µL.

12. The method according to any one of claims 1 to 11, wherein the incubation step is carried out in the microreactor for about 1 to 24 hours.

13. The method according to any one of claims 1 to 12, wherein the identification step is performed by fluorescence reading, luminescence reading or phosphorescence reading.

14. The method according to any one of claims 1 to 13, further comprising classifying the compound based on a comparison with one or more additional compounds, and / or ranking the compound based on a comparison of the binding affinity and / or internalization capacity of the compound to the target with the binding affinity and / or internalization capacity of one or more additional compounds to the target.

15. The method according to claim 14, wherein, The method according to any one of claims 1 to 13 analyzes the binding affinity and / or internalization capacity of the one or more additional compounds to the target in a parallel microreactor, optionally wherein the one or more additional compounds are classified in the same category as the compound.

16. The method according to any one of claims 1 to 15, further comprising calculating the affinity amount according to: (a) ,or (b) Wherein, "target signal (peak value)" and "compound signal (peak value)" are the peak signals of the target and the compound detected in the microreactor, respectively, and "target signal (average value outside the peak value)" and "compound signal (average value outside the peak value)" are the average signals of the target and the compound measured in the microreactor, respectively, with the corresponding peak signals optionally excluded.

17. The method of claim 16, wherein: Higher affinity corresponds to higher affinity and / or lower KD; The affinity level is related to the compound's affinity for the target and is negatively correlated with the KD. The target signal is obtained through direct or indirect target signal detection; and / or The compound signal is obtained through direct or indirect compound signal detection.

18. The method of claim 14 or claim 15, wherein the sorting is performed using an affinity measure calculated according to claim 16 or claim 17.

19. A system for screening compounds in an assay having a predetermined limit of detection (LOD), the system comprising: (a) The first module, which is configured to collect compounds and targets; (b) A second module configured to incubate the compound and the target; (c) A third module configured to detect the interaction between the compound and the target; (d) The fourth module, which is configured to screen the compounds.

20. The method according to any one of claims 1 to 18, wherein the detection agent is a reagent that directly or indirectly binds the compound.

21. The method according to any one of claims 1 to 18 and 20, wherein the detection agent is coupled to the reporter molecule.

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