Development of monoclonal antibodies and a competitive ELISA detection method for glycinin,an allergen in soybean

Soybean glycinin is a major food allergen causing anaphylaxis. A sensitive detection method for glycinin is needed to evaluate soybean allergies in food and feed products. In the present study, monoclonal antibodies (Mabs) against glycinin were prepared using purified glycinin as the immunogen. The generated Mabs, named 3B2 and 4B2, were identified as being IgG_2b and IgG_2a iso-types respectively, and exhibited high specificity to glycinin. Then we developed a competitive ELISA based on Mab 4B2 to measure glycinin which showed an IC₅₀ value of 1.7 ng/mL with a detection limit of 0.3 ng/mL, and the linear portion of the curve was 0.3–11.2 ng/mL. Recovery tests indicated that the competitive ELISA based on Mab 4B2 gave reliable reproducibility. The produced Mab 4B2 and the developed ELISA could provide a valuable tool for sensitive determination of glycinin and for future studies conducted to reveal the mechanism of how glycinin functions in anaphylaxis.     

废弃聚酯醇解液的回收与循环利用

为实现聚酯资源的可再生循环应用,采用乙二醇醇解法对废弃聚酯(PET)面料进行降解,得到产物对苯二甲酸乙二醇酯(BHET)。为了达到乙二醇(EG)的最大化利用率,在产物重结晶前后减压蒸馏回收醇解液,并用其代替EG再次用于废弃聚酯的降解。借助红外光谱、差示扫描量热法和热重分析法对醇解产物和醇解液进行测定。结果表明:重结晶前回收的醇解液可循环使用4~5次,EG回收率在57.1%~89.2%之间,BHET产率在58.9%~70.3%之间,醇解液使用的循环上限主要取决于醇解液中EG自聚程度;重结晶后回收的醇解液可循环使用1~2次,达到使用上限后,EG回收率由91.3%降至23.7%,BHET产率由71.2%降至50.2%。     

A robust capillary electrophoresis method for the determination of organic acids in wines

A robust capillary electrophoresis (CE) method for the determination of the six main organic acids in wines is described. The CE method is based on the use of 2,6- pyridinedicarboxylic acid (PDC) as background electrolyte (BGE) for the indirect UV detection of tartaric, malic, citric, succinic, acetic and lactic acids. Ethylenediaminetetracarboxylic acid (EDTA) was added to the BGE to eliminate interferences from trace metals on the determination of citric acid. Problems related to linearity reported in the literature using the same BGE were shown to be the result of the sample pH being lower than that of the buffer. Although this could be overcome simply by adjusting the pH of the sample, electrokinetic injection was found to be a better alternative, improving at the same time the sensitivity of the determination. Linearity over the range 100–5,000 mg/l (ppm) for all acids was achieved, requiring only 80× dilution of standards and samples prior to injection. Within day repeatabilitys (n=7) of migration times (RSD%<0.15) and peak areas (RSD%<5) as determined for a wine sample were found to be excellent. The method was applied to 20 South African wine samples, and the results are compared to data obtained by ion-exclusion liquid chromatography. Succinic acid was consistently overestimated using the latter technique, while for the other acids good agreement between the methods was noted.     

Identification of tetrodotoxin antigens and a monoclonal antibody

Two kinds of antigens for tetrodotoxin (TTX) were made, using carrier protein ovalbumin (OVA) and bovine serum albumin (BSA), by the formaldehyde method. Nondenaturing gel electrophoresis, gel filtration and ultraviolet spectrophotometry were employed to analyze the coupling of TTX to carrier proteins. The results indicate that nondenaturing gel electrophoresis and gel filtration can be employed to analyze the coupling of TTX to BSA and OVA qualitatively, and ultraviolet spectrophotometry can be employed to analyze the molecular coupling ratio of TTX to BSA and OVA quantitatively. Spleen cells from Balb/C mice immunized with an artificial TTX-formaldehyde-ovalbumin conjugate were fused with SP2/0 myeloma cells. A hybridoma cell line (4D5), which secreted IgG₁ subclass monoclonal antibody against TTX, was produced by “limiting dilution” cloning. The molecular weight, the affinity constant and the titre of the monoclonal antibody (mAb) secreted by 4D5 were 183.69 kDa, 0.98 × 10⁸ M⁻¹ and 3.6 × 10⁵, respectively. The number of the hybridoma chromosome was 88–104.     

应用抗黄曲霉毒素单链抗体检测酱油中黄曲霉毒素B1

利用本实验室制备的抗黄曲霉毒素B1的单链抗体(ScFv),通过棋盘实验确定了抗原抗体的最适工作浓度,在此之上根据间接竞争酶联免疫法(ELISA)绘制标准曲线,检测酱油中AFB1的含量;通过改变样品的盐浓度及pH来确定其对ELISA检测结果的影响。研究结果表明,利用ScFv检测黄曲霉毒素的最小检测值为0.10ng/mL,平均加标回收率在84%～109%之间,本文建立的ELISA方法在pH5～8,盐浓度小于10%时较稳定。本文建立的利用抗AFB1的ScFv检测黄曲霉毒素含量的方法方便快捷,稳定性较好,并且成本较低,适合于食品中黄曲霉毒素的检测。     

Comparison of contaminant and residue levels in organic and conventional milk and meat products from Northern Italy

Due to the growing interest in organic products, a comparison between the chemical safety of organic and conventional products was undertaken. Milk and meat were the products chosen for study. The parameters evaluated to assess chemical safety were organochlorine pesticides, polychlorinated biphenyls (PCBs), lead, cadmium and mycotoxin contamination. Pesticides and PCBs residues in both organic and conventional milk and meat were lower than legal limits. Lead and cadmium residues were very low and did not differ between organic and conventional products. However, aflatoxin M₁ contamination in some but not all samples of organic milk was significantly higher than those of conventional milk, although factors other than organic production might be implicated.     

Methoxy poly(ethylene glycol)-grafted-chitosan based microcapsules: Synthesis, characterization and properties as a potential hydrophilic wall material for stabilization and controlled release of algal oil

Methoxy poly(ethylene glycol)-grafted-chitosan (mPEG-g-CS) was synthesized in an attempt to increase the water solubility of CS, and it was applied as wall material to microcapsulate algal oil (AO). Both Fourier transformed-infrared (FT-IR) spectra and solubility test confirmed that mPEG had been successfully grafted onto CS. The larger particles showed smooth surfaces, whereas smaller ones presented surface folds. The amount of free and total oil were 2.73 and 47.87 g/100 g, respectively, with high encapsulation efficiency of 94.30%. Fourteen fatty acids were detected in free algal oil (FAO) and microcapsulated algal oil (MAO), and the important fatty acids were preserved after microcapsulation. AO contained within microcapsules was protected from UV-light and heat compared with FAO. Release rate of AO was initially rapid and then followed by controlled release with the erosion mechanism playing a dominant role. The mPEG-g-CS proved to be a promising wall material with excellent solubility and film forming ability, and therefore supplied great potential applications in food industry.     

Insulinoma cell line (MIN6) adhesion and spreading mediated by Arg-Gly-Asp (RGD) sequence conjugated in thermo-reversible gel

We have functionalized gels with a putative cell-binding (-Arg-Gly-Asp-) (RGD) domain in an effort to regulate mammalian cell behavior in cells entrapped with gel. Adhesion molecules composed of Gly-Arg-Gly-Asp-Ser (GRGDS) peptides and cell recognition ligands were inculcated into thermo-reversible hydrogel composed of N-isopropylacrylamide, with a small amount of succinyl poly(ethylene glycol) (PEG) acrylate (MW 2000) used as a biomimetic extracellular matrix (ECM). The GRGDS-containing p(NiPAAm-co-PEG) copolymer gel was studied in vitro for its ability to promote cell spreading and to increase the viability of cells by introducing PEG spacers. Hydrogel lacking the adhesion molecules proved to be a poor ECM for adhesion, permitting only a 20% spread of the seeded cells after 10 d. When PEG spacer arms, immobilized by a peptide linkage, had been integrated into the hydrogel, conjugation of RGD promoted cell spread by 300% in a 28-d trial. In addition, in a serum-free medium, only GRGDS peptides conjugated with the spacer arm were able to promote cell spread.     

NiO-/cotton- modified nanocomposite as a medication model for bacterial-related burn infections

Burn injuries are one of the major global health problems. Despite aggressive local and systemic treatment to minimize infection, severe burn wounds continue to become infected with environmental and nosocomial pathogens at relatively high rates. We developed an ultrasound assisted sol–gel technique to prepare treated cotton fibers by coating fabric surface with NiO/organic polymer/enzyme matrix for in vitro evaluation of antibacterial activity. The surface modification was performed to bond more efficiently NiO nanoparticles using cellulase enzyme, to reduce initial bacterial attachment on the hydrophilic surface using poly(ethylene glycol), and to promote ultrasonic generation and to overcome agglomeration of the nanoparticles, respectively. Disk diffusion method was used to quantify the efficacy of NiO-based cottons against Pseudomonas aeruginosa and Staphylococcus aureus as two most burn wound pathogens isolated from burn wound swabs and tissue biopsies of hospitalized burn patients. Almost bacterial isolates showed high resistance to the commonly used antibiotics for burn infection treatment. In vitro antibacterial evaluation showed that the modified cottons exhibited excellent biocidal action against both high-resistant isolated Gram-positive and Gram-negative bacteria.     

高有机溶剂耐受性的苯并(a)芘单克隆抗体制备及其免疫反应特性研究

目的 制备适应油脂样品中苯并(a)芘[Benzo(a)pyrene,BaP]检测需求的抗BaP单克隆抗体。方法 利用杂交瘤单克隆抗体技术制备抗BaP的抗体,在抗体筛选过程中使用高浓度的有机溶剂(50%甲醇)来选择具有较高有机溶剂耐受能力的抗体,并利用间接竞争酶联免疫吸附法考察了抗体的免疫反应特性。结果 筛选获得一株对有机溶剂有较高耐受能力的、稳定分泌BaP单克隆抗体的细胞株2H10,该抗体为IgG1亚型,可耐受高浓度的甲醇、丙酮、二甲亚砜(Dimethyl sulfoxide,DMSO)和二甲基甲酰胺(Dimethylformamide,DMF)。60%甲醇和60% DMSO作为样品稀释液时,抗体对BaP的IC₅₀分别为81.4和24.5 ng/mL,以60% DMSO作为样品稀释液,可以显著降低BaP抗体对其它多环芳烃化合物的交叉反应率。结论 本研究获得高特异性的BaP人工抗原及高有机溶剂耐受性的抗BaP单克隆抗体。     

Decaffeination of tea extracts by using poly(acrylamide-co-ethylene glycol dimethylacrylate) as adsorbent

Poly(acrylamide-co-ethylene glycol dimethacrylate) was synthesized by thermal polymerization. The adsorption properties of the polymers for catechins and caffeine were studied. Adsorption kinetics showed the polymers preferred to adsorb the catechins rather than caffeine, and adsorption of catechins and caffeine onto the polymers could be described by the pseudo-second-order model. Isothermal equilibrium curves of catechins and caffeine showed a good fit with Langmuir model. The maximum monolayer adsorption capacity (Q_m) of total catechins (192.85–171.11 mg g⁻¹) was 3.9–5.2-folds of caffeine. Adsorption of catechins and caffeine was spontaneous and exothermic process as the change in free energy and enthalpy were negative. Lower temperature was favorable for the adsorption process. Caffeine and catechins were well separated and recovered by eluting polymers-packed column with gradient ethanol, i.e., 30% ethanol elution for separating caffeine, and 80% ethanol elution for recovering catechins. Thus, the synthetic poly(acrylamide-co-ethylene glycol dimethacrylate) could be used as a new adsorbent candidate for producing the decaffeined catechins.     

Preparation and characterization of electrically semi-conductive polyfuran-coated poly(ethylene terephthalate) fibers

One of the most important textile materials, poly(ethylene terephthalate) (PET) fiber, was coated with a semi-conductive polyfuran (PFu) by in situ oxidative polymerization using FeCl₃ oxidant in solvent mixture of acetonitrile–chloroform. The effects of polymerization conditions such as volume ratios of acetonitrile/chloroform, monomer concentration, and oxidant/monomer mol ratio were investigated on PFu content (%) of the composites. It was observed that pretreatment of PET in dichloromethane increased PFu content and its coating continuity before polymerization. The highest PFu content (12.0%) was obtained using FeCl₃/furan mol ratios of 3.5 in acetonitrile/chloroform mixture (5/1). The density values of the composites with different PFu contents were measured. Composite fibers were also subjected to doping processes with HCl and I₂ vapors, separately, and it was observed that the surface resistivity of PFu/PET (10¹² Ω/cm²) reached to 53 Ω/cm² after doping with I₂. The structural, thermal, and morphological characterization was performed with FTIR, XRD, TGA, and SEM, respectively.     

Development of monoclonal antibody based sandwich ELISA for the rapid detection of pathogenic Vibrio parahaemolyticus in seafood

Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are considered important virulence factors of Vibrio parahaemolyticus and strains producing either of these or both are considered pathogenic. In this study, we generated monoclonal antibodies (mAbs) against purified TRH recombinant protein of pathogenic V. parahaemolyticus. Sandwich enzyme-linked immunosorbent assays (ELISA) using the hybridoma clone 4B10 showed higher sensitivity of detection compared to other clones. Using mAb 4B10 based sandwich ELISA, we could detect pathogenic V. parahaemolyticus in 41.18% (14 out of 34) of the seafood samples analyzed. PCR targeting the toxR gene showed the presence of V. parahaemolyticus in 64.7% (22 out of 34) seafood samples. Further, PCR targeting the virulence genes showed that 6 seafood samples harboured the tdh gene while 9 harboured the trh gene indicating the presence of pathogenic V. parahaemolyticus. Our results show that mAb 4B10 sandwich ELISA developed in this study could be used as a rapid method for screening seafood samples for the presence of pathogenic V. parahaemolyticus.     

A non-destructive method to monitor changes in a troponin T peptide in beef drip with a monoclonal antibody

To simplify the monitoring of postmortem beef aging, we established a system to detect a troponin T (TnT) peptide fragment in bovine muscle drip (natural exudates) with an original monoclonal antibody. The antibody was raised against a synthetic peptide APPPPAEVPEVHEEVH corresponding to the N-terminal region of bovine fast-type TnT. In a competitive enzyme-linked immunosorbent assay (ELISA), our antibody detected the standard peptide dose-dependently. According to the monitoring examination with a competitive ELISA during 22 days postmortem, the concentration of the peptide in both the drip and trichloroacetic acid extracts from the longissimus muscle (n = 4) significantly increased in parallel, up to 10 nmol/ml and 16.4 nmol/g at day 14 postmortem, respectively. These events were accompanied by an increase in the conventional 30 kDa fragment in western blot analysis and a decrease in the Warner–Bratzler shear force value of the beef from 5.0 to 2.4 N/cm². The peptide detection system using drips with the antibody has advantages applicable to a non-destructive, simple, quick, and on-site monitoring method, such as immunochromatography.     

Microstructure and model solute transport properties of transglutaminase‐induced soya protein gels: effect of enzyme dosage,protein composition and solute size

The diffusion of poly(ethylene glycol) (PEG) in transglutaminase (TGase) cross‐linked soya protein gels as affected by enzyme dosage and protein composition was investigated. PEG with different molecular weights (M_w) varying from 6000 to 400 000 g mol^?1 was selected. Results of this study revealed that the time‐related release of PEG from gel slices was found to be controlled by Fickian diffusion process. For PEG with M_w 6000 g mol^?1, the diffusion coefficients (D) were independent on the enzyme dosage and protein composition. However, for PEG with M_w ≥ 20 000 g mol^?1, there were strong dependences of diffusion on the solute size and on the gel network microstructure revealed by scanning electron microscopy (SEM), where more density gel network with smaller pores resulted in lower D of PEG. Lower D values of PEG with M_w ≥ 20 000 g mol^?1 were obtained in gels induced by 2 U g^?1 TGase, and the D values increased with increasing 11S ratio in gels. Moreover, data also showed that the diffusion of PEG with larger size was more sensitive to the variation of gel network.     

