Quantitative beads, kits and methods for immunoassay

Abstract

A surface-modified bead comprising streptavidin-biotinylated protein G is provided for quantitative flow cytometry. These quantitative streptavidin-Protein G-biotin beads (qBeads) can be used for qFCM analysis without limitations for a wide range of applications.

Description

Attorney Docket No: 122Z-405624-WOQUANTITATIVE BEADS, KITS AND METHODS FOR IMMUNOASSAYCROSS REFERENCE TO REEATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of the United States Provisional Application Serial No. 63 / 790,713, filed April 17, 2025, the content of which is hereby incorporated by reference in its entirety.TECHNICAL FILED

[0002] The present disclosure is directed to a surface-modified bead useful for immunoassay, a detection kit used in the immunoassay, and a method for carrying out an immunoassay using the kit.BACKGROUND

[0003] Flow cytometry is a powerful analytical technique used to measure the physical and chemical properties of cells or particles as they flow through a laser beam. It provides valuable insights into cell surface or intracellular protein expression, as well as cellular changes at the single-cell level by detecting light scatter and fluorescence signals emitted from cells or particles labeled with specific antibodies or dyes. Due to its versatility and high-throughput capabilities, flow cytometry has been widely adopted in biological assays, particularly in studying the functions and identity of cancer cells, stem cells, and immune cells.

[0004] Although most flow cytometry applications remain qualitative, quantitative flow cytometry (qFCM) has emerged as a critical tool in both research and clinical diagnostics, enabling the absolute quantification of molecules at the single-cell level. This quantification is achieved by using calibrated beads with known quantities of antibodies or fluorophores, expressed as molecules of equivalent soluble fluorophore (MESF) or antibody-binding capacity (ABC). Unlike conventional flow cytometry, which typically provides qualitative or semi-quantitative data, qFCM measures the absolute number of surface or intracellular molecules. This capability offers valuable insights into cellular dynamics, signaling pathways, and therapeutic responses, making qFCM an essential tool for biological analysis. As a result, qFCM has been actively developed for biomedical research, particularly in the fields ofAttorney Docket No: 122Z-405624-WOhematological malignancies, immune deficiencies, and cancer diagnostics. For example, qFCM plays a critical role in monitoring CD4 counts in HIV patients and assessing minimal residual disease (MRD) in leukemia.

[0005] Current quantitative flow cytometry techniques often rely on chemical conjugation methods to label fluorophores or antibodies onto beads. Several commercial kits, such as Quantibrite, Quantum Simply Cellular, and Quantum, have been broadly used for qFCM. In this process, a defined amount of activated anti-antibodies or fluorophores is incubated with chemically activated beads to enforce conjugation. The resulting beads are then used for quantitative analysis. However, these methods are prone to significant batch-to-batch variability, particularly due to the inefficiencies and inconsistencies inherent in the conjugation processes. An additional limitation of this approach includes the complexity of antibody binding stoichiometry. As each anti-antibody on the beads has two binding sites for antibody while each antibody has two antigen binding sites for each antigen on cells, accurately determining binding density can be challenging, often leading to overestimation or underestimation of binding capacity.SUMMARY

[0006] In one aspect, provided is a surface-modified bead comprising (i) a core bead; (ii) a first molecule conjugated to a surface of the core bead; and (iii) a second molecule binding to the first molecule, such that a complex is formed on the surface of the core bead, wherein the second molecule comprises a first moiety for binding to an antibody and a second moiety for binding to the first molecule, and the second moiety binds to the first molecule at a dissociation constant (KD) of not greater than IxlO12M; and at least one first molecule is not bound by the second molecule.

[0007] In another aspect, provided is a complex comprising (i) the surface-modified bead as provided herein, and (ii) a fluorochrome-conjugated antibody or an antigen-binding fragment thereof, wherein the fluorochrome-conjugated antibody is complexed to the second molecules.

[0008] In another aspect, provided is a kit comprising a first container and a second container, wherein the first container is provided with a first surface-modified bead as provided herein, and the second container is provided with a second surface-modified bead as provided herein,Attorney Docket No: 122Z-405624-WOand wherein the first surface-modified bead and the second surface-modified bead differ only in a copy number of the second molecule, wherein a ratio of the copy number of the second molecule for the first surface-modified bead to that for the second surface-modified bead is in a range of greater than 1x10° to IxlO8, preferably from IxlO1to IxlO6.

[0009] In another aspect, provided is a method for determining a copy number of a protein expressed by a cell, comprising providing a sample comprising the cell; providing a kit as provided herein; adding a fluorochrome-conjugated antibody that binds specifically to the protein expressed by the cell into the sample and each container of the kit; plotting a standard curve of mean fluorescence intensity versus copy number of the second molecule according to the kit; and determining the copy number of the protein by the mean fluorescence intensity obtained from the sample.

[0010] In another aspect, provided is a method for determining a copy number of a protein expressed by a cell, comprising providing a sample comprising the cell; providing a kit as provided herein; adding a primary antibody that binds specifically to the protein expressed by the cell into the sample; adding a fluorochrome-conjugated secondary antibody that binds specifically to the primary antibody into the sample and each container of the kit; plotting a standard curve of mean fluorescence intensity versus copy number of the second molecule according to the kit; and determining the copy number of the protein by the mean fluorescence intensity obtained from the sample.

[0011] In an example of this disclosure, the use of streptavidin-biotin conjugation was investigated as a more reliable and reproducible method for generating quantitative flow cytometry beads. The streptavidin-biotin interaction is one of the strongest noncovalent proteinligand interactions known, with a dissociation constant (KD) of less than 10"15M, ensuring nearly 100% conjugation efficiency, minimizing variability and uncertainties associated with current qFCM techniques. Further experiments validated that these quantitative streptavidin-Protein G-biotin beads (qBeads) can be used for qFCM analysis without limitations for a wide range of applications.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1A-F. Streptavidin-Biotin-FITC Conjugation and Analysis. A. Diagram ofAttorney Docket No: 122Z-405624-WOthe Streptavidin-Biotin-FITC conjugation process. Polystyrene beads coated with chemically conjugated streptavidin were incubated with different quantities of Biotin- FITC. B. After conjugation and washing, the samples were observed under UV light; or C. Flow cytometry analysis using an Accuri C6 flow cytometer. The mean fluorescence intensity (MFI) and presumed molecules per bead was listed on panel D. E. and F. The MFI was then plotted against the presumed of molecules of Biotin-FITC per bead.

[0013] FIG. 2A-C. Production of Protein G-Biotin. A. Diagram of the Protein G-Biotin production process. The Protein-G with Avitag at N- and C-temiini was co-expressed with GST-BirA, and purified with Ni-NTA affinity beads. After in vitro biotinylation, Protein-G-Biotin was further purified with Ni-NTA and Streptavidin- Mutein agarose. B. Coomassie blue staining of samples from the purification of Protein G-Biotin (samples 1-6) and GST-BirA (samples 7-9). Sample order: 1, before binding; 2, flow-through; 3, 50 mM imidazole fraction; 4. 250 mM imidazole fraction; 5, 500 mM imidazole fraction; 6, 1000 mM imidazole fraction; 7, before binding; 8. flow-through; 9, 50 mM glutathione fraction. C. In vitro biotinylation and purification of Protein G-Biotin. Samples: 1-2, before binding; 3-4, unbound fraction from Ni-NTA agarose; 5-6, unbound fraction from streptavidin-mutein agarose; 7-8, eluted fraction from streptavidin-mutein agarose (final Protein G-Biotin).

[0014] FIG. 3A-F. Quantitative Streptavidin-Protein G-Biotin Beads (qBeads) with defined copy numbers of binding ligands. A. Diagram of qBeads conjugation process. Streptavidin beads were mixed with varying quantities of Protein G-Biotin. B. The qBeads were incubated with fluorescence-labeled antibodies. C. The samples were analyzed using an Accuri C6 flow cytometer. D. The MFI and the presumed molecules per bead was listed. E. and F., the MFI was then plotted against the presumed of molecules of Biotin-FITC per bead.

[0015] FIG. 4A-F. Quantification of CD19 and CD20 on Raji and Daudi Cells using qBeads. A. Flow cytometry was performed to analyze bead samples conjugated with different amounts of anti-CD19-APC or anti-CD20-FITC using 10 pm qBeads. B. The mean fluorescence intensity (MFI) and the presumed molecules per bead are summarized. C. and D. The MFI was plotted against the presumed molecules per bead (C., anti-CD19-APC; D., anti-CD20-FITC). E. Flow cytometry analysis of Raji and Daudi cells using the same anti-CD19-APC and anti-CD20-FITC antibodies. F. Based on the MFI values obtained for Raji and DaudiAttorney Docket No: 122Z-405624-WOcells (E.), the copy numbers of CD 19 and CD20 were calculated.

[0016] FIG. 5A-F. Quantification of uPAR on MDA-MB-231 and A549 cells directly labeled with antibodies and qBeads. A. Flow cytometry analysis of bead samples conjugated with varying amounts of Alexa488-labeled uPar-Abl or uPar-Ab2 using 10 pm qbeads. B. The mean fluorescence intensity (MFI) and the presumed molecules per bead are summarized. C. and D. The MFI was plotted against the presumed molecules per bead (C., uPar-Abl, and D. uPar-Ab2). E. Based on the MFI values for MDA-MB-231 and A549 cells, the copy numbers of uPar were calculated using either antibody.

