A new method for the screening of solid-phase combinatorial libraries for affinity chromatography.
A new methodology for the rapid assessment of affinity ligands synthesized by combinatorial solid-phase chemistry is reported. This screening strategy utilizes the target protein conjugated to FITC, and represents an almost universal technique for the preliminary screening of solid-phase combinatorial libraries. The assessment of a triazine-scaffolded solid-phase combinatorial library of ligands, designed to bind to human IgG, was performed with FITC-human IgG, and the results compared with those obtained by conventional affinity chromatographic screening assays. The effect of different molar conjugation ratios of FITC-IgG (F/P) was evaluated. Independently of the F/P ratio, no false negative results were observed, although lower F/P ratios diminished non-specific interactions and the number of false positives. The nature of the substituents on the triazine scaffold was not related to the number of false positive IgG-binding ligands. The reproducibility of the FITC technique, using FITC-human IgG conjugates with low F/P ratio (F/P=2), was also evaluated. The FITC-based technique proved to be efficient and accurate in the identification of strongly binding ligands (binding >50% of loaded protein, by standard affinity chromatographic assays), and is envisaged as a versatile and cost-effective method to screen other systems, and evaluate several binding/elution conditions at small-scale, prior to scale-up to standard affinity chromatography.
Roque
A Cecília A
AC
Centre for Biological and Chemical Engineering, Instituto Superior Técnico, 1049-001 Lisbon, Portugal.
Taipa
M Angela
MA
Lowe
Christopher R
CR
D002846:Chromatography, Affinity / Q000379:methods*; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D014227:Triazines / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism
D007074:Immunoglobulin G; D008024:Ligands; D014227:Triazines; D016650:Fluorescein-5-isothiocyanate