Recognition of Schistosoma mansoni egg-expressed ovalbumin by T cell receptor transgenic mice.

Schistosoma mansoni eggs can influence immune responses directed at them, and the mechanisms by which this is achieved are being unravelled. Going towards, developing effective tools for the study of how S. mansoni influences naïve T cells, we have developed S. mansoni eggs expressing chicken ovalbumin (OVA), using a lentiviral transduction system. Indeed, such a parasite could be used in conjunction with cells from OT-II transgenic mice as a source of naïve, antigen-specific T cells. The expression of the transgenic protein was confirmed by real-time RT-PCR of OVA-specific mRNA and western blotting using polyclonal antibodies specific for OVA. T cells from OT-II transgenic mice expressing a T cell receptor specific for the OVA323-339 peptide recognised the OVA-transduced S. mansoni eggs. Using flow cytometry on CFSE-labelled OT-II splenocytes, we demonstrated that OVA-transduced eggs elicit higher OT-II proliferative responses than untransduced eggs. The OT-II T cells also produced TNF-α and IFN-γ following exposure to OVA-transduced eggs. In addition, moderate amounts of IL-6 and IL-17A were also detected. In contrast, no IL-10, IL-4 and IL-2 were detected in cultures, whether the cells were stimulated with transduced or untransduced eggs. Thus, the cytokine signatures showed the transfected eggs induced predominantly a Th1 response, with a small amount of IL-6 and IL-17.

Effects of dietary methionine on productivity, reproductive performance, antioxidant capacity, ovalbumin and antioxidant-related gene expression in laying duck breeders.

The study investigated whether dietary methionine (Met) affects egg weight and antioxidant status through regulating gene expression of ovalbumin (OVAL), nuclear factor erythroid 2 like 2 (Nrf2) and haem oxygenase 1 (HO-1) in laying duck breeders. Longyan duck breeders (n 540, 19 weeks) were randomly assigned to six treatments with six replicates of fifteen birds each. Breeders were fed diets with six Met levels (2·00, 2·75, 3·50, 4·25, 5·00 and 5·75 g/kg) for 24 weeks. The egg weight (g), egg mass (g/d), feed conversion ratio, hatchability, 1-d duckling weight, albumen weight, albumen proportion and OVAL mRNA level improved with dietary Met levels, whereas yolk proportion decreased (P<0·05). The weight of total large yellow follicles increased linearly (P<0·001) and quadratically (P<0·05) with dietary Met concentration, and their weight relative to ovarian weight showed a linear (P<0·05) effect. Dietary Met level had a linear (P<0·05) and quadratic (P<0·001) effect on the gene expression of glutathione peroxidase (GPX1), HO-1 and Nrf2, and quadratically (P<0·05) increased contents of GPX and total antioxidant capacity (T-AOC) in liver of duck breeders. In addition, maternal dietary Met enhanced gene expression of GPX1, HO-1 and Nrf2, increased contents of GPX and T-AOC and reduced carbonylated protein in the brains of hatchlings. Overall, dietary Met concentration affected egg weight and albumen weight in laying duck breeders, which was partly due to gene expression of OVAL in oviduct magnum. A diet containing 4·0 g Met/kg would achieve optimal hepatic GPX1 and Nrf2 expression, maximise the activity of GPX and minimise lipid peroxidation.

Chitosan and carboxymethylated derivative nanoparticles as delivery systems for biological products: preparation, characterization, stability and in vitro/in vivo evaluation / Nanopartículas de quitosana e derivado carboximetilado como sistemas de fornecimento (delivery) de produtos biológicos: preparo, caracterização, estabilidade e avaliação in vitro/in vivo

