Immunoassay reagents and methods of use thereof

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.

1 . (canceled) 
     
     
         2 . An antibody composition comprising:
 (a) an anti-p63 antibody;   (b) an antibody to at least one cytokeratin marker of myoepithelial cells; and   (c) an antibody to at least one cytokeratin marker of lumenal cells.   
     
     
         3 . The antibody composition of  claim 2 , wherein the antibody (b) is selected from the group consisting of CK5, CK14, and CK17. 
     
     
         4 . The antibody composition of  claim 2 , wherein the antibody (c) is selected from the group consisting of CK7, CK8, CK18, and CK19. 
     
     
         5 . The antibody composition of  claim 2 , wherein the antibody (b) is CK5 and the antibody (c) is CK8, CK18, or both. 
     
     
         6 . The antibody composition of  claim 2 , wherein at least one antibody is from a different species than at least a second antibody. 
     
     
         7 . The antibody composition of  claim 2 , wherein at least one antibody is a rabbit antibody. 
     
     
         8 . The antibody composition of  claim 2 , wherein the at least one rabbit antibody is a rabbit monoclonal antibody. 
     
     
         9 . The antibody composition of  claim 2 , wherein the p63 antibody and the at least one myoepithelial marker are stainable with one color stain and the at least one luminal marker is stainable with another color stain. 
     
     
         10 . A kit, comprising:
 a primary antibody cocktail comprising:   (a) an anti-p63 antibody as a first primary antibody;   (b) an antibody to at least one cytokeratin marker of myoepithelial cell as a second primary antibody; and   (c) an antibody to at least one cytokeratin marker of lumen al cells a third primary antibody;   a buffer aqueous solution for the primary antibody cocktail;   a composition comprising a first secondary antibody and a second secondary antibody to form at least three antigen-antibody complexes on the sample,   wherein the first secondary antibody is specific for one or two of the primary 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,   wherein   (i) the at least one first secondary antibody is coupled to a poly (alkaline phosphatase) (poly AP) moiety and the at least one second secondary antibody is coupled to a poly (horseradish peroxidase) (poly HRP) moiety, or   (ii) the at least one first secondary antibody is coupled to a poly (horseradish peroxidase) (poly HRP) moiety and the at least one second secondary antibody is coupled to a poly (alkaline phosphatase) (poly AP) moiety,   wherein the composition comprises a buffer for the composition; and   at least two different chromogens that result in at least two different colors,   wherein the at least two different chromogens are selected from the group consisting of DAB, 3-amino-9-ethylcarbazole (AEC), Fast Red, 4-chloro-l-naphthol (Bajoran Purple), and a combination of at least one diazonium salt with at least one naphthol phosphate, wherein diazonium is selected from the group consisting of 4-(4-diazonio-3-methoxy-phenyl)-2-methoxybenzenediazonium, 4-Benzoylamino-2,5-diethoxybenzenediazonium, and 4-Benzoylamino-2,5-dimethoxybenzenediazonium, and the naphthol phosphate is selected from the group consisting of 3-Hydroxy-2-naphthanilide phosphate, 7-Bromo-3-hydroxy-2-naphthoic-o-anisidide phosphate, [3-[(4-chloro-2-methyl-phenyl)carbamoyl]-2-naphthyl]dihydrogen phosphate, [3-[(2,4-dimethylphenyl)carbamoyl]-2-naphthyl]dihydrogen phosphate, [3[(3-nitrophenyl)carbamoyl]-2-naphthyl] dihydrogen phosphate, [3-[(4-chlorophenyl)carbamoyl]-2-naphthyl]dihydrogen phosphate, [3-[(2-methoxyphenyl)carbamoyl]-2-naphthyl]dihydrogen phosphate, and [3-(o-tolylcarbamoyl)-2-anthryl]dihydrogen phosphate,   wherein DAB is 3,3′-diaminobenzidine, and Fast Red is a combination of at least one diazonium salt with at least one naphthol phosphate, wherein diazonium is selected from the group consisting of 4-Chloro-2-methylbenzenediazonium, 5-Chloro-2-methoxybenzenediazonium, 2-Carbamoyl-5-methoxybenzenediazonium, 2-Methoxy-4-nitrobenzenediazonium, 5-Diethylaminosulfonyl-2-methoxybenzenediazonium, 5-Chloro-2-methylbenzenediazonium, 2-Methyl-4-nitrobenzenediazonium, and 5-Chloro-4-benzamido-2-methylbenzenediazonium, and the naphthol phosphate is selected from the group consisting of 3-Hydroxy-2-naphthanilide phosphate, 7-Bromo-3-hydroxy-2-naphthoic-o-anisidide phosphate, [3-[(4-chloro-2-methyl-phenyl)carbamoyl]-2-naphthyl]dihydrogen phosphate, [3-[(2,4-dimethylphenyl)carbamoyl]-2-naphthyl]dihydrogen phosphate, [3-[(3-nitrophenyl)carbamoyl]-2-naphthyl] dihydrogen phosphate, [3-[(4-chlorophenyl)carbamoyl]-2-naphthyl]dihydrogen phosphate, [3-[(2-methoxyphenyl)carbamoyl]-2-naphthyl]dihydrogen phosphate, and [3-(o-tolylcarbamoyl)-2-anthryl]dihydrogen phosphate,   wherein the p63 antibody and the at least one myoepithelial marker are stainable with one color stain and the at least one luminal marker is stainable with a different color stain.   
     
