Immunoassay reagents and methods of use thereof

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, 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.

2. The method of claim 1 , wherein the sample is contained in an automated staining device and wherein the contacting occurs in the automated staining device.

3. The method of claim 1 , wherein the first primary antibody is a rabbit monoclonal or polyclonal antibody.

4. The method of claim 1 , wherein the primary antibodies other than the first primary antibody are mouse antibodies.

5. The method of claim 4 , wherein the first secondary antibody is a goat anti-rabbit antibody and the second secondary antibody is a goat anti-mouse antibody.

6. The method of claim 1 , wherein the method is completed in not more than 15 steps including washing steps.

7. The method of claim 1 , wherein the buffered aqueous solution of the primary antibody cocktail comprises 2-methyl-4-isothiazolin-3-one as a preservative.

8. The method of claim 1 , wherein the method is completed in 2 to 2.5 hour.

9. The method of claim 1 , wherein the primary antibody cocktail consists of anti-P504S antibody, anti-p63 antibody, and anti-HMWCK antibody.

10. A detecting system for detecting antigens in a sample comprising at least four separately contained reagents:

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), and

wherein 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).

11. The composition of claim 10 , wherein the primary antibody cocktail consists of anti-P504S antibody, anti-p63 antibody, and anti-HMWCK antibody.

12. The detecting system of claim 10 , wherein the primary antibodies other than the first primary antibody are mouse antibodies.

13. The detecting system of claim 12 , wherein the first secondary antibody is a goat anti-rabbit antibody and the second secondary antibody is a goat anti-mouse antibody.