Affinity—based screening of combinatorial libraries using automated, serial-column chromatography

Abstract

We have developed an automated serial chromatographic technique for screening a library of compounds based upon their relative affinity for a target molecule. A "target" column containing the immobilized target molecule is set in tandem with a reversed-phase column. A combinatorial peptide library is injected onto the target column. The target-bound peptides are eluted from the first column and transferred automatically to the reversed-phase column. The target-specific peptide peaks from the reversed-phase column are identified and sequenced. Using a monoclonal antibody (3E-7) against beta-endorphin as a target, we selected a single peptide with sequence YGGFL from approximately 5800 peptides present in a combinatorial library. We demonstrated the applicability of the technology towards selection of peptides with predetermined affinity for bacterial lipopolysaccharide (LPS, endotoxin). We expect that this technology will have broad applications for high throughout screening of chemical libraries or natural product extracts.

Affinity chromatographic screening of soluble combinatorial peptide libraries

“…The divide, couple, and recombine (DCR) process was used for the synthesis of soluble peptide libraries (Evans et al, 1996;Lam et al, 1991). Peptide library I was constructed using only six amino acids: Y, N, F, E, V, and L. These amino acids were selected from the S-protein binding peptide: YNFEVL.…”

“…Domingo et al demonstrated an affinity chromatographic process for the screening of protease inhibitors from a peptide mixture containing 21 peptides constructed from the reactive site loop of natural protease inhibitors (Domingo et al, 1995). Other workers (Evans et al, 1996;Kaur et al, 1997) have used affinity techniques combined with mass spectrometry for screening libraries of no more than 1000 compounds. This supports the idea that affinity chromatography is effective in screening affinity peptide ligands.…”

Simultaneous identification of the anaphylactoid components from traditional Chinese medicine injections using rat basophilic leukemia 2H3 and laboratory of allergic disease 2 dual‐mixed/cell membrane chromatography model

“…In recent years, the increasingly popular chromatography/mass spectrometry, chip technology, genomics technology, computer virtual screening, and other techniques have been widely used in the screening of anaphylactoid components . High‐throughput screening is an important method of identifying target components, such as direct screening based on receptors or enzymes, and virtual screening via computer‐aided drug design method .…”

Immunoaffinity screening with capillary electrochromatography

“…One way to address this challenge is to exploit the specificity of bioaffinity interactions [3,[7][8][9][10][11][12]. Since biological activity almost always involves binding [6], potentially pharmacoactive compounds can be bound to a solid support, carrying an adequate affinity ligand (e.g., an antibody) and thereby be extracted from complex mixtures.…”

“…This method was used here in combination with CEC, for screening of cardiac glycosides in a mixture of eleven steroids. For screening of large libraries or complex samples, immunoaffinity binding and subsequent elution followed by chromatographic separation is appropriate [3]. Here, cardiac glycosides were selectively extracted from a urine sample, eluted and detected in submicromolar concentrations.…”