[0017] FIG. 6A-F. Quantification of uPar on MDA-MB-231 and A549 cells indirectly labeled with antibodies and qBeads. A. Flow cytometry analysis of bead samples conjugated with different amounts ofuPar-Abl or uPar-Ab2 using 10 pm qbeads in an indirect labeling method (see Methods and Materials). B. The mean fluorescence intensity (MFI) and the presumed molecules per bead are summarized. C. and D. The MFI was plotted against the presumed molecules per bead (C., uPar-Abl, and D. uPar-Ab2). E. Flow cytometry analysis of MDA-MB-231 and A549 cells using indirect labeling method (see Methods and Materials). F. Based on the MFI values for MDA-MB-231 and A549 cells, the copy numbers of uPar were calculated using either antibody.DETAILED DESCRIPTIONDefinition

[0018] As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0019] As used herein, the term “about” means that the recited numerical value is approximate and small variations would not significantly affect the practice of the disclosed embodiments. Where a numerical value is used, unless indicated otherwise by the context, the term “about” means the numerical value can vary by ±10% and remain within the scope of the disclosed embodiments.

[0020] As used herein, the term “comprising” may be replaced with “consisting of’ or “consisting essentially of’ in particular embodiments as desired.

[0021] “Binding affinity" as that term is known in the art, generally refers to the strength ofAttorney Docket No: 122Z-405624-WOthe sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule for its binding partner can generally be represented by the dissociation equilibrium constant (KD or KD). There is an inverse relationship between KD (molar) value and binding affinity, therefore the smaller the KD value (M), the higher the affinity. Thus “higher affinity” refers to antibodies that generally bind antigen stronger and / or faster and / or remain bound longer. Generally, a lower concentration (M) of antigen is needed to achieve the desired effect due to its stronger binding interaction. The term "KD" or “KD” (M) refers to the equilibrium dissociation constant of a particular interaction (e.g., antibody-antigen). Binding affinities can be determined by, e.g., surface plasmon resonance.

[0022] As used herein, the term “anti-Fc antibody” comprises an anti-Fcy antibody, an anti-Fca antibody, or an anti-Fcs antibody. The anti-Fcy antibody may comprise an anti-FcyRI antibody, an anti-FcyRIIA antibody, an anti-FcyRIIB antibody, an anti-FcyRIIB 1 antibody, an anti-FcyRIIB2 antibody, an anti-FcyRIIIA antibody, and / or an anti-FcyRIIIB antibody. The anti-Fca antibody may comprise an anti-FcaRI antibody. The anti-Fcs antibody may comprise an anti-FcsRI antibody or an anti-FcsRII antibody. As used herein, the temr “anti-Fc antibody” may also comprise an antigen binding fragment of an anti-Fc antibody.

[0023] As used herein, the term “Fc receptor” comprises an Fey receptor, an Fea receptor, and / or an Fes receptor. The Fey receptor may comprise an FcyRI receptor, an FcyRIIA receptor, an FcyRIIB receptor, an FcyRIIBl receptor, an FcyRIIB2 receptor, an FcyRIIIA receptor, an FcyRIIIB receptor, and / or an FcRn receptor. The Fea receptor may comprise an FcaRI receptor and / or an Fca / pR receptor. The Fes receptor may comprise an FcsRI receptor and / or an FcsRII receptor. The Fc receptor may also comprise a fragment of an Fc receptor capable of binding Fc.

[0024] As used herein, the term “antigen binding fragment” refers to a peptide fragment of a complete antibody that is capable of binding to the antigen independently. Such an antigen binding fragment is known in the art and includes, e.g., Fab, Fab’, F(ab’)2, Fv, and scFv.

[0025] As used herein, “Protein A” refers to a bacterial protein encoded by the spa gene andAttorney Docket No: 122Z-405624-WOwhich binds Fc and Fab regions of immunoglubulins. The DNA and polypeptide sequences for Protein A are known in the art, e.g., Staphylococcus Protein A (NCBI protein sequence WP_000728777.1, as of Sep. 8, 2022). Dozens of homologs of Protein A are known in the art and their sequences are available, e.g., in the NCBI database. Protein A comprises 4 to 5 Ig-binding domains, named Domain A, Domain B, Domain C, Domain D, and Domain F. The structure of Protein A is known in the art, e.g, for further discussion see Santos-Junior et al. BMC Microbiology 2016 16:143.

[0026] As used herein, “Protein G” refers to a bacterial protein which binds Fc and Fab regions of immunoglubulins. The DNA and polypeptide sequences for Protein G are known in the art, e.g., Protein G (UNIPROT protein sequence P06654, as of Sep. 8, 2022). Dozens of homologs of Protein G are known in the art and their sequences are available, e.g., in the NCBI and / or UNIPROT database. The structure of Protein G is known in the art.

[0027] As used herein, “Protein L” refers to a bacterial protein which binds Fc and Fab regions of immunoglubulins. The DNA and polypeptide sequences for Protein L are known in the art, e.g., Protein L (1YMH in the RCSB database, as of Sep. 8, 2022; see also Akerstrom et al. J Biol Chem 1989 264:19740-6, which is incorporated by reference herein in its entirety). Homologs of Protein L are known in the art and their sequences are available, e.g., in the NCBI and / or UNIPROT database. The structure of Protein L is known in the art.

[0028] Protein A / G is a recombinant protein that combines the IgG binding profiles of both Protein A and Protein G, offering a broadened specificity across various species and subclasses of IgG.

[0029] As used herein, the term “specific binding” refers to a chemical interaction between two molecules, compounds, cells and / or particles wherein the first entity binds to the second, target entity with greater specificity and affinity than it binds to a third entity which is a nontarget. In some embodiments of any of the aspects, specific binding can refer to an affinity of the first entity for the second target entity which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third nontarget entity. A reagent specific for a given target is one that exhibits specific binding for that target under the conditions of the assay being utilized. In some embodiments of any of the aspects, a reagent that binds specifically has the ability to bind to a target, such as an antigenAttorney Docket No: 122Z-405624-WOpresent on the cell-surface, with a KD 10’5M or less, e.g., IO’6M, IO’7M, IO’8M, IO’9M, IO10M, 10nM, 10'12M, or less. Specific binding can be influenced by, for example, the affinity and avidity of the antibody and the concentration of antibody.

[0030] As used herein, the term “molecule ratio” refers to the ratio of the number of a first molecule to the number of a second molecule and is equivalent to the molar ratio of the first molecule to the second molecule.

[0031] As used herein, the term “copy number” refers to the number of a molecule, e.g., expressed by a cell or conjugated on a solid support. Copy numbers can be calculated theoretically and stoichiometrically adjusted, which is known in the art.Surface-Modified Beads

[0032] In one aspect, provided is a surface-modified bead which comprises (i) a core bead; (ii) a first molecule conjugated to a surface of the core bead; and (iii) a second molecule binding to the first molecule, such that a complex is formed on the surface of the core bead, wherein the second molecule comprises a first moiety for binding to an antibody and a second moiety for binding to the first molecule, and the second moiety binds to the first molecule at a dissociation constant (KD) of not greater than about IxlO’12M; and at least one first molecule is not bound by the second molecule.

[0033] In the present disclosure, the second moiety and the first molecule constitute a binding pair, and the first molecule constitutes one component of the binding pair, and the second moiety constitutes the other component of the binding pair. In the present disclosure, the second moiety binds to the first molecule at a dissociation constant (KD) not greater than about IxlO’12M. In some embodiments, the second moiety binds to the first molecule at a KD of not greater than about IxlO’13M. In some embodiments, the second moiety binds to the first molecule at a KD of not greater than about IxlO’14M. In some embodiments, the second moiety binds to the first molecule at a KD of not greater than about IxlO’15M. For example, the second moiety binds to the first molecule at a KD of about IxlO’12M, about IxlO’13M, about 2xl0‘13M, about 3xl0‘13M, about 4xl0’13M, about 5xl0’13M, about 6xl0’13M, about 7xl0’13M, about 8xl0’13M, about 9xl0’13M, about IxlO’14M, about 2x10’14M, about 3x10’14M, about 4x10’14M, about 5xl0’14M, about 6xl0’14M, about 7xl0’14M, about 8xl0’14M, about 9xl0’14M, about IxlO’15Attorney Docket No: 122Z-405624-WOM, about 2xl0’13M, about SxlO-13M, about 4xl0-13M, about 5xl0'15M, about 6xl015M, about 7xl0’15M, about 8xl0’15M, about 9xl0’15M, or even less.

[0034] Exemplary binding pairs having a KD at pM (IO-12M) or fM (10’13M) scale include but are not limited to biotin-avidin binding pair, biotin-streptavidin binding pair, IL-2-CD25 binding pair, erythropoietin (EPO)-EPO receptor binding pair, anti-SARS-CoV-2 spike protein nanobody-SARS-CoV-2 spike protein binding pair, PDGF-PDGF aptamer binding pair, Spy Tag -SpyCatcher binding pair, and etc.

[0035] In some embodiments, the binding pair consists of a protein and a small molecule. In some embodiments, the binding pair consists of avidin and biotin. In some embodiments, the binding pair consists of streptavidin and biotin. In some embodiments, the first molecule is avidin, and the second moiety is biotin moiety. In some embodiments, the first molecule is biotin and the second moiety is avidin moiety. In some embodiments, the first molecule is streptavidin, and the second moiety is biotin moiety. In some embodiments, the first molecule is biotin and the second moiety is streptavidin moiety. Streptavidin- or avidin-coated beads or biotin-coated beads are all widely available in the art.

[0036] In some embodiments, the binding pair consists of two proteins. In some embodiments, the binding pair consists of SpyTag and SpyCatcher. In some embodiments, the first molecule is SpyTag, and the second moiety is SpyCatcher moiety. In some embodiments, the first molecule is SpyCatcher and the second moiety is SpyTag moiety.