Chitosan is a biocompatible and biodegradable mucoadhesive polymer with unique advantages, such as the distinct trait of opening the junctions to allow paracellular transport of antigen and good tolerability. However, the poor solubility of chitosan in neutral or alkalinized media has restricted its applications in the pharmaceutical field. Chitosan can be easily carboxymethylated to improve its solubility in aqueous media, while its biodegradability and biocompatibility are preserved. Apart from this, carboxymethyl chitosan (CMCS) can be easily processed into nanoparticles which highlight its suitability and extensive usage for preparing different drug delivery formulations. The present study deals with the development and characterization of a delivery system based on CMCS nanoparticles using ovalbumin as model protein. We demonstrated that ovalbumin loaded nanoparticles were successfully synthetized using calcium chloride as a cross-linker by ionic gelation. The nanoparticles exhibited an average size of approximately 169 nm and presented a pseudo-spherical shape. The nanoparticles size increased according to the addition of CaCl2 due to the strong electrostatic attraction. During storage the nanoparticles size increased was attributed to swelling and aggregation. The loading efficiency of ovalbumin was found to be 17%. Confocal microscopy clearly showed the association between ovalbumin and CMCS chains into nanoparticles. Therefore, we suggest these nanoparticles can be considered as an attractive and promising carrier candidate for proteins and antigens. The major challenge that limits the use of such carriers is their instability in an aqueous medium. Thus, the next step of this work was to determine the robustness of several formulations using distinct freeze-drying protocols. This study demonstrated that mannitol in concentration of 10% (w/v) is well suited to preserve ovalbumin loaded CMCS nanocapsules from aggregation during lyophilization and subsequent reconstitution. Importantly, the results showed that an annealing step has a huge impact on porosity of freeze-dried cake by nearly complete crystallization of mannitol, once the crystalline matrix prevents the partial collapse and the formation of larger pores observed without annealing. Therefore, the usual observation that annealing increases the pore size due to growth of ice crystal size does not always apply, at least when crystallization of solute is involved. Since all characterizations and stability studies had been performed, the main purpose of this study was to develop a stable antigen delivery system for oral immunization using CMCS and inactivated rabies virus (RV) as the antigen. RV loaded nanoparticles was found to enhance both systemic (IgG) and local (IgA) immune responses against RV after oral delivery in mice. The effective doses 50% were 50-times higher than the negative controls, indicating that the immune response started only after the third boosting dose. Furthermore, enough neutralizing antibodies was produced to be protected against the harmful effects of the rabies virus. It is therefore concluded, that the CMCS nanoparticles formulated in this study, are suitable for oral vaccine delivery, and can be suggested as a promising delivery system for a diverse range of antigens as well as a gene/protein delivery system, especially for those positively charged. Since several approaches show that effective intervention in airway allergic inflammation can be achieved with allergen-activated interleukin-10-secreting cells, the final part of this work was dedicated to assessing whether IL-10 loaded chitosan nanoparticles (IL10-CSNPs) could be used as a possible inhalable therapeutic tool for preventing exacerbations in asthmatic patients. As positive controls, we also assess whether interleukin 17A and interleukin 9 have the ability to stimulate human airway smooth muscle (HASM) cell contractility using magnetic twisting cytometry (MTC). Significant decreased baseline cell stiffness was observed in HASM cells pre-treated with IL-10, but not with IL10-CSNPs, whereas treatment with IL-17A significantly enhanced baseline cell stiffening. Our findings reveal a previously unknown mechanism underlying immunotherapy for prevention and treatment of asthma

