Multiplex screening with microcapillary arrays

Abstract

System and methods are described for multiplex screening of variant proteins via microcapillary arrays. Systems may include first and second surfaces having target molecules for exposure to the variant protein. The variant protein can associate with the immobilized target molecules at different affinities. The first and second surfaces can be optically distinguishable. A reporter element can associate with the variant protein to show location of the variant protein. The surface density of target molecules can be tuned to screen for high affinity / avidity variant proteins.

Description

OMAB2.014WO PATENTMULTIPLEX SCREENING WITH MICROCAPILLARY ARRAYSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U. S. Provisional Application No.63 / 747,768, filed January 21, 2025, and U. S. Provisional Application No. 63 / 792,806, filed April 22, 2025, the content of each of which is incorporated by reference in its entirety.BACKGROUNDField

[0002] The present disclosure relates to high-throughput biological screening and related devices.Description of the Related Art

[0003] The analysis of biological samples, including the identification, characterization, and re-engineering of proteins, nucleic acids, carbohydrates, and other important biomolecules, has benefited greatly from the scaling up of sample numbers and the scaling down of sample sizes. For example, the two-dimensional microarrays of biological materials, such as DNA microarrays, have enabled the development of high-throughput screening methods involving multiplexed approaches for processing samples and detecting results.

[0004] The above approaches have, in some cases, benefited from their combination with optical sensing technology to identify specimens of interest using fluorescent or other corresponding specific and sensitive labeling approaches.

[0005] While such techniques provide analytical information about a particular sample, for example the presence and potentially the amount of a particular biomolecule in a solution or the sequence of a particular nucleic acid or polypeptide, they typically do not allow for the recovery of a biological sample identified by the assay without inactivating or otherwise damaging the sample of interest.

[0006] There is therefore a continuing need to develop improved microscale screening and analysis methods and systems with high throughput capabilities, and particularly methods and systems that enable recovery of samples identified in the screening and analysis.SUMMARY

[0007] An aspect of the present disclosure relates to a system for screening a population of variant proteins present in a microcapillary array using a multiplex assay. The system can include: a microcapillary array including a plurality of microcapillaries that include a variant protein; a first bead, the first bead including a first target molecule immobilized on the surface of the first bead; a second bead, the second bead including a second target molecule immobilized on the surface of the second bead; and a first reporter element. The first bead and the second bead can be optically distinguishable. The variant protein can associate with the second immobilized target molecule with a second affinity or the variant protein may not bind to the second immobilized target molecule. The variant protein can associate with the first target molecule with a first affinity. The first reporter element associates with the variant protein,

[0008] In some examples, the first bead can include a first size and the second bead includes a second size, the first and second sizes being optically distinguishable. In some examples, the first bead includes a first fluorophore, wherein the second bead includes a second fluorophore, and wherein the first fluorophore and the second fluorophore are optically distinguishable. In some examples, the first bead includes a first fluorophore and a second fluorophore, and the second bead includes only the second fluorophore, and the first fluorophore and the second fluorophore are optically distinguishable. In some examples, the system includes a second reporter element, and the second reporter element includes the second fluorophore, and the first reporter element includes the first fluorophore.

[0009] In some examples, the system includes a third bead, the third bead including a third target molecule immobilized on the surface of the third bead, wherein the third bead is optically distinguishable from the first bead and the second bead, and the variant protein associates with the third immobilized target molecule with a third affinity. In further examples, the system includes a fourth bead, the fourth bead including a fourth target molecule immobilized on the surface of the fourth bead, wherein the fourth bead is opticallydistinguishable from the first bead, the second bead, and the third bead, and wherein the variant protein associates with the fourth immobilized target molecule with a fourth affinity.

[0010] An aspect of the present disclosure relates to a system for screening a population of variant proteins present in a microcapillary array using a multiplex assay. The system can include: a microcapillary array including a plurality of microcapillaries that include a variant protein; a first cell, the first cell expressing a first target molecule; a second cell; and a first reporter element. The first target molecule can be localized on a surface of the first cell. The variant protein can associate with the first target molecule with a first affinity. The second cell may not express the first target molecule. The first reporter element can associate with the variant protein

[0011] In some examples, the first cell includes an HEK cell, a CHO cell, or a yeast cell. In some examples, the second cell includes an HEK cell, a CHO cell, or a yeast cell. In some examples, the second cell is a wild type cell and the first cell is an engineered or transfected to express or overexpress the first target molecule. In such examples, the first cell can be an engineered or transfected version of the second cell. In alternative examples, the first cell is a wild type cell and the second cell is an engineered or transfected to not express or under-express the first target molecule. In such examples, the second cell can be an engineered or transfected version of the first cell. In some examples, the second cell is dyed.

[0012] An aspect of the present disclosure relates to a system for screening a variant protein in a microcapillary of a microcapillary array using a multiplex assay. The system can include: a first target molecule immobilized on a first surface; a second target molecule immobilized on a second surface; and a first reporter element. The first surface and the second surface can be optically distinguishable. The variant protein can associate with the first immobilized target molecule with a first affinity. The variant protein can associate with the second immobilized target molecule with a second affinity or the variant protein may not bind to the second immobilized target molecule. The first reporter element can associate with the variant protein

[0013] In some examples, the first surface is a surface of a first bead. In further examples, the second surface is a surface of a second bead. In yet further examples, the first bead includes a first size and wherein the second bead includes a second size, the first and second sizes being optically distinguishable. In some examples, the first surface includes a firstfluorophore, and the second surface includes a second fluorophore. The first fluorophore and the second fluorophore can be optically distinguishable. In other examples, the first surface is a surface of a first cell. In some examples, the second surface is a surface of a second cell. In some examples, the system includes a first fluorophore that associates with the first surface and a second fluorophore that associates with the second surface.

[0014] In some examples, the system includes a benchmark protein. The benchmark protein can associate with the first target molecule. In some examples, the benchmark protein can associate with the first target molecule at an affinity lower than the first affinity, and the benchmark protein and the variant protein can associate with the first target molecule at the same epitope on the first target molecule. In further examples, the benchmark protein can associate with the first target molecule at an affinity higher than the first affinity, and the benchmark protein and the variant protein associate with the first target molecule at the same epitope on the first target molecule. In other examples, the benchmark protein associates with a first epitope of the first target molecule and the variant protein associates with a second epitope of the first target molecule, wherein the first epitope and the second epitope are different. In some examples, the system includes a second reporter element. The second reporter element can bind to the benchmark protein, and the first reporter element can bind to the variant protein.

[0015] In some examples, the first reporter element includes a first fluorophore and the second reporter element includes a second fluorophore. The first fluorophore and second fluorophore can be optically distinguishable.

[0016] In some examples, the system includes a second benchmark protein, and the second benchmark protein can associate with the second target molecule at an affinity lower than the second affinity, and wherein the second benchmark protein and the variant protein associate with second target molecule at the same epitope on the second target molecule. In other examples, the system includes a second benchmark protein, the second benchmark protein associates with the second target molecule at an affinity higher than the second affinity, and wherein the second benchmark protein and the variant protein associate with second target molecule at the same epitope on the second target molecule. In other examples, the system includes a second benchmark protein, and the second benchmark protein can associate with afirst epitope of the second target molecule and the variant protein associates with a second epitope of the second target molecule.

[0017] In some examples, the first target molecule and second target molecule are the same. In some examples, the reporter element includes a labeled antibody or other binding molecule. In some examples, the reporter element includes a fluorophore. In some examples, the system includes a sample extraction device configured to isolate a sample of a microcapillary of interest based at least in part on localization of the reporter element. In some examples, the system includes a means for isolating a sample of a microcapillary of interest based at least in part on localization of the reporter element,

[0018] In some examples, the variant protein includes an antibody. In some examples, the system includes a cell expressing the variant protein. In some further examples, the cell is a B cell.

[0019] In some examples, the system includes a microcapillary chip, the microcapillary chip including the plurality of microcapillaries. In some examples, the system includes: an imaging device configured to image the contents of the plurality of microcapillaries; and an extraction device. In some further examples, the system includes a processor in communication with the imaging device and the extraction device. The processor can execute instructions to: identify the first bead or first surface and the second bead or second surface; determine a microcapillary of interest based at least in part on localization of the reporter element with a control reporter element; and cause the extraction device to eject the contents of the microcapillary of interest of the plurality of microcapillaries. In some examples, the imaging device is a fluorescence microscope.

[0020] An aspect of the present disclosure relates to a method of screening a population of variant proteins using a multiplex assay. The method includes: providing a microcapillary array including a plurality of microcapillaries that include: a variant protein; a first target molecule immobilized to a first surface; a second target molecule immobilized to a second surface; and a first reporter element; measuring optical signals from the first reporter element, the first surface, and the second surface that indicate association of the variant protein with the first immobilized target molecule and / or the second immobilized target molecule; and identifying at least one microcapillary of interest based at least in part on the measured signals. The first surface and the second surface can be optically distinguishable. The variant proteincan associate with the first immobilized target molecule with a first affinity and with the second immobilized target molecule with a second affinity or the variant protein may not bind to the second immobilized target molecule. The first reporter element can associate with the variant protein.

[0021] In some examples, the first surface can include a first bead. In some examples, the second surface can include a second bead. In some examples, the first surface includes a first size and wherein the second surface includes a second size, the first and second sizes being optically distinguishable. In some examples, the first surface includes a first cell. In some examples, the second surface includes a second cell. In some examples, the first surface includes a first fluorophore, the second surface includes a second fluorophore, and the first fluorophore and the second fluorophore are optically distinguishable.

[0022] In some alternative examples, the first surface includes a first fluorophore and a second fluorophore, the second surface includes only the second fluorophore, and wherein the first fluorophore and the second fluorophore are optically distinguishable. In some examples, each microcapillary of the plurality of microcapillaries can include a second reporter element. The second reporter element can include the second fluorophore, and the first reporter element can include the first fluorophore. In further examples, measuring optical signals can include using a plurality of optical filters to filter a first signal of the first fluorophore and a second signal of the second fluorophore. In further examples, identifying at least one microcapillary of interest is based at least in part on the measured signals of the first fluorophore and the second fluorophore.

[0023] In some examples, each microcapillary of the plurality of microcapillaries can include a third surface. The third surface can include a third target molecule immobilized on the third surface. The third surface can be optically distinguishable from the first surface and the second surface. The variant protein can associate with the third immobilized target molecule with a third affinity. The method can further include measuring a signal from the third surface. In some further examples, the third surface includes a third bead. In other further examples, the third surface includes a third cell. In some examples, the microcapillary includes a fourth surface, the fourth surface including a fourth target molecule immobilized to the fourth surface. The fourth surface can be optically distinguishable from the first surface, the second surface, and the third surface. The variant protein can associate with the fourth immobilizedtarget molecule with a fourth affinity. In such examples, the method can further include measuring a signal from the fourth surface.

[0024] In some examples, the method can include determining, using a processor, positions of the first surface and the second surface. In some examples, the identifying at least one microcapillary of interest is based at least in part on the sizes of the first surface and the second surface.

[0025] In some examples, each microcapillary of the plurality of microcapillaries includes a benchmark protein, and the benchmark protein associates with the first target molecule. In further examples, the benchmark protein and the variant protein both associate with the first target molecule at the same epitope. In other examples, the variant protein associates with the first target molecule at a second epitope, and wherein the first epitope and the second epitope are different. In some examples, each microcapillary of the plurality of microcapillaries includes a second reporter element, wherein the second reporter element associates with the benchmark protein. In some examples, the first target molecule and the second target molecule are the same.

[0026] An aspect of the present disclosure relates to a system for screening a population of variant proteins present in a microcapillary array using a multiplex assay, the system including: a microcapillary array including a plurality of microcapillaries that include a variant protein; a first bead, the first bead including a target molecule immobilized on the surface of the first bead at a first surface density, wherein the variant protein associates with the target molecule at an affinity; a second bead, the second bead including a target molecule immobilized on the surface of the second bead at a second surface density, wherein the first surface density and the second surface density are different, and wherein the first bead and the second bead are optically distinguishable; and a reporter element, wherein the reporter element associates with the variant protein.

[0027] In some examples, the first bead includes a first size and the second bead includes a second size, the first and second sizes being optically distinguishable. In some examples, the first bead includes a first fluorophore, the second bead includes a second fluorophore, and the first fluorophore and the second fluorophore are optically distinguishable. In alternative examples, the first bead includes a first fluorophore and a second fluorophore, wherein the second bead includes only the second fluorophore, and the first fluorophore andthe second fluorophore are optically distinguishable. In some examples, the system includes a second reporter element, where the second reporter element includes the second fluorophore, and where the reporter element includes the first fluorophore.

[0028] In some examples, the system can include a third bead, the third bead further including a third target molecule immobilized on the surface of the third bead, where the third bead is optically distinguishable from the first bead and the second bead, and wherein the variant protein associates with the third immobilized target molecule with a third surface density. In some examples, the system can include a fourth bead, the fourth bead further including a fourth target molecule immobilized on the surface of the fourth bead, where the fourth bead is optically distinguishable from the first bead, the second bead, and the third bead, and wherein the variant protein associates with the fourth immobilized target molecule with a fourth surface density.

[0029] In some examples, the first bead includes a non-target molecule immobilized on the surface of the first bead at a third surface density. In some examples, the second bead includes a non-target molecule immobilized on the surface of the second bead at a fourth surface density. In some examples, the third surface density and fourth surface density are different. In alternative examples, the third surface density and fourth surface density are approximately the same.

[0030] An aspect of the present disclosure relates to a system for screening a population of variant proteins present in a microcapillary array using a multiplex assay, the system including: a microcapillary array including a plurality of microcapillaries that include a variant protein; a bead, the bead including a target molecule immobilized on the surface of the bead at a first surface density, and a non-target molecule immobilized on the surface of the bead at a second surface density, where the variant protein associates with the target molecule with an affinity; and a reporter element, where the reporter element associates with the variant protein.