[0037] In the present disclosure, the first moiety of the second molecule is provided for binding to an antibody. In some embodiments, the antibody is an antibody binding specifically to a tumor specific antigen (TSA). In some embodiments, the antibody is an antibody binding specifically to a tumor associated antigen (TAA). In some embodiments, the antibody is an IgG antibody, such as IgGl, lgG2, lgG3, or lgG4 antibody. In some embodiments, the antibody is a human antibody, a murine antibody (such as a rat antibody or mouse antibody), a human- murine chimeric antibody, a rabbit antibody, a goat antibody, or a camel antibody. In some embodiments, the antibody has a Fc fragment. In some embodiments, the antibody is an IgG antibody binding specifically to a TSA or TAA and having a Fc fragment.

[0038] Thus, In some embodiments, the first moiety of the second molecule is protein G, protein A, protein A / G, protein L, an anti-Fc antibody, or an Fc receptor. In some embodiments,Attorney Docket No: 122Z-405624-WOthe first moiety is protein G. In some embodiments, the first moiety is protein A. In some embodiments, the first moiety is protein A / G. In some embodiments, the first moiety is protein L. In some embodiments, the first moiety is an anti-Fc antibody. In some embodiments, the first moiety is an Fc receptor.

[0039] In some embodiments, the second molecule comprises the first moiety and the second moiety at a molecule ratio of 1:1. In some embodiments, the second molecule comprises the first moiety and the second moiety at a molecule ratio of 1 :2.

[0040] In the present disclosure, in the second molecule, the second moiety can be attached to the first moiety directly or indirectly. In some embodiments, the second moiety is attached to the first moiety directly. In some embodiments, the second moiety is a biotin moiety and the first moiety is protein G and the biotin moiety is covalently attached to the protein G directly through a lysine residue.

[0041] In some embodiments, the second moiety is attached to the first moiety indirectly, such as through a tag. In these embodiments, the second molecule further comprises a tag. Suitable tags include but are not limited to avi-tag, halo-tag, sortase-tag, snap-tag and clip-tag. In some embodiments, the tag is avi-tag. In some embodiments, the tag is halo-tag. In some embodiments, the first moiety is fused with a tag at N- or C-terminus, or both. In some embodiments, the first moiety is fused to a tag at N-terminus of the first moiety. In some embodiments, the first moiety is fused to a tag at C-terminus of the first moiety. In some embodiments, the first moiety is fused to two tags at N- and C-termini of the first moiety.

[0042] In some embodiments, the second molecule comprises protein G and a biotinylated tag (such as a biotinylated avi-tag). In some embodiments, the second molecule comprises from N-to C- terminus protein G and a biotinylated tag (such as a biotinylated avi-tag). In some embodiments, the second molecule comprises from N- to C- terminus a biotinylated tag (such as a biotinylated avi-tag) and protein G. In some embodiments, the second molecule comprises from N- to C-terminus a first biotinylated tag (such as a biotinylated avi-tag), protein G, and a second biotinylated tag (such as a biotinylated avi-tag).

[0043] In some embodiments, the second molecule comprises protein A and a biotinylated tag (such as a biotinylated avi-tag). In some embodiments, the second molecule comprises from N-to C- terminus protein A and a biotinylated tag (such as a biotinylated avi-tag). In someAttorney Docket No: 122Z-405624-WOembodiments, the second molecule comprises from N- to C- terminus a biotinylated tag (such as a biotinylated avi-tag) and protein A. In some embodiments, the second molecule comprises from N- to C-terminus a first biotinylated tag (such as a biotinylated avi-tag), protein A, and a second biotinylated tag (such as a biotinylated avi-tag).

[0044] In some embodiments, the second molecule comprises protein A / G and a biotinylated tag (such as a biotinylated avi-tag). In some embodiments, the second molecule comprises from N- to C- terminus protein A / G and a biotinylated tag (such as a biotinylated avi-tag). In some embodiments, the second molecule comprises from N- to C- terminus a biotinylated tag (such as a biotinylated avi-tag) and protein A / G. In some embodiments, the second molecule comprises from N- to C-terminus a first biotinylated tag (such as a biotinylated avi-tag), protein A / G, and a second biotinylated tag (such as a biotinylated avi-tag).

[0045] In some embodiments, the second molecule comprises an Fc receptor and a biotinylated tag (such as a biotinylated avi-tag). In some embodiments, the second molecule comprises from N- to C- terminus an Fc receptor and a biotinylated tag (such as a biotinylated avi-tag). In some embodiments, the second molecule comprises from N- to C- terminus a biotinylated tag (such as a biotinylated avi-tag) and an Fc receptor. In some embodiments, the second molecule comprises from N- to C-terminus a first biotinylated a tag (such as a biotinylated avi-tag), an Fc receptor, and a second biotinylated tag (such as a biotinylated avi-tag).

[0046] In some embodiments, the second molecule comprises an anti-Fc antibody and a biotinylated tag (such as a biotinylated avi-tag). In some embodiments, the second molecule comprises an anti-Fc antibody and a biotinylated tag (such as a biotinylated avi-tag) attached to the C-terminus of a light chain (or a heavy chain) of the anti-Fc antibody. In some embodiments, the second molecule comprises an anti-Fc antibody and a biotinylated tag (such as a biotinylated avi-tag) attached to the N-terminus of a light chain (or a heavy chain) of the anti-Fc antibody. In some embodiments, the second molecule comprises a first biotinylated tag (such as a biotinylated avi-tag), an anti-Fc antibody, and a second biotinylated tag (such as a biotinylated avi-tag), in which the first and second biotinylated tags (such as the biotinylated avi-tags) are attached to the N- and / or C-terminus of a light chain (or a heavy chain) of the anti-Fc antibody.Attorney Docket No: 122Z-405624-WO

[0047] In the present disclosure, the core bead can be of any type available in the art for immunoassays. In some embodiments, the core bead can be a polymer microbead, such as a polystyrene microbead, an agarose microbead, a polyacrylamide microbead, a dextran microbead, a latex microbead, a cellulose microbead, a polyvinyl chloride (PVC) microbead, a polyethylene microbead, a hydrogel microbead, a poly(methyl methacrylate) (PMMA) microbead, a nylon microbead, or an acrylate microbead.

[0048] The core bead can also be made of other materials, such as a silica microbead or a glass microbead. In some embodiments, the core bead is a silica microbead. In some embodiments, the core bead is a glass microbead. In some embodiments, the core bead is a magnetic microbead such as an iron oxide magnetic bead. In some embodiments, the core bead is a fluorescent microsphere. In these embodiments, the microsphere can be made of any core beads as mentioned above coated, embedded or doped with fluorochrome.

[0049] In the present disclosure, the core bead can have a diameter suitable for an immunoassay. In some embodiments, the core bead is commercially available. Core beads that are commercially available have diameters ranging from about 25 nm to about 100 pm. In some embodiments, the core bead has a diameter ranging from about 1 pm to about 50 pm. In some embodiments, the core bead has a diameter ranging from about 5 pm to about 15 pm. For example, the core bead can have a diameter of about 25 nm, about 50 nm, about 100 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, about 1 pm, about 2 pm, about 3 pm, about 4 pm, about 5 pm, about 6 pm, about 7 pm, about 8 pm, about 9 pm, about 10 pm, about 11 pm, about 12 pm, about 13 pm, about 14 pm, about 15 pm, about 16 pm, about 17 pm, about 18 pm, about 19 pm, about 20 pm, about 30 pm, about 40 pm, about 50 pm, about 60 pm, about 70 pm, about 80 pm, about 90 pm, about 100 pm, or even larger.

[0050] In some embodiments, the core bead is a polystyrene microbead having a diameter ranging from about 1 pm to about 50 pm. In some embodiments, the core bead is a silica microbead having a diameter ranging from about 1 pm to about 50 pm. In some embodiments, the core bead is a glass microbead having a diameter ranging from about 1 pm to about 50 pm.

[0051] In some embodiments, the core bead is a polystyrene microbead having a diameter of about 6 pm, about 10 pm or about 14 pm. In some embodiments, the core bead is a polystyreneAttorney Docket No: 122Z-405624-WOmicrobead having a diameter of about 6 pm. In some embodiments, the core bead is a polystyrene microbead having a diameter of about 10 pm. In some embodiments, the core bead is a polystyrene microbead having a diameter of about 14 pm.

[0052] Beads chemically conjugated with reactive molecules (i.e., the first molecule in the present disclosure) are widely available in the art. Methods for conjugating a peptide or small molecule compound onto the surface of a bead are well known in the art. For example, the bead surface is activated or functionalized with carboxyl groups with EDC (l-ethyl-3-(3-dimethylaminopropyljcarbodiimide) and NHS (N-hydroxysuccinimide), followed by reaction with primary amines on streptavidin or biotin derivatives (e.g., biotin-amines). The bead surface may be functionalized with epoxy groups to react with amine groups on streptavidin or amine-modified biotin under alkaline conditions. Alternatively, maleimide groups are introduced on the bead surface via amine-reactive sulfo-SMCC, followed by conjugation with thiol-containing streptavidin (e.g., reduced cysteine residues) or thiolated biotin derivatives. Click chemistry (e.g. Azide-DBCO cycloaddition) can also be used to conjugate the first molecule onto the bead surface. For example, the bead is functionalized with dibenzocyclooctyne (DBCO) groups and then reacted with azide-modified streptavidin or biotin via strain-promoted alkyneazide cycloaddition (SPAAC). Beads may subject to plasma treatment and silanization before conjugation with the first molecule. For example, bead surfaces are modified via oxygen plasma to generate hydroxyl groups, then silanized with APTES (3-aminopropyltriethoxysilane) to introduce amines for subsequent EDC / NHS coupling.

[0053] For example, a polyethylene bead coated with streptavidin or biotin is commercially available, such as from Spherotech, Inc, under catalog numbers TP-60-5 and SVP-60-5, respectively.