A quitosana é um polímero mucoadesivo biocompatível e biodegradável, com vantagens únicas, tais como a característica distinta de abrir as junções que permitim o transporte paracelular de antígenos e boa tolerabilidade. No entanto, sua baixa solubilidade em meios neutros ou alcalinizados tem restringido suas aplicações no campo farmacêutico. A quitosana pode ser facilmente carboximetilada para melhorar de sua solubilidade em meios aquosos, enquanto sua biodegradabilidade e biocompatibilidade são preservadas. Além disso, a carboximetilquitosana (CMCS) pode ser facilmente processada na forma de nanopartículas, o que destaca sua adequabilidade para uso extensivo no preparo de sistemas de delivery de medicamentos. O presente estudo trata do desenvolvimento e caracterização de um sistema de delivery baseado em nanopartículas de CMCS utilizando ovalbumina como proteína modelo. Nós demonstramos que as nanopartículas carregadas com ovalbumina foram sintetizadas com sucesso utilizando cloreto de cálcio como agente de reticulação por gelificação iônica. As nanopartículas exibiram um tamanho médio de aproximadamente 169 nm e apresentaram uma forma pseudo-esférica. O tamanho das nanopartículas aumentou de acordo com a adição de CaCl2 devido à forte atração eletrostática. Durante o armazenamento, o tamanho aumentado das nanopartículas foi atribuído a incorporação de água e agregação. A eficiência de encapsulamento da ovalbumina foi de aproximadamente 17%. A microscopia confocal mostrou claramente a associação entre ovalbumina e a cadeias de CMCS nas nanopartículas. Sugerimos, portanto, que tal sistema pode ser considerado como candidato atraente e promissor para o carreamento de proteínas e antígenos. O principal desafio que limita o uso desses carreadores consiste na instabilidade em meio aquoso. Assim, o próximo passo deste trabalho foi determinar a robustez de várias formulações utilizandose diferentes protocolos de liofilização. Este estudo demonstrou que o manitol em uma concentração de 10% (p/v) é adequado para preservar da agregação as nanocápsulas de CMCS carregadas com ovalbumina durante a liofilização e subsequente reconstituição. Mais importante, os resultados mostraram que uma etapa de annealing tem um enorme impacto sobre a porosidade da amostra liofilizada devido a quase completa cristalização do manitol, uma vez que a matriz cristalina evita o colapso parcial e a formação de poros maiores observados na ausência do annealing. Portanto, a observação comum de que o annealing aumenta o tamanho doporos devido ao crescimento dos cristais de gelo nem sempre se aplica, pelo menos quando a cristalização de um soluto está envolvida. Uma vez que todas as caracterizações e estudos de estabilidade foram realizados, o principal objetivo deste estudo foi desenvolver um sistema estável de delivery de antígeno para imunização oral utilizando CMCS e vírus rábico inativado (RV) como antígeno. Verificou-se que as nanopartículas carregadas com RV aumentam as respostas imune sistêmica (IgG) e local (IgA) contra o RV após administração oral em camundongos. As doses efetivas 50% foram 50 vezes maiores que os controles negativos, indicando que a resposta imune foi iniciada apenas após a terceira dose da vacina. Além disso, foram produzidos anticorpos neutralizantes suficientes para proteção contra os efeitos nocivos do vírus rábico. Conclui-se, portanto, que as nanopartículas de CMCS formuladas neste estudo, são adequadas para o delivery oral de vacinas, e podem ser sugeridas como um sistema promissor de delivery para uma gama diversa de antígenos, bem como para o delivery de genes/proteínas, especialmente para aqueles carregados positivamente. Uma vez que diversas abordagens mostram que uma intervenção efetiva em casos de inflamação alérgica de vias aéreas pode ser conseguida por meio de células secretoras de interleucina 10 (IL-10) mediante ativação por alergenos, a parte final deste trabalho esteve dedicada a avaliação de nanopartículas de quitosana carregadas com IL-10 (IL10-CSNPs) como possível ferramenta terapêutica inalável para prevenção de exacerbações em pacientes asmáticos. Como controles positivos, avaliou-se adicionalmente se as interleucinas 17A (IL-17A) e 9 (IL-9) possuem a capacidade de estimular a contratilidade de células humanas de músculo liso de vias aéreas humanas (HASM) por meio de citometria de torção magnética (MTC). Uma diminuição significativa da rigidez celular basal foi observada em células HASM pré-tratadas com IL-10, mas não com IL10-CSNPs, enquanto que o tratamento com IL-17A aumentou significativamente a magnitude rigidez celular basal. Nossos resultados revelam um mecanismo previamente desconhecido subjacente à imunoterapia para prevenção e tratamento da asma

The Preparation and Identification of a Monoclonal Antibody against Domoic Acid and Establishment of Detection by Indirect Competitive ELISA.

