Elution of microparticles by size exclusion chromatography using water as a mobile phase

Abstract

The present disclosure relates to methods for eluting microparticles from a size exclusion chromatography column using water as a mobile phase. This method is useful for the detection of proteins associated with microparticles and provides an improved method to do so, as compared to using buffer as a mobile phase.

1 - 25 . (canceled) 
     
     
         26 . A method comprising eluting microparticles from a size exclusion chromatography column using water as a mobile phase. 
     
     
         27 . The method of  claim 26 , wherein the water is double distilled H 2 O (“ddH 2 O”). 
     
     
         28 . The method of  claim 26 , wherein elution with water elutes at least four times as many microparticles as elution with phosphate buffered saline. 
     
     
         29 . The method of  claim 26 , wherein elution with water elutes at least ten times as many microparticles as elution with phosphate buffered saline. 
     
     
         30 . The method of  claim 26 , wherein elution with water improves elution of microparticle-associated proteins as compared to elution with phosphate buffered saline as measured by absorbance at A280. 
     
     
         31 . The method of  claim 26 , comprising:
 a) loading a biological sample comprising the microparticles onto a size exclusion chromatography column; and   b) eluting the microparticles from the column using ddH 2 O as the mobile phase.   
     
     
         32 . The method of  claim 31 , comprising washing the column with ddH2O before loading the biological sample. 
     
     
         33 . The method of  claim 26 , wherein the size exclusion chromatography column comprises cross-linked agarose beads. 
     
     
         34 . The method of  claim 33 , wherein the agarose beads are 2% agarose. 
     
     
         35 . The method of  claim 26 , wherein the size exclusion chromatography column comprises a medium, and the medium comprises polystyrene beads. 
     
     
         36 . The method of  claim 35 , wherein the polystyrene beads comprise 100 nm polystyrene beads. 
     
     
         37 . The method of  claim 26 , comprising washing the size exclusion chromatography column with ddH2O before loading the microparticles onto the column. 
     
     
         38 . The method of  claim 26 , comprising loading the size exclusion chromatography column with a biological sample comprising the microparticles. 
     
     
         39 . The method of  claim 36 , wherein the biological sample comprises whole blood, serum or plasma. 
     
     
         40 . The method of  claim 36 , wherein elution dilutes a high molecular weight fraction of the biological sample 7-10 fold or 15-20 fold. 
     
     
         41 . The method of  claim 31 , wherein the ratio of load sample to bed volume of the size exclusion chromatography column is between 1-2.5% v/v. 
     
     
         42 . The method of  claim 26 , wherein the microparticles comprise exosomes or microvesicles.