[0054] Dependent on the size of the bead and the size of the first molecule, the copy number of the first molecule on the surface of the bead can be roughly determined. For example, a 6 pm diameter bead (e.g., polyethylene bead) may have roughly 3,140,000 copies of streptavidin when it is fully coated. A 10 pm diameter bead (e.g., polyethylene bead) may have roughly 8,722,000 copies of streptavidin when it is fully coated. When the diameter of the bead increases to 14 pm, the copy number of streptavidin when it is fully coated would increase to about 17, 095, 555. In some embodiments, the core bead is fully coated with the first molecule. In someAttorney Docket No: 122Z-405624-WOembodiments, the core bead is partly (e.g., at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99%, in relation to the full capacity of coating) coated with the first molecule.

[0055] In the present disclosure, at least one first molecule is not bound by the second molecule. That is, the binding sites of first molecule are unsaturated with respect to the binding sites of the second molecule. In the case that each first molecule has more than one binding site (for example, streptavidin has four binding sites for biotin), there are binding sites of the first molecule not bound by the second molecule. In the case that each first molecule has only one binding site (for example, biotin has only one binding site for streptavidin), there is at least one first molecule not bound by the second molecule. Preferably, a plurality of the first molecule is not bound by the second molecule. For example, about 1 % to about 99.9% of the first molecule or the binding sites of the first molecule is not bound by the second molecule. For example, about 1%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about 99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, about 99.9% of the first molecule or the binding sites of the first molecule is not bound by the second molecule.

[0056] In some embodiments, the present disclosure provides a surface-modified bead comprising (i) a polystyrene microbead having a diameter of about 6 pm, wherein the polystyrene microbead is conjugated with a plurality of streptavidin molecules, and (ii) a plurality of second molecules binding to the plurality of streptavidin molecules, wherein each of the second molecules comprises a fusion protein comprising from N- to C-terminus a first biotinylated avi-tag, a protein G, and a second biotinylated avi-tag, wherein both the first and the second biotinylated avi-tags bind to the plurality of streptavidin molecules; and wherein at least a part of the plurality of streptavidin molecules is not bound by the plurality of second molecules.

[0057] In some embodiments, the present disclosure provides a surface-modified bead comprising (i) a polystyrene microbead having a diameter of about 6 pm, wherein the polystyrene microbead is conjugated with a plurality of streptavidin molecules, and (ii) a plurality of second molecules binding to the plurality of streptavidin molecules, wherein eachAttorney Docket No: 122Z-405624-WOof the second molecules comprises a fusion protein comprising from N- to C-terminus a first biotinylated avi-tag, a protein A, and a second biotinylated avi-tag, wherein both the first and the second biotinylated avi-tags bind to the plurality of streptavidin molecules; and wherein at least a part of the plurality of streptavidin molecules is not bound by the plurality of second molecules.

[0058] In some embodiments, the present disclosure provides a surface-modified bead comprising (i) a polystyrene microbead having a diameter of about 6 pm, wherein the polystyrene microbead is conjugated with a plurality of streptavidin molecules, and (ii) a plurality of second molecules binding to the plurality of streptavidin molecules, wherein each of the second molecules comprises a fusion protein comprising from N- to C-terminus a first biotinylated avi-tag, a protein A / G, and a second biotinylated avi-tag, wherein both the first and the second biotinylated avi-tags bind to the plurality of streptavidin molecules; and wherein at least a part of the plurality of streptavidin molecules is not bound by the plurality of second molecules.

[0059] In some embodiments, the present disclosure provides a surface-modified bead comprising (i) a polystyrene microbead having a diameter of about 10 pm, wherein the polystyrene microbead is conjugated with a plurality of streptavidin molecules, and (ii) a plurality of second molecules binding to the plurality of streptavidin molecules, wherein each of the second molecules comprises a fusion protein comprising from N- to C-terminus a first biotinylated avi-tag, a protein G, and a second biotinylated avi-tag, wherein both the first and the second biotinylated avi-tags bind to the plurality' of streptavidin molecules; and wherein at least a part of the plurality of streptavidin molecules is not bound by the plurality of second molecules.

[0060] In some embodiments, the present disclosure provides a surface-modified bead comprising (i) a polystyrene microbead having a diameter of about 10 pm, wherein the polystyrene microbead is conjugated with a plurality of streptavidin molecules, and (ii) a plurality of second molecules binding to the plurality of streptavidin molecules, wherein each of the second molecules comprises a fusion protein comprising from N- to C-terminus a first biotinylated avi-tag, a protein A, and a second biotinylated avi-tag, wherein both the first and the second biotinylated avi-tags bind to the plurality of streptavidin molecules; and wherein atAttorney Docket No: 122Z-405624-WOleast a part of the plurality of streptavidin molecules is not bound by the plurality of second molecules.

[0061] In some embodiments, the present disclosure provides a surface-modified bead comprising (i) a polystyrene microbead having a diameter of about 10 pm, wherein the polystyrene microbead is conjugated with a plurality of streptavidin molecules, and (ii) a plurality of second molecules binding to the plurality of streptavidin molecules, wherein each of the second molecules comprises a fusion protein comprising from N- to C-terminus a first biotinylated avi-tag, a protein A / G, and a second biotinylated avi-tag, wherein both the first and the second biotinylated avi-tags bind to the plurality of streptavidin molecules; and wherein at least a part of the plurality of streptavidin molecules is not bound by the plurality of second molecules.

[0062] In some embodiments, the present disclosure provides a surface-modified bead comprising (i) a polystyrene microbead having a diameter of about 14 pm, wherein the polystyrene microbead is conjugated with a plurality of streptavidin molecules, and (ii) a plurality of second molecules binding to the plurality of streptavidin molecules, wherein each of the second molecules comprises a fusion protein comprising from N- to C-terminus a first biotinylated avi-tag, a protein G, and a second biotinylated avi-tag, wherein both the first and the second biotinylated avi-tags bind to the plurality of streptavidin molecules; and wherein at least a part of the plurality of streptavidin molecules is not bound by the plurality of second molecules.

[0063] In some embodiments, the present disclosure provides a surface-modified bead comprising (i) a polystyrene microbead having a diameter of about 14 pm, wherein the polystyrene microbead is conjugated with a plurality of streptavidin molecules, and (ii) a plurality of second molecules binding to the plurality of streptavidin molecules, wherein each of the second molecules comprises a fusion protein comprising from N- to C-terminus a first biotinylated avi-tag, a protein A, and a second biotinylated avi-tag, wherein both the first and the second biotinylated avi-tags bind to the plurality of streptavidin molecules; and wherein at least a part of the plurality of streptavidin molecules is not bound by the plurality of second molecules.

[0064] In some embodiments, the present disclosure provides a surface-modified beadAttorney Docket No: 122Z-405624-WOcomprising (i) a polystyrene microbead having a diameter of about 14 pm, wherein the polystyrene microbead is conjugated with a plurality of streptavidin molecules, and (ii) a plurality of second molecules binding to the plurality of streptavidin molecules, wherein each of the second molecules comprises a fusion protein comprising from N- to C-terminus a first biotinylated avi-tag, a protein A / G, and a second biotinylated avi-tag, wherein both the first and the second biotinylated avi-tags bind to the plurality of streptavidin molecules; and wherein at least a part of the plurality of streptavidin molecules is not bound by the plurality of second molecules.Kifs

[0065] Another aspect of the present disclosure provides a kit comprising the surface-modified bead provided herein. The kit is useful for an immunoassay, e.g., flow cytometry, immunohistochemistry (IHC), multiplex immunoassays, or neutralization assays.

[0066] In some embodiments, the kit comprises a first container and a second container, wherein the first container is provided with a first surface-modified bead as provided herein, and the second container is provided with a second surface-modified bead as provided herein, wherein the first and the second surface-modified beads differ only in a copy number of the second molecule, and wherein a ratio of the copy number of the second molecule for the first surface-modified bead to that for the second surface-modified bead is in a range of greater than 1x10° to IxlO8, preferably from IxlO1to IxlO6, such as 1.5, 2, 5, 10, 15, 20, 30, 40, 50, 100, 200, 500, 1,000, 2.000, 5,000, 10,000. 100,000, 1,000,000, or 10.000,000.

[0067] In some embodiments, the kit comprises a first container, a second container, and a third container, wherein the first container is provided with a first surface-modified bead as provided herein, the second container is provided with a second surface-modified bead as provided herein, and the third container is provided with a third surface-modified bead as provided herein, wherein the first surface-modified bead, the second surface-modified bead, and the third surface-modified bead differ only in a copy number of the second molecule, wherein a ratio of the copy number of the second molecule for the first surface-modified bead to that for the second surface- modified bead is in a range of greater than 1x10° to 1x10s, preferably from IxlO1to IxlO6, such as 1.5, 2, 5, 10, 15, 20, 30, 40, 50, 100, 200, 500, 1,000,Attorney Docket No: 122Z-405624-WO2,000, 5,000, 10,000, 100,000, 1,000,000, or 10,000,000, and a ratio of the copy number of the second molecule for the second surface-modified bead to that for the third surface-modified bead is in a range of greater than 1x10° to 1x10s, preferably from IxlO1to IxlO6, such as 1.5, 2, 5, 10, 15, 20, 30, 40, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 100,000, 1,000,000, or 10,000,000.