     
         11 . The kit of  claim 10 , wherein the antibody (b) is CK5 and the antibody (c) is CK8, CK18, or both. 
     
     
         12 . The kit of  claim 10 ,
 wherein the primary antibody cocktail comprises mouse anti-CK5 and anti-p63 antibodies and a rabbit anti-CK18 antibody,   wherein at least one of the secondary antibodies is a goat anti-mouse antibody coupled to the poly HRP moiety and at least one of the secondary antibodies is a goat anti-rabbit antibody coupled to the poly AP moiety, and   wherein the at least two different chromogens are DAB and Fast Red chromogens, which result in brown staining when applied, of basal myoepithelial cells containing CK5 and p63 and red staining of luminal cells containing CK18, respectively.   
     
     
         13 . A kit, comprising:
 a primary antibody cocktail comprising:   (a) an anti-p63 antibody as a first primary antibody;   (b) an antibody to at least one cytokeratin marker of myoepithelial cell as a second primary antibody; and   (c) an antibody to at least one cytokeratin marker of lumen al cells a third primary antibody; and   a buffer aqueous solution for the primary antibody cocktail.   
     
     
         14 . The kit of  claim 13 , wherein the antibody (b) is CK5 and the antibody (c) is CK8, CK18, or both. 
     
     
         15 . A kit comprising the antibody composition of  claim 2  and one or more reagents to detect an antibody-antigen complex. 
     
     
         16 . The kit of  claim 15 , wherein at least one antibody is a rabbit antibody. 
     
     
         17 . The kit of  claim 15 , wherein the at least one rabbit antibody is a rabbit monoclonal antibody. 
     
     
         18 . The kit of  claim 15 , wherein the antibody (b) is CK5 and the antibody (c) is CK8, CK18, or both. 
     
