Methods and kits for separation and detection of proteins in biological samples

Abstract

Methods and kits are provided for separating a mixture of proteins in a biological sample. Methods for detecting and profiling proteins in biological samples by the separation method and kits are also provided. These methods are particularly useful in assessing damage to cells such as cardiac and skeletal muscle cells and in the early clinical diagnosis of myocardial damage by detection of myofilament proteins in serum of a subject.

What is claimed is:  
     
         1 . A method of separating a mixture of proteins in a biological sample comprising: 
 (a) mixing the biological sample with a solution comprising a sulfhydryl reducing agent, an anionic detergent, and at least one detergent selected from the group consisting of an ionic detergent, a non-ionic detergent and a zwitterionic detergent, at concentrations sufficient to substantially denature albumin in the mixture; and    (b) subjecting the mixture of biological sample and solution to a separation technique to separate proteins in the mixture.    
     
     
         2 . The method of  claim 1  further comprising characterizing the separated proteins.  
     
     
         3 . The method of  claim 2  wherein the separated proteins are characterized by Western blot.  
     
     
         4 . The method of  claim 1  wherein the biological sample comprises serum.  
     
     
         5 . The method of  claim 1  further comprising heating the mixture from step (a) prior to separation in step (b).  
     
     
         6 . The method of  claim 5  wherein the mixture is boiled.  
     
     
         7 . The method of  claim 1  wherein said separation technique is performed using SDS-PAGE.  
     
     
         8 . The method of  claim 1  wherein the anionic detergent is sodium dodecyl sulfate.  
     
     
         9 . A kit for separating a mixture of proteins in a biological sample comprising: 
 (a) a solution containing a sulfhydryl reducing agent, an anionic detergent, and at least one detergent selected from the group consisting of an ionic detergent, a non-ionic detergent and a zwitterionic detergent; and    (b) instructions for separating proteins in said serum.    
     
     
         10 . A method of assessing cellular injury in a subject comprising: 
 (a) separating a mixture of proteins in a biological sample of the subject in accordance with the method of  claim 1;  and    (b) characterizing the separated proteins, wherein said characterization is indicative of cellular injury in the subject.    
     
     
         11 . The method of  claim 10  wherein the separated proteins are characterized by Western blot.  
     
     
         12 . The method of  claim 10  wherein the characterized protein is at least one of troponin I and troponin T.  
     
     
         13 . The method of  claim 10  wherein the cells are cardiac muscle cells.  
     
     
         14 . The method of  claim 10  wherein the cells are skeletal muscle cells.  
     
     
         15 . The method of  claim 14  wherein the characterized proteins comprise at least one of a fast and a slow isoform of TnI.  
     
     
         16 . A method of profiling proteins in a biological sample comprising: 
 (a) separating proteins of the biological sample in accordance with the method of  claim 1;  and    (b) characterizing proteins so as to produce a profile of proteins in said biological sample.    
     
     
         17 . The method of  claim 16  wherein the separated proteins are characterized by Western blot.  
     
     
         18 . A method for detecting myocardial damage in a subject comprising detecting a myofilament protein in serum of the subject by Western Blot-Direct Serum Analysis.  
     
     
         19 . The method of  claim 18  wherein detection of the myofilament protein in the serum of the patient provides an early clinical assessment or diagnosis of myocardial damage.  
     
     
         20 . A method for clinically assessing or diagnosing in a subject myocardial damage prior to detection by electrocardiogram or routine clinical testing showing significant elevations of biochemical cardiac markers in the subject, said method comprising detecting by Western Blot-Direct Serum Analysis a myofilament protein in serum of the subject.  
     
     
         21 . A method for monitoring the state of the myocardium in a subject, said method comprising monitoring myofilament protein modifications in serum of the subject by Western Blot-Direct Serum Analysis.  
     
     
         22 . The method of  claim 21  wherein monitoring is performed prior to detection by electrocardiogram or routine clinical testing showing significant elevations of biochemical cardiac markers in the subject.  
     
     
         23 . A method for assessing severity of skeletal muscle damage in a subject comprising measuring a ratio of two different isoforms of a myofilament protein in serum of the subject by Western Blot-Direct Serum Analysis.  
     
     
         24 . The method of  claim 23  wherein the two different isoforms of the myofilament protein are fast and slow troponin I or fast and slow troponin T.  
     
     
         25 . A method for diagnosing skeletal muscle damage in a subject comprising measuring a ratio of two different isoforms of a myofilament protein in serum of the subject by Western Blot-Direct Serum Analysis.  
     
     
         26 . The method of  claim 25  wherein the two different isoforms of the myofilament protein are fast and slow troponin I or fast and slow troponin T.  
     
     
         27 . A method for differentially diagnosing skeletal muscle damage in a subject comprising measuring a ratio of two different isoforms of a myofilament protein in serum of the subject by Western Blot-Direct Serum Analysis.  
     
     
         28 . The method of  claim 27  wherein the two different isoforms of the myofilament protein are fast and slow troponin I or fast and slow troponin T.