Domoic acid (DA) is a potent toxin, marine biotoxin, and primarily produced by Pseudo-nitzschia. The DA hapten was coupled with bovine serum albumin (BSA), and ovalbumin (OVA) as carrier proteins. DA-BSA conjugate was used as immunogen and DA-OVA as coating antigen. Cell fusion between spleen cells and sp2/0 myeloma cells developed 1C3 hybridoma clone producing 1C3 monoclonal antibody (mAb). Hybridoma was injected into the mice to produce ascites, and further purified by caprylic acid/ammonium sulfate method. The mAb was of IgG3 subclass, and was specific to DA with high affinity (2.5 × 108 L/mol). Moreover, western blot exhibited significant specificity to the DA antigens. Indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) showed DA working range of 0.006-0.2 ng/mL. The IC50 was 0.03 ng/mL with low limit of detection (LOD) of 0.006 ng/mL. Average DA recovery from spiked shellfish extract was 100.56% ± 2.8% with the coefficient variation of 0.01-0.1%. Hence, mAb producing 1C3 hybridoma was successfully developed and could be used to detect DA in contaminated samples.

Fabrication of micromagnetic beads with molecular recognition/electron-transfer peptides for the sensing of ovalbumin.

Electrochemical sensing of ovalbumin (OVA) was performed using magnetic beads with OVA recognition (RNRCKGTDVQAW)/electron-transfer (YYYYC) peptides. The focus of this study was to construct a highly sensitive and regenerative tool for OVA detection based on the interaction between a protein and peptide-1(RNRCKGTDVQAWYYYYC). The peptide-1 was introduced to the bead through four types of cross-linking reagents. Magnetic beads of different sizes with N-(6-maleimidocaproyloxy)sulfosuccinimide (Sulfo-EMCS) were also prepared. An oxidation peak due to tyrosine residues at 0.65 V depended on the distance of the electron-transfer peptide from the bead surface and on the surface area of the magnetic beads that contacted the electrode surface. The response of the electro-transfer peptide moiety was decreased because the protein was accumulated via the recognition peptide on the beads. When using Sulfo-EMCS and beads that were 6.0-6.9 µm in diameter, the calibration curve of OVA was linear and ranged from 8.0 × 10-13 to 2.0 × 10-11 M. To regenerate the magnetic beads, the measurements were achieved after removal of the OVA using a denaturing reagent. When OVA was added to fetal bovine serum containing a complex matrix, OVA was recovered at a rate of 98-100%. Consequently, these magnetic beads could be a powerful tool for the sensing of OVA in real samples.

Mechanisms of Antigen Adsorption Onto an Aluminum-Hydroxide Adjuvant Evaluated by High-Throughput Screening.

The adsorption mechanism of antigen on aluminum adjuvant can affect antigen elution at the injection site and hence the immune response. Our aim was to evaluate adsorption onto aluminum hydroxide (AH) by ligand exchange and electrostatic interactions of model proteins and antigens, bovine serum albumin (BSA), ß-casein, ovalbumin (OVA), hepatitis B surface antigen, and tetanus toxin (TT). A high-throughput screening platform was developed to measure adsorption isotherms in the presence of electrolytes and ligand exchange by a fluorescence-spectroscopy method that detects the catalysis of 6,8-difluoro-4-methylumbelliferyl phosphate by free hydroxyl groups on AH. BSA adsorption depended on predominant electrostatic interactions. Ligand exchange contributes to the adsorption of ß-casein, OVA, hepatitis B surface antigen, and TT onto AH. Based on relative surface phosphophilicity and adsorption isotherms in the presence of phosphate and fluoride, the capacities of the proteins to interact with AH by ligand exchange followed the trend: OVA < ß-casein < BSA < TT. This could be explained by both the content of ligands available in the protein structure for ligand exchange and the antigen's molecular weight. The high-throughput screening platform can be used to better understand the contributions of ligand exchange and electrostatic attractions governing the interactions between an antigen adsorbed onto aluminum-containing adjuvant.

A novel graphene-based label-free fluorescence 'turn-on' nanosensor for selective and sensitive detection of phosphorylated species in biological samples and living cells.

