PRECISE KD MEASUREMENTS FOR APTAMER-BASED AFFINITY ASSAYS USING GRADIENT ELUTION MOVING BOUNDARY ELECTROPHORESIS (GEMBE)

Catherine T. Lo, Matt S. Munson, Marc Salit, David J. Ross

Biochemical Science Division 

A method is described here for the precise measurement of the Kd affinity constant of aptamer-based affinity assays using gradient elution moving boundary electrophoresis (GEMBE).  GEMBE is an electrophoretic technique that utilizes a pressure-driven counterflow to separate the charged analytes as they come into the microchannel for detection; it is based on a balance between the electrophoretic mobility and the pressure-driven flow.  Highly charged analytes are detected in the sequence in which they overcome the counterflow.  Low charged analytes, such as biological proteins, are rejected from coming into the microchannel, which eliminates protein adsorption to the microchannel surface.  In this study, human immunodeficiency virus reverse transcriptase (HIVRT) was used as the target protein, with concentrations ranging from 0 nmol/L to 50 nmol/L.  The DNA binding aptamer was maintained constant at 1 nmol/L.  Using LIF with PMT, the Kd value of the DNA aptamer to the target protein was measured to be 5.5 nmol/L, with a 4 % RSD for the affinity assay (4 measurements were made for each protein concentration).   Compared to traditional affinity assays using ELISA (≈ 15 % to 20 % RSD) and CE-based aptamer assays (≈ 5 % to 10 % RSD), the new analytical technique presented here is much more precise and can potentially facilitate more accurate biomarker measurements for clinical applications.