Antisolvent micronization of BSA using supercritical mixtures carbon dioxide + organic solvent

In this work, expanded liquid antisolvent (ELAS) process has been used to micronize bovine serum albumin (BSA) solubilized in water. Carbon dioxide mixtures with ethanol, acetone or isopropyl alcohol, at expanded liquid conditions, have been used as the antisolvent. The effect of process parameters, such as the kind of co-antisolvent and the organic co-antisolvent/water/carbon dioxide mole fraction on the morphology and dimensions of the precipitates, was studied. Changing co-antisolvent and operating conditions, we obtained nanoparticles (with a mean diameter of about 60 nm ± 10 nm), sub-microparticles (with a mean diameter of 470 nm ± 130 nm), microparticles (with a mean diameter of 0.93 μm ± 0.37 μm) and expanded microparticles with an empty core (with a mean diameter of about 9 μm ± 5 μm). Fourier transform infrared analysis on BSA powders revealed that, using acetone as co-antisolvent, no modifications of the protein secondary structure were induced by ELAS processing.     

Specificity of a novel TaqMan PCR method for detection of poultry DNA

After the Bovine Spongiform Encephalopathy (BSE) crisis emerged in 1985/1986, all processed animal proteins (PAPs) were finally banned for use in animal feed in the European Union. To partially lift this feed ban, paths for re-introduction of PAPs from species other than ruminants e.g. pig and poultry, are described in the Transmissible Spongiform Encephalopathies (TSE) Roadmap 2. Cannibalism, however, is still not allowed. Specific detection methods for pig and poultry meal and PAPs are prerequisites for reintroduction of pig and poultry processed animal proteins into animal feed. Developing a sensitive PCR method that specifically detects the taxonomically diverse and therefore artificial group ‘poultry’ and that does not detect other birds at the same time is a challenge. Here, a novel TaqMan PCR method for poultry detection is presented. The specificity of the poultry method against target and non-target species has been extensively investigated. The efficiency, linearity and sensitivity was tested using dilution series of chicken, turkey, duck and goose DNA isolated from meat and autoclaved meat as a model system for PAPs.     

Mass spectrometry of peptides and proteins from human blood

It is difficult to convey the accelerating rate and growing importance of mass spectrometry applications to human blood proteins and peptides. Mass spectrometry can rapidly detect and identify the ionizable peptides from the proteins in a simple mixture and reveal many of their post‐translational modifications. However, blood is a complex mixture that may contain many proteins first expressed in cells and tissues. The complete analysis of blood proteins is a daunting task that will rely on a wide range of disciplines from physics, chemistry, biochemistry, genetics, electromagnetic instrumentation, mathematics and computation. Therefore the comprehensive discovery and analysis of blood proteins will rank among the great technical challenges and require the cumulative sum of many of mankind's scientific achievements together. A variety of methods have been used to fractionate, analyze and identify proteins from blood, each yielding a small piece of the whole and throwing the great size of the task into sharp relief. The approaches attempted to date clearly indicate that enumerating the proteins and peptides of blood can be accomplished. There is no doubt that the mass spectrometry of blood will be crucial to the discovery and analysis of proteins, enzyme activities, and post‐translational processes that underlay the mechanisms of disease. At present both discovery and quantification of proteins from blood are commonly reaching sensitivities of ～1 ng/mL. © 2010 Wiley Periodicals, Inc., Mass Spec Rev 30:685–732, 2011     

Portable Infrared Spectrometer to Characterize and Differentiate Between Organic and Conventional Bovine Butter

The performance of a portable infrared system combined with pattern recognition to discriminate between organically and conventionally produced bovine butter samples as well as to predict the levels of conjugated linoleic acid (CLA) were evaluated. Sixty butter (27 organic and 33 conventional) samples were used in this study. Bovine butter–fat were applied onto an attenuated total reflectance infrared (ATR‐IR) accessory equipped with a five‐bounce ZnSe crystal set at 65 °C for spectral collection. In addition, ATR‐IR spectra of bovine butter were directly collected at room temperature to avoid phase separation. The fatty acid profile and the levels of CLA were determined using reference FAME‐GC‐FID analysis. SIMCA models showed well separated clusters that discriminated between organic and conventional bovine butters due to C=C trans bending out of the plane vibration modes band at 967 cm^?1. Additionally, strong PLSR models were developed to predict CLA levels using butter–fat and bovine butter spectra with SEP of 0.05 % and RPD of 4.7, indicating that the models are suitable for quality control applications. Portable IR technology offers the ability for “in situ” analysis of butters that is much less time consuming than current analytical practices for authentication and quality control efforts by the industry.     

