Search

A boronate-modified magnetic affinity sorbent was prepared by adopting hyperbranched polyethyleneimine as the scaffold to amplify initiator sites. 3-Acrylamidophenylboronic acid was employed as monomer to proceed in situ free-radical polymerization on magnetite (Fe 3 O 4 ) nanoparticles. Due to the improved density of boronic acid polymers, the adsorbent exhibited high adsorption capacity, typically (134 ± 8) μg mg -1 for dopamine, (92 ± 7) μg mg -1 for catechol, (363 ± 14) μg mg -1 for ovalbumin and (464 ± 22) μg mg -1 for horseradish peroxidase. These capacities are much higher than those of other adsorbents. The sorbent was applied to the enrichment of catecholamines from spiked human urine. Owing to the high adsorption capacity, only 1.0 mg of adsorbent was sufficient to eliminate the interferences and enrich the targets (dopamine, norepinephrine and epinephrine) within 5 min. They were quantified by HPLC. The recoveries from spiked samples range between 83.5% ~106%, with relative standard deviations of 3.2 ~ 9.4% (n = 5). The separation of glycoproteins from egg white was also accomplished prior to their analysis by PAGE. In the authors' perception, this approach is promising in that the density of functional groups on the adsorbent is strongly increased. Graphical abstract The preparation routine of boronate affinity magnetic adsorbent (Fe 3 O 4 @HpAAPBA). The adsorbent is used for the magnetic solid phase extraction of cis-dol compounds from real sample.

The determination of specific IgE (sIgE) level is of great importance in IgE-mediated food allergies. Our aim was to develop a homogeneous immunoassay-light-initiated chemiluminescent assay (LICA)-for measuring allergen sIgE of a single component in egg white, thus evaluating the LICA-sIgE assay as a useful tool in the diagnosis of food allergy. The LICA-sIgE assay was performed by incubating serum sample with anti-human IgE antibody coated with chemiluminescer beads, streptavidin-coated sensitizer beads, and biotinylated antigens, which consist of four components in egg white. Serum samples from egg allergic patients (n = 70) and healthy volunteers (n = 30) were collected. For calibration, purified human IgE was used as the calibrator. Working conditions of this homogeneous immunoassay were optimized, analytical performance was determined, and correlation of the results between LICA and ImmunoCAP was evaluated. The assays were performed in 8-well plates with a sample volume diluted to 1:10 of 25 μl. Intra-assay precision (% coefficient of variation) ranged from 1.83 to 4.13%, and inter-assay precision ranged from 2.70 to 8.70%. It exhibited excellent sensitivity, which could distinguish between positive samples and negative samples even at a large dilution level. The sIgE-LICA and ImmunoCAP correlated well in patients allergic to single component (r 2  = 0.929). Also, the components ovomucoid and ovalbumin were best at predicting ImmunoCAP results, with the same area under the ROC curve (AUC) of 0.81, and a specificity of 90.0 and 93.3%, respectively. Our data show effective performance characteristics of LICA to detect sIgE in human serum based on component-resolved diagnostic tests (CRD). The homogeneous sIgE-LICA assay has the following key advantages: requires no washing, simplicity and rapidity, reproducibility, high-throughput, good performance in a liquid phase assay, and good suitability for sIgE diagnosis in food allergy based on CRD. Graphical abstract A light-initiated chemiluminescent assay was developed for the quantitation of sIgE against egg white allergens based on component-resolved diagnosis. Components Gal d 1 and Gal d 2 with the highest AUC values of 0.81 were considered the best at predicting egg allergy.

Corticosterone (CORT) is the dominant plasma glucocorticoid in birds. There has been increasing interest in the function of CORT in avian egg yolk and in the potential to use CORT concentrations in eggs to quantify stress and to assess the effect of maternal stress on offspring. The concentration of CORT in egg yolk is most frequently assessed using enzyme or radioimmunoassays, alone or in combination with high-performance liquid chromatography. However, the quantification of CORT is frequently hampered by the presence of high concentrations of other steroid hormones which cross-react with the CORT antibody. As an alternative, we developed a sensitive and specific LC-MS-MS method. The sample-preparation procedure consisted of a protein-lipid precipitation step, followed by defatting and clean-up using a C18 SPE column. Chromatography was performed on an Acquity C18 BEH column (50 mm × 2.1 mm i.d., dp: 1.7 μm, run-time: 6 min), using 0.1% formic acid in both water (A) and acetonitrile (B) as mobile phases. The MS-MS instrument was operated in the positive-electrospray-ionization mode. The method was validated in-house according to European Guidelines (linearity, accuracy and precision, limits of quantification and detection, specificity, stability) and the results fell within the accepted ranges. The method was successfully used for the analysis of CORT in yolk and albumen of eggs collected from eight breeding lesser black-backed gulls at a Flemish coastal colony. CORT concentrations were in the range 42.4-166.3 pg g(-1) in albumen and < LOQ (75 pg g(-1))-762.5 pg g(-1) in yolk.