A novel label-free fluorescence 'turn-on' nanosensor has been developed for highly selective and sensitive detection of phosphorylated species (Ps) in biological samples and living cells. The design strategy relies on the use of Ti(4+)-immobilized polydopamine (PDA) coated reduced graphene oxide (rGO@PDA-Ti(4+)) that serves as an attractive platform to bind riboflavin 5'-monophosphate molecules (FMNs) through ion-pair interactions between phosphate groups and Ti(4+). The as-prepared rGO@PDA-Ti(4+)-FMNs (nanosensor), fluoresce only weakly due to the ineffective Förster resonance energy transfer between the FMNs and rGO@PDA-Ti(4+). The experimental findings revealed that the microwave-assisted interaction of the nanosensor with α-, ß-casein, ovalbumin, human serum, non-fat milk, egg white, and living cells (all containing Ps) releases FMNs (due to the high formation constant between phosphate groups and Ti(4+)), leading to an excellent fluorescence 'turn-on' response. The fluorescence spectroscopy, confocal microscopy, and MALDI-TOF MS spectrometry were used to detect Ps both qualitatively and quantitatively. Under the optimized conditions, the nanosensor showed a detection limit of ca. 118.5, 28.9, and 54.8 nM for the tryptic digests of α-, ß-casein and ovalbumin, respectively. Furthermore, the standard addition method was used as a bench-mark proof for phosphopeptide quantification in egg white samples. We postulate that the present quantitative assay for Ps holds tremendous potential and may pave the way to disease diagnostics in the near future.

Distyrylbenzene-aldehydes: identification of proteins in water.

Three different, water soluble, aldehyde-appended distyrylbenzene (DSB) derivatives were prepared. Their interaction with different albumin variants (human, porcine, bovine, lactalbumin, ovalbumin) was investigated (pH 11). All three fluorophores exhibit graded, protein-dependent fluorescence turn-on at slightly differing wavelengths. Linear discriminant analysis (LDA) differentiated all of the investigated albumins and was used to discern commercially available protein shakes. The three DSB derivatives barely react with the constituting amino acids but cysteine. In the proteins significant fluorescence signals are generated, probably due to a combination of imine/N,S-aminal formation and hydrophobic interactions between the DSBs and the proteins.

Quantification of proteins using (13)C7-labeled and unlabeled iodoacetanilide by nano liquid chromatography/nanoelectrospray ionization and by selected reaction monitoring mass spectrometry.

The combination of cysteine-specific modifiers, iodoacetanilide (IAA) and (13)C7-labeled iodoacetanilide ((13)C7-IAA), has been applied to absolute quantification of proteins. The selected reaction monitoring (SRM) with the use of nano liquid chromatography/nanoelectrospray ionization ion trap mass spectrometry (nano LC/nano-ESI-IT-MS) analysis was applied to precise quantification of three commercial proteins. Good correlation was observed between the theoretical ratios and observed ratios for all these proteins both in a simple buffer solution and in a complex protein environment. Due to efficient tagging, this method does not require separate synthesis of isotope-labeled peptides for the SRM studies. Therefore, this method is expected to be a useful tool for proteomics research.

Construction of a peptide with an electroactive daunomycin like a pendant arm to detect ovalbumin.

In this study, a peptide-1 (RNRCKGTDVQAW) constructing lysozyme was conjugated with an electroactive daunomycin in order to voltammetrically detect ovalbumin (OVA). Hetero-bifunctional cross-linking agents with four kinds of ethylene chains in differing lengths were used to bind the peptide-1 and daunomycin. After a cross-linking agent had reacted with an amino group of daunomycin, the compound was introduced into the peptide to the cysteine residue in the peptide using a pendant arm. The OVA was sensed via a change in the electrode response of the daunomycin moiety, based on the binding between the peptide and the OVA. The adsorption of the peptide probe on the electrode increased with increases in the ethylene chain. The binding constants between the peptide probes and the OVA, however, did not depend on the length of the chain. This was because the ethylene chain influenced the binding. When the peptide and the daunomycin were bound using N-(6-maleimidocaproyloxy) sulfosuccinimide, the electrode response of the peptide probe was the most sensitive from among the four cross-linking agents. The calibration curve of the OVA using the peptide probe was linear and ranged from 1.5×10(-11) to 3.0×10(-10)M. Furthermore, this method could be applied to the electrochemical sensing of the OVA in egg whites and in fetal bovine serum.