[0031] In some examples, the ratio of the first surface density to the second density is from 100:1 to 1:100. In some examples, the ratio of the first surface density to the second density is from 10:1 to 1:10. In some examples, the ratio of the first surface density to the second density is from 2:1 to 1:2.

[0032] An aspect of the present disclosure relates to a system for screening a population of variant proteins present in a microcapillary array using a multiplex assay, the system including: a microcapillary array including a plurality of microcapillaries that include a variant protein; a bead, the bead including a target molecule immobilized on the surface of the bead at a first surface density, where the first surface density is sufficiently low such that the variant protein is not able to simultaneously bind to two target molecules; and a reporter element, where the reporter element associates with the variant protein.

[0033] In some examples, the reporter element includes a labeled antibody or other binding molecule. In some examples, the reporter element includes a fluorophore. In some examples, a system in accordance with the present disclosure includes a sample extraction device configured to isolate a sample of a microcapillary of interest based at least in part on localization of the reporter element. In some examples, the variant protein includes an antibody. In some examples, a system in accordance with the present disclosure can include a cell expressing the variant protein. In some examples, the cell is a B cell. In some examples, a system in accordance with the present disclosure includes a microcapillary chip, the microcapillary chip including the plurality of microcapillaries. In some examples, a system in accordance with the present disclosure includes: an imaging device configured to image the contents of the plurality of microcapillaries; and an extraction device. In some examples, a system in accordance with the present disclosure includes: a processor in communication with the imaging device and the extraction device; the processor executing instructions to: identify the first bead or first surface and the second bead or second surface; determine a microcapillary of interest based at least in part on localization of the reporter element with a control reporter element; and cause the extraction device to eject the contents of the microcapillary of interest of the plurality of microcapillaries. In some examples, the imaging device is a fluorescence microscope.

[0034] An aspect of the present disclosure relates to a method of screening a population of variant proteins using a multiplex assay, the method including: providing a microcapillary array including a plurality of microcapillaries, at least some of the plurality of microcapillaries including: a variant protein; a first bead; a second bead; a target molecule immobilized to the surfaces of the first bead at a first surface density and the second bead at a second density, wherein the variant protein associates with the target molecule at an affinity;and a reporter element, wherein the reporter element associates with the variant protein; measuring optical signals from the reporter element, the first bead, and the second bead that indicate association of the variant protein with the immobilized target molecule; and identifying at least one microcapillary of interest based at least in part on the measured signals.

[0035] In some examples, the method includes distinguishing the first bead and the second bead based on their respective sizes. In some examples, the first bead includes a first fluorophore, the second bead includes a second fluorophore, and the method includes distinguishing the first bead and the second bead based at least in part on the signal of the first fluorophore and the second fluorophore. In alternative examples, the first bead includes a first fluorophore and a second fluorophore, wherein the second bead includes only the second fluorophore, and the method includes distinguishing the first bead and the second bead based at least in part on the respective signal of the first fluorophore and the second fluorophore. In some examples, at least some of the microcapillaries include a second reporter element, where the second reporter element includes the second fluorophore, and where the reporter element includes the first fluorophore. In some examples, the method includes at least some of the microcapillaries include a third bead, the third bead including a third target molecule immobilized on the surface of the third bead, and where the variant protein associates with the third immobilized target molecule with a third surface density. In some examples, at least some of the microcapillaries include a fourth bead, the fourth bead including a fourth target molecule immobilized on the surface of the fourth bead, where the fourth bead is optically distinguishable from the first bead, the second bead, and the third bead, and where the variant protein associates with the fourth immobilized target molecule with a fourth surface density. In some examples, the first bead includes a non-target molecule immobilized on the surface of the first bead at a third surface density. In some examples, the second bead includes a non¬ target molecule immobilized on the surface of the second bead at a fourth surface density. In some examples, the third surface density and fourth surface density are different. In alternative examples, the third surface density and fourth surface density are approximately the same.

[0036] An aspect of the present disclosure relates to a method of screening a population of variant proteins using a multiplex assay, the method including: providing a microcapillary array including a plurality of microcapillaries, at least some of the plurality of microcapillaries including: a variant protein; a bead; a target molecule immobilized to the-10-surface of the bead at a first surface density and a non-target molecule immobilized on the surface of the bead at a second surface density, wherein the variant protein associates with the target molecule with an affinity; and a reporter element, wherein the reporter element associates with the variant protein; measuring optical signals from the reporter element and the bead that indicate association of the variant protein with the immobilized target molecule; and identifying at least one microcapillary of interest based at least in part on the measured signals.

[0037] In some examples, the ratio of the first surface density to the second is from 100:1 to 1:100. In some examples, the ratio of the first surface density to the second is from 10: 1 to 1:10. In some examples, the ratio of the first surface density to the second is from 2:1 to 1:2. In some examples, the variant protein is an antibody. In some examples, the association of the variant protein with the target molecule is indicative of smgle-arm affinity.BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIGS. 1A-1C schematically illustrate the steps of an exemplary microcapillary screening assay. The illustration on the left in each panel is a cross-sectional view from the side of a single microcapillary. The illustration on the right in each panel is a bottom view of a subsection of the array of microcapillaries. The shading in each case is intended to illustrate an electromagnetic signal, such as fluorescence.

[0039] FIGS. 2A-2E are different views of a microscope system designed to carry out the screening methods of the instant disclosure.

[0040] FIG. 3 A illustrates an exemplary screening array stage.

[0041] FIG. 3B illustrates an exemplary recovery array stage.

[0042] FIG. 4 shows exemplary positioning of a screening array and a recovery array relative to one another during the recovery of three samples of interest from the screening array, as facilitated by the instant sample recovery systems.

[0043] FIG. 5 illustrates a method of conducting a microcapillary-based multiplex assay in accordance with the present disclosure.

[0044] FIG. 6A shows an example microcapillary-based multiplex assay involving selective binding of a target to two different types of beads.

[0045] FIG. 6B shows another example microcapillary-based multiplex assay involving selective binding of a target to two different types of cells.

[0046] FIG. 7 A is a fluorescence microscopy image of a multiplex assay examining whether a variant protein binds to the same epitope of a target molecule as a benchmark protein.

[0047] FIG. 7B is a diagram show’ing the competition of the variant protein with the benchmark protein to bind with an antigen of a first bead in the assay of FIG. 7A.

[0048] FIG. 7C is a diagram showing the variant protein bound to the antigen of a second bead in the assay of FIG. 7A.

[0049] FIG. 7D is a fluorescence microscopy image of a multiplex assay examining whether a variant protein binds to a different epitope as a benchmark variant protein.

[0050] FIG, 7E is a diagram showing variant protein and benchmark protein simultaneously bound to the surface-bound antigens of a first bead of the assay of FIG, 7D.

[0051] FIG, 7F is a diagram showing only a variant protein 706 bound to the surface-bound antigen of a second bead of the assay of FIG. 7D,

[0052] FIGS. 8A-8C diagram multiplex schemes and shows fluorescence microscopy images of a 3-bead assay with beads of the same size but labeled with different fluorescent colors.

[0053] FIG. 9A depicts a multiplex assay scheme where a large bead is labeled with two undesired protein targets and the small bead is labeled with a desired protein target.

[0054] FIG. 9B depicts a multiplex assay scheme that reverses the assay of FIG.9A, where the large bead is labeled with the desired protein target and the small bead is labeled with two undesired targets.

[0055] FIG. 10A shows a scheme for a microcapillary-based multiplex assay where a small bead includes antibody-capture antibodies as target molecules.

[0056] FIG. 10B shows microscopy images of a multiplex assay carried out in accordance with the scheme depicted in FIG. 10A.

[0057] FIG. 11 A shows a scheme for a microcapillary-based multiplex assay with target cells expressing a target molecule on their surface.

[0058] FIG. 11B shows microscopy images of assays in accordance with the scheme of FIG. 11 A.

[0059] FIG. 12A illustrates an example scheme for examining affinity and avidity of an antibody.

[0060] FIG. 12B shows an example second bead that can include target molecules at a higher surface density than the bead.

[0061] FIG. 12C shows results of assays using beads including target molecules and non-target molecules at different ratios of surface densities.

[0062] FIGS. 12D and 12E plot results of a second example study employing the scheme of FIG. 12 A.

[0063] FIG. 13 A illustrates a scheme for a bead titration ladder assay.

[0064] FIG. 13B shows results of a bead titration ladder assay optimization, for determining which target molecule surface densities are suitable for use in the scheme discussed with respect to FIG, 13 A.

[0065] FIGS. 13C-13F show microscope images of a bead titration ladder screening.DETAWED DESCRIPTION

[0066] Previous microcapillary assays included single-assay readouts, eg. examining binding to target A or target B only, but not both. In certain other platforms, for example flow cytometry, multiplex readouts can be performed using primarily fluorescence intensity alone. In some examples, such flow cytometry multiplex readouts can exploit spatial segregation across a flow cell, among other potential approaches that may not be available in a microcapillary array-based assay platform. In the present disclosure provides for usage of homologous assays in microcapillary arrays. Multiplex approaches can allow assays to be performed in parallel. Different appearance / visualization of the beads (size, color, etc.) can be exploited to generate multiplex readouts.

[0067] Artifacts can be associated with multi-analyte assays and multiplex arrays in flow cytometry and other instrument systems that solely rely on fluorophore colors for multiplexing. Preexisting technologies may lack additional features or dimensions to increase specificity. The assays of the present disclosure can enhance specificity and provide additional dimensions that can increase multiplex capability by utilizing bead and / or cell location and sizes. Such enhanced specificity can reduce false positive rates.

[0068] A consideration in the evaluation of multiplex assays is the possibility that multiplexing itself can result in anomalies in the quantitation of some analytes, termed the “matrix effect.” The matrix effect can be caused by, for example, anti-cytokine antibodies thatmay cross-react with other cytokines, cross-species antibodies, and / or other interfering substances. These issues may cause an otherwise reliable uniplex assay to function poorly m a multiplex assay context. Each analyte must be tested for non-reactivity against all other antibodies used in the multiplex array, and the diluent must be carefully assessed to minimize unwanted cross-reactions. The assays described here improve accurate quantitation by analyzing different fluorescence overlap based on particle locations and sizes, thereby reducing the matrix effect.Overview of High-Throughput System

[0069] Microcapillary arrays have recently been employed in approaches for high-throughput analysis and protein engineering with large numbers of biological samples, for example in an approach that has been termed “microcapillary single-cell analysis and laser extraction” or “pSCALE”. See Chen et al. (2016) Nature Chem. Biol. 12:76-81; DOI: 10.1038 / NCHEMBI0.1978; see also US Pub. Nos. 2022 / 0162594, 2022 / 0373440, 2020 / 0080075, 2018 / 0188276 (each reference incorporated herein in its entirety). This approach relies on the spatial segregation of single cells within a microcapillary array, and thus enables repeated imaging, cell growth, and protein expression of the separate samples within each microcapillary (or a subset of microcapillaries) of the microcapillary array. Accordingly, the technique enables massively parallel, quantitative biochemical and biophysical measurements on millions or multi-millions of samples within a microcapillary array, for example, in the analysis of millions or multi-millions of protein variants expressed from yeast, bacteria, rodent, human, chicken, or other suitable cells distributed throughout the array. Advantageously, the approach allows for the simultaneous time-resolved kinetic analysis of the multiplexed samples, as well as the sorting of those cells based on targeted phenotypic features.

[0070] FIGS. 1A--1C illustrate an exemplary screening method for a soluble protein capable of associating with a target molecule, the target molecule being a cell-surface protein (e.g., the epidermal growth factor receptor (“EGFR”)) as an immobilized target molecule, in this case an immobilized target molecule within a microcapillary of a pSCALE system. FIG. 1 A (left panel) shows the target cell, which expresses the molecule on its surface. Also shown is a “library expressing cell”, which expresses a population of variant proteins(e.g., antibodies), and a number of fluorescent detection antibodies in the microcapillary solution. A bottom view of the microcapillary array is illustrated in the right panel. It is to be understood that other assays may be suitably implemented on the high-throughput system and that description of FIGS. 1A-1C is not intended to limit the assays w’hich can be performed on the high-throughput system.

[0071] Each capillary can include cells secreting the variant protein of interest (the “library expressing cell”), a target molecule immobilized on a surface, a reporter element, and a reaction buffer. The target molecule may be a target protein. The variant protein of interest can be a member of a population of variant proteins, i.e., a protein library. In some examples, the target molecule may be immobilized on the surface of a cell. The target molecule can be a native, cell-surface receptor (e.g., EGFR). Alternatively, the target molecule could be immobilized on another surface, such as a bead surface or an interior surface of the microcapillary itself. The reporter element may include a fluorescently-labeled antibody specific for the secreted protein (e.g., the “fluorescent detection antibodies”). The antibody can specifically localize to an epitope on the secreted protein but ideally does not interfere with the binding of the secreted protein to the target molecule on the target cell. Alternatively, the reporter element can be a signaling pathway within the cells that express the target molecule. If a secreted variant protein binds the target molecule on the cell surface and activates the signaling pathway within the target cell, the binding interaction will generate a fluorescent signal within the cell. The reaction buffer can be media for the library-expressing cells or for the target cells. The reaction buffer may additionally or alternatively be a mammalian imaging solution.

[0072] FIG. 1A illustrates the addition of the above-discussed components to a microcapillary. FIG. IB illustrates expression of a specific “secreted protein” by the library -expressing cell into the microcapillary. Secreted protein variants capable of binding to the target molecule (e.g., antibodies) can be localized to the target cell surface. FIG. 1C illustrates association of fluorescent detection antibodies associated with the bound secreted protein variants. Such association can indicate which microcapillary contains a sample of interest.

