Method of simultaneously detecting amplified nucleic acid sequences and cellular antigens in cells
AssigneeNorthwestern University
InventorsBruce Patterson, Steven Wolinsky, Michelle Till
Life of this patent
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Abstract
In one aspect, the present invention provides an in situ process of simultaneously detecting a specific predetermined nucleic acid sequence and a specific predetermined cellular antigen in the same cell. In accordance with that process, the antigen is labeled with a biotin- or DNP-tagged antibody that specifically immunoreacts with the antigen, the specific nucleic acid sequences in the cell are amplified, the amplified nucleic acid sequences are labeled with a fluorescently-tagged nucleic acid probe that specifically hybridizes to the amplified nucleic acid sequences, and the labeled nucleic acid sequences and labeled cellular antigen are detected.
Claims17
An in situ process of simultaneously detecting a specific predetermined nucleic acid sequence and a specific predetermined cellular antigen in the same intact cell, the process comprising the steps of:
- (a) labeling the antigen with a biotin- or DNP-tagged antibody that specifically binds to the antigen;
- (b) exposing the antigen-labeled cell to a water-soluble fixative and permeabilization agent;
- (c) amplifying the specific nucleic acid sequences in the cell in the presence of deoxyribonucleotide triphosphates coupled to a molecule that prevents diffusion of amplified sequences from the cell;
- (d) labeling the amplified nucleic acid sequences with a fluorescently-tagged nucleic acid probe that specifically hybridizes to the amplified nucleic acid sequences;
- and
- (e) detecting the labeled nucleic acid sequences and labeled cellular antigen, by determining the presence of the fluorescently-tagged probe and the biotin or DNP-tagged antibody, respectively.
An in situ process of simultaneously detecting HIV-1 proviral DNA and cell surface CD4 antigen in intact T cells, the process comprising the steps of:
- (a) labeling the CD4 cell surface antigen of the T cells with a biotin- or DNP-tagged antibody that specifically binds to the CD4 cell surface antigen;
- (b) exposing the antigen-labeled cells to a water-soluble fixative and permeabilization agent;
- (c) amplifying the HIV-1 proviral DNA nucleic acid sequences in the cells in the presence of deoxyribonucleotide triphosphates coupled to a molecule that prevents diffusion of amplified sequences from the cell;
- (d) labeling the amplified HIV-1 proviral DNA sequences with a fluorescently-tagged nucleic acid probe that is complementary to the HIV-1 proviral DNA sequences;
- and
- (e) detecting the labeled HIV-1 proviral DNA nucleic acid sequence and labeled CD4 cell surface antigen by fluorescence activated flow cytometry, by determining the presence of the fluorescently-tagged probe and the biotin or DNP-tagged antibody, respectively.
Dependencies are inferred from the claim text.