血清滤纸斑点免疫结合试验诊断棘球蚴病

以定性滤纸吸附血清样品,保存于室温和4℃冰箱内,3和6个月后对所保存的血清滤纸进行生物素-抗生物素系统斑点免疫结合试验,以检测血清样品中抗棘球蚴抗原的抗体.结果表明2种条件下保存的干血清滤纸的检测结果与新鲜血清样品检测结果基本一致(P＞0.05).证明在生物素-抗生物素系统斑点免疫结合试验检测棘球蚴病患者免疫应答时,干血清滤纸法可作为患者血清样品的保存方法.     

Inhibition binding studies of glycodendrimer/lectin interactions using surface plasmon resonance

Understanding protein/carbohydrate interactions is essential for elucidating biological pathways and cellular mechanisms but is often difficult due to the prevalence of multivalent interactions. Here, we evaluate the multivalent glycodendrimer framework as a means to describe the inhibition potency of multivalent mannose-functionalized dendrimers using surface plasmon resonance (SPR). Using highly robust, mannose-functionalized dithiol self-assembled monolayers on gold surfaces, we found that glycodendrimers were efficient inhibitors of protein/carbohydrate interactions. IC₅₀ values ranging from 260 nM to 13 nM were obtained for mannose-functionalized dendrimers with Concanavalin A.     

八肋游仆虫eRF1a的C端是肽链释放因子eRF3的结合区(英文)

真核生物蛋白质合成终止需要两类肽链释放因子,eRF1和eRF3.研究表明八肋游仆虫的两类肽链释放因子在体内和体外都能形成复合物,且第一类肽链释放因子eRF1a与第二类肽链释放因子eRF3的C端结合.为了确定eRF3在eRF1a上的结合区,本研究以八肋游仆虫第一类肽链释放因子eRF1a基因为模板,用PCR的方法获得了N端分别截短140和206个氨基酸的eRF1a片段,同时在这两个片段的3'端融合了编码6个组氨酸残基的核苷酸序列,将这两个序列分别插入原核表达载体pTWIN1,并构建重组表达质粒pTWIN1-eRF1aC1 his6和pTWIN1-eRF1 aC2his6,转入大肠杆菌BL21( DE3)中获得了可溶性表达,通过一步His60 Ni Superflow柱亲和层析,重组蛋白CBD-intein-eRF1 aC1 his6和CBD-intein-eRF1aC2 his6获得纯化.体外pull down分析显示eRF1aC1和eRF1aC2均能与八肋游仆虫第二类释放因子eRF3相互作用,这表明八肋游仆虫eRF1a的C端是肽链释放因子eRF3的结合区.     

CHO细胞微核的激光扫描细胞仪自动化检测方法

 运用激光扫描细胞仪,采用FITC和PI两种荧光染料,结合细胞胞质分裂阻断微核技术,对环磷酰胺(CP)诱导的中国仓鼠卵巢(CHO)细胞微核进行了检测,并将该方法与传统微核显微镜检法进行了比较,旨在探索CHO细胞体外微核的激光扫描细胞仪自动化检测方法.结果表明,激光扫描细胞仪方法能够准确识别胞质阻滞的双核细胞及双核细胞中的微核,环磷酰胺(CP)处理浓度与其诱导的双核细胞微核率成正相关,激光扫描细胞仪法与传统显微镜检法获得的微核率具有良好的相关性.激光扫描细胞仪检测方法能简便、经济、快速、高效地检测CHO细胞微核,适于体外培养细胞尤其是贴壁细胞微核的高通量检测.     

A G‐quadruplex/hemin DNAzyme‐based microchip electrophoresis chemiluminescence assay for highly sensitive detection of biotin in flour

Quantitative analysis of biotin in biological fluids, foods, and pharmaceutical is important for diagnosis and treatment of biotin‐related diseases and health maintenance. In this work, a novel G‐quadruplex/hemin DNAzyme‐based microchip electrophoresis chemiluminescence (CL) assay method was established for rapid and highly sensitive detection of biotin. This method is based on the specific binding between biotin and streptavidin, the catalytic CL characteristics of G‐quadruplex/hemin DNAzyme to the oxidation–reduction reaction of hydrogen peroxide with luminol, and the on‐line separation function of microchip electrophoresis. Under the optimal experimental conditions, on‐chip biotin analysis was achieved within 1 min. The CL intensity is linearly proportional to the concentration of biotin in the range of 13–630 nM with a detection limit of 6.4 nM. The proposed method has been applied for the detection of biotin in flour, biotin contents in three flour samples are found in the range of 199–223 ng/g with a mean value of 214 ng/g. The recoveries were in the range of 94–103%. With excellent sensitivity and good selectivity, the proposed method could be applied in a wide range of biological fluids, foods, and pharmaceutical analysis.     

