Microdialysis enhanced relative recovery of rat endocrine peptides using antibody-immobilized beads

by Fletcher, Heidi J.; McNay, Ewan; Stenken, Julie Ann

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The use of microdialysis sampling to collect proteins is challenging due to the diffusion limitations inherent in this process, resulting in low relative recovery values and sometimes difficulties with quantitation. An approach to improve protein collection would be to use antibody-immobilized beads to trap the protein within the dialysis perfusion fluid. This trapping process serves to increase the analyte diffusive mass transport driving force through the membrane causing an increase in relative recovery. Endocrine hormones, insulin, amylin, glucagon, GLP-1, and leptin were sampled using the antibody-immobilized bead approach using microdialysis with insulin as the target biomol. of interest to study. Insulins insufficiency is the result of many important diseases, such as Type 2 diabetes and Alzheimer's. A multiplexed bead assay for rat endocrine peptides was used to enhance the relative recovery of amylin (MW 3921), GLP-1 (MW 3297), glucagon (MW 3483), insulin (MW 5800) and leptin (MW 16162) during microdialysis sampling. A bead diluent in 0.1%BSA in PBS was perfused (2 and 1 µL/min) through a polyethersulfone probe (10 mm and 100 kDa MWCO) in vitro into a 3300 pM endocrine soln. at room temp. Samples were analyzed using a Luminex 100 IS instrument. Initial results show that the control and enhanced relative recoveries obsd. for all five analytes at 1.0 µL/min were: amylin, 11.6% and 31.4%, GLP-1, 3% and 32.5%, glucagons, 7.5% and 67.2%, insulin, 9.7% and 185.6%, and leptin, 2.7% and 43.8%. Using this approach, a 3 to 20-fold enhancement was achieved. The variations in enhancement for the five hormones may be due to differences in antibody binding and diffusive properties. Preliminary studies for the enhanced recovery of insulin during in vivo brain dialysis are underway and will be presented. This work was funded by NIH DA 20577. on SciFinder(R)]