Assay system for simultaneous detection and measurement of multiple modified cellular proteins

Abstract

A method and kit for simultaneous detection and/or determination of a plurality of modified proteins in a sample. The method comprises: a) contacting the sample under mild protein denaturation conditions with a plurality of first antibodies capable of binding to a specific target protein, the first antibodies being immobilized on solid support material, each first antibody being differentiable from others by a differentiation parameter, whereby the first antibodies bind to respective target proteins present in the sample; b) removing unbound materials from the locus of the first antibodies; c) contacting the materials from step (b) with one or more second antibodies, each of which is specific to a class or subclass of modified proteins or with a plurality of second antibodies, each of which is specific to a modified protein, so as to bind the second antibody or antibodies to modified proteins in the sample; and d) detecting and/or determining a plurality of modified proteins in the sample. The kit comprises a plurality of primary antibodies immobilized on the above-mentioned solid support material, one or more buffers for lysing and for washing cellular material samples to be assayed, an assay buffer for conducting the assay, the buffer containing a sulfate or sulfonate detergent, and one or more second antibodies specific to the modified proteins. Also disclosed is a process for mild denaturation of modified proteins for use in such a procedure.

1 - 23 . (canceled)  
   
   
       24 . A kit for simultaneous detection and/or determination of a plurality of modified proteins in a sample, comprising: 
 (a) a plurality of first antibodies, each capable of binding to a specific target protein, each first antibody being immobilized on a solid support material and each first antibody being differentiable from others by a differentiation parameter;    (b) one or more buffers for lysing and for washing cellular material samples to be assayed (c) an assay buffer for conducting the assay, said buffer containing from about 1 -10 mM of a sulfate or sulfonate detergent; and    (d) one or more second antibodies specific to classes or subclasses of modified proteins or to specific individual modified proteins.    
   
   
       25 . A kit according to  claim 24  wherein the solid support material comprises a series of subsets of solid particles, each subset being distinguishable from other subsets in accordance with a particular property or characteristic.  
   
   
       26 . A kit according to  claim 25  in which the solid particles are differentiable by specific color or emission spectra.  
   
   
       27 . A kit according to  claim 25  in which the solid particles comprise spherical particles formed from non-porous glass, polystyrene or latex.  
   
   
       28 . A kit according to  claim 24  in which the solid support material is a microchip, a plate having a multiplicity of wells, or a slide.  
   
   
       29 . A kit according to  claim 24  wherein the modified proteins are selected from phosphorylated proteins, glycosylated proteins, acetylated proteins, methylated proteins, ubiquinated proteins, and prenylated proteins.  
   
   
       30 . A kit according to  claim 24  wherein the modified proteins are phosphorylated proteins.  
   
   
       31 . A kit according to  claim 24  wherein the second antibodies comprise one or more antibodies that are specific to classes of modified proteins.  
   
   
       32 . A kit according to  claim 24  wherein the second antibodies comprise one or more antibodies that are specific to subclasses of modified proteins.  
   
   
       33 . A kit according to  claim 24  wherein the second antibodies are specific to phosphorylated proteins.  
   
   
       34 . A kit according to  claim 24  wherein the second antibodies comprise a plurality of antibodies, each of which is specific to an individual modified protein.  
   
   
       35 . A kit according to  claim 24  further comprising a labeled moiety.  
   
   
       36  and  37 . (canceled)