[0068] In some embodiments, the kit comprises a first container, a second container, a third container, and a fourth container, wherein the first container is provided with a first surface-modified bead as provided herein, the second container is provided with a second surface-modified bead as provided herein, the third container is provided with a third surface-modified bead as provided herein, and the fourth container is provided with a fourth surface-modified bead as provided herein, wherein the first surface-modified bead, the second surface-modified bead, the third surface-modified bead, and the fourth surface-modified bead differ only in a copy number of the second molecule, wherein a ratio of the copy number of the second molecule for the first surface-modified bead to that for the second surface-modified bead is in a range of greater than 1x10° to IxlO8, preferably from IxlO1to IxlO6, such as 1.5, 2, 5, 10, 15, 20, 30, 40, 50, 100, 200, 500, 1,000. 2,000, 5,000, 10,000, 100,000, 1,000,000, or 10,000,000, a ratio of the copy number of the second molecule for the second surface-modified bead to that for the third surface-modified bead is in a range of greater than 1x10° to 1x10s, preferably from IxlO1to IxlO6, such as 1.5, 2, 5, 10, 15, 20, 30, 40, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 100,000, 1,000,000, or 10,000,000, and a ratio of the copy number of the second molecule for the third surface-modified bead to that for the fourth surface-modified bead is in a range of greater than 1x10° to 1x10s, preferably from IxlO1to IxlO6, such as 1.5, 2, 5, 10, 15, 20, 30, 40, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 100,000, 1,000,000, or 10,000,000.

[0069] In some embodiments, the kit comprises a blank container, a first container and a second container, wherein the blank container does not contain a bead, the first container is provided with a first surface-modified bead as provided herein, and the second container is provided with a second surface-modified bead as provided herein, wherein the first and the second surface-modified beads differ only in a copy number of the second molecule, and wherein a ratio of the copy number of the second molecule for the first surface-modified bead to that for the second surface-modified bead is in a range of greater than 1x10° to 1x10s,Attorney Docket No: 122Z-405624-WOpreferably from IxlO1to IxlO6, such as 1.5, 2, 5, 10, 15, 20, 30, 40, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 100,000, 1,000,000, or 10,000,000.

[0070] In some embodiments, the kit comprises a blank container, a first container, a second container, and a third container, wherein the blank container does not contain a bead, the first container is provided with a first surface-modified bead as provided herein, the second container is provided with a second surface-modified bead as provided herein, and the third container is provided with a third surface-modified bead as provided herein, wherein the first surface-modified bead, the second surface-modified bead, and the third surface-modified bead differ only in a copy number of the second molecule, wherein a ratio of the copy number of the second molecule for the first surface-modified bead to that for the second surface-modified bead is in a range of greater than 1 x 10° to IxlO8, preferably from 1 x 101to 1 x 106, such as 1.5 , 2, 5 , 10, 15, 20, 30, 40, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 100,000, 1,000,000, or 10,000,000, and a ratio of the copy number of the second molecule for the second surface-modified bead to that for the third surface-modified bead is in a range of greater than 1x10° to IxlO8, preferably from IxlO1to IxlO6, such as 1.5, 2, 5, 10, 15, 20, 30, 40, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000. 100,000, 1,000,000, or 10.000,000.

[0071] In some embodiments, the kit comprises a blank container, a first container, a second container, a third container, and a fourth container, wherein the blank container does not contain a bead, the first container is provided with a first surface-modified bead as provided herein, the second container is provided with a second surface-modified bead as provided herein, the third container is provided with a third surface-modified bead as provided herein, and the fourth container is provided with a fourth surface-modified bead as provided herein, wherein the first surface-modified bead, the second surface-modified bead, the third surface-modified bead, and the fourth surface-modified bead differ only in a copy number of the second molecule, wherein a ratio of the copy number of the second molecule for the first surface-modified bead to that for the second surface-modified bead is in a range of greater than 1x10° to IxlO8, preferably from IxlO1to IxlO6, such as 1.5, 2, 5, 10, 15, 20, 30, 40, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 100,000, 1,000,000, or 10,000,000, a ratio of the copy number of the second molecule for the second surface-modified bead to that for the third surface-modified bead is in a range of greater than 1x10° to IxlO8, preferably from IxlO1to IxlO6, such as 1.5, 2, 5, 10, 15, 20, 30,Attorney Docket No: 122Z-405624-WO40, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 100,000, 1,000,000, or 10,000,000, and a ratio of the copy number of the second molecule for the third surface-modified bead to that for the fourth surface-modified bead is in a range of greater than 1x10° to IxlO8, preferably from 1x10’ to IxlO6, such as 1.5, 2, 5, 10, 15, 20, 30, 40, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 100,000, 1,000,000, or 10,000,000.

[0072] In any of the embodiments of the kit as described above, the kit may further comprise instraction for use. In any of the embodiments of the kit as described above, each of the containers may further be provided with a buffer solution, such as phosphate buffer saline (PBS). In any of the embodiments of the kit as described above, each of the containers is a tube. In any of the embodiments of the kit as described above, each of the containers is a plastic tube.

[0073] In some embodiments, provided is a kit comprising (i) a blank tube provided with a buffer solution and without a bead, (ii) a first tube comprising the buffer solution and a first bead, (ii) a second tube comprising the buffer solution and a second bead, and (iii) a third tube comprising the buffer solution and a third bead, wherein each of the first, second and third beads comprises (a) a polystyrene microbead having a diameter of about 6 pm, about 8 pm, about 10 pm or about 14 pm, wherein the polystyrene microbead is conjugated with a plurality of streptavidin molecules; and (b) a plurality of second molecules binding to the plurality of streptavidin molecules, wherein each of the second molecules comprises a fusion protein comprising from N- to C-terminus a first biotinylated avi-tag, a protein G, and a second biotinylated avi-tag, wherein both the first and the second biotinylated avi-tags bind to the plurality of streptavidin molecules; wherein a ratio of a copy number of the second molecule for the first bead to the copy number of the second molecule for the second bead is about 10:1, and a ratio of a copy number of the second molecule for the second bead to the copy number of the second molecule for the third bead is about 10:1.

[0074] For example, the copy number of the second molecule for the first bead is about 2,000,000, the copy number of the second molecule for the second bead is about 200,000, and the copy number of the second molecule for the third bead is about 20,000. In another example, the copy number of the second molecule for the first bead is about 1,000,000, the copy number of the second molecule for the second bead is about 100,000, and the copy number of the second molecule for the third bead is about 10,000. In another example, the copy number of the secondAttorney Docket No: 122Z-405624-WOmolecule for the first bead is about 1 ,500,000, the copy number of the second molecule for the second bead is about 150,000, and the copy number of the second molecule for the third bead is about 15,000. In another example, the copy number of the second molecule for the first bead is about 2, 000,000, the copy number of the second molecule for the second bead is about 100,000, and the copy number of the second molecule for the third bead is about 5,000. In another example, the copy number of the second molecule for the first bead is about 200,000, the copy number of the second molecule for the second bead is about 20,000, and the copy number of the second molecule for the third bead is about 2,000.Uses and Mef / iads

[0075] Another aspect of the present disclosure provides a method for determining a copy number of a protein expressed by a cell, comprising (i) providing a sample comprising the cell; (ii) providing a kit as provided herein; (iii) adding a fluorochrome-conjugated antibody that binds specifically to the protein expressed by the cell into the sample and each container of the kit; (iv) plotting a standard curve of mean fluorescence intensity versus copy number of the second molecule according to the kit; and (v) determining the copy number of the protein by the mean fluorescence intensity obtained from the sample.

[0076] In another aspect, the present disclosure provides a method for determining a copy number of a protein expressed by a cell, comprising (i) providing a sample comprising the cell; (ii) providing a kit as provided herein; (iii) adding a primary antibody that binds specifically to the protein expressed by the cell into the sample; (iv) adding a fluorochrome-conjugated secondary antibody that binds specifically to the primary antibody into the sample and each container of the kit; (iv) plotting a standard curve of mean fluorescence intensity versus copy number of the second molecule according to the kit; and (v) determining the copy number of the protein by the mean fluorescence intensity obtained from the sample.

[0077] In some embodiments, the cell is a tumor cell or an immune cell. In some embodiments, the cell is a tumor cell. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a T cell or an NK cell.

[0078] In some embodiments, the protein expressed by the cell is a tumor specific antigen (TSA), a tumor associated antigen (TAA), a therapeutic antibody, or a fusion protein. In someAttorney Docket No: 122Z-405624-WOembodiments, the protein expressed by the cell is a tumor specific antigen (TSA). In some embodiments, the protein expressed by the cell is a tumor associated antigen (TAA).

[0079] Illustrative TAA includes HER2 / ERBB2, CEA (Carcinoembryonic Antigen), MUC-1 (Mucin-1), PSA (Prostate-Specific Antigen), CA-125 (Cancer Antigen 125), CA19-9 (Carbohydrate Antigen 19-9), AFP (Alpha-Fetoprotein), EGFR (Epidermal Growth Factor Receptor), GD2 (Disialoganglioside GD2), NY-BR-1, WT1 (Wilms Tumor Protein 1), Mesothelin, EpCAM (Epithelial Cell Adhesion Molecule), CD19, CD20, CD22, CD33, CD47, CD52, CD117 (KIT), PD-L1 (Programmed Death-Ligand 1), TROP2 (Trophoblast Cell Surface Antigen 2), PSMA (Prostate-Specific Membrane Antigen), GPC3 (Glypican-3), hTERT (Human Telomerase Reverse Transcriptase), Survivin, Tyrosinase, MAGE-A3 (Melanoma-Associated Antigen A3), NY-ESO-1 (Cancer-Testis Antigen NY-ESO-1), FRAME (Preferentially Expressed Antigen in Melanoma), SOX2, ALK (Anaplastic Lymphoma Kinase), BCMA (B-Cell Maturation Antigen).

