Non-enzymatic immunohistochemical staining system and reagents
AssigneeImmunomedics, Inc.
InventorDan Shochat
Life of this patent
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Abstract
A non-enzymatic method of immunohistochemical staining uses a bound electron transfer agent to achieve amplification of antibody binding to specific antigens of interest in a histology or cytology specimen. A preferred embodiment uses a bound phenazine, which is reduced with a soluble reducing agent, and which in turn reduces a tetrazolium salt, to precipitate a formazan dye at the site of primary antibody binding to the antigen of interest. Reagents and kits for use in the method are also provided.
Claims25
In an immunohistochemical method for staining a histology or cytology specimen to reveal the presence therein of at least one immunologically detectable antigen, wherein (1) said specimen is contacted with a solution of a primary antibody or antibody fragment which specifically binds to said antigen, (2) unbound primary antibody or antibody fragment is removed, and (3) the presence of bound primary antibody or antibody fragment is revealed as a stain by reaction with a staining reagent system capable of transforming a chromogen to a colored dye which precipitates at the site of the bound primary antibody or antibody fragment,
- the improvement wherein said primary antibody or antibody fragment is directly or indirectly conjugated, or linked through one or more bridging antibodies or other specific binding couples, to an electron transfer agent capable of transforming said chromogen to said dye in the presence of a soluble oxidizing or reducing agent;
- and wherein the staining reaction effected by said staining reagent system is effected without the use of an enzyme.
An enzyme-free immunohistochemical staining kit for staining histology or cytology specimens, comprising in suitable containers
- (A) at least one primary antibody or antibody fragment which specifically binds at least one immunologically detectable antigen whose presence in said specimen is to be detected and visualized:
- (B) an electron transfer agent capable of transforming a soluble chromogen to an insoluble dye in the presence of a soluble oxidizing or reducing agent, said electron transfer agent being directly or indirectly conjugated to said antibody or antibody fragment, or to a second antibody or antibody fragment which specifically binds to said primary antibody at a site which does not comprise its immunological reactivity, or to another carrier capable of linking with said primary antibody through the intermediacy of one or a plurality of specific binding couples;
- (C) a soluble chromogen;
- and
- (D) a soluble oxidizing or reducing agent;
- wherein the amounts of said antibody or antibody fragment, electron transfer agent, chromogen and oxidizing or reducing agent are each sufficient to enable said antigen to be stained if present in said specimen.
Dependencies are inferred from the claim text.