The efficient isolation and concentration of protein antigens from complex biological samples is a critical step in several analytical methods, such as mass spectrometry, flow cytometry and immunochemistry. These techniques take advantage of magnetic microspheres as immunosorbents. The focus of this study was on the development of new superparamagnetic polymer microspheres for the specific isolation of the tumor suppressor protein p53. Monodisperse macroporous poly(glycidyl methacrylate) (PGMA) microspheres measuring approximately 5 μm and containing carboxyl groups were prepared by multistep swelling polymerization of glycidyl methacrylate (GMA), 2-[(methoxycarbonyl)methoxy]ethyl methacrylate (MCMEMA) and ethylene dimethylacrylate (EDMA) as a crosslinker in the presence of cyclohexyl acetate as a porogen. To render the microspheres magnetic, iron oxide was precipitated within their pores; the Fe content in the particles received ∼18 wt%. Nonspecific interactions between the magnetic particles and biological media were minimized by coating the microspheres with poly(ethylene glycol) (PEG) terminated by carboxyl groups. The carboxyl groups of the magnetic PGMA microspheres were conjugated with primary amino groups of mouse monoclonal DO-1 antibody using conventional carbodiimide chemistry. The efficiency of protein p53 capture and the degree of nonspecific adsorption on neat and PEG-coated magnetic microspheres were determined by western blot analysis.

• MeSH
• kyseliny polymethakrylové chemie   MeSH
• mikrosféry * MeSH
• myší monoklonální protilátky chemie   MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádorový supresorový protein p53 chemie   izolace a purifikace   MeSH
• polyethylenglykoly chemie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Magnetic macroporous PGMA and PHEMA microspheres containing carboxyl groups are synthesized by multi-step swelling and polymerization followed by precipitation of iron oxide inside the pores. The microspheres are characterized by SEM, IR spectroscopy, AAS, and zeta-potential measurements. Their functional groups enable bioactive ligands of various sizes and chemical structures to couple covalently. The applicability of these monodisperse magnetic microspheres in biospecific catalysis and bioaffinity separation is confirmed by coupling with the enzyme trypsin and huIgG. Trypsin-modified magnetic PGMA-COOH and PHEMA-COOH microspheres are investigated in terms of their enzyme activity, operational and storage stability. The presence of IgG molecules on microspheres is confirmed.

• MeSH
• biokatalýza MeSH
• chromatografie afinitní metody   MeSH
• imobilizované proteiny chemie   MeSH
• imunoglobulin G chemie   MeSH
• kyseliny polymethakrylové chemická syntéza   MeSH
• lidé MeSH
• magnety MeSH
• mikrosféry MeSH
• mikroskopie elektronová rastrovací MeSH
• polyhydroxyethylmethakrylát chemická syntéza   MeSH
• polymerizace MeSH
• spektrofotometrie infračervená MeSH
• trypsin chemie   MeSH
• železité sloučeniny chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• DNA genetika   izolace a purifikace   MeSH
• elektroforéza v agarovém gelu MeSH
• finanční podpora výzkumu jako téma MeSH
• genom MeSH
• methakryláty chemie   MeSH
• mikrosféry MeSH
• mikroskopie elektronová rastrovací MeSH
• polymerázová řetězová reakce MeSH
• techniky in vitro MeSH

• MeSH
• Bifidobacterium chemie   MeSH
• finanční podpora výzkumu jako téma MeSH
• methakryláty chemie   MeSH
• mikrosféry MeSH
• polymerázová řetězová reakce s reverzní transkripcí metody   MeSH

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.

• MeSH
• imobilizované proteiny chemie   imunologie   MeSH
• imunoglobulin G imunologie   izolace a purifikace   MeSH
• lidé MeSH
• magnetismus metody   MeSH
• magnety chemie   MeSH
• mikrosféry MeSH
• myosin typu I chemie   imunologie   MeSH
• myši MeSH
• polyhydroxyethylmethakrylát chemie   MeSH
• protein - isoformy chemie   imunologie   MeSH
• roztroušená skleróza diagnóza   imunologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Magnetic non-porous hydrophilic poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) microspheres prepared by the dispersion polymerization and modified with iminodiacetic acid (IDA) were employed for the IMAC separation of phosphopeptides. Fe(3+) and Ga(3+) ions immobilized on IDA-modified magnetic microspheres were used for the enrichment of phosphopeptides from the proteolytic digests of two model proteins differing in their physico-chemical properties and phosphate group content: porcine pepsin A and bovine α-casein. The optimum conditions for phosphopeptide adsorption and desorption in both cases were investigated and compared. The phosphopeptides separated from the proteolytic digests were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The ability of the prepared Fe(3+)- and Ga(3+)-IDA-modified magnetic microspheres to capture phosphopeptides from complex mixtures was shown on an example of bovine milk proteolytic digest.

