Development of peptide receptor binding assays: methods to avoid false negatives.
Selection of appropriate ligand receptor binding assay conditions is critical for peptides, where the possibility of obtaining false negative results is pertinent due to their inherent adsorption and instability characteristics, as well as high response-sensitivity to operational conditions. The aim of this study was thus to develop a cost-effective multivariate screening method for determination of the influence of different factors on the outcome of such studies, using (125)I-labelled vasoactive intestinal peptide binding on lung homogenate as a model. The study was divided into two parts: investigation of filtration for bound-unbound ligand separation, and screening of sample incubation variables. Experimental designs were used (including Plackett-Burman) to evaluate adsorption, total binding, non-specific binding, specific binding and (non-)specific/total binding ratio. Several significant factors were identified. For filtration, a combination of polyethylenimine and BSA filter pretreatment was best, whereas albumin-containing washing solvent negatively influenced the amount of specific bound radioligand. For sample incubation, significant effects on one or more of the studied responses were observed for several factors. Bacitracin protease inhibitor also decreased adsorption. We report here multivariate experimental designs for screening of peptide (radio)ligand receptor binding assay conditions. This approach efficiently minimizes the risk on false negative results due to inappropriate operational conditions.
Vergote
Valentijn
V
Drug Quality and Registration (DruQuaR) group, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium.
Van Dorpe
Sylvia
S
Verbeken
Mathieu
M
Burvenich
Christian
C
Van de Wiele
Christophe
C
Banks
William A
WA
De Spiegeleer
Bart
B
D000595:Amino Acid Sequence; D000818:Animals; D005188:False Negative Reactions; D005374:Filtration; D008024:Ligands; D008168:Lung / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D018000:Receptors, Peptide / Q000378:metabolism*; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism
D008024:Ligands; D018000:Receptors, Peptide; D014660:Vasoactive Intestinal Peptide