A reliable and sensitive time-resolved fluorescent immunochromatographic assay (TRFICA) for ochratoxin A in agro-products

Immunochromatographic assays (ICAs) are considered as a suitable diagnostic tool for the detection of mycotoxins. Mycotoxins and especially, ochratoxin A are analytes with more demanding sensitivity requirements. To enhance the sensitivity of current immunochromatographic assays for ochratoxin A (OTA), a novel sensitive ICA was developed in this study. In the assay, microspheres enclosing fluorescent europium (III) [Eu(III)] nanoparticles (EuNPs) were used as a label for OTA monoclonal antibody (OTA-mAb) conjugation. Accordingly, assay was called time-resolved fluorescent immunochromatographic assay (TRFICA). The test strip was composed of three parts: a sample pad, nitrocellulose membrane and an absorbent pad. As for detection, a proper concentration of conjugated microspheres was pipetted into the microtube and sample extract was added to it. Then the strip was inserted into the tube and the fluid flow along the strip. The TRFICA results were obtained in 8 min and read by a portable TRFICA strip reader. The established method allows quantitative determination of OTA with limit of detection as low as 1.0 μg kg⁻¹ in the samples. For validation, spiked samples including wheat, maize, soybean and rice were respectively assayed by TRFICA and a standard high performance liquid chromatography equipped with a fluorescence detector (HPLC-FLD), and good agreement of results was obtained between two methods.     

Thermal markers arising from changes in the protein component of milk

Classification of heat load applied to milk requires the detection of parameters appropriately related to the intensity of the heat treatment. Current analytical methods based on heat-induced changes in the protein component of milk have been directed either to determine the amount of protein-derived products arised from heat treatments or to evaluate the extent of thermal denaturation of milk proteins. Lately, a new analytical strategy has been developed according to the occurrence of three major whey proteins, namely bovine serum albumin (BSA), beta-lactoglobulin (βlg) and alfa-lactalbumin (αla), normally soluble at pH 4.6 in raw milk, in the pH 4.6 insoluble protein fraction recovered from heat-treated milk. The results have shown that pH 4.6 insoluble BSA, βlg and αla, as detected by ELISA in milk, can be regarded as thermal markers suited for either dairy process control or regulation purposes.     

Isolation of recombinant antibody fragments (scFv) by phage display technology for detection of almond allergens in food products

Tree nut allergies are considered an important health issue in developed countries. To comply with the regulations on food labeling, reliable allergen detection methods are required. In this work we isolated almond-specific recombinant antibody fragments (scFv) from a commercial phage display library bypassing the use of live animals, hence being consistent with the latest policies on animal welfare. To this end an iterative selection procedure employing the Tomlinson I phage display library and a crude almond protein extract was carried out. Two different almond-specific scFv (named PD1F6 and PD2C9) were isolated after two rounds of biopanning, and an indirect phage ELISA was implemented to detect the presence of almond protein in foodstuffs. The isolated scFvs demonstrated to be highly specific and allowed detection of 40 ng mL⁻¹ and 100 ng mL⁻¹ of raw and roasted almond protein, respectively. The practical detection limit of the assay in almond spiked food products was 0.1 mg g⁻¹ (110–120 ppm). The developed indirect phage ELISA was validated by analysis of 92 commercial food products, showing good correlation with the results obtained by a previously developed real-time PCR method for the detection of almond in foodstuffs. The selected phage clones can be affinity maturated to improve their sensitivity and genetically engineered to be employed in different assay formats.     

老年高血压患者脉压指数与尿微量白蛋白/尿肌酐、血清视黄醇结合蛋白的关系

目的 探讨老年原发性高血压（EH）患者脉压指数（PPI）与尿微量白蛋白/尿肌酐（尿mAlb/尿Cr）、血清视黄醇结合蛋白（RBP）的关系.方法 选取血肌酐（Cr）正常的老年EH患者50例（EH组,根据PPI分为PPI＆lt;0.5组及≥0.5组）及健康体检的老年人20例（对照组）.比较各组尿mAlb/尿Cr比值、血清RBP的变化及与PPI的相关性.结果 尿mAlb/尿Cr比值、血清RBP值比较：EH组高于对照组,PPI≥0.5组高于PPI＆lt;0.5组（均P＆lt;0.05）;尿mAlb/尿Cr比值联合血清RBP值阳性率比较：PPI≥0.5组高于PPI＆lt;0.5组（63.64%比28.57%,P＆lt;0.05）;PPI与尿mAlb/尿Cr比值、血清RBP值呈正相关（r=0.425,P=0.019;r=0.401,P=0.032）.结论 老年EH患者PPI与尿mAlb/尿Cr、血清RBP密切相关,联合检测可作为老年EH患者早期肾功能损害的一个评估指标.     

吡唑羧酸类金属配合物的合成、晶体结构及其与牛血清白蛋白的相互作用

以吡唑-3-甲酸、吡唑-4-甲酸为配体,采用溶液法分别与Cu Cl_2·2H_2O、Zn(NO_3)_2·6H_2O反应,合成了两种金属配合物并通过IR、X-射线单晶衍射仪对其晶体结构进行了表征。利用紫外-可见吸收光谱和荧光光谱研究了它们与牛血清白蛋白(BSA)的相互作用。实验结果表明,配合物与BSA发生强烈的结合,这些研究为人类研究药物分子与BSA相互作用的机理提供有价值的信息。