Voltammetric detection of ovalbumin using a peptide labeled with an electroactive compound.

For this study, a new method was developed to electrochemically detect ovalbumin via its binding with the peptide-1(RNRCKGTDVQAW) in lysozymes. The peptide that exists at the C-terminal of a lysozyme was combined with ovalbumin. When an electroactive compound was introduced to the N-terminal side of the peptide through ethylene gycolbis(sulfosuccinimidyl succinate), the labeled peptide-1 served as a probe for the detection of ovalbumin. The electrode responses of labeled peptide-1 were measured after the labeled peptide-1 and ovalbumin were incubated in a 0.1 M phosphate buffer (pH 5.6). As a result, the electrode response decreased as the concentration of ovalbumin increased. The detection limit of ovalbumin was 2.3 × 10(-11) M as estimated at 3-fold the standard deviation (3σ) (n = 5). Because the steric structure of the peptide and some of the amino acid residues were related to the binding, we prepared a peptide-2, to which the N- and C-terminals of peptide-1 were alternated. The decrease in the response for the labeled peptide-2 was less than that for the labeled peptide-1. In addition, the peak current of a peptide-3, for which the D of peptide-1 was replaced with S, was hardly changed with or without ovalbumin. Therefore, it was clear that the binding was influenced by the steric factors and by the sequence of the peptide. However, a peptide-1 with bis(sulfosuccinimidyl) suberate was designed to investigate the hydrophobic influences on the probe. The change in the peak current was smaller than that of peptide-1 with ethylene gycolbis(sulfosuccinimidyl succinate), which was due to the hydrophobic properties of the alkyl chain between the peptide and the ovalbumin. The proposed method could be applied to the determination of ovalbumin in egg whites. Consequently, the concept becomes an electrochemical sensing method for proteins based on the protein-peptide interaction.

A magnetic particles-based chemiluminescence enzyme immunoassay for rapid detection of ovalbumin.

Egg allergy is an important public health and safety concern, so quantification and administration of food or vaccines containing ovalbumin (OVA) are urgently needed. This study aimed to establish a rapid and sensitive magnetic particles-chemiluminescence enzyme immunoassay (MPs-CLEIA) for the determination of OVA. The proposed method was developed on the basis of a double antibodies sandwich immunoreaction and luminol-H2O2 chemiluminescence system. The MPs served as both the solid phase and separator, the anti-OVA MPs-coated polyclonal antibodies (pAbs) were used as capturing antibody, and the horseradish peroxidase (HRP)-labeled monoclonal antibody (mAb) was taken as detecting antibody. The parameters of the method were evaluated and optimized. The established MPs-CLEIA method had a linear range from 0.31 to 100ng/ml with a detection limit of 0.24ng/ml. The assays showed low reactivities and less than 5% of intraassay and interassay coefficients of variation (CVs), and the average recoveries were between 92 and 97%. Furthermore, the developed method was applied in real samples analysis successfully, and the correlation coefficient with the commercially available OVA kit was 0.9976. Moreover, it was more rapid and sensitive compared with the other methods for testing OVA.

Quantitative protein analysis using (13)C7-labeled iodoacetanilide and d5-labeled N-ethylmaleimide by nano liquid chromatography/nanoelectrospray ionization ion trap mass spectrometry.

We have developed a methodology for quantitative analysis and concurrent identification of proteins by the modification of cysteine residues with a combination of iodoacetanilide (IAA, 1) and (13)C7-labeled iodoacetanilide ((13)C7-IAA, 2), or N-ethylmaleimide (NEM, 3) and d5-labeled N-ethylmaleimide (d5-NEM, 4), followed by mass spectrometric analysis using nano liquid chromatography/nanoelectrospray ionization ion trap mass spectrometry (nano LC/nano-ESI-IT-MS). The combinations of these stable isotope-labeled and unlabeled modifiers coupled with LC separation and ESI mass spectrometric analysis allow accurate quantitative analysis and identification of proteins, and therefore are expected to be a useful tool for proteomics research.

Protein quantitation using various modes of high performance liquid chromatography.