[0073] In some of the method embodiments, for example in the screening methods illustrated in FIGS. 1A-1C, the variant protein mediates the association of a reporter element with a target molecule, in this example, a target molecule on the surface of a target cell. Asshowii in FIG. IB, where the variant protein (here designated as a “secreted protein”) has sufficient affinity for its target molecule on the target cell that the variant proteins associate with the target cell under the conditions of the microcapillary solution. The reporter element (here designated as “fluorescent detection antibodies”) binds to the variant protein, ideally at an epitope that does not affect the affinity of the variant protein for the target molecule, as shown in FIG. 1 C.

[0074] In other embodiments, the reporter element is an intracellular reporter element that generates a detectable signal in connection with a binding event, such as, for example, the association of a variant protein with an immobilized target molecule, for example, a receptor or other target molecule on the surface of the cell. In these embodiments, the reporter element may include an entire cellular pathway, such as, for example, an intracellular signaling pathway. Such a pathway can include, or be engineered to include, a detectable signal as the downstream readout of the pathway. In contrast to the assays illustrated in FIGS. 1A-1C, where the detectabl e signal is bound to the outer surfa ce of the target cell, the detectable signal in these embodiments would typically be generated inside the target cell.

[0075] Various views of an exemplary system capable of performing the steps laid out in FIGS. 1 A- l C and then recovering samples of interest are provided in FIGS. 2A--6E. In particular, FIG. 2E illustrates an exploded view of a screening array stage 12, a recovery array 14, a recovery array holder 16, a first recovery array stage 18, a second recovery array stage 20, and a microscope objective 22. The optical pathways of an extraction beam, m this case a laser beam, and of the screening array image, are illustrated from the three perspectives shown in FIGS. 2B-2D as “laser beam path” and “imaging path”, respectively. The screening array stage is preferably configured to accommodate an array of microscale sample vessels (e.g., microcapillary array), in particular within an aperture that allows for the transmission of the optical beams through the associated array. An example screening stage is shown m more detail in FIG. 3A. An exemplary recoveiy array stage is illustrated in FIG. 3B. At least one recovery array stage is preferably connected to a recoveiy array holder, for example as illustrated m FIG 2E, to facilitate the reversible association of the recovery array (e.g., a multi¬ well collection plate as described herein) with the recovei ’ array stage. Reversible association refers to the ability of the recoveiy' array to be able to associate and dissociate with the recoveiy array stage (e.g., a first recovei ' stage) before, during, or after the sample recovery process. Insome embodiments, reversible association indicates that the recovery’ array can be placed into the system and / or removed from the system, in some cases more than once. In some embodiments, the recovery array is reversibly associated with the recovery array stage via spring tension, gravity', magnetic forces, friction, screws / fasteners, and / or Velcro.

[0076] In preferred embodiments, the multi-stage sample recovery systems include both a screening array reversibly associated with the screening array stage and a recovery array reversibly associated with the first recovery array stage. More specifically, the screening array includes a plurality of microscale sample vessels, and the recovery array includes a plurality of recovery vessels (e.g., wells in a multi-well plate),

[0077] As previously noted, the instant multi-stage sample recovery systems typically include an optical source and an optical detector to identify samples of interest within a screening array. In some cases, for example where a bioluminescent signal is being monitored, a separate optical source may not be required, and the systems may include only an optical detector. In either case, the optical detector is typically configured to monitor optical signals emitted from samples in a screening array by optically coupling the screening array to the detector through an aperture in the screening array stage. As described above, observation of optical signals from reporter elements within the sample vessels of the screening array enables the identification of specific sample vessels holding samples of interest, and the contents of those sample vessels can then be recovered by a pulse from the extraction beam generator. The optical detector, for example an imaging camera such as a charge-coupled device (CCD) or a complementary metal-oxide-semiconductor (CMOS) imaging sensor, is ideally' capable of imaging large numbers of sample vessels from the screening array' within a single field. In some embodiments, the optical detector is a charge-coupled device (CCD). In some embodiments, the optical detector is complementary' metal-oxide-semiconductor (CMOS) imaging sensor. In some embodiments, the optical detector is a photodiode. Where fluorescent labels are used m the reporter elements, imaging detectors are typically chosen for their sensitivity in the visible range of the electromagnetic spectrum. Fluorescence emission from the screening array is directed to the optical detector, typically through a microscope objective, via the imaging path of the system. Commercial microscopes, such as, for example, Nikon Eclipse series inverted microscopes and the like, can be suitably adapted for use in the instant systems, as would be understood by those of ordinary skill in the art.

[0078] In some embodiments, the multi-stage sample recovery systems further include an extraction beam generator optically coupled through an aperture in the screening array stage to one microscale sample vessel within the screening array. More specifically, the extraction beam can be a laser beam, for example a beam emitted by a diode laser, a diode- pumped Q-switched laser, such as a diode- pumped Q-switched Nd: YLF laser, or another appropriate laser device. In some embodiments, the laser is a diode laser. In some embodiments, the laser is a nanosecond pulsed laser. In some embodiments, the laser is a picosecond pulsed laser. Where the system includes an array of microcapillaries, the extraction beam can be directed at the water-glass interface between the microcapillary wall and the sample contained m the microcapillary. Use of lasers to isolate the contents of specific microcapillaries identified by fluorescence imaging within an array of microcapillaries has been described previously. See, e.g,, Chen et al. (2016) Nature Chem. Biol. 12:76-81; DOI: 10.1038 / NCHEMBIO.1978 and U. S. Patent Application Publication No, 2016 / 0244749 Al, which are incorporated herein by reference in their entirety.

[0079] In some embodiments, the extraction beam is directed from below the targeted microscale sample vessel. It should also be understood, however, that the extraction beam can alternatively be directed from above the targeted microscale sample vessel if so desired.

[0080] In specific embodiments, the system further includes a second recovery array stage. The first and second recovery array stages can reside in a horizontal plane. In more specific embodiments, the second recovery array stage can be oriented orthogonally to the first recovery array stage while still in the horizontal plane. According to these embodiments, samples can be recovered automatically from a screening array into a recovery array having recovery vessels arranged in orderly grids, in particular grids with x rows and y columns, where x and y can independently be 3, 8, 10, 12, 30, 100, or even more, or a value within a range defined by any two of the previous values.

[0081] In some embodiments, the screening array stage and the recovery array stage or stages are controllable by one or more electronic motors as would be understood by those of ordinary skill in the art.

[0082] In some embodiments, the screening array and the recovery’ array of the instant systems are configured so that at least one microscale sample vessel (e.g., amicrocapillary array) and at least one recovery’ vessel (e.g., a collection plate in accordance with the present disclosure) are positioned within a working distance of the microscope objective. In some embodiments, the working distance of the microscope objective is from about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 mm, or within a range defined by any two of the proceeding values, though in some examples other values may be suitably implemented. In some embodiments, the working distance, including the vertical distance, is from about 1 mm to 40 mm. In some embodiments, the working distance, including the vertical distance, is from about 2 mm to 30 mm. In some embodiments, the working distance, including the vertical distance, is from about 1,5 mm to 30 mm. In some embodiments, the working distance, including the vertical distance, is from about 2.5 mm to 30 mm. In some embodiments, the working distance, including the vertical distance, is from about 2 mm to 25 mm. In some embodiments, the working distance, including the vertical distance, is from about 3 mm to 30 mm. In some embodiments, the working distance, including the vertical distance, is from about 3 mm to 25 mm. More specifically, the working distance is from about 2.5 mm to about 25 mm. In these embodiments, the systems allow for the simultaneous imaging of the contents of a microscale sample vessel of interest and the associated recovery vessel. In more specific embodiments, the working distance of the microscope objective is from about 4 mm to about 10 mm or even from about 6 mm to about 8 mm, for example about 7.4 mm. In some embodiments, the recovery array is a first recovery array. In some embodiments, the recovery array is a second recovery array.

[0083] As previously noted, in preferred embodiments the screening arrays of the instant multi-stage sample recovery systems include a plurality of microcapillaries. More specifically, the screening arrays include at least 100,000, at least 300,000, at least 1,000,000, at least 3,000,000, at least 10,000,000, or even more microcapillaries. In some embodiments, the array includes at least 100,000, at least 200,000, at least 300,000, at least 400,000, at least 500,000, at least 600,000, at least 700,000, at least 800,000, at least 1,000,000, at least 1,500,000, at least 2,000,000, at least 2,500,000, or at least 3,000,000 or more microcapillaries.

[0084] As also previously noted, in some examples the recovery arrays of the instant multi-stage sample recovery systems can include one or more recovery vessels (e.g., collection plates in accordance with the present disclosure). Accordingly, in such systems, therecovery arrays may include at least 1 recovery vessel, at least 2 recovery vessels, at least 3 recovery vessels, at least 10 recovery vessels, at least 30 recovery vessels, at least 100 recovery vessels, or even more recovery vessels.

[0085] In preferred embodiments, the recovery array of the instant systems is positioned below the screening array. In some embodiments, the recovery array and the screening array are at least 25 mm, at least 30 mm, at least 35 mm, at least 40 mm, at least 45 mm, or at least 50 mm or more apart. In some embodiments, the recovery array and the screening array are at least 30 mm, at least 35 mm, or at least 40 mm apart. In some embodiments, the recovery array and the screening array are at least at least 35 mm or at least 40 mm apart. In some embodiments, the recovery7array and the screening array are at least at least 35 mm apart. In some embodiments, the recovery array is at least 25 mm, at least 30 mm, at least 35 mm, at least 40 mm, at least 45 mm, or at least 50 mm below the screening array. In some embodiments, the recovery array is at least 30 mm, at least 35 mm, or at least 40 mm below the screening array. In some embodiments, the recovery array is at least 35 mm or at least 40 mm below the screening array. In some embodiments, the recovery array is at least 35 mm below the screening array.

[0086] It will be readily apparent to one of ordinary skill in the relevant arts that other suitable modifications and adaptations to the methods and applications described herein can be made without departing from the scope of the invention or any embodiment thereof. Having now described the present invention in detail, the same will be more clearly understood by7reference to the following Examples, which are included herewith for purposes of illustration only7and are not intended to be limiting of the invention.

[0087] As noted above, the sample recovery7system consists of an X-Y stage, as illustrated m FIG. 3A, and at least one X-Y stage, as illustrated in FIG. 3B. The stages interface with a Nikon Ti-E Motorized microscope or the like. The X-Y stage holds a screening array, such as an array of microcapillaries, and X / Y stage or stages are configured to hold a sample recovery array, such as 96- well plate or the like.

[0088] Light from the associated microscope travels through both tiers of stages for purposes of visualizing the contents of each sample in the screening array, for example each microcapillary of the plurality of microcapillaries in an array of microcapillaries held on the screening array stage. Because of the close proximity between the screening array stageand the recovery array stage, the objective is also able to image vessels associated with the recovery array, for example an 18-well slide.

[0089] These stages work independently of one another to position the desired microscale sample vessel, for example a microcapillary within an array of microcapillaries, and the desired capture surface, for example a recovery vessel within a recovery array, at the desired location relative to the microscope objective. For example, as illustrated in FIG. 4, if screening array 10 is found to contain three sample vessels of interest, for example the three sample vessels labeled 1, 2, and 3 in the drawing, the screening array stage is moved to position the first sample vessel in line with the light path of the extraction beam, and the recovery array stage is likewise independently moved to position the first recovery vessel of recovery array 14 in line with the light paths as shown in the top left panel of FIG. 4.

[0090] After the first sample of interest has been transferred into the first recovery vessel, the screening array stage is moved in the X and Y directions to position the second sample of interest in line with the extraction beam, and the recovery array stage is independently moved to position the second recovery vessel in line with the beam, as shown in the top right panel of FIG. 4. After the second sample of interest has been transferred into the second recovery vessel, the process is repeated by moving the screening array stage in the X and Y directions as necessary to position the third sample of interest in line with the extraction beam. The recovery stage is independently moved to position the third recovery vessel in line with the beam, as shown in the bottom panel of FIG. 4, and the sample is transferred into the third recovery vessel by' the extraction beam.Systems for Microcapillary-Based Multiplex Assay

[0091] The present disclosure provides for systems capable of carrying out microcapillary-based multiplex assays. Microscopy can be used to image the microcapillaries. Such assays may use a high-throughput system as discussed with reference to FIGS. 1A–4 for imaging and isolation of samples of interest.

[0092] A system for screening a population of variant proteins present in a microcapillary array using a multiplex assay can include an array including a plurality of microcapillaries. Each microcapillary of the plurality of microcapillaries can include a variant protein to be tested. Each microcapillary of the plurality of microcapillaries can include a firstbead and a second bead. The first bead can include a first target molecule immobilized on the surface of the first bead. Whether the variant protein is of interest may depend on whether or to what degree the variant protein specifically associates with the first target molecule. The variant protein may be capable of associating with the first target molecule at a first affinity. The second bead can include a second target molecule immobilized on the surface of the second bead. The second target molecule can be used to assess, for example, nonspecific binding of the variant protein. The variant protein can associate with the second immobilized target molecule or does not bind to the second immobilized target molecule. The first bead and the second bead can be optically distinguishable. Each microcapillary of the plurality of microcapillaries can also include a first reporter element, that can associate specifically with the variant protein. The first reporter element can be used to tag the variant protein to allow for fluorescence microscopy.

