High-throughput serotyping and antibody profiling assays
Inventors
Okerberg, Eric • Gunderson, Kevin L. • MURANAKA, Norihito • Kim, Jongbum • LIM, Byung Joon • Shi, Lei • GANGULY, Soumya • SULLIVAN, Devin
Assignees
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Publication Number
US-12043862-B2
Publication Date
2024-07-23
Expiration Date
Abstract
Provided herein are high-throughput, population-wide serotyping and antibody profiling assays. Disclosed variants of a Digital Serotyping assay employ next generation sequencing to measure the “serotyping profile” of barcoded subject serum antibodies tested against a range of DNA-tagged pathogen-derived antigens. The disclosed assay setup enables multiplexing in both the sample and antigen dimensions, generating a large multi-dimensional serotyping data set for more comprehensive serotyping profiling of large populations across a large number of antigens and possible pathogens. Moreover, the ability to easily scale and multiplex the number of peptide epitopes allows rapid updating of the assay content to monitor the ever-changing spectrum of pathogens. Additional applications of this technology include cancer immunology and autoimmune conditions (e.g., neoantigen or autoimmune profiling), screening for toxins, antibody therapeutics development, biosecurity, and veterinary medicine.
Core Innovation
The invention provides a method for sample analysis that detects antibodies in a plurality of samples using nucleic-acid-associated recording and coding tags. A plurality of different antibodies from each sample is separately contacted with a binding element that binds an antibody constant region and is not configured to bind an antibody variable region, thereby obtaining binding element-antibody conjugates or complexes associated with a sample-specific barcode. The binding element or the binding element-antibody conjugate is attached to a bead with a recording tag associated with a sample-specific barcode.
After the beads from the plurality of samples are mixed, the mixture is contacted with a binding agent comprising an antigen and a coding tag attached thereto. The coding tag comprises an encoder sequence that comprises identifying information regarding the antigen. Following antigen binding to an antibody attached to a bead, identifying information is transferred between the coding tag and the recording tag of the bead, generating an extended coding tag or an extended recording tag or extended nucleic-acid tag.
The identifying information transfer occurs through a primer extension reaction and/or ligation, so that the extended tag encodes both antigen-identifying information and the sample-specific barcode. The analyzing step determines the antigen and the sample that contains the antibody by analyzing the encoder sequence or a complement and the sample-specific barcode or a complement using nucleic acid sequencing. The method thereby detects the antibody in the sample via sequencing-readable extended tags that couple antigen identity to sample identity.
Claims Coverage
The independent claims are directed to a multiplex sample analysis workflow with two alternative implementations for carrying the sample-specific barcode via a recording-tag-associated binding element or via a nucleic acid tag within an antibody complex. Across both independent claims, at least five inventive aspects are present: constant-region binding restriction, bead attachment with sample-specific barcodes, antigen-binding-agent coding tags with encoder sequences, transfer of identifying information via primer extension and/or ligation, and sequencing-based analysis of extended tags to identify both antigen and sample of origin.
Constant-region binding element with bead-associated sample barcode
The binding element binds an antibody constant region and is not configured to bind an antibody variable region, and is associated with a recording tag comprising a sample-specific barcode.
Bead-multiplexed mixing of sample-tagged antibody conjugates
Beads comprising attached binding element-antibody conjugates from the plurality of samples are mixed to obtain a mixture of beads.
Antigen binding agent carrying coding tag with antigen-identifying encoder
The mixture of beads is contacted with a binding agent comprising an antigen and a coding tag attached thereto, where the coding tag comprises an encoder sequence that comprises identifying information regarding the antigen.
Tag information transfer between coding tag and recording tag
Following binding of the antigen to an antibody attached to a bead of the mixture of beads, identifying information is transferred between the coding tag and the recording tag, thereby generating an extended coding tag or an extended recording tag, where the transfer occurs through a primer extension reaction and/or ligation.
Sequencing-based analysis of extended tags to identify antigen and sample
Analyzing the encoder sequence or a complement thereof and the sample-specific barcode or a complement thereof in the extended coding tag or the extended recording tag, where analyzing comprises nucleic acid sequencing to identify the antigen and the sample that contains the antibody, thereby detecting the antibody in the sample.
Nucleic acid tag sample barcode carried in antibody complex
A nucleic acid tag comprising a sample-specific barcode is obtained as part of a complex from each sample, where the binding element binds antibody constant regions and not antibody variable regions.
Mixing sample complexes and attaching to beads for nucleic-acid-tag-associated antibodies
The pluralities of complexes from the plurality of samples are mixed and attached on beads, thereby obtaining a mixture of beads comprising attached antibodies each associated with a nucleic acid tag.
Transfer from antigen coding tag to nucleic-acid sample tag
Following binding of the antigen to an antibody attached to a bead, identifying information is transferred between the coding tag attached to the antigen and the nucleic acid tag associated with the antibody attached to the bead, thereby generating an extended coding tag or an extended nucleic acid tag, where the transfer occurs through a primer extension reaction and/or ligation.
The independent claims share the same core scheme: bead-associated antibodies with sample-specific barcodes are combined, antigen-binding agents carry coding tags with antigen-identifying encoder sequences, and an identifying-information transfer via primer extension and/or ligation generates extended tags that are read by nucleic acid sequencing to identify both the antigen and the sample containing the antibody.
Stated Advantages
Enables multiplexed detection and quantification of antigen- and sample-associated antibodies via encoding-based information transfer and nucleic acid sequencing.
Scales sample multiplexing and antigen multiplexing.
Allows updating antigen panels.
Compatible with multiple antibody isotypes.
Supports pathogen serotyping and antigen-epitope profiling.
Provides stated ability for low limit of detection.
Provides stated assessment of sample crosstalk and reproducibility and supports multiplexing up to 96 samples.
Allows detection of antibodies in the sample by identifying antigen and the sample that contains the antibody using nucleic acid sequencing of extended tags.
Documented Applications
Pathogen serotyping, including stated examples such as SARS-CoV-2 and influenza H1N1 peptide epitopes.
Cancer immunology/neoantigen profiling.
Autoimmune profiling.
Toxin screening.
Therapeutics development.
Biosecurity.
Veterinary medicine.
Multiplex sample analysis to detect multiple different antibodies across a plurality of samples using extended coding/recording tags and nucleic acid sequencing.
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