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.
Affinity isolation of anti-histone immunoglobulins from blood serum of systemic lupus erythematosus patients using histone-conjugated magnetic poly(2-oxoethyl methacrylate) microspheres (IO-iron oxide, HIS-histone).
In order to find novel molecular markers of multiple sclerosis we developed a scheme of oligopeptides' isolation including their extraction from blood serum with 10 % trichloroacetic acid, followed by precipitation of soluble substances with acetone in ratio 6:1. Oligopeptides were dissolved in water and their characteristics was determined by gel filtration under HPLC conditions and thin layer chromatography. Obtained data have shown that blood serum of MS patients contains two oligopolypeptides with average molecular masses of 300-500 Da. We also studied biological activity of TCA-soluble peptides toward some eukaryotic and prokaryotic cells in comparison with phosphopeptides isolated from casein hydrolysates. It was found that TCA-soluble peptides are capable of effective inhibiting HeLa cells' proliferation, while their inhibitory effect was expressed toward Jurkat T-cells and was not detectable toward U373 cells. The casein's phosphopeptides were capable to stimulate proliferation of Jurkat cells and effectively inhibited growth of cells. Neither antibacterial, nor antifungal activities of these oligopeptides were detected.
The review is focused on the analysis of published data and the results obtained by the authors about the catalytic activity of antibodies (abzymes) at norm and pathology. Potential pathogenic and beneficial role of natural abzymes is discussed.Abzymes, possessing protease activity were named as "protabzymes". As was mentioned above, protabzymes, capable of hydrolyzing intestinal
Monitoring of multiple sclerosis (MS) requires of molecular markers. Recently, using TCA-precipitation/ extraction and MALDI TOF/TOF mass-spectrometry, we identified in blood serum of multiple sclerosis (MS) patients earlier unknown 46 kDa form of human unconventional myosin IC isoform b (Myo1C) (Myronovskij et al., 2015). During SDS-electrophoresis of TCA-extracted proteins of blood serum of 28 MS patients, 3 additional polypeptides with 55, 50 and 25 kDa molecular masses were detected. Western-blot analysis using monospecific anti-human IgG rabbit HRP-conjugated antibodies showed that 55 and 25 kDa polypeptides belong to heavy and light chains of IgG molecules, while 50 kDa protein corresponds to free heavy chains of IgGs. None of these polypeptides was found in fractions of TCAextracted polypeptides isolated from blood serum of healthy human donors or patients with systemic lupus erythematosis and rheumatoid arthritis.
The aim of this study was to characterize the proliferative activity of the anti-histone H1 IgGs towards human T-leukaemia CEM cells. Materials and Methods: Anti-histone H1 IgGs were purified from blood serum of systemic lupus erythematosus patients by precipitation of serum proteins with 50% ammonium sulfate followed by a sequential affinity chromatography on Protein GSepharose and histone H1-Sepharose columns. To avoid contamination with other proteins, anti-histone H1 IgGs were subjected to strongly acidic pH 2.0 during gel filtration through HPLC column. The effects of the anti-histone H1 IgGs on cell viability and cell cycle were tested by MTS-assay and flow cytometry, correspondingly. The cross-reactivity of the anti-histone H1 antibodies towards heterogenetic and cellular antigens was evaluated by Western-blot analysis. Results: It was found that incubation of CEM cells with the HPLC-purified anti-histone H1 IgGs resulted in significant stimulation of cell growth by 46% after 48 h of incubation. These IgGs possess an antigenic poly-specificity to positively charged heterogenetic antigens and different cellular antigens. FITC-labeled and biotinylated anti-histone H1 IgGs are internalized by CEM cells and preferentially accumulated in the cytoplasm. Conclusion: The anti-histone H1 IgGs are shown to internalize human T-leukemia CEM and stimulate their proliferation. These IgGs are polyspecific toward cellular antigens.
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