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A new method for the screening of solid‐phase combinatorial libraries for affinity chromatography
Author(s) -
Roque A. Cecília A.,
Taipa M. Ângela,
Lowe Christopher R.
Publication year - 2004
Publication title -
journal of molecular recognition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.401
H-Index - 79
eISSN - 1099-1352
pISSN - 0952-3499
DOI - 10.1002/jmr.661
Subject(s) - triazine , elution , affinity chromatography , chemistry , conjugate , combinatorial chemistry , chromatography , solid phase synthesis , biochemistry , organic chemistry , peptide , enzyme , mathematical analysis , mathematics
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. Copyright © 2004 John Wiley & Sons, Ltd.