# US 11,307,206 B2 · Functionalized metal-labeled beads for mass cytometry > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,307,206 B2 - Document ID: US11307206B2 - Status: Granted - Kind: B2 - Application number: 16/395,022 - Filed: 2019-04-25 - Published: 2019-10-31 - Granted: 2022-04-19 - Earliest claimed: 2018-04-27 - Assignees: Deutsches Rheuma-Forschungszentrum Berlin - Applicants: Deutsches Rheuma-Forschungszentrum Berlin - Inventors: Henrik Mei; Axel Ronald Schulz; Lisa Budzinski - Web page: https://patentgrep.com/patent/US11307206B2 ## CPC - G01N 33/585: {with a particulate label, e.g. coloured latex} - G01N 33/54313: {the carrier being characterised by its particulate form} - G01N 33/566: using specific carrier or receptor proteins as ligand binding reagents {where possible specific carrier or receptor proteins are classified with their target compounds} - G01N 33/56972: {White blood cells} - G01N 2458/15: Non-radioactive isotope labels, e.g. for detection by mass spectrometry - G01N 2560/00: Chemical aspects of mass spectrometric analysis of biological material - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract Beads for use as a control, calibration and/or quantification probe in a mass cytometry assay, wherein the beads are labeled with a heavy metal selected from the group comprising osmium or ruthenium. Also disclosed are beads labeled with a heavy metal exhibiting a surface functionalization that allows for the binding of an affinity reagent, such as a metal-conjugated antibody. Methods are described for the labeling of the beads and usage of the beads for quantification of cell surface receptors or for a compensation of channel crosstalk in mass cytometry assays. ## Claims ### Claim 1 Method of analyzing beads as control, calibration and/or quantification probes in a mass cytometry assay, comprising the steps of a. providing a mass cytometer b. providing cells to be probed in said mass cytometer c. providing beads, the beads are labeled with a heavy metal selected from the group consisting of osmium and ruthenium, wherein the beads are synthetic polymeric beads and have been labelled with osmium by incubating the beads with an osmium tetroxide dilution or have been labeled with ruthenium by incubation the beads with a ruthenium tetroxide dilution, d. performing a mass cytometry assay including the steps of incubating the cells with one or more mass tagged affinity reagents, wherein the one or more mass tagged affinity reagents are metal conjugated antibodies configured for a specific binding to cellular target molecules of said cells such that a quantity of mass tagged affinity reagents allows for a determination of the presence of the cellular target molecules on said cells, and conducting an elemental analysis of said cells and beads using the mass cytometer, wherein said beads are analyzed as a control, calibration and/or quantification probe in said mass cytometry assay. ### Claim 2 (depends on 1) The method according claim 1, wherein the beads exhibit a surface functionalization allowing for the binding of the one or more mass tagged affinity reagents to the surface functionalization of said beads. ### Claim 3 (depends on 2) The method according to claim 2, wherein the one or more affinity reagents allowing for the binding to the surface functionalization of the beads is/are an antibody and the surface functionalization refers to the presence of antibody capturing sites. ### Claim 4 (depends on 1) The method according to claim 1, wherein the beads are polystyrene beads. ### Claim 5 (depends on 1) The method according to claim 1, wherein the beads have a size of 10 nm to 100 μm. ### Claim 6 (depends on 1) The method according to claim 1, wherein the concentration of the heavy metal tetroxide diluted in the buffer solution is in between 0.0001 wt.-% to 0.01 wt.-%. ### Claim 7 (depends on 1) The method to claim 1, wherein the beads exhibit a surface functionalization allowing for the binding of an affinity reagent to the surface functionalization of said beads and that the step of providing the beads includes a step of providing beads labeled with the affinity reagent and/or the method further includes a step of labelling the beads with the affinity reagent and wherein the affinity reagent is preferably an antibody, most preferably a metal-conjugated antibody. ### Claim 8 (depends on 1) The method according to claim 1 wherein: the beads are provided in one or more groups of the beads labeled with the heavy metal, and the mass cytometry assay is performed by introducing said beads and said cells into the mass cytometer for the elemental analysis, wherein data acquired from said beads is used for the compensation of the channel-cross talk or the spill-over in the data acquired from said cells. ### Claim 9 (depends on 1) The method according to claim 1 wherein: the beads are provided in at least two groups of the beads labeled with the heavy metal and exhibiting the surface functionalization allowing for the binding of the one or more mass tagged affinity reagents to the surface functionalization of the beads, wherein the beads of each of the at least two groups exhibit as the surface functionalization a different defined amount of antibody capturing sites and are labeled with a different defined amount of osmium or ruthenium, the one or more mass tagged affinity reagents are one or more metal-conjugated antibody/antibodies targeting a cell surface receptor or cell surface receptors, the least two groups of beads are incubated with the one or more metal-conjugated antibody/antibodies under saturating conditions, the mass cytometry assay is performed by introducing a mixture of said beads and said cells into the mass cytometer for the elemental analysis, and wherein said method further comprises: calculating a reference curve from the data acquired from the at least two groups of beads yielding a correlation between signal intensity detected in the mass cytometer and absolute amount of metal-conjugated antibodies, and using said reference curve for an absolute quantification of cell surface receptors, wherein the method is used for an absolute quantification of cell surface receptors in the mass cytometry assay.