     
         19 . A method of detecting three antigens in a sample, comprising:
 simultaneously or sequentially contacting a sample with a primary antibody cocktail comprising:   (a) an anti-p63 antibody as a first primary antibody,   (b) an antibody to at least one cytokeratin marker of myoepithelial cell as a second primary antibody, and   (c) an antibody to at least one cytokeratin marker of lumen al cells a third primary antibody,   wherein the sample is a tissue or cell;   simultaneously contacting the sample, which has been previously simultaneously or sequentially contacted with the primary antibody cocktail comprising the first, the second and the third primary antibodies in a buffered aqueous solution for the primary antibody cocktail, with a composition comprising a first secondary antibody and a second secondary antibody to form three antigen-antibody complexes on the sample, thereby forming three antigen-antibody complexes,   wherein the first secondary antibody is specific for the anti-p63 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   (i) 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, or   (ii) the at least one first secondary antibody is coupled to a poly (horseradish peroxidase) (poly HRP) moiety and the at least one second secondary antibody is coupled to a poly (alkaline phosphatase) (poly AP) moiety,   wherein the composition comprises a buffer for the first and second secondary antibodies; and   applying two different chromogens that result in two different colors, and detecting the three antigen-antibody complexes in the sample,   wherein the two different chromogens are (i) 3,3′-diaminobenzidine (DAB) and (ii) a combination of at least one diazonium salt with at least one naphthol phosphate (Fast Red), wherein diazonium is selected from the group consisting of 4-Chloro-2-methylbenzenediazonium, 5-Chloro-2-methoxybenzenediazonium, 2-Carbamoyl-5-methoxybenzenediazonium, 2-Methoxy-4-nitrobenzenediazonium, 5-Diethylaminosulfonyl-2-methoxybenzenediazonium, 5-Chloro-2-methylbenzenediazonium, 2-Methyl-4-nitrobenzenediazonium, and 5-Chloro-4-benzamido-2-methylbenzenediazonium, and the naphthol phosphate is selected from the group consisting of 3-Hydroxy-2-naphthanilide phosphate, 7-Bromo-3-hydroxy-2-naphthoic-o-anisidide phosphate, [3-[(4-chloro-2-methyl-phenyl)carbamoyl]-2-naphthyl]dihydrogen phosphate, [3-[(2,4-dimethylphenyl)carbamoyl]-2-naphthyl]dihydrogen phosphate, [3-[(3-nitrophenyl)carbamoyl]-2-naphthyl] dihydrogen phosphate, [3-[(4-chlorophenyl)carbamoyl]-2-naphthyl]dihydrogen phosphate, [3-[(2-methoxyphenyl)carbamoyl]-2-naphthyl]dihydrogen phosphate, and [3-(o-tolylcarbamoyl)-2-anthryl]dihydrogen phosphate.   
     
     
         20 . The method of  claim 19 , wherein the antibody (b) is CK5 and the antibody (c) is CK8, CK18, or both. 
     
     
         21 . The method of  claim 19 ,
 wherein the primary antibody cocktail comprises mouse anti-CK5 and anti-p63 antibodies and a rabbit anti-CK18 antibody,   wherein at least one of the secondary antibodies is a goat anti-mouse antibody coupled to the poly HRP moiety and at least one of the secondary antibodies is a goat anti-rabbit antibody coupled to the poly AP moiety, and   wherein the at least two different chromogens are DAB and Fast Red chromogens, which result in brown staining when applied, of basal myoepithelial cells containing CK5 and p63 and red staining of luminal cells containing CK18, respectively.   
     
     
         22 . The method of  claim 19 , wherein the sample is contained in an automated staining device and wherein the contacting occurs in the automated staining device. 
     
     
         23 . The method of  claim 19 , wherein the first primary antibody is a rabbit monoclonal or polyclonal antibody. 
     
     
         24 . The method of  claim 19 , wherein the method is completed in not more than 15 steps including washing steps. 
     
     
         25 . The method of  claim 19 , wherein the buffered aqueous solution of the primary antibody cocktail comprises 2-methyl-4-isothiazolin-3-one as a preservative. 
     
     
         26 . The method of  claim 19 , wherein the method is completed in 2 to 2.5 hour. 
     
     
         27 . The method of  claim 19 , wherein the method is a double-staining method. 
     
     
         28 . A detecting system for detecting antigens in a sample comprising at least four separately contained reagents:
 a first separately contained reagent being a primary antibody cocktail comprising a buffer aqueous solution of:   (a) anti-p63 antibody from a first animal species as a first primary antibody,   (b) an antibody to at least one cytokeratin marker of myoepithelial cell from a second animal species different from the first animal species as a second primary antibody, and   (c) an antibody to at least one cytokeratin marker of lumen al cells from the second animal species as a third primary antibody,   a 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-p63 primary antibody 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, and   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; or   the first secondary antibody is coupled to a poly (horseradish peroxidase) (poly HRP) moiety and the second secondary antibody is coupled to a poly (alkaline phosphatase) (poly AP) 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-1-naphthol (Bajoran Purple), and   wherein a second separately contained chromogen reagent is for detecting the poly AP moiety and is selected from the group consisting of 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).   
     
     
         29 . The detecting system of  claim 28 , wherein the primary antibodies other than the first primary antibody are mouse antibodies. 
     
     
         30 . The detecting system of  claim 28 , wherein the first secondary antibody is a goat anti-rabbit antibody and the second secondary antibody is a goat anti-mouse antibody.