[0093] In some examples, the first and second beads can be distinguished based on their respective sizes. The first bead can be a first size and the second bead can be a second size, the first and second sizes being optically distinguishable. In some examples, the first size is larger than the second size. In alternative examples, the first size is smaller than the second size. In some examples a size of the first bead or the second bead may be 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14.0, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15.0, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16.0, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17.0, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18.0, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19.0, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 μm or within a range defined by any two of the previous values. In some examples, a size of the first bead or the second bead may from 1.4 to 12 μm. In some examples, a size of the smaller of the first bead or the second bead may be from 1.4 to 5 μm and a size of the larger of the first bead or the second bead may befrom 5 μm to 12 μm. In some examples, a size of the smaller of the first bead or the second bead may be about 1.4 μm and a size of the larger of the first bead or the second bead may be about 12 μm. In some examples, a size of the smaller of the first bead or the second bead may be about 2 μm and a size of the larger of the first bead or the second bead may be about 12 μm. In some examples, a size of the smaller of the first bead or the second bead may be about 1.4 μm and a size of the larger of the first bead or the second bead may be about 10 μm. In some examples, a size of the smaller of the first bead or the second bead may be about 2 μm and a size of the larger of the first bead or the second bead may be about 10 μm. In some examples, a size of the smaller of the first bead or the second bead may be about 1.4 μm and a size of the larger of the first bead or the second bead may be about 5 μm. In some examples, a size of the smaller of the first bead or the second bead may be about 2 μm and a size of the larger of the first bead or the second bead may be about 5 μm. In some examples, a size of the smaller of the first bead or the second bead may be about 5 μm and a size of the larger of the first bead or the second bead may be about 12 μm. In some examples, a size of the smaller of the first bead or the second bead may be about 5 μm and a size of the larger of the first bead or the second bead may be about 10 μm.

[0094] In some examples, the first and second beads can be distinguished based on their different fluorescent signals. The first bead can include a first fluorophore, and the second bead can include a second fluorophore. The first fluorophore and the second fluorophore can optically distinguishable. For instance, the first fluorophore may fluoresce at a first wavelength and the second fluorophore may fluoresce at a second wavelength. The first wavelength and the second wavelength may be sufficiently different that they are distinguishable via an optical system, for example a fluorescence microscope. For instance, a filter set of such an optical system may be capable of isolating signal of the first wavelength from signal of the second wavelength.

[0095] The system can also include a benchmark protein. The benchmark protein may be capable of associating with the first target molecule. The benchmark protein and the variant protein may both be capable of associating with a first target molecule at the same epitope site. In such examples, the variant protein may be filtered based on whether it is capable of out-competing the benchmark protein for binding at the epitope site of the first target molecule. In some examples, the benchmark protein may be capable of associating with thefirst target molecule at an affinity lower than the first affinity. In alternative examples, the benchmark protein can associate with the first target molecule at an affinity higher than the first affinity. In other examples, such as in a blocking assay, the benchmark protein may block the epitope site of the first target molecule to which the variant protein is capable of binding. Such a blocking assay can compare the variant protein’s failure to bind to a benchmark-bound antigen to the variant proteins successful binding to a unblocked antigen to confirm that the variant protein binds to the particular blocked epitope.

[0096] In other examples, the benchmark protein associates with a first epitope of the first target molecule and the variant protein associates with a second epitope of the first target molecule. In such examples, the first epitope and the second epitope are different. In further examples, the system can include a second benchmark protein. The second benchmark protein may be used to assess nonspecific binding of the variant protein, for example binding to a molecule (e.g., the second target molecule) for which binding is undesirable. The second benchmark protein may be capable of associating with the second target molecule at an affinity lower than the second affinity. The second benchmark protein and the variant protein may associate with second target molecule at the same epitope. In such examples, the variant protein may be undesirable for its high propensity for non-specific binding. In some alternative examples, the second benchmark protein can associate with the second target molecule at an affinity higher than the second affinity. In such examples, the variant protein may be desirable for its low propensity for non-specific binding.

[0097] In some examples, the second benchmark protein associates with a first epitope of the second target molecule and the variant protein associates with a second epitope of the second target molecule. Such examples may be used for assessing steric effects of the first epitope and the variant protein. Additionally or alternatively, such examples may be used for validating co-localization of the second benchmark protein and the variant protein.

[0098] The system can include a third bead. The third bead can include a third target molecule. The third target molecule can be immobilized on the surface of the third bead. The third bead can be optically distinguishable from the first bead and the second bead. The third bead may be optically distinguishable from the first bead and the second bead based on bead size and / or fluorescent signal as discussed herein. The variant protein can associate with the third immobilized target molecule with a third affinity. In further examples, the systemmay include a fourth bead. The fourth bead can include a fourth target molecule immobilized on the surface of the fourth bead. The fourth bead is optically distinguishable from the first bead, the second bead, and the third bead. The variant protein associates with the fourth immobilized target molecule with a fourth affinity. The system may include additional beads and target molecules.

[0099] The first reporter element can include a labeled antibody or other binding molecule. The first reporter element can include a fluorophore. In some examples, the system may include a second reporter element in addition to the first reporter element. In such examples, the first reporter element may include a first fluorophore and a second reporter element may include a second fluorophore, and the first and second fluorophores may be different such that the first and second fluorophores are optically distinguishable. The first reporter element can bind to the variant protein. The second reporter element can bind to a benchmark protein. In certain examples, the first reporter element can associate with the first bead and the second reporter element can associate with the second bead, for instance via binding to the variant protein or to the benchmark protein, respectively. In other examples, the first reporter element and the second reporter element can both associate with the first bead, and only the first reporter element can associate with the second bead, for instance via binding to the variant protein or to the benchmark protein. The system can include an extraction device for isolating a microcapillary of interest based at least in part on localization of the first reporter element. For instance, as discussed herein with reference to the high-throughput systems of FIG. 1–4, the system may include a laser for extracting the contents of a microcapillary of interest. The variant protein can include an antibody. The system can include a cell expressing the variant protein. In some examples, the cell can be a B cell.

[0100] A system for screening a population of variant proteins present in a microcapillary array using a multiplex assay can include an array of microcapillaries. Each of the microcapillary can include including a variant protein. The microcapillaries may include a first cell and a second cell. The first cell can express a first target molecule. The first target molecule can be localized on a surface of the first cell. The variant protein can associate with the first target molecule with a first affinity. The second cell does not express the first target molecule. Each microcapillary of the plurality of microcapillaries can also include a firstreporter element. The first reporter element can associate with the variant protein. In some examples, the second cell can be dyed to allow for fluorescence imaging.

[0101] The system can include an imaging device that can image the contents of the plurality’ of microcapillaries. The system can include an extraction device. The extraction device can isolate a sample from a microcapillary of interest. The system can include a processor in communication with the imaging device and the extraction device. The processor can execute instructions to identify the first bead (or first surface) and the second bead (or second surface) to determine a microcapillary of interest based at least in part on localization of the first reporter element with a control reporter element; and cause the extraction device to eject the contents of the microcapillary of interest of the plurality of microcapillaries.

[0102] A system for screening a variant protein using a multiplex assay can include a first target molecule immobilized on a first surface and a second target molecule immobilized on a second surface, and a first reporter element. The variant protein associates with the first immobilized target molecule with a first affinity. The variant protein associates with the second immobilized target molecule with a second affinity or the variant protein does not bind to the second immobilized target molecule. The first surface and the second surface can be optically distinguishable.

[0103] In some examples, the first surface is a surface of a first bead. In some examples, the second surface is a surface of a second bead. In some examples, the first bead can include a first size and the second bead can include a second size, the first and second sizes can be optically distinguished. In some examples, the first surface is a surface of a first cell. In some examples, the second surface is a surface of a second cell. In some examples, the first surface can include a first fluorophore. The second surface can include a second fluorophore. In some examples, the system includes a first fluorophore that associates with the first surface. In some examples, the system includes a second fluorophore that associates with the second surface. The first fluorophore and the second fluorophore can be optically distinguished.

[0104] The system can include a benchmark protein. The benchmark protein can associate with the first target molecule. In some examples, the benchmark protein can associate with the first target molecule at an affinity lower than the first affinity. The benchmark protein and the variant protein may be capable of associating with a first target molecule at the same epitope. In alternative examples, the benchmark protein can associate with the first targetmolecule at an affinity higher than the first affinity. In some examples, the benchmark protein and the variant protein associate with a first target molecule at the same epitope. In alternate examples, the benchmark protein can associate with a first epitope of the first target molecule and the variant protein can associate with a second epitope of the first target molecule. The first epitope and the second epitope can be different. A second reporter element (e.g., a control reporter element) can bind to the benchmark protein. The second reporter element can include a second fluorophore to allow for fluorescence imaging of the benchmark protein. In some examples, the first target molecule and second target molecule are the same.

[0105] The system may include a second benchmark protein. In some examples, the second benchmark protein can associate with the second target molecule at an affinity lower than the second affinity, and wherein the second benchmark protein and the variant protein associate with second target molecule at the same epitope. In alternative examples, the second benchmark protein associates with the second target molecule at an affinity higher than the second affinity. In further alternative examples, the second benchmark protein associates with a first epitope of the second target molecule and the variant protein associates with a second epitope of the second target molecule.

[0106] In some examples, the system includes a third bead. The third bead can include a third target molecule immobilized on the surface of the third bead. The third bead can be optically distinguished from the first bead and the second bead. The variant protein can associate with the third immobilized target molecule with a third affinity.

[0107] In some examples, the system includes a fourth bead. The fourth bead can include a fourth target molecule. The fourth target molecule can be immobilized on the surface of the fourth bead. The fourth bead can be optically distinguished from the first bead, the second bead, and the third bead. The variant protein can associate with the fourth immobilized target molecule with a fourth affinity.Methods for Microcapillary-Based Multiplex Assay

[0108] FIG. 5 illustrates a method 500 of conducting a microcapillary-based multiplex assay in accordance with the present disclosure.

[0109] At step 502, a plurality of microcapillaries are provided, each microcapillary of the plurality of microcapillaries can include a variant protein, a first targetmolecule, a second target molecule, and a reporter element. The plurality of microcapillaries can include a portion of or all of a microcapillary array. The first target molecule can be immobilized to a first surface. The second target molecule can be immobilized to a second surface. The first surface and the second surface can be optically distinguishable. The first surface and second surface can be optically distinguishable based on their respective sizes and / or association with different fluorophores, etc. The variant protein can associate with the first immobilized target molecule with a first affinity and w’ith the second immobilized target molecule with a second affinity.

[0110] In some examples, the first surface is a surface of a first bead. In some examples, the second surface is a surface of a second bead. In such examples, the first bead can include a first size and the second bead can include a second size. The first size and second size can be optically distinguished using a microscope.

[0111] In other examples, the first surface may be a surface of a first cell. In some examples, the second surface may be a surface of a second cell. In some examples, one of the first surface or second surface is a surface of a bead while the other of the first surface or second surface is a surface of a cell.

[0112] In some examples, the first surface can include a first fluorophore. The second surface can include a second fluorophore. The first fluorophore and the second fluorophore can be optically distinguishable (e.g., the first fluorophore can emit fluorescence at different wavelength(s) than the second fluorophore).

[0113] In some examples, each microcapillary of the plurality of microcapillaries can include a third surface. The third surface can include a third target molecule immobilized on the third surface. The third surface can be optically distinguishable from the first surface and the second surface. The variant protein can associate with the third immobilized target molecule with a third affinity. In some examples, the third surface can be a surface of a third bead. In some examples, each microcapillary of the plurality of microcapillaries includes a fourth surface. The fourth surface can include a fourth target molecule immobilized to the fourth surface. The fourth surface can be optically distinguishable from the first surface, the second surface, and the third surface. The variant protein can associate with the fourth immobilized target molecule with a fourth affinity.

[0114] At step 504, the optical signals of the plurality of microcapillaries are measured. Optical signals can be generated from the reporter element, the first fluorophore associated with the first surface, and / or the second fluorophore associated with the second surface. Positioning (e.g. lack or presence of colocalization) of the reporter element signal, first surface signal, and / or second surface signal can indicate association of the variant protein with the first immobilized target molecule or the second immobilized target molecule. In some examples, step 504 can include using a plurality of optical filters to filter a first signal of the first fluorophore and a second signal of the second fluorophore.

[0115] In examples where the microcapillary includes a third surface, step 504 can include measuring a signal of the third surface. In examples where the microcapillary includes a fourth surface, step 504 can include measuring a signal from the fourth surface.

[0116] At step 506, one or more microcapillaries of interest of the plurality of microcapillaries are identified. Identification of the one or more microcapillaries of interest can be based at least in part on the measured signals. For instance, the measured signals may indicate whether the variant protein associates with the first surface, the second surface, both, or neither. Identifying the at least one microcapillary of interest can be based at least in part on the measured signals of the first fluorophore and the second fluorophore. In some examples, a processor can be used to determining positions of the first surface and the second surface as part of step 506.

[0117] In some examples, the identifying at least one microcapillary of interest is based at least in part on the sizes of the first surface and the second surface. The sizes of the first surface and second surface can be helpful for identifying which signals originate from the first surface and which signals originate from the second surface in examples where fluorescent signals of the first surface and second surface are present in the same fluorescence channel.

[0118] At step 508, samples from the one or more microcapillaries of interest can optionally be isolated. In some examples, samples from the one or more microcapillaries of interest can be isolated as discussed with reference to FIGS. 1 A-4.

[0119] At step 510, further analysis can be conducted on the samples isolated in step 508. Further analysis may include, for example, nucleic acid sequencing. In some examples, nucleic acid sequencing may be used to determine a cDNA sequence corresponding to the amino acid sequence of the variant protein.Example Microcapillary-Based Multiplex Assays

[0120] Discussed herein are a number of working examples of microcapillary-based multiplex assays. The working examples are not intended to limit the scope of the disclosure, but are intended to illustrate various aspects. Any features discussed with reference to the working examples below can be combined with embodiments discussed elsewhere this disclosure, and vice versa. Persons having skill in the art will appreciate that aspects of two or more of the working examples discussed with respect to FIGS. 6A-11B can be combined to the extent they are compatible.Multiple Bead Sizes

[0121] FIG. 6A shows an example microcapillary-based multiplex assay involving selective binding of a target to two different types of beads. In the example of FIG. 6A, the two different bead types are different with respect to size (e.g., small and large). In this example, the small bead is shown as the control bead while the large bead is shown as the target bead. It is to be understood that, in alternative examples, the small bead can act as the target bead and the large bead can act as the control bead. Antibody-secreting cells (e.g., B cells) within the same space (e.g., within the same microcapillary) can be assayed based on the labeled beads. A specific antibody-secreting cell binding to a target antigen can be screened based on the fluorescence of the large beads. Microscope images are also included in FIG. 6A, showing bright field (“BF”) and fluorescence (“binding”) images showing binding of the antibodies to both the target bead and the control bead. In the fluorescent images, the target beads appear noticeably larger than the control beads.

