Monodisperse magnetic poly(glycidyl methacrylate) microspheres for isolation of autoantibodies with affinity for the 46 kDa form of unconventional Myo1C present in autoimmune patients

Abstract

Monodisperse nonmagnetic macroporous poly(glycidyl methacrylate) (PGMA) microspheres were synthesized by multistep swelling polymerization of glycidyl methacrylate, ethylene dimethacrylate and 2-[(methoxycarbonyl)methoxy]ethyl methacrylate (MCMEMA). This was followed (a) by ammonolysis to modify the microspheres with amino groups, and (b) by incorporation of iron oxide (γ-FeO) into the pores to render the particles magnetic. The resulting porous and magnetic microspheres were characterized by scanning and transmission electron microscopy (SEM and TEM), atomic absorption and Fourier transform infrared spectroscopy (AAS and FTIR), elemental analysis, vibrating magnetometry, mercury porosimetry and Brunauer-Emmett-Teller adsorption/desorption isotherms. The microspheres are meso- and macroporous, typically 5 μm in diameter, contain 0.9 mM · g of amino groups and 14 wt.% of iron according to elemental analysis and AAS, respectively. The particles were conjugated to p46/Myo1C protein, a potential biomarker of autoimmune diseases, to isolate specific autoantibodies in the blood of patients suffering from multiple sclerosis (MS). The p46/Myo1C loaded microspheres are shown to enable the preconcentration of minute quantities of specific immunoglobulins prior to their quantification via SDS-PAGE. The immunoglobulin M (IgM) with affinity to Myo1C was detected in MS patients. Graphical abstract Monodisperse magnetic poly(glycidyl methacrylate) microspheres were synthesized, conjugated with 46 kDa form of unconventional Myo1C protein (p46/Myo1C) via carbodiimide (DIC) chemistry, and specific autoantibodies isolated from blood of multiple sclerosis (MS) patients; immunoglobulin M (IgM) level increased in MS patients.

Isolation and identification in human blood serum of the proteins possessing the ability to bind with 48 kDa form of unconventional myosin 1c and their possible diagnostic and prognostic value

“…These data show that the polypeptides with the molecular masses of 87, 85 and 25 kDa are unknown, while two other polypeptides with molecular masses of 58 and 54 kDa showed high similarity to component 3 of the complement isoform CRA and the antithrombin‐III isoform 1, respectively (Figure 2b). Previously, when using monospecific antibodies, we identified 82 and 25 kDa polypeptides as heavy and light chains of the immunoglobulin M sub‐class (Zasońska et al, 2018). In order to verify the identified 58 and 54 kDa proteins, we applied western blot analysis using monospecific commercial antibodies (Figure 2).…”

“…Previously, we used novel monodisperse magnetic poly(glycidyl methacrylate) (mag‐PGMA–NH 2 ) microspheres bearing 48/Myo1C, which allowed us to detect IgM and IgG immunoglobulins in blood serum of patients with multiple sclerosis, while other proteins remained uncharacterized (Zasońska et al, 2018). A separation of the desired protein when using the affinity chromatography relies on the reversible interactions between the protein to be purified and the affinity ligand coupled to the chromatographic matrix (Manko et al, 2019).…”

“…The p48/Myo1C protein was isolated from human blood serum (Myronovkij et al, 2015). It was immobilized on the mag‐PGMA–NH 2 microspheres via carbodiimide (Zasońska et al, 2018), resulting in mag‐PGMA‐48/Myo1C microparticles.…”

“…Previously, we used magnetic poly(glycidyl methacrylate) (mag‐PGMA–NH 2 ) microspheres with immobilized 48/Myo1C for isolation of several blood serum proteins capable of binding to these proteins. The IgM and IgG immunoglobulins were among them (Zasońska et al, 2018), while other proteins remained uncharacterized. A combination of the magnetic microparticle‐based affinity chromatography with the MALDI–TOF mass spectrometry allowed us to identify potential interacting partners of the 48/Myo1C protein, which was earlier detected in the bloodstream of some autoimmune, hemato‐oncological and neurodegenerative patients.…”

Biosensors and nanobiosensors for rapid detection of autoimmune diseases: a review

“…In addition to AAbs, some proteins [16,17], chemokines [18], cytokines [19], cells [20], and ions [21] have also been found as promising biomarkers for autoimmune diseases diagnosis or monitoring. A list of common autoimmune diseases and related biomarkers are presented in Table 1.…”