Fluorescent hybridization of antibody-oligonucleotide for multiplexing and signal amplification
Inventors
Uytingco, Cedric • Bent, Zachary
Assignees
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Publication Number
US-12553898-B1
Publication Date
2026-02-17
Expiration Date
Abstract
Provided herein are methods of determining a location or presence of a target analyte in a biological sample by detecting oligonucleotide-conjugated binding agents.
Core Innovation
The invention relates to multiplex and signal-amplified immunostaining strategies that determine a location or the presence of a target protein in a tissue sample. Oligonucleotide-conjugated analyte-binding moieties, including antibodies or antigen-binding fragments, bind target proteins in the sample and carry oligonucleotides. Detection probes then hybridize to nucleic acid sequences on the conjugates and provide detectable labels, enabling location or presence determination based on where hybridization occurs in the tissue.
Hybridization is performed between nucleic acid sequences on detection probes and oligonucleotide sequences on the oligonucleotide-conjugated binding agents. Signal amplification is enabled via oligonucleotides that include repeated or concatenated sequences, and multiplexing is enabled by using different probe labels and/or different oligonucleotide sequence sets. The approach also accommodates oligonucleotide design variations, including using the same oligonucleotide sequences versus different oligonucleotide sequences and using the same detectable label versus different detectable labels.
The described workflow includes optional fixation and washing, followed by contacting the tissue sample with the detection probes and detecting the detectable labels. Imaging and detection modalities include epifluorescence microscopy, confocal microscopy, and multiphoton microscopy. The document further discusses related spatial analysis and barcoding context, and includes embodiment parameters for oligonucleotide sequence structures and for selecting fixation conditions, including FFPE and fresh frozen tissue sample.
Claims Coverage
The partial content includes three independent claims directed to determining the location of a target protein, a similar location-determination method using a single plurality of oligonucleotide-conjugated binding agents, and determining the presence of a protein. Across the independent claims, the core coverage includes oligonucleotide-conjugated antibody or antigen-binding fragments, detection probes with hybridizing nucleic acid sequences and detectable labels, and detection to yield spatial location or presence in a tissue sample. The claims also require fixation and detection of the labels after contacting with detection probes.
Oligonucleotide-conjugated binding agent subsets for location determination with paired antibody conjugates
Deliver a first subset of antibodies or antigen-binding fragments that bind the target protein, and deliver a first plurality of oligonucleotide-conjugated binding agents comprising a second subset of antibodies or antigen-binding fragments that bind to the first subset bound to the target protein, wherein the second antibody or antigen-binding fragment is conjugated to an oligonucleotide comprising at least two oligonucleotide sequences capable of hybridizing to a first detection probe; deliver a second plurality of oligonucleotide-conjugated binding agents comprising a third subset that binds the target protein, wherein the third antibody or antigen-binding fragment is conjugated to an oligonucleotide comprising at least two oligonucleotide sequences capable of hybridizing to a second detection probe.
Fixation and hybridizing detection probes with detectable labels for spatial location
Fix the tissue sample; contact the tissue sample with a plurality of detection probes wherein at least two detection probes each comprises a nucleic acid sequence that hybridizes to a portion of one of the oligonucleotide sequences of the first and second pluralities of oligonucleotide-conjugated binding agents and a detectable label; detect the detectable labels to determine the locations of the target protein in the tissue sample.
Oligonucleotide-conjugated binding agents with at least two oligonucleotide sequences for location determination
Deliver a plurality of oligonucleotide-conjugated binding agents to the tissue sample, wherein an oligonucleotide-conjugated binding agent comprises an antibody or antigen-binding fragment that binds the target protein and is conjugated to an oligonucleotide comprising at least two oligonucleotide sequences.
Hybridizing detection probes with detectable labels for determining protein location
Fix the tissue sample; contact the tissue sample with a plurality of detection probes wherein at least two detection probes each comprises a nucleic acid sequence that hybridizes to a portion of one of the at least two oligonucleotide sequences and a detectable label; detect the detectable labels of the at least two detection probes to determine the location of the target protein in the tissue sample.
Oligonucleotide-conjugated binding agents and hybridizing detection probes for protein presence determination
Deliver a plurality of oligonucleotide-conjugated binding agents to the tissue sample, wherein an oligonucleotide-conjugated binding agent comprises an antibody or antigen-binding fragment that binds the protein and is conjugated to an oligonucleotide comprising at least two oligonucleotide sequences; fix the tissue sample; contact the tissue sample with a plurality of detection probes wherein at least two detection probes each comprises a nucleic acid sequence that hybridizes to a portion of one of the at least two oligonucleotide sequences and a detectable label; detect the detectable labels of the at least two detection probes to determine the presence of the protein in the tissue sample.
The independent claims cover methods that determine either location or presence of a target protein in a tissue sample by delivering oligonucleotide-conjugated antibody or antigen-binding fragments, fixing the tissue sample, contacting with detection probes that hybridize to oligonucleotide sequences and carry detectable labels, and detecting the labels to produce the location or presence result. One claim additionally structures the delivery into subsets and first and second pluralities linked to different detection probes.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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