[0122] FIG. 6B shows another example microcapillary-based multiplex assay involving selective binding of a target to two different types of cells. In some examples, the cells can be stained with different dyes to allow for multiplexing. In some examples, control cells can be dyed, and cells expressing target molecules may be free of dye. Other schemes to distinguish different types of cells (e.g., control cells from target cells) using fluorescent images may be suitably implemented. For the microscopy images shown in FIG. 6B, two different types of reporter cells were used to assay for antibody-secreting cells (e.g. B cells). The false-red color represents a reporter element (e.g., an antibody) binding to target reporter cells. The false green color represents control cells. In this example, the control cells were dyedwith fluorescein (FITC). Antibody binding to target reporter cells appears not to overlap with the fluorescein-dyed control cells. In examples, the reporter cells may be transiently or permanently expressing a reporter element. In some examples, the reporter element is an antibody such as a secondary antibody.Multiple Fluorophores

[0123] FIGS. 7A-7F depict multiplex assays including two beads of the same size that are labeled with different and / or overlapping fluorescent colors. Specifically, the beads are labelled via the variant proteins or benchmark proteins able to bind to the beads. The assays can examine whether variant proteins (e.g., variant antibodies) bind to the same or different epitope of a target molecule as a benchmark protein (e.g., a benchmark antibody). In these assays, the benchmark protein can be tagged with a different fluorophore color with respect to the variant protein.

[0124] FIG. 7A is a fluorescence microscopy image of an assay examining whether a variant protein (e.g., a variant antibody) can bind to the same epitope as a benchmark protein (e.g., a benchmark antibody). The type of assay shown in FIG. 7 A may be referred to as a “blocking assay.” In this assay, all beads 702 and 704 include surface-bound antigens 710. The variant protein 706 can compete with the benchmark protein 708 for binding to an isotope of the bound antigen 710. That is to say, the variant protein 706 and the benchmark protein 708 can bind to the same epitope of the antigen. In some embodiments, the benchmark-bound antigen beads 704 are prepared prior to mixing with beads 702 containing only antigen 710 and variant proteins 706. For example, benchmark-bound antigen beads can be made by combining beads with bound antigen 710 with benchmark protein 708 and any free benchmark protein 708 removed prior to mixing with beads containing only antigen 710 and variant protein 706. In other embodiments, the beads containing bound antigen 710 (i.e., a single type of bead) may be mixed with both benchmark protein 708 and variant protein 706 such that through binding competition between benchmark protein 708 and variant protein 706, two distinguishable beads are formed in situ (i.e., one containing bound benchmark protein 708 and one containing bound variant protein 706). FIG. 7B is a diagram showing the competition of the variant protein 706 with the benchmark protein 708 to bind with an antigen 710 of bead 702 in the assay of FIG. 7A. FIG 7C is a diagram showing the variant protein 706 bound tothe antigen 710 of bead 704. Two different reporter elements are used, a first reporter element 716 and a second reporter element 714. The first reporter element 716 can include an antibody with a first type of bound fluorophore (depicted as green) that binds to the benchmark protein 708. The second reporter element 714 can include an antibody with a second type of bound fluorophore (depicted as red) that binds to the variant protein 706. In FIG. 7 A, the beads 702 bound to a benchmark protein are shown in false green, while the beads 704 bound to a variant protein are shown in false red. The beads 702 appear in the green channel only, and the beads 704 appear in the red channel only. The presence of both green and red beads indicates that the variant protein 706 (i.e., secreted antibody) binds to the same epitope as the benchmark protein 708.

[0125] FIG, 7D is a fluorescence microscopy image of an assay examining whether a variant protein (e.g., a variant antibody) has a different epitope as a benchmark protein (e.g,, a benchmark antibody).

[0126] The assay of FIG. 7D allows for screening for variant proteins (e.g., antibodies) that bind to a different epitope of the surface-bound antigen 710 as the benchmark antibody. In this assay, all beads 704 and 712 include surface-bound antigens 710. The variant protein 706 does not compete with the benchmark protein 708 for binding to the surface-bound antigens 710, as each of the variant protein 706 and benchmark protein 708 can bind to different epitopes of the surface-bound antigens 710. In some embodiments, the benchmark¬ bound antigen beads 712 are prepared prior to mixing with beads 704 containing only antigen 710 and variant protein 706. For example, benchmark-bound antigen beads 712 can be made by combining beads with bound antigen 710 with benchmark protein 708 and any free benchmark protein 708 removed prior to mixing with beads 704 containing only antigen 710 and variant proteins 706. In other embodiments, the beads containing bound antigen 710 (i.e., a single type of bead) may be mixed with both benchmark protein 708 and variant protein 706 such that through binding of benchmark protein 708 and variant protein 706, two distinguishable beads are formed in situ (i.e., beads 712 containing both bound benchmark protein 708 and bound variant protein 706, as well as beads 704 containing bound variant protein 706). FIG. 7E is a diagram showing variant protein 706 and benchmark protein 708 simultaneously bound to the surface-bound antigens 710 of bead 712. FIG. 7F is a diagram showing only a variant protein 706 bound to the surface-bound antigen 710 of bead 704. In theimage of FIG. 7D, the variant protein 706 are tagged with a fluorophore shown in false red, while the benchmark protein 708 tagged with a fluorophore shown in false green. The beads 712 appear in both the green and red channels (appearing yellow in the image of FIG. 7D), and the beads 704 appear in only the red channel. The presence of false yellow beads (both green and red channels) and false red beads indicates that the variant protein 706 binds to antigen 710 at a different epitope than benchmark protein 708.

[0127] The assay illustrated in FIGS. 7A-7F will indicate whether a particular microcapillary contains a variant protein that binds the target antigen (presence of red beads), and if so, whether the variant proteins binds the same epitope as the benchmark protein (additional presence of green beads) or whether the variant protein binds to a different epitope as the benchmark protein (additional presence of yellow beads).Epitope-Specific Binding with Multiple Benchmark Proteins

[0128] Screening of antibodies that compete for epitope sites with multiple benchmark antibodies can be scaled by simultaneously using different panels of fluorophores or varying bead sizes. FIGS. 8A-8C diagram schemes and show fluorescence microscopy images of a 3 -bead assay with beads of the same size but labeled with different fluorescent colors. The assay of FIGS. 8A-8C include two different benchmark proteins. In the examples depicted in FIGS. 8A-8C the benchmark proteins are SIF antibodies and RON antibodies, though it is to be understood that two other antibodies or benchmark proteins may be suitably implemented. In some alternative examples, more than two benchmark proteins may be suitably implemented. The assay includes a first bead 802, a second bead 804, and a third bead 806. The first bead 802 includes a surface-bound antigen that is not blocked at any epitope site. The second bead 804 includes a surface-bound antigen blocked at a first epitope site (in the depicted example, at a RON site). The third bead 806 includes a surface-bound antigen blocked at second epitope site (in the depicted example, at a SIF site). The second bead 804 and third bead 806 may be collectively referred to as benchmark-bound antigen beads 804 and 806 herein. Inclusion of the first bead 802, the second bead 804, and third bead 806 in a microcapillary-based assay allows for characterization of variant protein (e.g., variant antibodies) by examining the overlap of fluorescent colors. In example microscopy images shown in FIGS. 8A-8C, the variant protein is tagged with a fluorophore in the false redchannel, the second bead 804 is tagged with a fluorophore in the false blue channel, and the third bead 806 is tagged with a fluorophore in the false green channel. In some embodiments, the benchmark-bound antigen beads 804 and 806 are prepared prior to mixing with beads 802 containing only antigen and variant protein. For example, benchmark-bound antigen beads 804 and 806 can be made by combining beads with bound antigen with respective benchmark proteins and any free benchmark proteins removed prior to mixing with beads 802 containing only antigen and variant proteins. In other embodiments, the beads containing bound antigen (i.e,, a single type of bead) may be mixed with one or both types benchmark protein and / or variant protein such that through binding of benchmark proteins and / or variant protein, more than one type of distinguishable bead is formed in situ (e.g., beads 804 containing both a first bound benchmark protein and bound variant protein, beads 806 containing both a second bound benchmark protein and a bound variant protein, and beads 802 containing bound variant protein).

[0129] In the scheme shown in FIG. 8A, the variant protein is capable of binding the antigen at a site other than the first epitope site or the second epitope site. Accordingly, the variant protein can bind to the first bead 802, the second bead 804, and the third bead 806. Fluorescent microscopy shows beads colored red (i.e., first beads 802), beads colored purple / magenta (i.e., second beads 804 labeled with red and blue reporter elements binding to the variant protein and the RON antibody, respectively), and beads colored yellow (i.e., third beads 806 labeled with red and green reporter elements binding to the variant protein and the SIF antibody, respectively). There are no beads colored only blue or only green.

[0130] In the scheme shown in FIG. 8B, the variant protein is capable of binding to the second epitope site. Accordingly, the variant protein can bind to the first bead 802 and the second bead 804, but not the third bead 806, as the second epitope site of the antigen of third bead 806 is blocked. The third beads 806a of FIG. 8B therefore do not include a bound variant protein and do not generate any signal in the false red channel. Fluorescent microscopy shows beads colored red (i.e., first bead 802), beads colored purple / magenta (i.e., second beads 804 labeled with red and blue reporter elements binding to the variant protein and the RON antibody, respectively), and beads colored green (i.e., third beads 806 that do not include a bound variant protein and are only labeled with the green reporter element binding to the SIF antibody).

[0131] In the scheme shown in FIG. 8C, the variant protein is capable of binding to the first epitope site. Accordingly, the variant protein can bind to the first bead 802 and the third bead 806 but not the second bead 804, as the first epitope site of the second bead 804 is blocked. The second beads 804a of FIG. 8C therefore do not include a bound variant protein and do not generate any signal in the false red channel. Fluorescent microscopy shows beads colored red, (i.e., first bead 802), beads colored blue (i.e., second beads 804a that do not include a bound variant protein and are only labeled with the green reporter element binding to the RON antibody), and beads colored yellow (i.e., third beads 806 labeled with red and green reporter elements binding to the variant protein and the SIF antibody, respectively).Multiplex Assay for Analyzing Non-Specific Binding

[0132] It may be desirable to select a variant protein with high specificity to a target molecule and / or minimal cross-reactivity with one or more non-target molecules.

[0133] FIGS. 9 A and 9B show two example assays with two different bead sizes and three different protein targets a 912, P 914, and y 916 attached to the beads. In some examples, a large bead 902a or 902b may be an aldehyde bead. In some examples, a small bead 904a or 904b may be a M-450 bead. In both assays, only the variant protein 908 (e.g., the variant antibody) is tagged fluorescently. FIGS. 9A and 9B depict the fluorescent tag as a secondary antibody 906, but it is to be understood that any suitable fluorescent tag can be used. FIG. 9A depicts a first assay scheme, the large bead 902a is labeled with two undesired protein targets (proteins P 914 and y 916) while the small bead 904a is labeled with a desired protein target (protein a 912), FIG. 9B depicts a reverse scheme, where the large bead 902b is labeled with the desired protein target (protein a 912) and the small bead 904b is labeled with two undesired targets (proteins p 914 and y 916). The fluorescence microscopy images show desired and undesired hits. For the scheme shown in FIG. 9A, a sample of interest (alternatively referred to as a “desired hit”) is a sample where only large beads appear in fluorescent microscopy. / Additionally, for the scheme shown m FIG. 9A, a sample that is not of interest (alternatively referred to as an “undesired hit”) is one where both large beads and small beads appear in fluorescent microscopy. Such an undesired hit demonstrates non-specific binding of the variant protein 908 to both beads 902a and 904a. For the scheme shown in FIG. 9B, a sample of interest is one where only large beads appear in the fluorescence channel.Additionally, for the scheme shown in FIG. 9B, a sample that is not of interest is one where both large beads and small beads appear in the fluorescence channel, showing non-specific binding of the variant protein 908 to both beads 902b and 904b. The assays of FIG. 9A and 9B may each include a variant protein-expressing cell 910 (e.g., a B cell) capable of expressing the variant protein 908.Target Molecules

[0134] Surfaces, for example bead surfaces or cell surfaces, can include target molecules. Target molecules can be antigens, as discussed with respect to FIGS. 6A-9B. Target molecules may alternatively be capture antibodies or expressed proteins.

[0135] FIG. 10A shows an example scheme for a microcapillary-based assay where a small bead 1010 is labeled with antibody-capture antibodies 1004 as an alternative to an antigen. The antibody-capture antibodies 1004 can capture the variant protein 1002 (e.g., a variant antibody). The variant protein 1002 can be tagged with a secondary antibody 1006 containing a fluorophore. In the example scheme of FIG 10A, the small bead 1010 (e g., IgG capture beads) includes the antibody-capture antibodies 1004. The antibody-capture antibodies 1004 can bind to portions of the variant protein 1002 other than the site at which the variant protein 1002 binds to the target molecule 1008. For example, if the variant protein 1002 is an antibody, the antibody-capture antibodies 1004 can bind to sites of the variant protein 1002 other than the antigen-binding site of the variant protein 1002. A large bead 1012 is coated with a target molecule 1008 (e.g., an antigen such as P-Gal), It is to be understood that any suitable combination of bead sizes, fluorescence tags, and / or target molecules may be suitably implemented. That is to say, in some examples, a large bead may include antibody-capture antibodies while a small capture bead may include a target molecule (e.g., an antigen). The specific antibody can be detected by either the bead size or the antigen labeled with a specific fluorophore. Other suitable types of target molecules may be suitably implemented. A free target molecule 1014 may also be used in the assay of FIG. 10A. The free target molecule 1014 may be fluorescently tagged, for example with a fluorophore that fluoresces at a different wavelength than the secondary antibody 1006. The site of the variant protein 1002 may or may not be capable of binding to the free target molecule 1014. For instance, in examples where the variant protein 1002 is a variant antibody, the variant protein 1002 can bind to the freetarget molecule 1014 via an antigen binding site of the variant protein 1002. The free target molecule 1014 can help demonstrate specificity of binding of the variant protein 1002. Inclusion of the free target molecule 1014 and the target molecule 1008 in the assay can, for example, show whether the variant protein 1002 can bind to both the target molecule 1008 and the free target molecule 1014 or just one of the target molecule 1008 or free target molecule 1014. In some examples, the free target molecule 1014 may be an undesired target (e.g., an albumin). In such examples, lack of binding between the variant protein 1002 and free target molecule 1014 with binding between the variant protein 1002 and the target molecule 1008 can indicate high specific binding of the variant protein 1002 to the target molecule 1008, The microcapillary can also include a variant protein expressing-cell 1016 (e.g., a B cell).