• MeSH
• adsorpce MeSH
• chromatografie afinitní přístrojové vybavení   metody   MeSH
• fosfopeptidy analýza   izolace a purifikace   MeSH
• iminokyseliny chemie   MeSH
• magnetismus MeSH
• mikrosféry MeSH
• mléko chemie   MeSH
• polyhydroxyethylmethakrylát chemie   MeSH
• polymerizace MeSH
• skot MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH

Magnetic microspheres P(HEMA-co-EDMA) were used for PCR-ready phage DNA isolation from lysogenic strains of Staphylococcus aureus, including two new clinical isolates. The conditions of phage particle lysis were optimized. The quality of eluted phage DNA was evaluated by PCR. It was demonstrated that PCR-ready phage DNA can be isolated from small volumes of phage lysates (150 microl) by magnetic microspheres. The reported method is very expeditious without using toxic compounds such as phenol or chloroform. It can be used for phage identification and phage gene detection.

• MeSH
• DNA virů genetika   izolace a purifikace   MeSH
• extrakce na pevné fázi metody   MeSH
• financování organizované MeSH
• genetické techniky MeSH
• magnetismus MeSH
• mikrosféry MeSH
• polymerázová řetězová reakce MeSH
• stafylokokové bakteriofágy genetika   izolace a purifikace   MeSH
• Staphylococcus aureus virologie   MeSH

Monodisperse (4 μm) macroporous crosslinked poly(glycidyl methacrylate) (PGMA) microspheres for use in microfluidic immunomagnetic cell sorting, with a specific application to the capture of circulating tumor cells (CTCs), were prepared by multistep swelling polymerization in the presence of cyclohexyl acetate porogen and hydrolyzed and ammonolyzed. Iron oxide was then precipitated in the microspheres to render them magnetic. Repeated precipitation made possible to raise the iron oxide content to more than 30 wt %. To minimize nonspecific adsorption of the microspheres in a microchannel and of cells on the microspheres, they were coated with albumin crosslinked with glutaraldehyde. Antibodies of epithelial cell adhesion molecule (anti-EpCAM) were then immobilized on the albumin-coated magnetic microspheres using the carbodiimide method. Capture of breast cancer MCF7 cells as a model of CTCs by the microspheres with immobilized anti-EpCAM IgG was performed in a batch experiment. Finally, MCF7 cells were captured by the anti-EpCAM-immobilized albumin-coated magnetic microspheres in an Ephesia chip. A very good rejection of lymphocytes was achieved. Thus, albumin-coated monodisperse magnetic PGMA microspheres with immobilized anti-EpCAM seem to be promising for capture of CTCs in a microfluidic device.

• MeSH
• acetoacetáty chemie   MeSH
• antigeny nádorové imunologie   MeSH
• epitelové buňky účinky léků   patologie   MeSH
• hydrolýza účinky léků   MeSH
• imobilizační protilátky farmakologie   MeSH
• kyseliny polymethakrylové chemie   MeSH
• lidé MeSH
• magnetické jevy * MeSH
• methakryláty chemie   MeSH
• MFC-7 buňky MeSH
• mikrofluidní analytické techniky MeSH
• mikrosféry * MeSH
• mikroskopie elektronová rastrovací MeSH
• molekuly buněčné adheze imunologie   MeSH
• nádory prsu patologie   MeSH
• poréznost MeSH
• sérový albumin chemie   MeSH
• spektrofotometrie infračervená MeSH
• železité sloučeniny chemie   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Identification and evaluation of small changes in β-amyloid peptide (Aβ) levels in cerebrospinal fluid is of crucial importance for early detection of Alzheimer's disease. Microfluidic detection methods enable effective preconcentration of Aβ using magnetic microparticles coated with Aβ antibodies. Poly(glycidyl methacrylate) microspheres are coated with α-amino-ω-methoxy-PEG5000 /α-amino-ω-Boc-NH-PEG5000 Boc groups deprotected and NH2 succinylated to introduce carboxyl groups. Capillary electrophoresis with laser-induced fluorescence detection confirms the efficient capture of Aβ 1-40 peptides on the microspheres with immobilized monoclonal anti-Aβ 6E10. The capture specificity is confirmed by comparing Aβ 1-40 levels on the anti-IgG-immobilized particles used as a control.

• MeSH
• adsorpce MeSH
• amyloidní beta-protein izolace a purifikace   MeSH
• chromatografie afinitní MeSH
• elektroforéza kapilární MeSH
• imunoglobulin G metabolismus   MeSH
• kyseliny karboxylové chemie   MeSH
• kyseliny polymethakrylové chemie   MeSH
• magnetické jevy MeSH
• mikrosféry * MeSH
• mikroskopie atomárních sil MeSH
• peptidové fragmenty izolace a purifikace   MeSH
• polyethylenglykoly chemie   MeSH
• skot MeSH
• termogravimetrie MeSH
• velikost částic MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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-Fe2O3) 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-1 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.

• MeSH
• autoimunitní nemoci imunologie   MeSH
• autoprotilátky krev   chemie   imunologie   izolace a purifikace   MeSH
• kyseliny polymethakrylové chemie   MeSH
• lidé MeSH
• magnety chemie   MeSH
• mikrosféry * MeSH
• molekulová hmotnost MeSH
• myosin typu I chemie   imunologie   MeSH
• roztroušená skleróza imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH