Methods of identifying immune cells in PD-L1 positive tumor tissue
AssigneeVentana Medical Systems, Inc.
InventorChandler Morgan Birch
Life of this patent
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Abstract
The present disclosure is directed to multiplex assays, kits and methods, including automated methods, for identifying PD-L1 positive immune cells, PD-L1 positive tumor cells, and PD-L1 negative immune cells within a tissue sample using two dyes selected from diaminobenzidine (DAB), 4-(dimethylamino) azobenzene-4′-sulfonamide (DABSYL), tetramethylrhodamine (DISCOVERY Purple), N,N′-biscarboxypentyl-5,5′-disulfonato-indo-dicarbocyanine (Cy5), and Rhodamine 110 (Rhodamine).
Claims13
An immunohistochemical method of identifying tumor infiltrating PD-L1 positive immune cells in a PD-L1 positive tumor via brightfield microscopy, said method comprising:
- contacting a tissue sample with a first primary antibody specific to PD-L1 protein in a manner to effect binding of the first primary antibody to the tissue sample in proximity to PD-L1 protein expressed by the tissue sample;
- depositing a first enzyme in proximity to the first primary antibody bound to the tissue sample;
- reacting the first enzyme with a first substrate to effect deposition of a first brightfield microscopy-detectable dye on the tissue sample in proximity to the first primary antibody;
- contacting the tissue sample with a second primary antibody specific to at least one immune cell marker protein in a manner to effect binding of the second primary antibody to the tissue sample in proximity to immune cell marker protein expressed by the tissue sample;
- depositing a second enzyme in proximity to the second primary antibody bound to the tissue sample and
- reacting the second enzyme with a second substrate to effect deposition of a second brightfield microscopy-detectable dye on the tissue sample in proximity to the second primary antibody,
- wherein (i) the first brightfield microscopy-detectable dye is 4-(dimethylamino) azobenzene-4′-sulfonamide and the second brightfield microscopy-detectable dye is tetramethylrhodamine, or (ii) the first brightfield microscopy-detectable dye is tetramethylrhodamine and the second brightfield microscopy-detectable dye is 4-(dimethylamino) azobenzene-4′-sulfonamide.
An immunohistochemical method of identifying PD-L1 positive immune cells in a tumor tissue section, said method comprising:
- contacting a tissue sample with a first primary antibody specific to PD-L1 protein in a manner to effect binding of the first primary antibody to the tissue sample in proximity to PD-L1 protein expressed by the tissue sample;
- depositing a first enzyme in proximity to the first primary antibody bound to the tissue sample;
- reacting the first enzyme with a first substrate to effect deposition of 4-(dimethylamino) azobenzene-4′-sulfonamide on the tissue sample in proximity to the first primary antibody, wherein the 4-(dimethylamino) azobenzene-4′-sulfonamide is deposited via a signaling conjugate comprising tyramide directly conjugated to 4-(dimethylamino) azobenzene-4′-sulfonamide;
- contacting the tissue sample with a second primary antibody specific to at least one immune cell marker protein in a manner to effect binding of the second primary antibody to the tissue sample in proximity to immune cell marker protein expressed by the tissue sample;
- depositing a second enzyme in proximity to the second primary antibody bound to the tissue sample;
- and
- reacting the second enzyme with a second substrate to effect deposition of tetramethylrhodamine on the tissue sample in proximity to the second primary antibody, wherein the tetramethylrhodamine is deposited via a signaling conjugate comprising tyramide directly conjugated to the tetramethylrhodamine, and
- detecting by brightfield microscopy cells that are co-stained with tetramethylrhodamine and 4-(dimethylamino) azobenzene-4′-sulfonamide in the tissue sample, thereby identifying PD-L1 positive immune cells.
Dependencies are inferred from the claim text.