Capillary electrophoresis‐based enzyme assays for β‐lactamase enzymes

Generic in‐capillary as well as offline CE‐based enzyme assays were developed for serine‐β‐lactamases and metallo‐β‐lactamases. The hydrolysis of benzylpenicillin to benzylpenicilloic acid was analyzed using 100 mM sodium phosphate solution, pH 6.0, as a background electrolyte. In‐capillary assays employed an uncoated as well as a polyethylene oxide‐coated capillary, while the offline assays employing long end and short end injection were performed in an uncoated capillary. Using procaine hydrochloride or 4‐hydroxybenzoic acid as internal standard, the respective assays were validated with regard to linearity, LOD and LOQ, repeatability, precision, and accuracy. The assays were applied to the determination of the Michaelis‐Menten parameters K_m and V_max of Bacillus cereus penicillinase as well as New Delhi metallo‐β‐lactamase 1 and Verona integrin‐encoded metallo‐β‐lactamase 2. Furthermore, the inhibition of the enzymes by irreversible and competitive inhibitors was evaluated. Comparable data were obtained with all assays. The use of a simple substrate ensured broad applicability to the various types of β‐lactamases.     

基于碳点-铕的比率荧光探针可视化检测四环素

结合四环素对碳点(CDs)的荧光猝灭及对铕离子(Eu ³⁺)的荧光增强作用, 建立了一种肉眼可辨的比率荧光分析方法用于水样中四环素残留的便捷检测. 通过优化CDs/Eu ³⁺比例、 溶液pH值等实验条件, 使检测体系对四环素的荧光响应呈现由蓝色→粉色→红色的颜色转变过程, 易于通过肉眼进行分辨和半定量分析. 实验结果表明, 2种荧光探针响应比(I₆₁₈/I₄₄₀)的对数值与四环素的浓度在20~100 nmol/L范围内呈线性关系, 方法的检出限为1 nmol/L. 将该方法应用于江水样品中四环素的加标检测, 获得了较好结果.     

Production of thermostable multiple enzymes from Bacillus amyloliquefaciens KUB29

A strain of Bacillus amyloliquefaciens KUB29 was identified by 16S ribosomal RNA sequencing (Genbank: MF772779.1). Production of thermostable protease, amylase and lipase were done by the isolated strain. The produced enzymes were partially purified by ammonium precipitation followed by dialysis process. Protease and lipase enzymes are effectively used in bio-oil extraction from proteinaceous sample followed by transesterification to produce methyl ester. Amylase enzyme is widely used in food and laundry industry. The produced enzymes are active at thermophilic condition of 55 °C. Use of these enzymes in biofuel production process will make the process cleaner and greener.     

Triple Amplification Strategy for the Improved Efficiency of a Microplate-Based Assay for the Chemiluminescent Detection of DNA

Significant research efforts are currently focused on advancing DNA detection methodology. Various nanoparticles (NPs), which are currently the most widely used solid-phase carriers for nucleic acid assays, have a number of essential drawbacks. Microtiter plates provide a simple and economical alternative to the NPs. This article reports the development of a sandwich assay for DNA detection using a microtiter plate as the solid carrier. A capture oligonucleotide modified with fluorescein was bound to the anti-fluorescein antibody adsorbed on the polystyrene microplate surface. A hepatitis B virus DNA fragment was used as the model analyte. To improve the assay sensitivity, the biotinylated reporter oligonucleotide and streptavidin-horseradish polyperoxidase (polyHRP) conjugate were used to provide an amplified detection system. Additional amplification was achieved because the peroxidase activity was measured by a chemiluminescent method using the 3-(10′-phenothiazinyl)propane-1-sulfonate/N-morpholinopyridine pair as an enhancing reagent. The detection limit of the developed assay was 0.9?pM with a linear dynamic range from 0.9 to 100?pM. This method may be used as a platform for the development of sensitive DNA assays.     

Damages to the extracellular matrix in articular cartilage due to cryopreservation by microscopic magnetic resonance imaging and biochemistry

To investigate the damages to the extracellular matrix in articular cartilage due to cryopreservation, the depth-dependent concentration profiles of glycosaminoglycans (GAGs) in 34 cartilage specimens from canine humeral heads were imaged at 13-μm pixel resolution using the in vitro version of the dGEMRIC protocol in microscopic MRI (μMRI). In addition, a biochemical assay was used to determine the GAG loss from the tissue to the solution where the tissue was immersed. For specimens that had been frozen at −20°C or −80°C without any cryoprotectant, a significant loss of GAG (as high as 56.5%) was found in cartilage, dependent upon the structural zones of the tissue and the conditions of cryopreservation. The cryoprotective abilities of dimethyl sulfoxide (DMSO) as a function of its concentration in saline and storage temperature were also investigated. A 30% DMSO concentration was sufficient in preventing the reduction of GAG in the tissue at the −20°C storage temperature, but a 50% concentration of DMSO was necessary for the −80°C cryopreservation. These imaging results were verified by the biochemical analysis.