# US 11,305,289 B2 · Method for automated detection of antibodies in a liquid biological sample using an antigen chip, and antigen chip therefor > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,305,289 B2 - Document ID: US11305289B2 - Status: Granted - Kind: B2 - Application number: 17/250,216 - Filed: 2019-06-17 - Published: 2021-08-12 - Granted: 2022-04-19 - Earliest claimed: 2018-07-06 - Assignees: EUROIMMUN Medizinische Labordiagnostika AG - Applicants: EUROIMMUN Medizinische Labordiagnostika AG - Inventors: Katja Morgenroth; Vanessa Viertel; Ulf Steller; Stefan Gerlach; Christian Marzahl; Jörn Voigt; Winfried Stöcker - Web page: https://patentgrep.com/patent/US11305289B2 ## CPC - B01L 3/50855: {using modular assemblies of strips or of individual wells} - G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)} - G01N 21/6447: {by visual observation} - G01N 33/543: with an insoluble carrier for immobilising immunochemicals - G01N 33/58: involving labelled substances (G01N33/53 takes precedence) - G01N 33/6854: {Immunoglobulins} - G01N 2021/6439: {with indicators, stains, dyes, tags, labels, marks} - B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE - 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 A method is useful for automated detection of antibodies in a liquid biological sample with an antigen chip having antigen spots which are applied thereon and which have an identical, common dye. The antigen spots form respective antigen spot sets which form corresponding respective, regular antigen spot patterns. Furthermore, reference spots comprising the identical dye are applied, and form a reference spot set which forms a regular reference pattern. The reference pattern differs with respect to its regularity from the antigen spot patterns. Through a first image information item which represents a color of the reference spots and of the antigen spots due to the identical, first dye and through a second image information item which represents a potential color of the antigen spots due to a second dye after an incubation, binding of antibodies of the biological sample to respective antigen types is then determined by image processing. ## Claims ### Claim 1 An antigen chip, comprising: a level substrate surface with antigen spots applied thereon, wherein the antigen spots are spaced apart and comprise an identical, common dye, wherein the antigen spots form respective antigen spot sets, wherein the antigen spot sets form corresponding respective, regular antigen spot patterns, wherein the antigen spots of a same antigen spot set comprise an identical, common antigen type, and wherein two or more of the antigen spot sets comprise different antigen types, wherein reference spots are applied on the level substrate surface, wherein the reference spots form a reference spot set, wherein the reference spot set forms a reference pattern having a regularity, wherein the regularity of the reference pattern differs from a regularity of the antigen spot patterns, and wherein the reference spots comprise the identical, common dye. ### Claim 2 (depends on 1) The antigen chip as claimed in claim 1, wherein the regularity of the reference pattern differs from regularity of the antigen spot patterns by one or more of the following criteria: within the reference pattern and within the antigen spot patterns, the reference spots and the antigen spots follow one another at respective regular distances, wherein a regular distance of the reference spots of the reference pattern differs from a regular distance of the antigen spots of the antigen spot patterns; the reference pattern has, in relation to an immediately adjacent antigen spot pattern, a neighboring distance which differs from other neighboring distances of the antigen spot patterns; and/or within the reference pattern and within the antigen spot patterns, the reference spots and the antigen spots have a respective, identical size, such that a size of the reference spots of the reference pattern differs from a size of the antigen spots of the antigen spot patterns. ### Claim 3 (depends on 1) The antigen chip as claimed in claim 1, wherein the reference pattern and the antigen patterns are arranged along an arrangement direction, and wherein the reference pattern and the antigen patterns have respective spreading directions, wherein the respective spreading directions run substantially perpendicularly to the arrangement direction. ### Claim 4 (depends on 1) The antigen chip as claimed in claim 1, wherein the reference spots of the reference spot set form a reference line pattern, and wherein the antigen spots of the respective antigen spot sets form respective antigen line patterns, and wherein the reference line pattern and antigen line patterns run substantially parallel to one another. ### Claim 5 (depends on 1) The antigen chip as claimed in claim 1, wherein the reference spots comprise first reference spots, wherein the first reference spots form a first reference spot set, wherein the first reference spot set forms a first regular reference pattern, wherein the reference spots comprise second reference spots, wherein the second reference spots form a second reference spot set, wherein the second reference spot set forms a second regular reference pattern, wherein the second reference spots comprise the identical, common