Immunoassay reagents and methods of use thereof
First Claim
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1. A method of detecting presence and/or distribution of three antigens in a sample, comprising:
- simultaneously contacting a sample with a primary antibody cocktail comprising a buffer aqueous solution of anti-P504S antibody from a first animal species as a first primary antibody, anti-p63 antibody from a second animal species different from the first animal species as a second primary antibody, and anti-high molecular weight cytokeratin (anti-HMWCK) antibody from the second animal species as a third primary antibody, wherein the sample is a tissue or cell, under conditions sufficient for the primary antibodies to specifically bind to antigens in the sample;
simultaneously contacting the sample, which has been previously simultaneously contacted with the primary antibody cocktail with a composition comprising a first secondary antibody and a second secondary antibody under conditions sufficient to form secondary antibody complexes with the primary antibodies to antigens in the contacted sample,wherein the first secondary antibody is specific for the anti-P504S primary antibody in the primary antibody cocktail and the second secondary antibody is specific for the rest of the primary antibodies in the primary antibody cocktail,wherein the first secondary antibody is coupled to a poly (alkaline phosphatase) (poly AP) moiety and the second secondary antibody is coupled to a poly (horseradish peroxidase) (poly HRP) moiety, andwherein the composition comprises a buffer for the first and second secondary antibodies; and
applying two different chromogens that result in two different colors for separately detecting the bound poly HRP moiety and the bound poly AP moiety on the secondary antibody-contained sample, thereby detecting the secondary antibody complexes on the contacted sample as indicative of the presence and/or distribution of the P504S, p63, and/or HMWCK antigens in the sample,wherein the two different chromogens are (i) 3,3′
-diaminobenzidine (DAB) for detecting the poly HRP moiety and (ii) a combination of at least one Fast Red salt with at least one naphthol phosphate (Fast Red) for detecting the poly AP moiety.
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Abstract
The present invention provide reagents and methods of using the reagents, for example, on automated staining devices, that facilitate detection of two or more antigens in a sample simply and efficiently.
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Citations
13 Claims
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1. A method of detecting presence and/or distribution of three antigens in a sample, comprising:
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simultaneously contacting a sample with a primary antibody cocktail comprising a buffer aqueous solution of anti-P504S antibody from a first animal species as a first primary antibody, anti-p63 antibody from a second animal species different from the first animal species as a second primary antibody, and anti-high molecular weight cytokeratin (anti-HMWCK) antibody from the second animal species as a third primary antibody, wherein the sample is a tissue or cell, under conditions sufficient for the primary antibodies to specifically bind to antigens in the sample; simultaneously contacting the sample, which has been previously simultaneously contacted with the primary antibody cocktail with a composition comprising a first secondary antibody and a second secondary antibody under conditions sufficient to form secondary antibody complexes with the primary antibodies to antigens in the contacted sample, wherein the first secondary antibody is specific for the anti-P504S primary antibody in the primary antibody cocktail and the second secondary antibody is specific for the rest of the primary antibodies in the primary antibody cocktail, wherein the first secondary antibody is coupled to a poly (alkaline phosphatase) (poly AP) moiety and the second secondary antibody is coupled to a poly (horseradish peroxidase) (poly HRP) moiety, and wherein the composition comprises a buffer for the first and second secondary antibodies; and applying two different chromogens that result in two different colors for separately detecting the bound poly HRP moiety and the bound poly AP moiety on the secondary antibody-contained sample, thereby detecting the secondary antibody complexes on the contacted sample as indicative of the presence and/or distribution of the P504S, p63, and/or HMWCK antigens in the sample, wherein the two different chromogens are (i) 3,3′
-diaminobenzidine (DAB) for detecting the poly HRP moiety and (ii) a combination of at least one Fast Red salt with at least one naphthol phosphate (Fast Red) for detecting the poly AP moiety. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A detecting system for detecting antigens in a sample comprising at least four separately contained reagents:
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the first separately contained reagent being a primary antibody cocktail comprising a buffer aqueous solution of anti-P504S antibody from a first animal species as a first primary antibody, anti-p63 antibody from a second animal species different from the first animal species as a second primary antibody, and anti-high molecular weight cytokeratin (anti-HMWCK) antibody from the second animal species as a third primary antibody; the second separately contained reagent being a buffer composition comprising a first secondary antibody and a second secondary antibody, wherein the first secondary antibody is specific for the anti-P504S primary antibody antibodies in the primary antibody cocktail and the second secondary antibody is specific for the rest of the primary antibodies in the primary antibody cocktail, and wherein the first secondary antibody is coupled to a poly (alkaline phosphatase) (poly AP) moiety and the a second secondary antibody is coupled to a poly (horseradish peroxidase) (poly HRP) moiety; and at least two different separately contained chromogen reagents that result in at least two different colors, wherein a first separately contained chromogen reagent is for detecting the poly HRP moiety and is selected from the group consisting of 3,3′
-diaminobenzidine (DAB), 3-amino-9-ethylcarbazole (AEC), and 4-chloro-l-naphthol (Bajoran Purple), andwherein a second separately contained chromogen reagent is for detecting the poly AP moiety and is a combination of at least one Fast Red salt with at least one naphthol phosphate (Fast Red), and a combination of at least one Fast Blue salt with at least one naphthol phosphate (Fast or Ferangi Blue). - View Dependent Claims (11, 12, 13)
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Specification