Method for measurement of total protein content and detection of protein via immunoassay in a microfluidic device

Abstract

Some embodiments described herein relate to systems and methods operable to combine immunoassay and Total Protein techniques in a single sample run. Some embodiments described herein allow for multiple sequential immunoassays to be performed in the same microfluidic device. Some embodiments described herein relate to stripping reagents operable to remove primary antibodies associated with immunoassays. Such stripping reagents can allow for additional immunoassays and/or Total Protein assays to be performed on the same sample.

What is claimed is: 
     
         1 . A method, comprising:
 electrophoretically separating a sample containing a first analyte and a second analyte in a microfluidic device;   immobilizing the first analyte and the second analyte in the microfluidic device;   introducing a first primary antibody configured to bind to the first analyte;   introducing a first secondary antibody configured to bind to the first primary antibody;   detecting the first analyte based on an optical characteristic associated with the first secondary antibody;   introducing a stripping reagent configured to remove the first primary antibody from the first analyte while the first analyte and the second analyte remain immobilized in the microfluidic device;   introducing a second primary antibody configured to bind to the second analyte;   introducing a second secondary antibody configured to bind to the second primary antibody; and   detecting the second analyte based on an optical characteristic associated with the second secondary antibody.   
     
     
         2 . The method of  claim 1 , wherein:
 the first primary antibody is introduced before the stripping agent is introduced; and   the second primary antibody is introduced after the stripping agent is introduced.   
     
     
         3 . The method of  claim 1 , wherein the first secondary antibody and the second secondary antibody are the same secondary antibody. 
     
     
         4 . The method of  claim 1 , wherein:
 the first secondary antibody and the second secondary antibody are configured to produce indistinguishable optical characteristics; and   the stripping reagent is introduced after detecting the first analyte and before introducing the second secondary antibody.   
     
     
         5 . The method of  claim 1 , wherein the first secondary antibody is conjugated to horseradish peroxidase (HRP) such that the optical characteristic associated with the first secondary antibody is a chemiluminescense reaction associated with the horseradish peroxidase. 
     
     
         6 . The method of  claim 1 , wherein the first secondary antibody is conjugated to horseradish peroxidase (HRP), the method further comprising:
 Introducing a chemiluminescent substrate into the capillary, the optical characteristic associated with the first secondary antibody being a chemiluminescent signal associated with an interaction between the chemiluminescent substrate and HRP conjugated to the first secondary antibody.   
     
     
         7 . The method of  claim 1 , wherein:
 the first secondary antibody is conjugated to horseradish peroxidase (HRP) such that the optical characteristic associated with the first secondary antibody is a chemiluminescent signal; and   the second secondary antibody is conjugated to a fluorescent dye such that the optical characteristic associated with the second secondary antibody is a fluorescent signal.   
     
     
         8 . The method of  claim 7 , wherein the first analyte and the second analyte are detected prior to introducing the stripping reagent. 
     
     
         9 . The method of  claim 1 , further comprising washing unbound first secondary antibodies before detecting the first analyte. 
     
     
         10 . The method of  claim 1 , further comprising washing the first primary antibody and the second primary antibody from the microfluidic device after introducing the stripping reagent, before introducing the second primary antibody, and while the first analyte and the second analyte remain immobilized in the microfluidic device. 
     
     
         11 . The method of  claim 1 , wherein the stripping agent is configured to remove the first secondary antibody. 
     
     
         12 . The method of  claim 1 , wherein electrophoretically separating the sample causes the first analyte to migrate to a first portion of the microfluidic device and the second analyte to migrate to a second portion of the microfluidic device, the first analyte and the second analyte immobilized in the first portion of the microfluidic device and the second portion of the microfluidic device, respectively. 
     
     
         13 . The method of  claim 1 , wherein introducing the first primary antibody, introducing the first secondary antibody, detecting the first analyte, and introducing the stripping reagent occur before introducing the second primary antibody and introducing the second secondary antibody. 
     
     
         14 . The method of  claim 1 , wherein introducing the first primary antibody, introducing the first secondary antibody, detecting the first analyte, and introducing the stripping reagent, introducing the second primary antibody, and introducing the second secondary antibody occur in that order and without user intervention. 
     
     
         15 . The method of  claim 1 , wherein the stripping reagent includes Tris(2-carboxyethyl)phosphine hydrochloride and has a pH between 3 and 4.5. 
     
     
         16 . The method of  claim 1 , wherein the stripping reagent has a composition selected from Table 2, has a stripping efficiency of greater than 95%, and does not exhibit precipitation. 
     
     
         17 . A method, comprising:
 electrophoretically separating a sample containing a plurality of proteins in a microfluidic device;   immobilizing the plurality of proteins in the microfluidic device after electrophoretic separation;   introducing a molecule with a reactive moiety configured to non-specifically bind to proteins;   introducing a primary antibody configured to bind to at least a subset of proteins from the plurality of proteins;   introducing a secondary antibody configured to bind to the primary antibody;   detecting the subset of proteins based on an optical characteristic associated with the secondary antibody;   introducing a stripping reagent configured to remove the primary antibody from the subset of proteins;   introducing an optically detectable agent configured to bind to the molecule;   detecting an optical signal associated with the optically detectable agent; and   normalize the optical characteristic associated with the secondary antibody based on the optical signal associated with the optically detectable agent.   
     
     
         18 . A stripping reagent, comprising:
 a buffer;   Tris(2-carboxyethyl)phosphine hydrochloride (TCEP); and   a detergent,   the stripping reagent having a pH below 5.   
     
     
         19 . The stripping reagent of  claim 18 , wherein the pH is between 3 and 4.5. 
     
     
         20 . The stripping reagent of  claim 18 , wherein the buffer is selected from the group consisting of:
 Trizma having a pH greater than 8;   Glycine HCl having a pH less than 5;   4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); and   Bicine.   
     
     
         21 . The stripping agent of  claim 20 , wherein a concentration of a buffer species is between 100 and 200 millimolar. 
     
     
         22 . The stripping agent of  claim 18 , wherein the stripping reagent has a composition selected from Table 2 and has a stripping efficiency of greater than 95%. 
     
     
         23 . The stripping agent of  claim 18 , wherein the stripping reagent has a composition selected from Table 2, has a stripping efficiency of greater than 95%, and does not exhibit precipitation.