dye, and wherein regularity of the second regular reference pattern differs from regularity of the antigen spot patterns of the antigen spot sets, and from regularity of the first regular reference pattern of the first reference spots. ### Claim 6 (depends on 1) The antigen chip as claimed in claim 1, wherein the reference spots further comprise an antigen. ### Claim 7 (depends on 1) The antigen chip as claimed in claim 1, wherein the reference spots further comprise an antibody. ### Claim 8 (depends on 1) A method for automated detection of antibodies in a liquid biological sample by means of an antigen chip, the method comprising: providing the antigen chip as claimed in claim 1, incubating the reference spots and the antigen spots of the antigen chip with the biological sample, incubating the reference spots and the antigen spots with a conjugate, wherein the conjugate comprises a secondary antibody labeled with a second dye, acquiring or providing at least one first image information item, wherein the at least one first image information item represents a color of the reference spots and of the antigen spots due to a first dye, detecting the reference pattern and an associated reference position on the basis of the at least one first image information item, detecting the respective antigen spot patterns and respective associated further positions on the basis of the at least one first image information item, generating an assignment information item, wherein the assignment information item indicates an assignment of the respective antigen spot patterns to respective antigen types, on the basis of detecting the associated reference position and the respective associated further positions, acquiring or providing a second image information item, wherein the second image information item represents a potential color of the antigen spots due to the second dye, and determining respective measures of binding, wherein the respective measures of binding indicate respective bindings of antibodies of the biological sample to the respective antigen types, on the basis of the second image information item and the assignment information item. ### Claim 9 (depends on 1) A method for automated image processing for detection of antibodies in a liquid biological sample, the method comprising: providing or acquiring at least one first image information item, wherein the at least one first image information item represents a color of the reference spots and of the antigen spots of the antigen chip as claimed in claim 1 due to a first dye, detecting the reference pattern and an associated reference position, detecting the respective antigen spot patterns and respective associated further positions, on the basis of the at least one first image information item, generating an assignment information item, wherein the assignment information item indicates an assignment of the respective antigen spot patterns to respective antigen types, on the basis of detecting the associated reference position and the respective associated further positions, providing or acquiring a second image information item, wherein the second information item represents a potential color of the antigen spots of the antigen chip due to a second dye after incubation of the reference spots and the antigen spots of the antigen chip with the biological sample and with a conjugate, wherein the conjugate comprises a secondary antibody labeled with the second dye, and determining respective measures of binding, wherein the respective measures of binding indicate respective bindings of antibodies of the biological sample to the respective antigen types on the basis of the second image information item. ### Claim 10 (depends on 8) The method as claimed in claim 8, wherein the associated reference position is an associated first reference position, the method further comprising: detecting a second regular reference pattern and an associated second reference position on the basis of the first image information item, and generating the assignment information item wherein the antigen chip comprises the reference spots, wherein the reference spots comprise first reference spots, wherein the first reference spots form a first reference spot set, wherein the first reference spot set forms a first regular reference pattern, wherein the reference spots comprise second reference spots, wherein the second reference spots form a second reference spot set, wherein the second reference spot set forms the second regular reference pattern, wherein the second reference spots comprise the identical, common dye, and wherein regularity of the second regular reference pattern differs from regularity of the antigen spot patterns of the antigen spot sets, and from regularity of the first regular reference pattern of the first reference spots. ### Claim 11 (depends on 8) The method as claimed in claim 8, wherein the assignment of the respective antigen spot patterns to respective antigen types is achieved by determining a spatial sequence of the associated reference position and of the respective associated further positions, providing a sequence data set which indicates a sequence of antigen types, and generating the assignment information item, wherein the assignment information item indicates the assignment of the respective antigen spot patterns to respective antigen types, on the basis of detecting the associated reference position and the respective associated further positions, and the sequence data set. ### Claim 12 (depends on 1) A method for producing an antigen chip for immunodiagnostics, the method comprising: providing a substrate having a level substrate surface, applying reference spots to the level substrate surface, and applying antigen spots to the level substrate surface, wherein application of the reference spots and of the antigen spots is carried out such that the reference spots and the antigen spots of the antigen chip are configured as per the antigen chip as claimed in claim 1. ### Claim 13 (depends on 12) The method as claimed in claim 12, further comprising: fragmenting the substrate in order to obtain multiple antigen chips. ### Claim 14 (depends on 12) The method as claimed in claim 12, wherein the level substrate surface is a surface of a glass substrate or of a glass substrate coated with a membrane and/or film. ### Claim 15 (depends on 1) A device for automated image processing for detection of antibodies in a liquid biological sample, comprising: at least one image acquisition unit configured to acquire at least one first image information item, wherein the at least one first image information item represents a color of the reference spots and of the antigen spots of the antigen chip as claimed in claim 1 due to a first dye, and acquire a second image information item, wherein the second information item represents a potential color of the antigen spots of the antigen chip as claimed in claim 1 due to a second dye, after incubation of the reference spots and the antigen spots of the antigen chip with the biological sample and with a conjugate, wherein the conjugate comprises a secondary antibody labeled with the second dye, and at least one computing unit configured to detect the reference pattern and an associated reference position, on the basis of the at least one first image information item, detect the respective antigen spot patterns and respective associated further positions, on the basis of the at least one first image information item, generate an assignment information item, wherein the assignment information item indicates an assignment of the respective antigen spot patterns to respective antigen types, on the basis of the associated reference position and the respective associated further positions, and determine respective measures of binding, wherein the respective measures of binding indicate respective bindings of antibodies of the biological sample to respective antigen types, on the basis of the second image information item. ### Claim 16 (depends on 15) The device as claimed in claim 15, further comprising: at least one illumination unit for emitting excitation light for exciting an emission of first fluorescence radiation of a first wavelength range due to the first dye. ### Claim 17 (depends on 1) A data network device for automated image processing for detection of antibodies in a liquid biological sample, comprising: at least one data interface configured to receive at least one first image information item, wherein the at least one first image information item represents a color of the reference spots and of the antigen spots of the antigen chip as claimed in claim 1 due to a first dye, and receive a second image information item, wherein the second information item represents a potential color of the antigen spots of the antigen chip as claimed in claim 1 due to a second dye, after incubation of the reference spots and the antigen spots of the antigen chip with the biological sample and with a conjugate, wherein the conjugate comprises a secondary antibody labeled with the second dye, and at least one computing unit configured to detect the reference pattern and an associated reference position, on the basis of the at least one first image information item, detect the respective antigen spot patterns and respective associated further positions, on the basis of the at least one first image information item, generate an assignment information item, wherein the assignment information item indicates an assignment of the respective antigen spot patterns to respective antigen types, on the basis of the associated reference position and the respective associated further positions, and to determine respective measures of binding, wherein the respective measures of binding indicate respective bindings of antibodies of the biological sample to respective antigen types, on the basis of the second image information item. ### Claim 18 (depends on 9) A computer readable medium having commands stored thereon, the commands being executable to cause a computer to carry out the method as claimed in claim 9. ### Claim 19 (depends on 1) A slide comprising a multiplicity of the antigen chip as claimed in claim 1. ### Claim 20 (depends on 1) A kit for detection of antibodies in a sample, comprising: at least one of the antigen chip as claimed in claim 1, and a conjugate, wherein the conjugate comprises a secondary antibody labeled with a second dye. ### Claim 21 (depends on 20) The kit as claimed in claim 20, wherein the second dye is selected from the group consisting of a fluorescent dye, a chromogenic substrate, and an enzyme or a substrate for a chemiluminescence reaction.