Pharmaceuticals based on proteins (biologicals), such as monoclonal antibodies (mAb), attain more and more relevance since they were established as potent drugs in anticancer therapy or for the treatment of autoimmune based diseases. Due to their high efficiency it is essential to have accurate and precise methods for protein quantitation and the detection of protein aggregates, which in some cases may lead to adverse effects after application. Selectivity and precision of traditional protein quantification methods such as the Bradford assay or SDS-PAGE are insufficient for quality control (QC) purposes. In this work several HPLC separation modes, which can significantly improve these important parameters, were compared for their application in this field. High performance size exclusion (HP-SEC), strong anion exchange (SAX), weak cation exchange (WCX) as well as reversed phase chromatography are all already successfully applied in protein analysis. Good precision (SEC: <1.9%, SAX: <5%, RP: <2% and WCX: <3.5% - RSD% for peak areas day-to-day), high selectivity and low quantitation limits (<15µg/ml) for the model proteins ovalbumin, myoglobin and bovine serum albumin (BSA), respectively cytochrome c and lysozyme in the cation exchange mode, could be achieved. Consecutively, the four separation modes were compared to each other and to electrophoretic techniques in terms of precision, selectivity, analysis time, effort of sample and mobile phase preparation as well as separating capacity. Moreover, the analysis of an IgG1-type antibody was included in this study.

Fast protein detection using absorption properties of gold nanoparticles.

In this study we describe the use of gold nanoparticles as a fast detection system for the sensitive analysis of proteins. The immunological method allows for protein analysis at the nanogram level, as required for clinical diagnosis. Initially a test protein is used for the development of the assay. The system is subsequently adopted for alpha-fetoprotein, which is a relevant tumor marker. This work demonstrates that antibody functionalized gold nanoparticles can be used for the detection of proteins by forming gold nanoparticle aggregates. The influence of the size of the gold nanoparticles on the sensitivity of the assay is investigated in the range from 20-60 nm particles; the larger particles show here the highest relative changes. The formation of antigen-gold nanoparticle aggregates is detected by an increase in hydrodynamic diameter by dynamic light scattering (DLS). UV/Vis spectroscopy also allows assay monitoring by quantifying the red shift of the plasmon resonance wavelength. Alpha-fetoprotein can be analysed in the concentration range of 0.1-0.4 µg ml(-1). The influence of pH, ionic strength and ratio of sample to Au-NP solution is studied. With this method, the protein AFP can be rapidly detected as demanded for clinical diagnosis.

Surface chemical analysis and chromatographic characterization of polyethylenimine-coated hydroxyapatite with various amount of polyethylenimine.

Polyethylenimine (PEI) has been widely used as a coating material to produce stationary phase for ion-exchange chromatography of biomolecules. However, a precise study of the PEI coating fraction has been lacking, despite such quantification being very important for fundamental research as well as identifying further industrial applications. In this study, we produced four types of PEI-coated hydroxyapatite (PEI-HAp) with various fractions of PEI (0.16%, 0.5%, 1.0%, 1.5%) using a spray-drying system to evaluate correlations between coating fractions and the thermochemical or chromatographic behaviors of theses products. The thermal analyses of these matrices showed two exothermic peaks when the PEI coating fraction exceeded 1.0%. The one peak indicated a PEI decomposition peak and the other would indicate bond dissociation of PEI layers formed over the HAp surface as the PEI concentration increased. Furthermore, the chromatographic analysis for the surface chemical characteristics showed the correlation between coating fraction and the retention time of protein or nucleotide. Acidic or phosphorylated proteins were more strongly adsorbed as the PEI coating fraction increased when the initial coating fraction was low, but at fraction exceeding 0.5%, constant retention was observed. The retention time of nucleotides increased in proportion to the fraction of PEI added. The good selectivity of PEI-HAp may be attributable to multifunctional interactions of electrostatic and bare Ca sites on HAp, not just the amino sites of PEI. These precise studies of PEI coating fraction are our original novel contributions, which could be achieved by quantitative consideration using thermal analysis and chromatography.

The effect of maternal state on the steroid and macronutrient content of lesser black-backed gull eggs.

