Method for evaluation of target in histological sample

Abstract

The present invention lies in the field of visualization and quantification of immobilized targets in samples using immunochemical means. In particular, the invention relates to a method and reagents for detection, visualization and quantification of a molecular target in immunosiained histological samples using particular compositions of the target specific binding agent. Methods and compositions of the invention are suitable for any assay that uses a target visualization system in histological samples based on detection of the target by the target specific binding agent. The methods and compositions tire useful for evaluation of targets that are biomarkers of diseases in medical diagnostic.

1 - 16 . (canceled) 
     
     
         17 . A kit-of-parts comprising a composition comprising a binding agent which is capable of specifically binding to the binding partner comprised in said one or more target sites,
 wherein the amount of the binding agent is sufficient to bind to substantially all units of the binding partner present in the sample, and   wherein the binding agent comprises first binding molecules and second binding molecules,
 wherein the first binding molecules comprise a binding part and a detectable part, and the second binding molecules comprise a binding part, 
 wherein the binding part of both first and second binding molecules is capable of specifically binding to the binding partner and competing for said binding, and 
 wherein the second binding molecules do not comprise a part that is substantially identical to the detectable part of the first binding molecules. 
   
     
     
         18 . The kit-of-parts of  claim 17 , further comprising one or more of the following
 (i) reagents for visualizations of the detectable part of the first binding molecule;   (ii) protocols for visualizations of the detectable part of the first binding molecule;   (iii) guidance for quantification of a target in a test sample;   (iv) reference materials   (v) instrument(s) for visual analysis of the sample and/or image capture, or a reference to such instruments;   (vi) software for controlling the instruments;   (vii) software for image analysis;   (viii) locked image analysis algorithms;   (ix) standards for evaluating the samples, e.g. scoring standards and scoring guidelines;   (x) instructions for use.   
     
     
         19 . The kit-of-part of  claim 17 , for evaluation of a target in a histological sample. 
     
     
         20 . The kit-of-parts of  claim 17 , wherein the binding agent comprises an antibody, or a fragment or a derivative thereof, or a nucleic acid or a nucleic acid analog sequence. 
     
     
         21 . The kit-of-parts of  claim 17 , wherein the first binding molecules and the second binding molecules are antibodies that are (i) specific for the same antigen; (ii) capable of inhibiting each other binding to said antigen, and (iii) have different Fc regions. 
     
     
         22 . The kit-of-parts of claim, wherein the first binding molecule is an antibody and the second binding molecule is the F(ab)2 fragment of the same antibody. 
     
     
         23 . The kit-of-parts of  claim 19 , wherein the target is a biological or chemical target molecule, particle, molecular or cellular complex, molecular or cellular structure, virus or microorganism, or a fragment of said target molecule, particle, complex, structure, virus or microorganism. 
     
     
         24 . The kit-of-parts of  claim 17 , comprising the reference material which is one or more histological samples comprising cells with predetermined amounts of the target. 
     
     
         25 . The kit-of-parts of  claim 17 , for the quantitative evaluation of the target. 
     
     
         26 - 30 . (canceled) 
     
     
         31 . A kit-of-parts comprising a composition comprising a binding agent which is capable of specifically binding to the binding partner comprised in said one or more target sites,
 wherein the amount of the binding agent in the incubation medium is sufficient to bind to at least 51% of the binding partners present in the sample, and   wherein the binding agent comprises a population of first binding molecules and a population of second binding molecules,   wherein the first binding molecule comprises a binding part and a detectable part, and the second binding molecule comprises a binding part,   wherein the binding part of the first binding molecule and the binding part of the second binding molecule are both capable of specifically binding to the binding partner and competing against each other for said binding,   wherein the first binding molecule comprises an antibody molecule, or a derivative thereof, comprising an Fc region, and   wherein the second binding molecule does not comprise a part that can be detected by a detector of the detectable part of the first binding molecule and the second binding molecule does not comprise the Fc region of the first binding molecule.   
     
     
         32 . The kit-of-parts of  claim 31 , wherein the binding partner is the target or a substance associated with the target. 
     
     
         33 . The kit-of-parts of  claim 31 , wherein the binding agent and the binding partner are members of a specific binding pair. 
     
     
         34 . The kit-of-parts of  claim 31 , wherein the first and second binding molecules each comprise an antibody or an antigen-binding portion of an antibody, and the target comprises an antigen. 
     
     
         35 . The kit-of-parts of  claim 31 , wherein the antibody molecule, or a derivative thereof, is specific for an epitope comprised in the binding partner, and
 the second binding molecule comprises an antigen binding portion that is specific for the same epitope as said antibody or is capable of inhibiting the first binding molecule binding to said epitope.   
     
     
         36 . The kit-of-parts of  claim 34 , wherein the binding part of the first binding molecule and the binding part of the second binding molecule comprise an antigen binding part of the same antibody or antigen binding parts of two different antibodies that are specific for the same binding partner. 
     
     
         37 . The kit-of-parts of claim  1 , wherein the concentration of the binding agent in the incubation medium is greater than the Kd (dissociation constant) value of the binding agent-binding partner complex. 
     
     
         38 . A kit-of-parts comprising a composition comprising:
 a first binding molecule comprising an antibody molecule, or a derivative thereof, comprising an Fc region, wherein said antibody or derivative thereof is capable of specifically binding said target,   a second binding molecule capable of specifically binding said target, wherein said second binding molecule does not comprise the Fc region of the first binding molecule, and   a detectable agent specific for said Fc region of said first binding molecule.   
     
     
         39 . The kit-of-parts of  claim 38 , wherein said second binding molecule is an F(ab)2 fragment. 
     
     
         40 . The method of  claim 39 , wherein the amounts of the first binding molecule and the second binding molecule together are sufficient to bind to at least 51% of the target present in the sample. 
     
     
         41 . The method of  claim 40 , wherein said target is the Her2 protein.