[0136] FIG. 10B shows microscopy images of an assay carried out in accordance with the scheme depicted in FIG. 10A, The assay included large p-Gal-coated beads and small IgG-coated beads. The assay also included a fluorescently-tagged free target molecule, OVA-AF647 (Invitrogen), The images show brightfield and fluorescent signal at two different wavelengths, in an IgG / β-Gal channel and an OVA channel. In this example, a microcapillary of interest (referred to in the figure as a “desirable hit”) would include fluorescent signals from both small beads 1010 and large beads 1012 (e.g., in the IgG / p-Gal channel) but no signal from the free target molecules 1014 (e.g., in the OVA channel). An undesirable hit may have signal from only the small beads 1010 (e.g., the IgG capture beads) and / or may include signal in the free target molecule 1014 channel (e.g., in the OVA channel).Target Molecule-Expressing Cells

[0137] FIG. 11 A shows an example scheme for a microcapillary-based assay with target cells 1102 expressing a target molecule 1104 (e.g., an antigen, for example GPRC5D) on their surface. In a microcapillary of interest, a variant protein 1108 (e.g., a variant antibody) may be capable of binding to the target molecule 1104. The cell type used for the target cell 1102 expressing the target molecule 1104 can be wild type, engineered, or transfected. The example depicted in FIGS. 11 A and 1 I B used HEK cells to express the target molecule 1104. It is to be understood that any suitable cell type may be used for expression of a target molecule 1104. In examples, HEK293 cells, CHO cells, yeast cells, insect cells (e.g., Sf9 cells), or various cancer cell lines may be used to express target molecules. Similar cells to the targetcells 1102 may be used as a control cell 1106. However, the control cell 1106 may not express the target molecule 1104. For example, the control cell 1106 may be a wild type of the target cell 1102 in assays where the target cell 1102 is engineered or transfected. In other examples, the control cell 1106 may be engineered to transfected in assays where the target cell 1102 is a wild type cell (e.g., control cell 1106 may be engineered or transfected to prevent and / or inhibit expression of the target molecule 1104). The assay can also include a secondary antibody 1110 that can tag the target molecule 1104. The secondary antibody 1110 can include a fluorophore to allow for fluorescent imaging of the variant protein 1108, The control cell 1106 can be stained with a cell tracker dye. Fluorescence images of the control cell 1106 can be compared with fluorescence images in the secondary antibody 1110 channel. In the example specific antibodies binding to the transfected cells can be identified.

[0138] FIG. 11 B shows microscopy images of assays in accordance with the scheme of FIG. 11 A, Images from four different microcapillaries, numbered 1-4, are shown. All of microcapillaries 1-4 represent “hits”: microcapillaries where the variant protein 1108 was capable of specific binding to the target molecule 1104. The microscopy images in the brightfield channel, the secondary antibody 1110 channel (referred to in FIG. 1 IB as the “GPRC5D HEK Binding” channel), and the stained control cell 1106 channel (referred to m FIG. 11B as the “WT HEK Celltracker” channel). In microcapillaries 1-4, there appears to be no overlap between the cells tagged in the GPRC5D HEK Binding channel and the WT HEK Celltracker channel, which may indicate that the variant protein 1108 binds specifically to the target molecule 1104 of the target cell 1102. Microcapillaries that would not be of interest in this assay would be those where (1) there was some or substantial overlap between the signals in the secondary antibody 1110 channel and the stained control cell 1106 channel or (2) where there was no signal or minimal signal in the secondary antibody 1110 channel.Single- Arm Affinity and Avidity

[0139] Certain assays may be particularly suited for screening the binding ability of single arms of bispecific antibodies to target molecules. For instance, certain assays may tune the surface density of target molecules on a surface (e.g., a bead surface) so as to increase or decrease opportunities for antibodies to bind to a single target molecule.

[0140] FIG. 12A shows an example scheme for examining affinity and avidity of an antibody 1206. A bead 1202 can include a surface 1204, on which target molecules 1208 and non-target molecules 1210 can be attached. The target molecule 1208 can be a molecule to which the antibody 1206 is likely to bind. The non-target molecule 1210 can be a molecule to which the antibody 1206 is not likely to bind.

[0141] The surface density of each of the target molecules 1208 and the non-target molecules 1210 can be tuned such that the antibody 1206 is likely to bind to a single target molecule 1208. For instance, FIG. 12B shows an example second bead 1212 that can include target molecules 1208 at a higher surface density than on the bead 1202. Though FIG 12B depicts the second bead 1212, it is to be understood that an assay in accordance with the present disclosure can include a single type of bead (e.g., either of the beads 1202 or 1212) or multiple types of beads (e.g., both of beads 1202 and 1212 or more).

[0142] Though FIGS. 12A and 12B depict beads 1202 and 1212 including non-target molecules 1210, it is to be understood that the beads 1202 and 1212 need not include the non-target molecules 1210. In such examples, the surface density of the target molecules 1208 on the beads 1202 or 1212 can be tuned through other means so that the antibody 1206 only binds to a single target molecule 1208 via a single arm of the antibody 1206.

[0143] In some examples, the non-target molecules 1210 can be relatively inert or unlikely to bind a screened antibody. In some examples, the non-target molecules 1210 can include proteins. In some examples, the non-target molecules 1210 can be biotin, BSA, the like, or a combination.

[0144] Beads in accordance with the assay for FIGS. 12A and 12B may include target molecules and non-target molecules. In some examples, the ratio of target molecules to non-target molecules on the surface of the bead is 1000:1, 500:1, 250:1, 200:1, 100:1, 50:1, 25:1, 20:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:20, 1:25, 1:50, 1:100, 1:200, 1:250, 1:500, or 1:1000, or within a range defined by any two of the previously- enumerated ratios, though in some instances other ratios may be suitably implemented. In some examples, the ratio of target molecules to non-target molecules is from 100: 1 to 1: 100. In some examples, the ratio of target molecules to non-target molecules is from 10:1 to 1:10. In some examples, the ratio of target molecules to non-target molecules is from 2:1 to 1:2.

[0145] FIG. 12C shows results of assays using beads including target molecules and non-target molecules at different ratios of surface densities. Three conditions were examined: Standard; Condition 1; and Condition 2. The standard beads included only target proteins at the standard surface density. Condition 1 and Condition 2 included beads having molecules bound to the surface at the same overall surface density as the standard condition. However, the beads of Condition 1 included target molecules and non-target (e.g., inert) molecules at a ratio of 2 target molecules to every 1 inert molecule. The beads of Condition 2 included target molecules and non-target (e g., inert) molecules at a ratio of 1 target molecule to every 2 inert molecules,

[0146] Fluorescence intensity was measured in the ELISA Pos channel (a clone that was positive in ELISA but had poor affinity) and the benchmark (e.g,, a variant protein with a relatively high level of binding to the target molecule) channel. The signal to noise ratio of each of Condition 1 and Condition 2 appears higher than that of the standard.

[0147] For screening assays, suitable ratios of target molecule to non-target molecule can be empirically determined. Suitable ratios of target molecule to non-target molecule may vary depending on the particular target molecule and / or the particular non-target molecule used. It may be desirable to choose a ratio of target molecule to non-target molecule that allows for a signal-to-noise ratio of at least about 2.5 or at least about 3, though m some instances other signal-to-noise ratios may be suitable. Additionally or alternatively, a signal-to-noise ratio of 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0 or greater may be suitable. As the target molecule to non-target molecule ratio decreases, the signal corresponding to binding of the variant protein to the target may begin to decrease. It may be desirable to choose a ratio of target molecule to non-target molecule that does not result in a high decrease in target molecule signal. It may be desirable that the control and the benchmark have a separation resolution of 2 or greater for such an assay, though in some instances other separation resolutions may be suitable. In some examples, the control may be a known binder of the target molecule. In some such examples, the control may be a clinically-validated benchmark.

[0148] FIGS. 12D and 12E plot results of a second example study employing the scheme of FIG. 12A, after optimization of the beads, and measured by surface plasmon resonance (SPR). FIG. 12D shows a quartile plot of KDfor the screened antibodies. FIG. 12Eplots kaas a function of kdfor the screened antibodies. The study subjected 341 variant proteins (e.g., antibodies) to screening assays in accordance with the present disclosure. Of these, 78% of the variant proteins that passed the screening process subsequently passed SPR validation. The median affinity of the antibodies was 3 nM. Most clones are between 1 nM and 10 nM, with some down to 100 pM. These clones were considered to be “high affinity” for the particular target.

[0149] FIG. 13A illustrates a scheme for a bead titration ladder assay. Such an assay can identify particularly high-affinity or avidity variant proteins. In such an assay, a microcapillary can include a first bead 1302 and a second bead 1304. Each of the first bead 1302 and the second bead 1304 can include respective surfaces 1306, on which target molecules 1308 are bound. The first bead 1302 can have a different surface density of target molecules 1308 as compared to the surface density of target molecules 1308 of the second bead 1304. For example, the surface density of target molecules 1308 on the second bead 1304 may be higher than the surface density of target molecules 1308 of the first bead 1302. A variant protein 1310 (e.g., a variant antibody) can be introduced to examine whether the variant protein 1310 binds to one or more of the first bead 1302 and second bead 1304. Exceedingly low- affinity variant proteins 1310 may be unable to bind to either the first bead 1302 or the second bead 1304. Low-affinity variant proteins 1310 may be able to bind to the bead having the higher surface density of target molecules 1308 (e.g., the second bead 1304 as shown in FIG.13 A), but unable to bind to the bead having the lower surface density of target molecules 1308 (e.g., the first bead 1302 as shown in FIG. 13 A). High-affinity variant proteins 1310 may be able to bind to both the bead having the higher surface density of target molecules 1308 (e.g., the second bead 1304 as shown in FIG. 13 A) and the bead having the lower surface density of target molecules 1308 (e.g., the first bead 1302 as shown in FIG. 13A). Though the scheme of FIG. 13 A discusses the first bead 1302 and second bead 1304 within the same microcapillary, it is to be understood that, in some instances, the first bead 1302 and the second bead 1304 can be in separate microcapillaries. In some examples, the separation of the first bead 1302 and second bead 1304 between separate microcapillaries may allow the first bead 1302 and second bead 1304 to be optically distinguished.

[0150] The first bead 1302 and the second bead 1304 can be optically distinguishable, as discussed herein. In some examples, the surface density of target molecules1308 on the first bead 1302 may be sufficiently low that the variant protein 1310 is likely to bind to only a single target molecule 1308. In some such examples, a surface density of the target molecules 1308 on the second bead 1304 may be sufficiently high such that the variant protein 1310 is likely to bind to two target molecules 1308.

[0151] In some examples, there may be more than two types of beads, each type of bead having a different surface density of target molecules 1308. In some examples, there may be 3, 4, 5, 6, 7, 8, 9, 10, or more different types of beads, each having a different surface density of target molecules 1308. Higher-affinity variant protein 1310 would be likely to show signal on more of the types of beads present in such an assay. Lower-affinity variant protein 1310 would show binding to fewer of the types of beads in such an assay. Each of the beads may be optically distinguishable. It may be desirable to choose a total number of bead types that may still be optically distinguishable.

[0152] FIG, 13B shows experimental results of a bead titration ladder assay optimization, for determining which target molecule surface densities are suitable for use in the scheme discussed with respect to FIG. 13 A. In the experiments reflected in FIG. 13B, the concentration of the target molecule (EGFR) on the bead surface was varied to identify antibodies in sera (“sera polyclonal”) that bind to EGFR with greater affinity than Panitumumab (5 x 10-11M), an approved anti-EGFR antibody. Three types of beads were coated with three different surface densities of EGFR: 0.2 pg / mg coated bead (Condition 1); 0.04 pg / mg coated bead (Condition 2); or 0.008 pg / mg coated bead (Condition 3). Condition 2, representing a concentration of 5x less than the standardly-used beads, clearly demonstrates that the sera polyclonal antibodies bind with a significantly higher affinity than the Panitumumab monoclonal antibodies. Condition 2 appears to allow for surface density where only a single arm of the variant antibody can bind, like the first bead 1302 as discussed with reference to FIG. 13 A.

[0153] FIGS. 13C-13F show microscope images of a bead titration ladder screening. Using two of the target surface densities examined in FIG. 13B, small beads were coated with the standard amount of target molecule (EGFR at 0.2 pg / mg coated bead; referred to as high-density target beads) and larger beads were coated with optimized amount (EGFR at 0.04 pg / mg coated bead, referred to as low-density target beads). Imaging can be used detect whether an antibody can bind to both the high-density target beads and the low-density targetbeads (i.e., a high-affinity antibody), or just the high-density target beads (i.e., a low-affinity antibody).

[0154] FIGS. 13C and 13D show brightfield and fluorescence images taken of a particularly high-affinity antibody. The high affinity antibody was able to bind to the high- density target beads (i.e., the small beads) and the lower-density target beads (i.e., the large beads, which are indicated with arrows).