Distribution of POPs,pesticides and antibiotic residues in organic honeys from different production areas

Demand for honey is increasing, especially if it is organic and if its nutritional properties are linked to untreated environments in order to guarantee quality for health. Sources of contamination of honey can be divided into environmental and apicultural. Therefore, the distribution of persistent organic pollutants, pesticides and antibiotic residues from geographical areas with different contamination sources (high anthropic impact, intensive farming, husbandry and low anthropic impact) was investigated in order to confirm the potential transfer of xenobiotics into the supply chain and to give beekeepers tools for the selection of areas dedicated to organic production. The presence of polychlorinated biphenyls, polybrominated diphenyl ether and polycyclic aromatic hydrocarbons was confirmed, not only in proximity to highly urbanised centres, where the concentrations were higher, but in all environment contexts, confirming their ubiquity. No antibiotics or neonicotinoids were detected in 95 organic honeys, demonstrating the absence of apicultural treatments and consequently the good quality of honey of different areas. These results are important due to the undefined regulatory European situation on honey antibiotic limits.     

Understanding consumer resistance to the consumption of organic food. A study of ethical consumption,purchasing, and choice behaviour

Although the demand for organic food is growing globally, the mainstream consumption of organic food is far less. The present study attempts to understand the underlying reasons for consumer resistance toward consuming organic food using the theoretical framework of innovation resistance theory (IRT). The study further examines the association between different consumer barriers and purchase decisions (purchase intentions, ethical consumption intentions, and choice behaviour) at different levels of buying involvement and environmental concerns. The collected data, consisting of 452 consumers, were analyzed by structural equation modeling approach. The results showed that value barrier shared a negative association with purchase intentions and ethical consumption intentions. Ethical consumption and purchase intention were found to have a direct influence on choice behaviour. Additionally, the relationship between ethical consumption intention and choice behaviour is mediated by purchase intention. However, no significant differences have emerged based on the level of buying involvement and environment concerns. The findings of the study provide insight into public policymakers, marketers, suppliers, and consumer associations by enhancing their current understanding of buying behaviour of the growing organic food community.     

Determination of caffeine content in tea based on poly(safranine T) electroactive film modified electrode

Safranine T was electropolymerised on a glassy carbon electrode and then characterised by scanning electron microscope (SEM), X-ray diffraction (XRD) and electrochemical impedance spectroscopy (EIS). This uniform electropolymerised film was crystallisable and showed a high electrocatalytic ability towards the oxidation of caffeine. To avoid the interferences of the anions, Nafion was covered on the surface of poly(safranine T) film modified glassy carbon electrode. As a new voltammetric sensor, this modified electrode is sensitive, selective and stable to determine caffeine content in tea. The peak current increased linearly with the concentration of caffeine in the range of 3 × 10⁻⁷–1 × 10⁻⁴ M, with a detection limit of 1 × 10⁻⁷ M. All of these make it a useful tool for determining caffeine content in tea. What’s more, it produces much less organic waste compared with other analytical techniques.     

非水相酶促酯交换法合成抗坏血酸脂肪酸酯

研究了一种L-抗坏血酸脂肪酸酯（AFAE）合成新途径。实验结果表明，氢化棕榈油、大豆油和海狗油等食用油脂作为脂肪酸基团供体，在固定化脂肪酶的催化下，在有机溶荆中与L-抗坏血酸直接进行酯交换反应形成L-抗坏血酸脂肪酸酯。对反应介质体系的筛选发现，在叔戊醇中用Novozym435催化此反应所得的产物浓度最高。对影响合成抗坏血酸脂肪酸酯的因素进行了探讨，确定了最适反应条件：初始油脂底物浓度为200-600mmol／L，温度为55℃，反应时间为9h。此条件下产物质量浓度可达43．51g／L。     

Recent Trends in Hydrogels Based on Starchgraft‐Acrylic Acid: A Review

This paper presents a reviewal profile of water absorbing resins based on graft copolymers of acrylic acid and gelatinised starch. It first elaborates on the synthesis of these hydrogels, use of these hydrogels in a variety of commercial applications, and the role of hydrogen bonding in the water absorbing capacity of these hydrogels. It describes the exact experimental conditions for producing hydrogels based on gelatinised maize starch grafted with acrylic acid showing maximum water absorption of 260 g/g.