It has been proposed that female birds can influence the phenotype of their offspring by provisioning eggs with variable amounts of nutrients and maternal hormones. Egg quality is strongly influenced by maternal body reserves and the amount of food available at the time of egg formation. This study investigated the effects of maternal state and food availability on the capacity of female lesser black-backed gulls Larus fuscus to provision their eggs with macronutrients and steroid hormones. Maternal state was reduced by increasing egg-production effort, whereas extra food was provided to reverse this effect. Compared with eggs of first clutches, eggs of experimentally induced replacement clutches exhibited a lower yolk/albumen ratio and contained more yolk testosterone. During one of the three years in which the study was performed, replacement eggs also contained more 17ß-estradiol. Food provisioning during the relaying interval did not affect changes in yolk/albumen ratio or steroid concentrations, but fed females produced bigger eggs in their replacement clutch. This study demonstrates significant within-female consistency in egg size, macronutrient content, and yolk steroid concentration, and it shows that these egg characteristics are influenced by maternal state, food availability, and the timing of breeding.

Relation of structure to performance characteristics of monolithic and perfusive stationary phases.

Commercially available polymer-based monolithic and perfusive stationary phases were evaluated for their applicability in chromatography of biologics. Information on bed geometry, including that from electron microscopy (EM), was used to interpret and predict accessible volumes, binding capacities, and pressure drops. For preparative purification of biologics up to at least 7 nm in diameter, monoliths and perfusive resins are inferior to conventional stationary phases due to their low binding capacities (20-30 g/L for BSA). For larger biologics, up to several hundred nanometers in diameter, calculations from EM images predict a potential increase in binding capacity to nearly 100 g/L. The accessible volume for adenovirus calculated from the EM images matched the experimental value. While the pores of perfusive resins are essentially inaccessible to adenovirus under binding conditions, under non-adsorbing conditions the accessible intrabead porosity is almost as large as the interbead porosity. Modeling of breakthrough curves showed that the experimentally observed slow approach to full saturation can be explained by the distribution of pore sizes.

Acid hydrolysis of proteins in matrix assisted laser desorption ionization matrices.

Sample preparation is crucial to the success of experiments in biological mass spectrometry. In proteomics, digestion of the proteins into peptides is a key step for "bottom-up" approaches. Often, the use of enzymes requires physiological conditions, producing peptides that must be extracted or further purified before mass analysis. Chemical cleavage reagents offer more flexibility and can be more compatible with downstream mass analysis. Expanding on prior work using acid hydrolysis, proteolysis with matrix-assisted laser desorption ionization (MALDI) matrices is presented. This sample preparation can be performed rapidly with a minimum of reagents and sample handling, but it must first be evaluated in terms of digestion efficiency, missed cleavages, and side reactions before implementation for in-gel digestion and in-solution digestion using minimal volumes of protein. Time courses of acid hydrolysis are shown for protein standards, illustrating the sensitivity of this type of sample preparation, minimization of side reactions, and performance for proteins in mixtures. To illustrate the potential for sensitive detection of a specific protein, MALDI matrix hydrolysis is used to digest a protein immunoprecipitated from cell lysate.

Identification of protein binders in works of art by high-performance liquid chromatography-diode array detector analysis of their tryptic digests.

Proteins in works of art are generally determined by the relative amounts of amino acids. This method, however, implies a loss of information on the protein structure and its modifications. Consequently, we propose a method based on the analysis of trypsin digests using high-performance liquid chromatography (HPLC) UV diode array detection (DAD) for painting binder studies. All reaction steps are done in the same vial; no extraction methods or sample transfer is needed, reducing the risk of sample losses. A collection of pure binders (collagen, ovalbumin, yolk and casein) as well as homemade and historical paint samples have been investigated with this method. Chromatograms of unknowns at 214 nm and 280 nm are compared with those of the reference samples as a fingerprint. There is a good agreement between many peptides, but others seem to have been lost or their retention time shifted due to small compositional changes because of ageing and degradation of the paint. The results are comparable with the results of other techniques used for binder identification on the same samples, with the additional advantage of differentiation between egg yolk and glair.