[0080] Illustrative TSA includes neoantigens (e.g . , BRAF V600E, KRAS G 12D / V, EGFRvIII, IDH1 R132H), HPV E6 / E7 (Human Papillomavirus oncoproteins), EBV LMP1 / 2 (Epstein-Barr Virus Latent Membrane Proteins), MAGE-A1 (Melanoma-Associated Antigen Al), SSX2 (Synovial Sarcoma X Breakpoint 2), HERV-K (Human Endogenous Retro virus-K), CTAG1B (NY-ESO-1), PML-RARA (ProMyelocytic Leukemia- Retinoic Acid Receptor Alpha fusion), BCR-ABL (Breakpoint Cluster Region-Abelson fusion), ETV6-RUNX1 (ETS Variant 6-RUNX Family Transcription Factor 1 fusion), NPM-ALK (Nucleophosmin-Anaplastic Lymphoma Kinase fusion), KRAS neoantigens, TP53 neoantigens, PIK3C A neoantigens, MSL H (Microsatellite Instability-High) frameshift peptides.

[0081] In some embodiments, the protein expressed by the cell is a membrane protein. In some embodiments, the protein expressed by the cell is an intracellular protein.

[0082] The fluorochrome-conjugated antibody (or in specific aspect, the fluorochrome-conjugated secondary antibody) used in the methods as provided in the present disclosure is commercially available in the art. Fluorochrome suitable for labeling an antibody includes but is not limited to FITC, TRITC, Cy3, Cy5, Texas Red, PE (Phycoerythrin), APC (Allophycocyanin), PerCP (Peridinin-Chlorophyll-Protein), Alexa Fluor Series (such as Alexa Fluor 488, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 750, DyLight Series (e.g., DyLightAttorney Docket No: 122Z-405624-WO488, 550, 650), ATTO Dyes (e.g., ATTO 488, 532, 647), tandem dyes such as PE-Cy5, PE-Cy7, PE-Alexa Fluor 700, APC-Cy7, APC-Alexa Fluor 750, polymer dyes such as Brilliant Violet Series (including BV421, BV510, BV605, BV711, BV786) and Brilliant Ultra Violet (BUV Series), near-infrared (NIR) dyes including APC-Cy7, Alexa Fluor 790, and IR Dye 800CW, quantum dots (QDs) such as Qdot 525, 605, 655, 705, 800, and other specialty dyes such as CF Dyes (e.g., CF488A, CF594), mNeonGreen, or mCherry.

[0083] The addition of the fluorochrome-conjugated antibody (or in specific aspect, the fluorochrome-conjugated secondary antibody) into the container of the kit results in the formation of a complex within the container. The complex is formed by the binding of the fluorochrome-conjugated antibody to the surface-modified bead via the second molecule, e.g., through a non-covalent interaction between the fluorochrome-conjugated antibody and the second molecule. In one embodiment, the complex is formed by the binding of the fluorochrome-conjugated antibody to the surface-modified bead via the protein G-Fc interaction, in which the Fc is the Fc fragment of the fluorochrome-conjugated antibody. In one embodiment, the complex is formed by the binding of the fluorochrome-conjugated antibody to the surface-modified bead via the protein A-Fc interaction, in which the Fc is the Fc fragment of the fluorochrome-conjugated antibody. In one embodiment, the complex is formed by the binding of the fluorochrome-conjugated antibody to the surface-modified bead via the protein A / G-Fc interaction, in which the Fc is the Fc fragment of the fluorochrome-conjugated antibody. In one embodiment, the complex is formed by the binding of the fluorochrome-conjugated antibody to the surface-modified bead via the protein L-kappa light chain interaction, in which the kappa light chain is the kappa light chain of the fluorochrome-conjugated antibody.EXAMPLESMaterials and MethodsReagents and cell lines

[0084] Streptavidin beads (6.6 pm, Spherotech SVP-60-5), Biotin-FITC, Protein G, and biotinylation signal peptides were obtained from Spherotech Inc. The following monoclonal antibodies were used in this study: anti-CD19-APC (ThermoFisher, Catalog no. 17-0198-42); anti-CD20-FITC (Thermo Fisher, Catalog no. 11-0209-42); Biotin-FITC (ThermoFisher,Attorney Docket No: 122Z-405624-WOCatalog no. B1370); Goat anti-human Alexa647 (Invitrogen, Catalog no. A21445); Goat antihuman Alexa488 (Invitrogen); uPar-Abl and uPar-Ab2, chimeric antibodies consisting of a mouse anti-human uPar Fab region and a human IgGl Fc region (Huabio).

[0085] Raji and Daudi cells were cultured in RPMI-1640 complete medium supplemented with 10% fetal bovine serum (FBS) and 1% Pen-Strep. MDA-MB-468 and A549 cells were maintained in DMEM complete medium supplemented with 10% FBS and 1% Pen-Strep. Protein G with Avitag Purification

[0086] The gene encoding Protein G with Avi-tags at both the N- and C-termini was synthesized and cloned into a pET28a plasmid. The BirA gene was cloned into a pGEX6T-l plasmid with an N-terminal GST fusion. Both plasmids were transformed into BL21 E. coli cells. Protein expression was induced by adding IPTG to a final concentration of 0.1 mM and D-Biotin at 0. ImM, followed by incubation at 37°C for 24 hours. The cells were then harvested, and the cell pellet was washed once with PBS, centrifuged, and stored at -80°C until further purification.

[0087] For protein purification, the cell pellets were thawed and resuspended in 5 mL lysis buffer (50 mM Tris-HCl, pH 8.0; 500 mM NaCl; 10 mM imidazole; 0.1 mM EDTA; 1 mM DTT) supplemented with protease inhibitors. The samples were then sonicated using a Branson Digital Sonifier (set at 50% amplitude) with five 15-second pulses each, allowing for at least one minute on ice between pulses. The lysate was centrifuged at 13,000 x g for 15 minutes at 4°C, and the supernatant was collected for affinity purification.

[0088] For Ni-NTA purification, the supernatant was applied to a Ni-NTA column and eluted with a lysis buffer containing increasing concentrations of imidazole (50 mM, 250 mM, 500 mM, and 1000 mM). The 500 mM imidazole elution fraction was dialyzed against ice-cold PBS and stored at -80°C for further usage.GST-BirA Purification

[0089] The unbound fraction from the Ni-NTA purification was incubated with Glutathione beads (ThermoFisher, Catalog no. 16101), and GST-BirA was purified according to the manufacturer’s instructions.Biotinylation of Protein G

[0090] To enzymatically biotinylate the purified Protein G, the BirA enzymatic biotinylationAttorney Docket No: 122Z-405624-WOreaction was performed as follows. A reaction with a total volume of 1 mL was set up with the following components: 100 pL GST-BirA enzyme (1 mg / mLin TE buffer), 50 pL ATP (10 mM, final concentration 0.4 mM), 100 pL Tris buffer (500 mM Tris, pH 8.0; 100 mM MgOAc; 500 pM d-biotin), 100 pL Protein G-avitag (0.16 mM in TE buffer), 650 pL ddH2O. The mixture was incubated at 25°C for 2 hours, followed by 37°C for 30 minutes.Purification of Biotinylated Protein G

[0091] To purify the biotinylated Protein G, the reaction mixture was first incubated with 100 pL Ni-NTA agarose and washed with PBST. The biotinylated Protein G was then eluted with 0.5 mL lysis buffer (50 mM Tris-HCl, pH 8.0; 250 mM NaCl; 0.5 mM EDTA) containing 500 mM imidazole. The eluate was mixed with 0.25 mL Streptavidin-Mutein beads (Sigma, Catalog no. 03708152001) and incubated with rotation at 25°C for 30 minutes. After washing the beads three times with 0.5 mL PBST, the biotinylated Protein G was eluted three times using 400 pL PBST supplemented with 4 mM D-biotin. The sample was then dialyzed against PBST containing 5% glycerol. The resulting fraction represents the final Protein G-Biotin product. Streptavidin-Protein G-Biotin beads preparation

[0092] To prepare Streptavidin-Biotinylated Protein G beads, 100,000 Streptavidin beads of varying sizes were used: 6.6 pm beads (Spherotech SVP-60-5) for Biotin-FITC and Protein G-Biotin testing; 10 pm beads (Spherotech SVP-100-4) for testing with Daudi and Raji cells; and 14 pm beads (Spherotech SVP-150-4) for testing with Daudi cells, Raji cells, MDA-MB-231 cells, and A549 cells.

[0093] The beads were washed once with PBST and resuspended in 500 pL PBST. Different amounts of biotinylated Protein G in 100 pL PBST were added to the beads and immediately mixed by vigorous vertexing. The mixture was then rotated at room temperature (RT) for 2 hours and stored at 4°C until use. For storage periods longer than overnight, sodium azide was added to a final concentration of 0.05%.Streptavidin Biotinylated Protein G beads binding to antibodies

[0094] For flow cytometry analysis, 25,000 Streptavidin beads were used for each preparation. Different amounts of biotinylated Protein G beads were aliquoted into individual tubes, and PBST was added to a final volume of 1 mL. The samples were centrifuged at 2,000 x g for 2 minutes at room temperature (RT), and the supernatant was carefully removed. Antibodies,Attorney Docket No: 122Z-405624-WOeither with or without fluorescent probes, were then added at a final concentration of 1 pg / mL (providing over 109antibodies per bead) and mixed immediately by vertexing. The mixture was rotated at RT for 0.5 hours, followed by two washes with PBST. After washing, the beads were either incubated with a secondary antibody or subjected directly to flow cytometry analysis. Flow cytometry analysis of Raji and Daudi Cells

[0095] Both Raji and Daudi cells were grown in RPMI-1640 complete medium. When the cells reached the exponential growth phase, they were harvested, washed once with cold PBS, and plated on a 96-well plate at a density of 1 x 105cells per well in 50 pL of medium. The cells were kept on ice, and anti-CD19-APC and anti-CD20-FITC antibodies were added individually or combined at a concentration of 1 pg / mL each. For bead samples, the antibodies were added individually to each tube and mixed thoroughly. After 30 minutes of incubation on ice, the plate was centrifuged at 200 x g for 5 minutes at 4°C, and the supernatant was carefully removed. The cells and beads were resuspended in 200 pL of ice-cold PBST buffer, and flow cytometry analysis was performed.Flow cytometry analysis of MDA-MB-231 and A549 Cells

