Material matters: evaluating polymer 96-well plates to reduce DAMGO loss in LC-MS/MS-based in vitro μ-opioid structure affinity assay

• First report of DAMGO sticking to laboratory consumables made of a polymer • Evaluation protocol to identify suitable 96-well plates for receptor affinity assays • Eight different plates tested • Plates by Thermo Scientific do not depict non-specific adsorption The peptide DAMGO has been used for decades to determine binding affinity at the μ-opioid receptor in numerous types of receptor binding assays utilizing radiolabeled DAMGO, and more recently, liquid chromatography tandem mass spectrometry (LC-MS/MS)-based procedures with unlabeled DAMGO. While previously there have been numerous reports of non-specific adsorption (NSA) at plastic surfaces of various peptides, this study reports for the first time the NSA of DAMGO at polymer 96-well plates used in receptor binding affinity assays. In the present study, seven different types of polypropylene and one modified polystyrene 96-well plates were assessed for DAMGO NSA. The number of solution transfers, two different incubation temperatures and times were evaluated. NSA and DAMGO signal loss were observed for six out of eight tested plates. Best results were obtained with Thermo Scientific (Cat. No. 60180-P133) 96-well deep-well plates, for which no significant signal deviation could be observed compared to the blanks even after six sequential transfers. Coated microtiter plates from Greiner Bio-One (Cat. No. 655901) showed significant signal enhancement compared to the blanks, which may also cause issues when performing quantitative analysis of results. This study highlights the necessity of testing laboratory equipment for NSA during assay development, since peptide or analyte adsorption may not be immediately apparent, leading to the issue going unnoticed.