Magnetic poly(2-hydroxyethyl methacrylate) microspheres for affinity purification of monospecific anti-p46 kDa/Myo1C antibodies for early diagnosis of multiple sclerosis patients.

Daniel Horák,Severyn Myronovsky,Helena Hlídková,Yurii Kit,Volodymyr Antonyuk,Rostyslav Stoika

doi:10.1042/bsr20160526

Abstract

The aim of the present study is to develop new magnetic polymer microspheres with functional groups available for easy protein and antibody binding. Monodisperse macroporous poly(2-hydroxyethyl methacrylate) (PHEMA-COOH) microspheres ~4 µm in diameter and containing ∼1 mmol COOH/g were synthesized by multistep swelling polymerization of 2-hydroxyethyl methacrylate (HEMA), ethylene dimethacrylate (EDMA), and 2-[(methoxycarbonyl)methoxy]ethyl methacrylate (MCMEMA), which was followed by MCMEMA hydrolysis. The microspheres were rendered magnetic by precipitation of iron oxide inside the pores, which made them easily separable in a magnetic field. Properties of the resulting magnetic poly(2-hydroxyethyl methacrylate) (mgt.PHEMA) particles with COOH functionality were examined by scanning and transmission electron microscopy (SEM and TEM), static volumetric adsorption of helium and nitrogen, mercury porosimetry, Fourier transform infrared (FTIR) and atomic absorption spectroscopy (AAS), and elemental analysis. Mgt.PHEMA microspheres were coupled with p46/Myo1C protein purified from blood serum of multiple sclerosis (MS) patients, which enabled easy isolation of monospecific anti-p46/Myo1C immunoglobulin G (IgG) antibodies from crude antibody preparations of mouse blood serum. High efficiency of this approach was confirmed by SDS/PAGE, Western blot, and dot blot analyses. The newly developed mgt.PHEMA microspheres conjugated with a potential disease biomarker, p46/Myo1C protein, are thus a promising tool for affinity purification of antibodies, which can improve diagnosis and treatment of MS patients.

Highlights

Rapid and effective magnetic separation and manipulation of various biological entities, including proteins and cells, as well as drug targeting and delivery, require involvement of biocompatible magnetic carriers [1]
Starting monodisperse macroporous poly(2-hydroxyethyl methacrylate) (PHEMA) particles, 4.3 μm in diameter, were developed employing multistep swelling polymerization of hydroxyethyl methacrylate (HEMA), 2-[(methoxycarbonyl)methoxy]ethyl methacrylate (MCMEMA), and ethylene dimethacrylate (EDMA) according to the Ugelstad method, where inert solvents, such as Cyclohexyl acetate (CyAc) and dibutyl phthalate (DBP), served as the porogen [30]
Subsequent hydrolysis of MCMEMA-containing PHEMA microspheres introduced COOH functionalities (∼1 mmol/g according to titration with NaOH)

Summary

Introduction

Rapid and effective magnetic separation and manipulation of various biological entities, including proteins (antibodies and enzymes) and cells, as well as drug targeting and delivery, require involvement of biocompatible magnetic carriers [1]. To avoid detrimental effects, such as Fe ion leaching, aggregation in aqueous media, insufficient compatibility with living tissues, and absence of functional groups available for attachment of a target biomolecule, proper coating of the magnetic particles with polymers is needed [4]. This coating can be achieved by simple adsorption [5], graft polymerization [6,7] and co-polymerization from the particle surface [8] or c 2017 The Author(s).

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