[0096] MDA-MB-231 and A549 cells were grown in DMEM complete medium. When the cells reached 70-90% confluency, they were trypsinized, harvested, washed once with cold PBS, and plated on a 96-well plate at a density of 1 x 105cells per well in 50 pL of medium. The cells were then kept on ice.Indirect Flow Cytometry

[0097] Anti -uPar Antibody 1 (uPar-Abl) or Anti -uPar Antibody 2 (uPar-Ab2) was added to each well (cells and beads) at a concentration of 1 pg / mL. After 30 minutes of incubation on ice, the plate was centrifuged at 200 x g for 5 minutes at 4°C, and the supernatant was removed. A secondary antibody (goat anti-human Alexa647) was added to each well and mixed thoroughly. After another 30 minutes of incubation on ice with occasional mixing, the plate was centrifuged again, and the supernatant was removed. The cells and beads were resuspended in 200 pL of ice-cold PBST buffer, and flow cytometry analysis was performed.Direct Flow Cytometry

[0098] uPar-Abl and uPar-Ab2 were conjugated with Alexa488 using a conjugation kit (ThermoFisher, Catalog no. A30006) according to the manufacturer’s protocol. The cells andAttorney Docket No: 122Z-405624-WObeads were prepared as described above, and the conjugated antibodies were added to each well at a concentration of 1 pg / mL. After 30 minutes of incubation on ice, the plate was centrifuged, and the supernatant was removed. The cells and beads were resuspended in 200 pL of ice-cold PBST buffer, and flow cytometry analysis was performed using either an Accuri C6 (for Biotin-FITC and Protein G-Biotin testing) or a SONY SA3800 (for cell experiments).Data Analysis

[0099] The raw mean fluorescence intensity (MFI) was obtained for each sample based on fluorescence signals from flow cytometry instruments. Background noise was subtracted to calculate the final MFI for each of qBead and cell samples. The MFI values and predefined copy numbers of Protein G-Biotin were plotted using Excel to generate a standard curve. The MFI of cell binding was used to calculate the copy number for each antibody. All data presented in this study represents one of three independent replicates.Examp lei. Testing Streptavidin-Bio fin Conjugated Beads for Quantitative Flow Cytometry Analysis

[0100] To evaluate whether streptavidin-biotin conjugation can be used to produce quantitative flow cytometry beads, different amounts of Biotin-FITC were conjugated to streptavidin beads (Fig. 1A). After conjugation, the beads with different amounts of Biotin-FITC were visualized under UV light (Fig. IB). The fluorescence intensity increased with the increased amount of Biotin-FITC conjugated to the beads (Fig. 1 B, C, D), indicating that streptavidin-biotin conjugation is suitable for producing quantitative flow cytometry beads.

[0101] To further assess the correlation between signal intensity and Biotin-FITC molecules per bead, the mean fluorescence intensity (MFI) was measured using flow cytometry (Fig. 1C). The MFI values were plotted against the presumed copy numbers of Biotin-FITC (Fig. ID and Fig. IE). When the beads were oversaturated with Biotin-FITC (exceeding the maximum binding capacity of 2-5 million sites per bead for the 6.6 pm beads), the linear correlation between MFI and Biotin-FITC copy numbers was weak (R2= 0.6023) (Fig. IE). However, when the binding sites were not saturated, under 200,000 molecules per bead, a strong linear correlation was observed (R2= 0.9992) (Fig. IF). These results demonstrate that streptavidin-biotin conjugated beads can be effectively used for quantitative flow cytometry analysis.Attorney Docket No: 122Z-405624-WOExample 2. Production of Biotinylated Protein G

[0102] Antibodies are widely used in flow cytometry to quantify or observe protein expression on the cell surface or within cells. To facilitate a broad use of quantitative analysis, Protein G was selected due to its high affinity for a broad range of antibodies from various species, instead of a species-specific antibody. Traditional chemical conjugation of biotin to Protein G often leads to batch-to-batch variability and uncontrolled conjugation sites on Protein G. To avoid these limitations, a biochemical biotinylation approach was employed to introduce biotin at both the N- and C-tcrmini of Protein G.

[0103] To produce Protein G with biotinylation, Avi-tag sequences, which enable enzymatic biotinylation in vivo and in vitro using the BirA enzyme, were incorporated at the N- and C-termini of Protein G (Fig. 2A). The plasmid encoding Protein G-Avi-tag was co-transformed into BL21 E. coli with a plasmid encoding GST-BirA. Following induction with biotin in the culture medium, Protein G was purified using Ni-NTA agarose affinity chromatography (Fig.2B), and GST-BirA was purified using glutathione agarose affinity chromatography (Fig. 2B).

[0104] For further in vitro biotinylation, the purified Protein G and GST-BirA were incubated in the presence of Biotin (Fig. 2C). The biotinylated Protein G was subsequently purified using Streptavidin-Mutein affinity chromatography to ensure that Protein G was fully biotinylated (Fig. 2C).Example 3. Quantification of Protein Binding Using Strep tavidin-Protein G Beads

[0105] To evaluate whether Protein G-biotin can be used to quantitatively bind antibodies, defined amounts of Protein G-biotin were mixed with streptavidin beads to make quantitative Streptavidin Protein G beads (qBeads) (Fig. 3A), and then incubated with FITC-conjugated antibodies (Fig. 3B). The beads with different quantities of FITC-labeled antibodies were then analyzed by flow cytometry. The flow cytometry analysis confirmed that the Protein G-biotin conjugates exhibited quantitative binding to the antibodies (Fig. 3C).

[0106] Similar to the results observed with Biotin-FITC, the maximum binding capacity of the beads was under 2 million sites per bead for the 6.6 pm beads. When the beads were oversaturated with Protein G-biotin (e.g., >2 million molecules per bead), correlation between mean fluorescence intensity (MFI) and the presumed copy number of Protein G-biotin wasAttorney Docket No: 122Z-405624-WOweak (R2= 0.5435). However, when the binding sites were not saturated (below 200,000 molecules per bead), a strong linear correlation was observed (R2= 0.9894). These results demonstrate that Streptavidin-Protein G-biotin conjugated beads can be effectively used for quantitative flow cytometry analysis.Example 4. Quantification of Surface Proteins on Cells

[0107] To test if the qBeads can be used to quantify protein copy numbers on cell surfaces, Raji and Daudi cells were labeled with APC-conjugated anti-CD19 (anti-CD19-APC) and FITC-conjugated anti-CD20 (anti-CD20-FITC) antibodies individually or in combination. qBeads (10 pm beads) were also made and incubated with varying amounts of the same antibodies.

[0108] Flow cytometry analysis was performed to measure mean fluorescence intensity (MFI) on both the bead and cell samples (Fig. 4A and 4E). The MFI values for anti-CD19-APC or anti-CD20-FITC antibodies with qBeads were acquired and summarized with the presumed molecules per bead (Fig. 4, panel B). Standard curves were generated for either anti-CD19-APC or anti-CD20-FITC by plotting the MFI vs molecules per bead with linear correlation (Fig.4C, 4D). Based on the standard curves using beads, the copy numbers of CDI9 and CD20 on Raji and Daudi cells were calculated (Fig. 4, panel F), with Raji cells, CD19 at 67639 molecules per cells, CD20 at 48139 molecules per cell, and Daudi cells, CD 19 at 44275 molecules per cells, CD20 at 63187 molecules per cell. The results were consistent with previously published data (around 13,000-70,000 copies per cell). Similar results were also obtained with 14 pm streptavidin beads.

[0109] To further validate the system, MDA-MB-231 and A549 cell lines, which express high and low levels of the uPar, a cell surface protein, were analyzed using two monoclonal anti-uPar antibodies (uPar-Ab 1 and uPar-Ab2). For direct labeling, both antibodies were conjugated with Alexa488, and then used to bind cells and qBeads. Flow cytometry analysis was performed using qBeads (Fig. 5A, panel 5B), and a linear correlation between molecules per bead and MFI was established (Fig. 5C and 5D). Based on the MFI values from cell binding (Fig. 5E and panel 5F), the copy numbers of uPar on both cell lines were calculated. Both antibodies yielded similar uPar copy numbers for the two cell lines.Attorney Docket No: 122Z-405624-WO

[0110] In many applications, indirect labeling of cells is preferred, but is not possible with current qFCM beads. To test if the qBeads can be used with the indirect labeling approach, unlabeled anti-uPar antibodies were first bound to cells and beads, followed by incubation with a fluorescence labeled secondary antibody (Goat anti -human Alexa647). Flow cytometry analysis was performed (Fig. 6A), and a linear correlation between copy number and MFI was observed (Fig. 6B, 6C, 6D). Using the MFI values from cell surface binding (Fig. 6E, panel 6F), the uPar copy numbers calculated from indirect labeling were consistent with those obtained from direct labeling (within the range of two-fold). High expression of uPar on MDA-MB-231 cells are confirmed as described before, demonstrating the flexibility of the Streptavidin-Protein G-biotin system for both direct and indirect methods.

[0111] Through a series of studies, this report demonstrated that streptavidin-biotin conjugated Protein G beads can be effectively used in quantitative flow cytometry. The ability to quantify surface proteins such as CD 19, CD20, and uPar on various cell lines directly or indirectly highlights the potential of qBeads for a wide range of applications in biomedical research.

