Ce-western applications for antibody development

Abstract

Methods for detecting and/or discriminating between variants of an antibody contaminating protein or multiple antibodies in a sample by a physical parameter, in which the method includes: separating protein components of a sample by molecular weight or charge in one or more capillaries using capillary electrophoresis; immobilizing the protein components of the sample within the one or more capillaries; contacting the protein components within the one or more capillaries with one or more primary antibodies that specifically bind to the antibody, the contaminating protein or multiple antibodies in the sample, thereby detecting and/or discriminating between variants in the sample.

What is claimed is: 
     
         1 . A method for detecting and/or discriminating between variants of an antibody in a sample by a physical parameter, comprising:
 separating protein components of a sample comprising an antibody of interest by molecular weight or charge in one or more capillaries using capillary electrophoresis, wherein the antibody of interest is conjugated to biotin;   immobilizing the protein components of the sample within the one or more capillaries;   contacting the protein components within the one or more capillaries with one or more primary antibodies that specifically bind to the antibody of interest and with streptavidin conjugated to a detectable label; and   detecting the binding of the one or more primary antibodies and the binding of the streptavidin conjugated to a detectable label, thereby detecting and/or discriminating between size variants or charge variants of the antibody of interest in the sample.   
     
     
         2 . The method of  claim 1 , wherein the one or more primary antibodies comprise at least one antibody that specifically binds a heavy chain of the antibody of interest. 
     
     
         3 . The method of  claim 1 , wherein the one or more primary antibodies comprise at least one antibody that specifically binds a light chain of the antibody of interest. 
     
     
         4 . The method of  claim 1 , wherein the one or more primary antibodies are labeled with a detectable label, and wherein detecting the binding of the one or more primary antibodies comprises detecting the detectable label. 
     
     
         5 . The method of  claim 1 , wherein detecting the binding of the one or more primary antibodies comprises:
 contacting the one or more primary antibodies with a secondary antibody that specifically binds at least one of the one or more primary antibodies, and wherein the secondary antibody has a detectable label; and   detecting the detectable label.   
     
     
         6 . The method of  claim 1 , wherein the protein components of a sample are separated by charge and the method is a method of detecting and/or discriminating between charge variants of the antibody of interest. 
     
     
         7 . The method of  claim 1 , wherein the protein components of a sample are separated by molecular weight and the method is a method of detecting and/or discriminating between size variants of the antibody of interest. 
     
     
         8 . The method of  claim 1 , wherein the sample comprises one or more additional antibodies of interest. 
     
     
         9 . The method of  claim 8 , wherein the one or more additional antibodies of interest are detected. 
     
     
         10 . The method of  claim 1 , further comprising determining a relative or absolute amount of the variants of the antibody in a sample. 
     
     
         11 . The method of  claim 1 , wherein the antibody of interest comprises a bispecific antibody. 
     
     
         12 . The method of  claim 1 , wherein the detectable label comprises a chemiluminescent label, a fluorescent label, or a bioluminescent label. 
     
     
         13 . The method of  claim 12 , wherein the chemiluminescent label comprises horseradish peroxidase. 
     
     
         14 . The method of  claim 1 , wherein the immobilizing comprises photo-immobilizing, chemically immobilizing, or thermally immobilizing. 
     
     
         15 . The method of  claim 1 , wherein the one or more capillaries comprise a separation matrix. 
     
     
         16 . The method of  claim 15 , wherein the separation matrix comprises carrier ampholytes. 
     
     
         17 . The method of  claim 15 , wherein the separation matrix comprises a sieving matrix configured to separate proteins by molecular weight.