Enhanced microdialysis relative recovery of inflammatory cytokines using antibody-coated microspheres analyzed by flow cytometry

Abstract

Achieving high relative recovery (RR) of proteins during microdialysis sampling is difficult due to the diffusion limitations inherent to this sampling process. This often causes low microdialysis RR for proteins with molecular weight >10 kDa. A RR enhancement process for microdialysis sampling of proteins has been developed that can be readily used with flow cytometers. Multiplexed RR enhancement and detection of five different cytokines (TNF-alpha, IFN-gamma, IL-2, IL-4, and IL-5) was achieved by including antibody-coated microspheres in the microdialysis perfusion fluid. Inclusion of these antibody-coated microspheres causes an increase in the analyte diffusive driving force across the dialysis membrane and a subsequent increase in the relative recovery. For the five cytokines, typical control and enhanced relative recoveries at a 1.0 microL/min flow rate were as follows (n = 3): TNF-alpha, 5.5 +/- 0.6% and 60.4 +/- 5.8%; IFN-gamma, 2.6 +/- 0.3% and 25.8 +/- 2.3%; IL-5, 1.4 +/- 0.2% and 4.9 +/- 0.1%; IL-4, 10.9 +/- 0.6% and 78.8 +/- 8.0%; and IL-2, 4.1 +/- 0.4% and 19.8 +/- 2.5%. Using this approach, a four- to 12-fold enhancement of microdialysis RR was achieved for the five cytokines from a quiescent solution. The enhancement varies among the five cytokines and may be due to different diffusive and antibody binding properties. TNF-alpha exhibited the highest RR enhancement, while IL-5 exhibited the lowest. Experimental parameters that affect the enhancement, such as flow rate, sample collection volume, and bead density, were studied.