[0112] The qBeads using streptavidin-biotin conjugation offer several advantages over current quantification kits that rely on chemical conjugation. Traditional kits, such as Quantum™ Simply Cellular®, Quantibrite™, and Quantum™ MESF Beads, use chemical conjugation to attach a defined number of antibodies or fluorophores to beads. However, chemical conjugation is often inefficient and prone to batch-to-batch variability. Since qFCM requires high precision for accurate quantification, even minor variations in bead preparation can compromise results. Consequently, rigorous and burdensome quality control is necessary for each batch, and specific specifications must be provided to ensure consistency.

[0113] In contrast, the streptavidin-biotin conjugation used in this study is highly efficient and easy to produce, with nearly 100% binding efficiency due to the extremely high affinity of the streptavidin-biotin interaction (Kd < IO-13M). This minimizes batch-to-batch variability and eliminates the need for extensive quality control. Moreover, the 1:1 binding stoichiometry between Protein G and antibodies simplifies quantification and reduces variability.

[0114] Furthermore, current qFCM kits are often limited to specific antibody species (e.g., mouse, rat, rabbit, goat), making them unsuitable for antibodies from other species. The use ofAttorney Docket No: 122Z-405624-WOProtein G in the system addresses these issues, as it binds to antibodies from a wide range of species with high affinity.

[0115] Another significant advantage of the qBeads is their compatibility with indirect labeling. Current qFCM kits are typically limited to direct labeling, where fluorophore-conjugated primary antibodies are used for detection. The indirect approach involves binding unlabeled primary antibodies to the target, followed by detection with fluorophore-conjugated secondary antibodies. This method is particularly advantageous when working with unlabeled primary antibodies or when signal amplification is required for low-abundance antigens. Traditional anti-Fc beads are incompatible with indirect labeling because the secondary antibody competes with the beads for primary antibody binding. Using qBeads, Protein G does not interfere with secondary antibody binding, enabling signal amplification and accurate quantification (Fig. 6).

[0116] This study incorporated two biotinylation sites on Protein G to enhance the binding stability between streptavidin and Protein G. To ensure efficient biotinylation, Protein G was co-expressed with the BirA enzyme in vivo and further biotinylated in vitro. The biotinylated Protein G was then purified using streptavidin-mutein beads.

[0117] In summary, the qBeads system using streptavidin-biotin conjugated Protein G offers significant improvements in consistency, reproducibility, and ease of use compared to traditional chemical conjugation methods. By minimizing batch-to-batch variability and providing flexibility in both antibody selection and labeling approaches, this system has great potential to facilitate basic research as well as clinical applications.

[0118] The qBeads system using Streptavidin-Biotin conjugation provides a robust and standardized approach for quantitative flow cytometry. This system offers a powerful tool for measuring cell surface protein expression, receptor-ligand interactions, and other molecular processes with high precision, bridging the gap between qualitative and quantitative biological measurements. By enabling accurate and reproducible quantification, this approach is poised to play a significant role in advancing both basic research and clinical diagnostics, paving the way for new discoveries and improved therapeutic strategies.

Claims

Attorney Docket No: 122Z-405624-WOWHAT IS CLAIMED IS:

1. A surface-modified bead comprising(i) a core bead:(ii) a first molecule conjugated to a surface of the core bead; and(iii) a second molecule binding to the first molecule, such that a complex is formed on the surface of the core bead, whereinthe second molecule comprises a first moiety for binding to an antibody and a second moiety for binding to the first molecule, and the second moiety binds to the first molecule at a dissociation constant (KD) of not greater than about IxlO12M; andat least one first molecule is not bound by the second molecule.

2. The surface-modified bead according to claim 1, wherein the first molecule and the second moiety constitute a binding pair selected from a group consisting of(a) avidin and biotin;(b) streptavidin and biotin; and(c) SpyTag and SpyCatcher;preferably, the first molecule comprises avidin or streptavidin, and the second moiety comprises biotin.

3. The surface-modified bead according to claim 1 or 2, wherein the second molecule further comprises a tag; preferably, the tag is selected from a group consisting of avi-tag, halo-tag, sortase-tag, snap-tag and clip-tag; more preferably, the tag is avi-tag.

4. The surface-modified bead according to any one of claims 1 to 3, wherein the first moiety is selected from a group consisting of protein G, protein A, protein A / G, protein L, an anti- Fc antibody, and an Fc receptor: preferably, the first moiety is protein G.

5. The surface- modified bead according to any one of claims 1 to 4, wherein the second molecule comprises the first moiety and the second moiety at a molecule ratio of 1:1 or 1:2.

6. The surface- modified bead according to any one of claims 1 to 5, wherein the second molecule comprises (a) a protein G fused to a biotinylated avi-tag or (b) a protein G fused to two biotinylated avi-tags, wherein the two biotinylated avi-tags are located at N- and C-Attorney Docket No: 122Z-405624-WOtermini of the protein G.

7. The surface-modified bead according to any one of claims 1 to 6. wherein the core bead:(a) is a polymer microbead such as a polystyrene microbead, a silica microbead, a glass microbead, a magnetic microbead, or a fluorescent microsphere; and / or(b) has a diameter ranging from about 25 nm to about 100 pm, preferably about 1 pm to about 50 pm, more preferably about 5 pm to about 15 pm, such as about 6 pm, about 10 pm or about 14 pm.

8. A surface-modified bead comprising(i) a polystyrene microbead having a diameter of about 6 pm, wherein the polystyrene microbead is conjugated with a plurality of streptavidin molecules, and (ii) a plurality of second molecules binding to the plurality of streptavidin molecules, wherein each of the second molecules comprises a fusion protein comprising from N- to C-terminus a first biotinylated avi-tag, a protein G, and a second biotinylated avi-tag,wherein both the first and the second biotinylated avi-tags bind to the plurality of streptavidin molecules; and wherein at least a part of the plurality of streptavidin molecules is not bound by the plurality of second molecules.

9. A complex comprising (i) the surface-modified bead according to any one of claims 1 to 8, and (ii) a fluorochrome-conjugated antibody or an antigen-binding fragment thereof, wherein the fluorochrome-conjugated antibody is complexed to the second molecules.

10. A kit comprising a first container and a second container, whereinthe first container is provided with a first surface-modified bead according to any one of claims 1 to 8, and the second container is provided with a second surface-modified bead according to any one of claims 1 to 8, and whereinthe first surface-modified bead and the second surface-modified bead differ only in a copy number of the second molecule, wherein a ratio of the copy number of the second molecule for the first surface-modified bead to that for the second surface- modified bead is in a range of greater than 1x10° to lxl()8, preferably from IxlO1to IxlO6.

11. The kit according to claim 10, wherein the kit further comprises a third container, wherein the third container is provided with a third surface-modified bead according to any one ofAttorney Docket No: 122Z-405624-WOclaims 1 to 8, and wherein the first surface-modified bead, the second surface-modified bead, and the third surface-modified bead differ only in a copy number of the second molecule, wherein a ratio of the copy number of the second molecule for the first surface- modified bead to that for the second surface-modified bead is in a range of greater than 1x10° to 1x10s, preferably from IxlO1to IxlO6, and a ratio of the copy number of the second molecule for the second surface-modified bead to that for the third surface-modified bead is in a range of greater than 1x10° to 1x10s, preferably from IxlO1to IxlO6.

12. The kit according to claim 11, wherein the kit further comprises a fourth container, wherein the fourth container is provided with a fourth surface-modified bead according to any one of claims 1 to 8, and wherein the first surface-modified bead, the second surface-modified bead, the third surface-modified bead, and the fourth surface-modified bead differ only in a copy number of the second molecule, wherein a ratio of the copy number of the second molecule for the first surface-modified bead to that for the second surface-modified bead is in a range of greater than 1x10° to 1x10s, preferably from IxlO1to IxlO6, a ratio of the copy number of the second molecule for the second surface-modified bead to that for the third surf ace- modified bead is in a range of greater than 1x10° to 1x10s, preferably from IxlO1to IxlO6, and a ratio of the copy number of the second molecule for the third surface- modified bead to that for the fourth surface-modified bead is in a range of greater than 1x10° to 1x10s, preferably from IxlO1to IxlO6.

13. The kit according to any one of claims 10 to 12, wherein:(a) the kit further comprises a blank container, wherein the blank container does not contain a surface-modified bead according to any one of claims 1 to 8; and / or (b) the kit further comprises instruction for use; and / or(c) each of the containers further contains a buffer solution, such as Phosphate Buffer Saline.

14. A method for determining a copy number of a protein expressed by a cell, comprising (i) providing a sample comprising the cell;(ii) providing a kit according to any one of claims 10 to 13;(iii) adding a fluorochrome-conjugated antibody that binds specifically to the protein expressed by the cell into the sample and each container of the kit;Attorney Docket No: 122Z-405624-WO(iv) plotting a standard curve of mean fluorescence intensity versus copy number of the second molecule according to the kit; and(v) determining the copy number of the protein by the mean fluorescence intensity obtained from the sample.

15. A method for determining a copy number of a protein expressed by a cell, comprising (i) providing a sample comprising the cell;(ii) providing a kit according to any one of claims 10 to 13;(iii) adding a primary antibody that binds specifically to the protein expressed by the cell into the sample;(iv) adding a fluorochrome-conjugated secondary antibody that binds specifically to the primary antibody into the sample and each container of the kit;(v) plotting a standard curve of mean fluorescence intensity versus copy number of the second molecule according to the kit; and(vi) determining the copy number of the protein by the mean fluorescence intensity obtained from the sample.

16. The method of claim 14 or 15, wherein(a) the protein expressed by the cell is a tumor specific antigen, a tumor associated antigen, a therapeutic antibody, or a fusion protein, preferably the protein is a tumor associated antigen; and / or(b) the cell is a tumor cell or an immune cell, preferably the immune cell is a T cell or NK cell; and / or(c) the protein expressed by the cell is a membrane protein or an intracellular protein.

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