[0155] FIGS. 13E and 13F show brightfield and fluorescence images taken of a low-affinity antibody. The low-affinity antibody was only able to bind to the high-density target beads (i.e., the small beads), but not the low-density target beads (i.e., the large beads, which are indicated in the brightfield images with arrows). Thus, the large beads are not apparent in the fluorescence images of FIG. 13E and 13F.Definitions

[0156] In the examples discussed herein, it is to be understood that the term “variant protein” can refer to a variant antibody or other protein. Variant proteins and / or variant polypeptides to be analyzed after library’ preparation in accordance with the present disclosure can include but are not limited to secreted proteins. In some embodiments, the secreted proteins are from a recombinant protein and / or polypeptide library. In some embodiments, the secreted proteins are from a recombinant protein and / or polypeptide library. In some embodiments, the secreted proteins are from a recombinant protein and / or polypeptide library from a mammalian cell line. In some embodiments, the recombinant protein and / or polypeptide library includes full length mammalian antibodies. In some embodiments, the recombinant protein and / or polypeptide library includes full length mammalian antibodies, including IgGl, IgG2, and IgG4 antibodies and variants thereof. In some embodiments, the recombinant protein and / or polypeptide library includes full length human antibodies. In some embodiments, the recombinant protein and / or polypeptide library includes full length human antibodies, including IgGl, IgG2, and IgG4 antibodies. In some embodiments, the recombinant protein and / or polypeptide library includes full length mouse antibodies. In some embodiments, the recombinant protein and / or polypeptide library includes full length mouse antibodies, including IgGl, IgG2, and IgG4 antibodies. In some embodiments, the recombinant protein and / or polypeptide library includes full length rat antibodies. In some embodiments, therecombinant protein and / or polypeptide library includes full length rat antibodies, including IgGl, IgG2, and IgG4 antibodies. In some embodiments, the recombinant protein and / or polypeptide library includes antibody fragments (Fab). In some embodiments, the recombinant protein and / or polypeptide library includes single chain variable fragments (scFv). In some embodiments, the recombinant protein and / or polypeptide library includes natural protein ligands. In some embodiments, the recombinant protein and / or polypeptide library includes natural protein ligands to a defined target molecule and / or polypeptide. In some embodiments, the recombinant protein and / or polypeptide library includes target antibodies and / or fragments thereof. In some embodiments, the recombinant protein and / or polypeptide library includes target antibody heavy chains and / or fragments thereof, such as variable heavy chains. In some embodiments, the recombinant protein and / or polypeptide library includes target antibody light chains and / or fragments thereof, such as variable light chains. In some embodiments, the system of the present invention allows for accurate pairing of VH / VL (variable heavy chains and variable light chains).

[0157] The term “antibody” is used in the broadest sense and includes, for example, an intact immunoglobulin or an antigen binding portion. Antigen binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. The term “antibody” can include tetrameric antibodies of two heavy chains and two light chains, as well as antigen binding fragments such as Fv, Fab and scFvs. In some cases, the present disclosure provides for bispecific antibodies that include at least one antigen binding domain as outlined herein.

[0158] The population of variant proteins is typically generated using a genetic library in a biological expression system, for example in an in vitro (i.e., cell-free) expression system or in an in vivo or cellular expression system. Exemplary cellular expression systems include, for example, animal systems (e.g., mammalian systems), fungal systems (e.g., yeast systems), bacterial systems, insect systems, or plant systems. In specific embodiments, the expression system is a mammalian system or a yeast system. In specific embodiments, the expression system is an avian system (for example, a chicken system). The expression system, whether cellular or cell-free, typically includes a library of genetic material encoding the population of variant proteins. Cellular expression systems may offer an advantage that cells with a desirable phenotype, for example cells that express a particular variant protein ofinterest, such as a variant protein capable of associating with an immobilized target molecule with high affinity, can be grown and multiplied, thus facilitating and simplifying the identification and characterization of the proteins of interest expressed by the cells. In some embodiments, the biological expression system includes a mammalian cell line. In some embodiments, the mammalian cell line is selected from the group consisting of CH0-K1, CHO-S, HEK293T, and / or any derivatives of these cell types. In some embodiments, the mammalian cell line is CH0-K1. In some embodiments, the mammalian cell line is CHO-S. In some embodiments, the mammalian cell line is HEK293T. In some embodiments, the mammalian cell line is selected from the group consisting of human, mouse, and / or rat hybridoma cell lines. In some embodiments, the mammalian cell line is a human hybridoma cell line. In some embodiments, the mammalian cell line is a mouse hybridoma cell line. In some embodiments, the mammalian cell line is a rat hybridoma cell line.

[0159] Genetic libraries encoding large populations of variant proteins are often utilized in systems relying on the process of directed evolution to identify proteins with advantageous properties, such as high-affinity binding to target molecules, stability, high expression, or particular spectroscopic, e.g., fluorescence, or enzymatic activities. Often the libraries include genetic fusions with sequences from the host expression system, for example fragments of proteins directing subcellular localization, where the expressed population of variant fusion proteins are directed by the targeting fragment to a particular location of the cell or virus particle for purposes of activity screening of the variant protein population. Large numbers of variant proteins (e.g., 106variants, 108variants, 1010variants, 1012variants, or even more variants) can be generated using routine bioengineering techniques, as is well known in the art. Such libraries can include any of the variant proteins described herein, including antibodies, antibody fragments, single chain variable fragments, or natural protein ligands. In some embodiments, the system of the present invention allows for accurate pairing of VH / VL (variable heavy chains and variable light chains).

[0160] Accordingly, in some embodiments, the variant proteins to be sequenced using the present methods are soluble proteins, for example soluble proteins that are secreted by a cellular expression system. Exemplary soluble variant proteins include antibodies and antibody fragments, alternative protein scaffolds, such as disulfide-bonded peptide scaffolds, extracellular domains of cell-surface receptor proteins, receptor ligands, such as, for example,G-protein coupled receptor ligands, other peptide hormones, lectins, and the like. Advantageously, when using a microcapillary array, the variant proteins screened for binding activity do not need to be covalently attached to the cell or virus that expresses them in order to be identified following a screening assay, since a variant protein with a desired binding activity’ and the cell that expressed it remain co-localized within the same microcapillary of a microcapillary array throughout a screening assay. Isolation of the contents of the desired microcapillary, followed by library preparation in accordance with the present disclosure and / or propagation of the cell or virus clone responsible for expression of the desired variant protein, thereby enables the identification and characterization of that protein. Unlike screening assays where a variant protein of interest is displayed by fusion of the protein to a molecule on the surface of a cell or virus particle, the variant proteins identified in the screening methods need not be altered in any way following their identification. The observed activities of the variant proteins in the screens are thus more likely to represent the actual activities of those proteins in their subsequent applications,

[0161] In other embodiments, however, it may be desirable for the variant proteins to be membrane-associated proteins, for example proteins remaining associated with the surface of a cell or a viral particle in an expression system. Screening of cell-associated variant proteins may be desirable where the variant protein and its target molecule mediate interactions between two cells within a biological tissue. The ability to screen against cell-associated variant proteins may also be desirable in screening for interactions with traditionally “non¬ druggable” protein targets, such as, for example, G-protein coupled receptors or ion channels.

[0162] In addition to a variant protein, each microcapillary of the plurality of microcapillaries in screening methods using microcapillary arrays also includes an immobilized target molecule. Such immobilized target molecules are immobilized to a surface. Such surfaces are discussed herein and may include the surface of a bead or a surface of a cell, though it should be understood that target molecules may be suitably immobilized to other surfaces. For instance, a target molecule may be immobilized to an interior surface of a microcapillary. The immobilized target molecule serves as the potential binding partner for the variant protein of the screening assay. Unlike the population of variant proteins, where each microcapillary of the plurality of microcapillaries ideally contains a variant protein of slightly different sequence, the immobilized target molecules ideally have the same molecular structurein each microcapillary of the plurality of microcapillaries of the array. In some embodiments, there is no binding or other interaction between the variant protein and another agent or molecule (e g., the target molecule) prior to the addition of the variant protein to the microcapillary. In some embodiments, the interaction between the variant protein and the target molecule occurs within the microcapillary and / or microcavity. As discussed herein, target molecules may include antigens and / or antibody-capture antibodies, among other suitable molecules.

[0163] In the examples discussed herein, it is to be understood that “reporter element” can refer to molecules capable of binding to a variant protein so as to indicate localization of the variant protein within a microcapillary. In some examples, such reporter elements may be secondary antibodies. Reporter elements may include a fluorophore.

Claims

WHAT IS CLAIMED IS:

1. A system for screening a population of variant proteins present in a microcapillary array using a multiplex assay, the system comprising:a microcapillary array comprising a plurality of microcapillaries that comprise a variant protein;a first bead, the first bead comprising a first target molecule immobilized on the surface of the first bead, wherein the variant protein associates with the first target molecule with a first affinity;a second bead, the second bead comprising a second target molecule immobilized on the surface of the second bead, wherein the variant protein associates with the second immobilized target molecule with a second affinity or the variant protein does not bind to the second immobilized target molecule, and wherein the first bead and the second bead are optically distinguishable; anda first reporter element, wherein the first reporter element associates with the variant protein.

2. The system of claim 1, wherein the first bead comprises a first size and wherein the second bead comprises a second size, the first and second sizes being optically distinguishable.

3. The system of claim 1 or 2, wherein the first bead comprises a first fluorophore, wherein the second bead comprises a second fluorophore, and wherein the first fluorophore and the second fluorophore are optically distinguishable.

4. The system of claim 1 or 2, wherein the first bead comprises a first fluorophore and a second fluorophore, wherein the second bead comprises only the second fluorophore, and wherein the first fluorophore and the second fluorophore are optically distinguishable.

5. The system of claim 3 or 4, comprising a second reporter element, wherein the second reporter element comprises the second fluorophore, and wherein the first reporter element comprises the first fluorophore.

6. The system of any one of claims 1-5, comprising a third bead, the third bead comprising a third target molecule immobilized on the surface of the third bead, wherein the third bead is optically distinguishable from the first bead and the second bead, and wherein the variant protein associates with the third immobilized target molecule with a third affinity.

7. The system of claim 6, comprising a fourth bead, the fourth bead comprising a fourth target molecule immobilized on the surface of the fourth bead, wherein the fourth bead is optically distinguishable from the first bead, the second bead, and the third bead, and wherein the variant protein associates with the fourth immobilized target molecule with a fourth affinity.

8. A system for screening a population of variant proteins present in a microcapillary array using a multiplex assay, the system comprising:a microcapillary array comprising a plurality of microcapillaries that comprise a variant protein;a first cell, the first cell expressing a first target molecule, wherein the first target molecule is localized on a surface of the first cell, wherein the variant protein associates with the first target molecule with a first affinity;a second cell, wherein the second cell does not express the first target molecule; anda first reporter element, wherein the first reporter element associates with the variant protein.

9. The system of claim 8, wherein the first cell comprises an HEK cell, a CHO cell, or a yeast cell.

10. The system of claim 8 or 9, wherein the second cell comprises an HEK cell, a CHO cell, or a yeast cell.

11. The system of any one of claims 8-10, wherein the second cell is a wild type cell and the first cell is an engineered or transfected to express or overexpress the first target molecule.

12. The system of any one of claims 8—11, wherein the first cell is a wild type cell and the second cell is an engineered or transfected to not express or under-express the first target molecule.

13. The system of claim 12, wherein the second cell is dyed.

14. A system for screening a variant protein in a microcapillary of a microcapillary array using a multiplex assay, the system comprising:a first target molecule immobilized on a first surface, wherein the variant protein associates with the first immobilized target molecule with a first affinity; a second target molecule immobilized on a second surface, wherein the variant protein associates with the second immobilized target molecule with a second affinity or the variant protein does not bind to the second immobilized target molecule, and wherein the first surface and the second surface are optically distinguishable; and a first reporter element, wherein the first reporter element associates with the variant protein.

15. The system of claim 14, wherein the first surface is a surface of a first bead.

16. The system of claim 15, wherein the second surface is a surface of a second bead.

17. The system of claim 16, wherein the first bead comprises a first size and wherein the second bead comprises a second size, the first and second sizes being optically distinguishable.

18. The system of any one of claims 14-17, wherein the first surface comprises a first fluorophore, wherein the second surface comprises a second fluorophore, and wherein the first fluorophore and the second fluorophore are optically distinguishable.

19. The system of claims 15 or 18, wherein the first surface is a surface of a first cell.

20. The system of claim 14-15 and 19, wherein the second surface is a surface of a second cell.

21. The system of claim 19 or 20, comprising a first fluorophore that associates with the first surface and a second fluorophore that associates with the second surface.

22. The system of any one of claims 1-21, comprising a benchmark protein, wherein the benchmark protein associates with the first target molecule.

23. The system of claim 22, wherein the benchmark protein associates with the first target molecule at an affinity lower than the first affinity, and wherein the benchmark protein and the variant protein associate with the first target molecule at the same epitope on the first target molecule.

24. The system of claim 22, wherein the benchmark protein associates with the first target molecule at an affinity higher than the first affinity, and wherein the benchmark protein and the variant protein associate with the first target molecule at the same epitope on the first target molecule.

25. The system of claim 22, wherein the benchmark protein associates with a first epitope of the first target molecule and the variant protein associates with a second epitope of the first target molecule, wherein the first epitope and the second epitope are different.

26. The system of any one of claims 22-25, comprising a second reporter element, wherein the second reporter element binds to the benchmark protein, and wherein the first reporter element binds to the variant protein.

27. The system of claim 26, wherein the first reporter element comprises a first fluorophore, wherein the second reporter element comprises a second fluorophore, and wherein the first fluorophore and second fluorophore are optically distinguishable.

