Development of Immunoaffinity Restricted Access Media for Rapid Extractions of Low-Mass Analytes

Abstract

Restricted access media using antibodies as immobilized ligands were developed for the rapid and selective capture of small analytes by immunoextraction, giving rise to materials referred to as immunoaffinity restricted access media (IA-RAM). To make such a material, intact antibodies for the desired target were first immobilized onto porous silica, with antibodies at or near the outer surface of the support then being treated with papain (or a related agent) to release and remove their binding domains. The result was a support in which only antibodies deep within the pores remained intact and able to bind to the target. Items evaluated in the development of such media included the immobilization method used for the antibodies, the pore size of the support, and the amount of papain and time that were used for support treatment. A theoretical model was also developed to describe the extent of binding domain removal based on the measured polypeptide content of the IA-RAM support before and after treatment with papain. The final optimized conditions for making the IA-RAM supports were used to prepare columns that contained anti-fluorescein antibodies. Injections of fluorescein and fluorescein-labeled bovine serum albumin onto these IA-RAM columns gave selective and quantitative extraction of fluorescein in 1-2 s. This approach can be used with other antibodies and low mass targets and should be valuable for such applications as the rapid separation of drugs from drug-protein complexes or the isolation of labeled/modified peptides from intact proteins that contain the same modification or label.

Sensitivity Enhancement for Separation-Based Analytical Techniques Utilizing Solid-Phase Enrichment Approaches and Analyte Derivatization for Trace Analysis in Various Matrices

“…Protein exclusion is frequently achieved using physical barrier layers based on pore size or a chemical diffusion barrier composed of hydrophilic polymer networks. The modification of adsorbent surfaces with hydrophilic polymers for the manufacture of modified RAMs has received more attention [119][120][121][122]. Several kinds of RAMs as SPEs have been designed that utilize different functional groups on the solid particles' outer surface.…”

Preparation and application of an immunoaffinity column based on an antibody with strong affinity and packing material with good stability

“…In most IACs, antibodies are generally immobilised on support materials, such as carbohydrate-related materials, for example, Sepharose (Esteve-Turrillas et al 2011), silica (Wa et al 2008), chitosan (Wang et al 2011) and sol-gel (Klinglmayr et al 2010), or synthetic organic supports (Liang et al 2010) by chemical covalent bonding. Most of the IACs reported in the literature have either used harsh elution conditions and are used only once (Xu et al 2005) or used gentle elution conditions and are reused many times (Brenn-Struckhofova et al 2007).…”

Synthesis of a novel restricted access chiral stationary phase based on atom transfer radical polymerization and click chemistry for the analysis of chiral drugs in biological matrices

“…Restricted-access materials (RAMs) have been recognized as a suitable approach for direct HPLC injection of biological samples in pharmaceutical analysis to improve the analytical efficiency and accuracy of separations [1][2][3][4][5][6]. RAMs generally possess dual surface configurations.…”