An in vitro comparison of microdialysis relative recovery of Met- and Leu-enkephalin using cyclodextrins and antibodies as affinity agents

Abstract

Cyclodextrins and antibodies have been used as affinity agents to improve relative recovery during microdialysis sampling. Two neuropeptides, methionine-enkephalin (ME) and leucine-enkephalin (LE), were chosen to compare the use of cyclodextrins and antibodies as possible affinity agents for improving their relative recovery across polycarbonate and polyethersulfone membranes during in vitro sampling. Cyclodextrins (CD) including beta-CD, 2-hydroxypropyl-beta-cyclodextrin (2HPbeta-CD), and gamma-CD gave improvements of relative recovery for both peptides of less than 2-fold as compared to controls. Comparisons of relative recovery between tyrosine-glycine-glycine, tyrosine, and phenylalanine using different cyclodextrins in the perfusion fluid were also obtained. Inclusion of an antibody against met-enkephalin in the microdialysis perfusion fluid resulted in relative recovery increases of up to 2.5-fold. These results show that using antibodies as affinity agents during microdialysis sampling may be more effective agents to improve the relative recovery of these opioid neuropeptides.

Figure 1

A schematic of a microdialysis probe with various affinity agents included in the perfusion fluid. Analytes can freely diffuse into the semi-permeable probe and can be trapped by the affinity agent.

Figure 2

In vitro ME antibody RR enhancement of 62.5 μM FITC-ME quiescent solution at room temperature using various antibody dilutions as an affinity agent through a PC probe: 0.75 μM (grid), 0.15 μM (white), 75 nM (dots), 15 nM (lines), and 7.5 nM (diagonal lines), aCSF (black) at flow rates 1.0, 1.5 and 2.0 μL min⁻¹ (n = 3). * Indicates there is no significant difference in recoveries compared to the control (p = 0.05).