Taurine is one of the osmolytes that maintain the structure of proteins in cells exposed to denaturing environmental stressors. Recently, cryoelectron tomographic analysis of eukaryotic cells has revealed that their cytoplasms are crowded with proteins. Such crowding conditions would be expected to hinder the efficient folding of nascent polypeptide chains. Therefore, we examined the role of taurine on the folding of denatured and reduced lysozyme, as a model protein, under a crowding condition. The results confirmed that taurine had a better effect on protein folding than did β-alanine, which has a similar chemical structure, when the protein to be folded was present at submillimolar concentration. NMR analyses further revealed that under the crowding condition, taurine had more interactions than did β-alanine with the lysozyme molecule in both the folded and denatured states. We concluded that taurine improves the folding of the reduced lysozyme at submillimolar concentration to allow it to interact more favorably with the lysozyme molecule. Thus, the role of taurine, as an osmolyte in vivo, may be to assist in the efficient folding of proteins.

An organic-inorganic hybrid monolithic column based on 1-vinyl-3-dodecylimidazolium bromide (VC12Im(+)Br(-)) has been prepared in a single step by combining radical copolymerization with a non-hydrolytic sol-gel (NHSG) process. The NHSG process was significantly shortened to 6 h by using formic acid as catalyst. For comparison, we also prepared polymeric ionic liquid (PIL) monolithic columns by hydrolytic sol-gel and organic polymeric process, respectively. The resulting monolithic columns were characterized by Fourier transform infrared spectra, scanning electron microscopy, and Brunauer-Emmett-Teller. Under the capillary electrochromatography mode, these columns were applied to separate alkylbenzenes, anilines, and proteins, respectively. The results indicated that the NHSG-based hybrid PIL monolithic column exhibited the highest column efficiency among the three types of columns; organic solvent, commonly required by the traditional columns to achieve satisfactory separation efficiency for proteins, was absent in the NHSG-based hybrid PIL monolithic column because of the biocompatibility of the VC12Im(+)Br(-), which was beneficial to analysis of protein containing samples. In order to demonstrate its application potential, the developed NHSG-based hybrid PIL monolithic column was also employed to separate egg white sample.

A nanocomposite is prepared by encapsulating silica nano-spheres with polymerized ionic liquid in aqueous medium without use of any organic solvents. Vinyl groups are covalently introduced on to the surface of silica nano-spheres, which are then encapsulated by copolymerization of 1-vinyl-3-ethylimidazolium bromide (monomer) and 1,4-butanediyl-3,3'-bis-l-vinylimidazolium dibromide (cross-linker) at room temperature. The derived nanocomposite, PIL@SiO2, provides a green adsorbent for protein sorption. PIL@SiO2 is selective toward acidic proteins, and its selectivity can be controlled via varying the amount of monomer used in the copolymerization process. At pH 6.0, use of 5 mg PIL@SiO2 nanocomposite results in a sorption efficiency of up to 95 % for 200 mg L(-1) ovalbumin in 1 mL sample solution. Electrostatic and hydrophobic interactions between PIL@SiO2 and protein species dominate the adsorption process. The ovalbumin adsorption behavior is consistent with the Langmuir model, giving a sorption capacity of 333.3 mg g(-1). The retained ovalbumin is recovered by elution with 0.2 % SDS solution. Circular dichroism spectra reveal virtually no change to the α-helix content of ovalbumin after elimination of SDS by use of dialysis. In summary, high-purity ovalbumin is isolated from chicken egg-white by use of the PIL@SiO2 nanocomposite as adsorbent.

This study describes a highly efficient method for the selective precipitation of phosphoproteins by trivalent europium, terbium, and erbium metal ions. These metal cations belong to the group of lanthanides and are known to be hard acceptors with an overwhelming preference for oxygen-containing anions such as phosphates to which they form very tight ionic bonds. The method could be successfully applied to specifically precipitate phosphoproteins from complex samples including milk and egg white by forming solid metal-protein complexes. Owing to the low solubility product of the investigated lanthanide salts, the produced metal-protein complexes showed high stability. The protein pellets were extensively washed to remove nonphosphorylated proteins and contaminants. For the analysis of proteins the pellets were first dissolved in 30 % formic acid and subjected to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS. For peptide mass-fingerprint analysis the precipitated phosphoproteins were enzymatically digested using microwave-assisted digestion. The method was found to be highly specific for the isolation and purification of phosphoproteins. Protein quantification was performed by colorimetric detection of total precipitated phosphoproteins and revealed more than 95 % protein recovery for each lanthanide salt.

Immunofluorescence microscopy offers a highly specific analytical tool for unambiguous recognition and mapping of proteins in complex matrices. In the present work, the analytical potentials of immunofluorescence microscopy have been exploited to provide recognition of proteinaceous binders in painting cross-sections. An optimised analytical protocol is proposed for the identification of ovalbumin and of bovine serum albumin as markers of egg white and casein, respectively. The study has been carried out on laboratory model samples simulating both easel and mural paintings. The obtained results demonstrated the effectiveness of the method, suggesting the potential future use of immunofluorescence microscopy as a routine diagnostic tool in conservation science. Possible developments of the proposed methodology in order to improve the specificity of the method and its detection sensitivity are presented and discussed.