28. The system of any one of claims 23-25, comprising a second benchmark protein, wherein the second benchmark protein associates with the second target molecule atan affinity lower than the second affinity, and wherein the second benchmark protein and the variant protein associate with second target molecule at the same epitope on the second target molecule.

29. The system of any one of claims 23 to 25, comprising a second benchmark protein, wherein the second benchmark protein associates with the second target molecule at an affinity higher than the second affinity, and wherein the second benchmark protein and the variant protein associate with second target molecule at the same epitope on the second target molecule.

30. The system of any one of claims 23 to 25, comprising a second benchmark protein, wherein the second benchmark protein associates with a first epitope of the second target molecule and the variant protein associates with a second epitope of the second target molecule.

31. The system of any one of claims 1-30, wherein the first target molecule and second target molecule are the same.

32. The system of any one of claims 1-31, wherein the reporter element comprises a labeled antibody or other binding molecule.

33. The system of any one of claims 1-32, wherein the reporter element comprises a fluorophore.

34. The system of any one of claims 1-33, comprising a sample extraction device configured to isolate a sample of a microcapillary of interest based at least in part on localization of the reporter element.

35. The system of any one of claims 1-33, comprising a means for isolating a sample of a microcapillary of interest based at least in part on localization of the reporter element.

36. The system of any one of claims 1-35, wherein the variant protein comprises an antibody.

37. The system of any one of claims 1–36, comprising a cell expressing the variant protein.

38. The system of claim 37, wherein the cell is a B cell.

39. The system of any one of claims 1–38, comprising a microcapillary chip, the microcapillary chip comprising the plurality of microcapillaries,40. The system of any one of claims 1-39, comprising:an imaging device configured to image the contents of the plurality of microcapillaries; andan extraction device.

41. The system of claim 40, comprising:a processor in communication with the imaging device and the extraction device; the processor executing instructions to:identify the first bead or first surface and the second bead or second surface;determine a microcapillary of interest based at least in part on localization of the reporter element with a control reporter element; and cause the extraction device to eject the contents of the microcapillary of interest of the plurality of microcapillaries.

42. The system of claim 40 or 41, wherein the imaging device is a fluorescence microscope.

43. A method of screening a population of variant proteins using a multiplex assay, the method comprising:providing a microcapillary array comprising a plurality of microcapillaries that comprise:a variant protein;a first target molecule immobilized to a first surface;a second target molecule immobilized to a second surface, wherein the first surface and the second surface are optically distinguishable, wherein the variant protein associates with the first immobilized target molecule with a first affinity and with the second immobilized target molecule with a second affinity or the variant protein does not bind to the second immobilized target molecule; anda first reporter element, wherein the first reporter element associates with the variant protein;measuring optical signals from the first reporter element, the first surface, and the second surface that indicate association of the variant protein with the first immobilized target molecule and / or the second immobilized target molecule; and identifying at least one microcapillary of interest based at least in part on the measured signals.

44. The method of claim 43, wherein the first surface comprises a first bead.

45. The method of claim 43 or 44, wherein the second surface comprises a second bead.

46. The method of any one of claim 43-45, wherein the first surface comprises a first size and wherein the second surface comprises a second size, the first and second sizes being optically distinguishable.

47. The method of claim 43 or 45, w’herein the first surface comprises a first cell.

48. The method of any one of claims 43-44 or 46-47, wherein the second surface comprises a second cell.

49. The method of any one of claims 43-48, wherein the first surface comprises a first fluorophore, wherein the second surface comprises a second fluorophore, and wherein the first fluorophore and the second fluorophore are optically distinguishable.

50. The method of any one of claims 43-48, wherein the first surface comprises a first fluorophore and a second fluorophore, wherein the second surface comprises only the second fluorophore, and wherein the first fluorophore and the second fluorophore are optically distinguishable.

51. The method of claim 49 or 50, each microcapillary of the plurality of microcapillaries comprising a second reporter element, wherein the second reporter element comprises the second fluorophore, and wherein the first reporter element comprises the first fluorophore,52. The method of claim 49, wherein the measuring optical signals comprises using a plurality of optical filters to filter a first signal of the first fluorophore and a second signal of the second fluorophore.

53. The method of claim 52, wherein the identifying at least one microcapillary of interest is based at least in part on the measured signals of the first fluorophore and the second fluorophore.

54. The method of any one of claims 43-53,wherein the microcapillary comprises a third surface, the third surface comprising a third target molecule immobilized on the third surface, wherein the third surface is optically distinguishable from the first surface and the second surface, and wherein the variant protein associates with the third immobilized target molecule with a third affinity,the method further comprising measuring a signal from the third surface.

55. The method of claim 54, wherein the third surface comprises a third bead.

56. The method of claim 54, wherein the third surface comprises a third cell.

57. The method of any one of claims 54-56, whereinthe microcapillary comprises a fourth surface, the fourth surface comprising a fourth target molecule immobilized to the fourth surface, wherein the fourth surface is optically distinguishable from the first surface, the second surface, and the third surface, and wherein the variant protein associates with the fourth immobilized target molecule with a fourth affinity,the method further comprising measuring a signal from the fourth surface,58. The method of any one of claims 43-57, comprising determining, using a processor, positions of the first surface and the second surface.

59. The method of any one of claims 43-58, wherein the identifying at least one microcapillary of interest is based at least in part on the sizes of the first surface and the second surface.

60. The method of any one of claims 43 -59, wherein the microcapillary comprises a benchmark protein, wherein the benchmark protein associates with the first target molecule.

61. The method of claim 60, wherein the benchmark protein and the variant protein both associate with the first target molecule at the same epitope.

62. The method of claim 60, wherein the benchmark protein associates with the first target molecule at a first epitope, wherein the variant protein associates with the first target molecule at a second epitope, and wherein the first epitope and the second epitope are different.

63. The method of any one of claims 60-62, each microcapillary of the plurality of microcapillaries comprising a second reporter element, wherein the second reporter element associates with the benchmark protein.

64. The method of any one of claims 43-63, wherein the first target molecule and the second target molecule are the same.

65. A system for screening a population of variant proteins present in a microcapillary array using a multiplex assay, the system comprising:a microcapillary’ array comprising a plurality of microcapillaries that comprise a variant protein;a first bead, the first bead comprising a target molecule immobilized on the surface of the first bead at a first surface density, wherein the variant protein associates with the target molecule at an affinity;a second bead, the second bead comprising a target molecule immobilized on the surface of the second bead at a second surface density, wherein the first surface density and the second surface density are different, and wherein the first bead and the second bead are optically distinguishable; anda reporter element, wherein the reporter element associates with the variant protein,66. The system of claim 65, wherein the first bead comprises a first size and wherein the second bead comprises a second size, the first and second sizes being optically distinguishable.

67. The system of claim 65 or 66, wherein the first bead comprises a first fluorophore, wherein the second bead comprises a second fluorophore, and wherein the first fluorophore and the second fluorophore are optically distinguishable.

68. The system of claim 65 or 66, wherein the first bead comprises a first fluorophore and a second fluorophore, wherein the second bead comprises only the second fluorophore, and wherein the first fluorophore and the second fluorophore are optically distinguishable.

69. The system of claim 67 or 68, comprising a second reporter element, wherein the second reporter element comprises the second fluorophore, and wherein the reporter element comprises the first fluorophore.

70. The system of any one of claims 65-69, comprising a third bead, the third bead comprising a third target molecule immobilized on the surface of the third bead, wherein thethird bead is optically distinguishable from the first bead and the second bead, and wherein the variant protein associates with the third immobilized target molecule with a third surface density.

71. The system of claim 70, comprising a fourth bead, the fourth bead comprising a fourth target molecule immobilized on the surface of the fourth bead, wherein the fourth bead is optically distinguishable from the first bead, the second bead, and the third bead, and wherein the variant protein associates with the fourth immobilized target molecule with a fourth surface density.

72. The system of any one of claims 65-71, wherein the first bead comprises a nontarget molecule immobilized on the surface of the first bead at a third surface density.

73. The system of claim 72, wherein the second bead comprises a non-target molecule immobilized on the surface of the second bead at a fourth surface density,74. The system of claim 73, wherein the third surface density and fourth surface density are different.

75. The system of claim 73, wherein the third surface density and fourth surface density are approximately the same.

76. A system for screening a population of variant proteins present in a microcapillary array using a multiplex assay, the system comprising:a microcapillary array comprising a plurality of microcapillaries that comprise a variant protein;a bead, the bead comprising a target molecule immobilized on the surface of the bead at a first surface density, and a non-target molecule immobilized on the surface of the bead at a second surface density, wherein the variant protein associates with the target molecule with an affinity; anda reporter element, wherein the reporter element associates with the variant protein.

77. The system of claim 76, wherein the ratio of the first surface density to the second density is from 100:1 to 1:100.

78. The system of claim 77, wherein the ratio of the first surface density to the second density is from 10:1 to 1:10.

79. The system of claim 78, wherein the ratio of the first surface density to the second density is from 2:1 to 1:2.

80. A system for screening a population of variant proteins present in a microcapillary array using a multiplex assay, the system comprising:a microcapillary array comprising a plurality of microcapillaries that comprise a variant protein;a bead, the bead comprising a target molecule immobilized on the surface of the bead at a first surface density, wherein the first surface density is below a threshold density at which the variant protein is able to simultaneously bind to two target molecules; anda reporter element, wherein the reporter element associates with the variant protein.

81. The system of any one of claims 65-80, wherein the reporter element comprises a labeled antibody or other binding molecule.

82. The system of any one of claims 65-81, wherein the reporter element comprises a fluorophore.

83. The system of any one of claims 65-82, comprising a sample extraction device configured to isolate a sample of a microcapillary of interest based at least in part on localization of the reporter element.

84. The system of any one of claims 65-83, wherein the variant protein comprises an antibody.

85. The system of any one of claims 65-84, comprising a cell expressing the variant protein.

86. The system of claim 85, wherein the cell is a B cell.

87. The system of any one of claims 65-86, comprising a microcapillary chip, the microcapillary chip comprising the plurality of microcapillaries.

88. The system of any one of claims 65-87, comprising:an imaging device configured to image the contents of the plurality of microcapillaries; andan extraction device.

89. The system of claim 88, comprising:a processor in communication with the imaging device and the extraction device; the processor executing instructions to:identify the first bead or first surface and the second bead or second surface;determine a microcapillary of interest based at least in part on localization of the reporter element with a control reporter element; and cause the extraction device to eject the contents of the microcapillary of interest of the plurality of microcapillaries.

90. The system of claim 88 or 89, wherein the imaging device is a fluorescence microscope.

91. A method of screening a population of variant proteins using a multiplex assay, the method comprising:providing a microcapillary array comprising a plurality of microcapillaries, at least some of the plurality of microcapillaries comprising:a variant protein;a first bead;a second bead;a target molecule immobilized to the surfaces of the first bead at a first surface density and the second bead at a second density, wherein the variant protein associates with the target molecule at an affinity; anda reporter element, wherein the reporter element associates with the variant protein;measuring optical signals from the reporter element, the first bead, and the second bead that indicate association of the variant protein with the immobilized target molecule; andidentifying at least one microcapillary of interest based at least in part on the measured signals,92. The method of claim 91, comprising distinguishing the first bead and the second bead based on their respective sizes.

93. The method of claim 91 or 92, wherein the first bead comprises a first fluorophore, wherein the second bead comprises a second fluorophore, wherein the method comprises distinguishing the first bead and the second bead based at least in part on the signal of the first fluorophore and the second fluorophore.

94. The method of claim 91 or 92, wherein the first bead comprises a first fluorophore and a second fluorophore, wherein the second bead comprises only the second fluorophore, and wherein the method comprises distinguishing the first bead and the second bead based at least in part on the respective signal of the first fluorophore and the second fluorophore.

95. The method of claim 93 or 94, wherein at least some of the microcapillaries comprise a second reporter element, wherein the second reporter element comprises the second fluorophore, and wherein the reporter element comprises the first fluorophore.

96. The method of claim 91–95, wherein at least some of the microcapillaries comprise a third bead, the third bead comprising a third target molecule immobilized on the surface of the third bead, and wherein the variant protein associates with the third immobilized target molecule with a third surface density.

97. The method of claim 96, wherein at least some of the microcapillaries comprise a fourth bead, the fourth bead comprising a fourth target molecule immobilized on the surface of the fourth bead, wherein the fourth bead is optically distinguishable from the first bead, the second bead, and the third bead, and wherein the variant protein associates with the fourth immobilized target molecule with a fourth surface density.

98. The method of claim 91–97, wherein the first bead comprises a non-target molecule immobilized on the surface of the first bead at a third surface density,99. The method of claim 98, wherein the second bead comprises a non-target molecule immobilized on the surface of the second bead at a fourth surface density.

100. The method of claim 99, wherein the third surface density and fourth surface density are different.

101. The method of claim 99, wherein the third surface density and fourth surface density are approximately the same.

102. A method of screening a population of variant proteins using a multiplex assay, the method comprising:providing a microcapillary array comprising a plurality of microcapillaries, at least some of the plurality of microcapillaries comprising:a variant protein;a bead;a target molecule immobilized to the surface of the bead at a first surface density and a non-target molecule immobilized on the surface of the bead at a second surface density, wherein the variant protein associates with the target molecule with an affinity; anda reporter element, wherein the reporter element associates with the variant protein;measuring optical signals from the reporter element and the bead that indicate association of the variant protein with the immobilized target molecule; and identifying at least one microcapillary of interest based at least in part on the measured signals.

103. The method of claim 102, wherein the ratio of the first surface density to the second is from 100:1 to 1:100.

104. The method of claim 103, wherein the ratio of the first surface density to the second is from 10:1 to 1:10,105. The method of claim 104, wherein the ratio of the first surface density to the second is from 2:1 to 1:2.

106. The method of any one of claims 102 to 105, wherein the variant protein is an antibody.

107. The method of claim 106, wherein the association of the variant protein with the target molecule is indicative of single-arm affinity.

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