改良双抗体夹心酶联免疫法测定重组人活性蛋白C含量的研究

研究用ELISA法直接定量检测细胞培养液中的重组人活性蛋白C(rhAPC)。用改良双抗体夹心酶联免疫检测法进行定量分析。用棋盘滴定法对包被抗体、反应一抗和辣根过氧化物酶(HRP)标记二抗的适宜工作浓度进行了选择,并对实验条件进行优化,参考品标准曲线线性相关性较好。改良双抗体夹心酶联免疫检测法可用于直接定量分析细胞培养液中的rhAPC水平。     

宽泛底物谱色氨酸脱羧酶体外合成卤代色胺衍生物和血清素

【背景】色氨酸脱羧酶在自然界有高度特异性,行使催化色氨酸为色胺的功能。一个色氨酸脱羧酶BaTDC参与南海海绵共生菌Bacillus atrophaeus C89次级代谢产物bacillamides的生物合成过程。【目的】探究BaTDC酶学特征和底物谱,建立体外合成色胺衍生物的方法。【方法】通过构建系统发育树揭示BaTDC在进化中的地位。在温度梯度和pH梯度下进行酶反应,利用不同的色氨酸衍生物为底物,通过HPLC和UPLC-MS检测酶反应过程,表征BaTDC活性。【结果】系统发育分析显示BaTDC与肠道菌Ruminococcus gnavus亲缘关系相近。纯化重组BaTDC的最适温度为40?45 °C,最适pH值为8.0。BaTDC可以催化羟代色氨酸和卤代色氨酸包括4-氟色氨酸和5,6,7-氯色氨酸及4-溴色氨酸,得到相应的卤代色胺衍生物和血清素。【结论】本研究分析了BaTDC的特性,发现BaTDC表现出宽泛的底物耐受性,可为前体喂养或定向生物合成新型药用色胺衍生物和下游复杂天然产物奠定基础。     

3种检测吡咯喹啉醌的方法比较

目的:研究和比较3种检测吡咯喹啉醌(PQQ)的方法,确定各种方法的特点和适用范围。方法:设计和改进了3种检测PQQ的方法,分别为活性电泳法、光谱法和NBT-Gly法,探讨其检测限和线性范围、精密度、样品检测和加样回收率。结果:活性电泳法专一性好,具有很高的灵敏度,可检测到12.6 ng/mL PQQ,可靠但准确性较差;NBT-Gly法操作简便,可用于大量样品的检测,但重复性不佳;光谱法精密度较好,但样品中存在吸光物质时对检测结果影响较大。结论:活性电泳法、光谱法和NBT-Gly法均可用于PQQ的检测,活性电泳法适于培养上清等复杂样品的粗略定量,NBT-Gly法适于大量样品的检测,光谱法适于纯度较高的PQQ的定量。     

二恶英反应增强子调控的虫荧光素酶报告基因质粒的构建

为加强二恶英类化学物质的快速筛选和半定量检测,我们构建了一在二恶英反应增强子调控下的虫荧光素酶报告基因质粒。二恶英反应增强子来源于pHAV质粒,MMTV启动子来源于pCatM质粒,上述两者连接后与虫荧光素酶载体连接,转染人HepG2肝癌细胞,以2,3,7,8四氯代二苯并二恶英(TCDD)诱导报告基因表达后检测虫荧光素酶活性。结果表明该质粒中虫荧光素酶的表达受二恶英反应增强子的调控,且在一定浓度范围内虫荧光素酶的活性与TCDD的量呈线性关系。研究显示该质粒转染的细胞株有望用于快速筛选及半定量检测二恶英,可进一步加强研究作为二恶英类化学物质监测的常规方法。     

宽泛底物谱色氨酸脱羧酶体外合成卤代色胺衍生物和血清素（英文）

【背景】色氨酸脱羧酶在自然界有高度特异性,行使催化色氨酸为色胺的功能。一个色氨酸脱羧酶BaTDC参与南海海绵共生菌Bacillus atrophaeus C89次级代谢产物Bacillamides的生物合成过程。【目的】探究BaTDC酶学特征和底物谱,建立体外合成色胺衍生物的方法。【方法】通过构建系统发育树揭示BaTDC在进化中的地位。在温度梯度和pH梯度下进行酶反应,利用不同的色氨酸衍生物为底物,通过HPLC和UPLC-MS检测酶反应过程,表征BaTDC活性。【结果】系统发育分析显示BaTDC与肠道菌Ruminococcusgnavus亲缘关系相近。纯化重组BaTDC的最适温度为40-45°C,最适pH值为8.0。BaTDC可以催化羟代色氨酸和卤代色氨酸包括4-氟色氨酸和5,6,7-氯色氨酸及4-溴色氨酸,得到相应的卤代色胺衍生物和血清素。【结论】本研究分析了BaTDC的特性,发现BaTDC表现出宽泛的底物耐受性,可为前体喂养或定向生物合成新型药用色胺衍生物和下游复杂天然产物奠定基础。     

荧光内参在校正Caspase-3／7活性检测偏差中的应用

目的改进Caspase-3／7活性检测方法。方法在顺铂诱导的HeLa细胞凋亡模型中,分别利用传统的和改良的实验方法检测Caspase-3／7的活性变化,并与Western印迹实验结果进行方法学比较。结果改良的实验方法显示不同剂量顺铂诱导下,HeLa细胞Caspase-3／7活性有剂量依赖性增高,与Western印迹实验结果相一致,但传统实验方法显示HeLa细胞Caspase-3／7活性呈现先增高后降低的趋势。结论由于参测细胞数不同,导致这种Caspase-3／7活性检测方法不能真实反映细胞凋亡程度。本研究成功建立了一种新的改良型Caspase-3／7活性检测方式,这种检测方法可以排除不同凋亡诱导方式诱导细胞凋亡时所产生的因参测细胞数不同所造成的Caspase-3／7活性检测的误差。     

用ELISA定量测定破伤风毒素

<正>破伤风毒素的测定一般是按MLD(最小致死量)试验检定其毒力;或者按絮凝反应法用标准抗血清(ATS)检测其免疫反应性(Lf活性)。MLD测定法需要大量动物,经过很多时日,且受到实验动物的影响,测定依赖于毒素活性,而不是直接定量测定样品的毒素蛋白,尽管絮凝反应法操作简便,但它仅仅是毒素量的间接测定,是半定量的方法,不具备在酶免疫测定中可以获得的灵敏度。 本文介绍了破伤风毒素的夹心ELISA定量测定法,并与常规絮凝法进行了比较。     

加工产品中转基因玉米Bt11成分实时荧光PCR定量(性)检测

实验在玉米自身基因和外源基因的边界序列之间设计了具有品种和品系特异性的引物和探针 ,并以实时荧光PCR技术 ,建立了加工产品中转基因玉米Bt1 1成分品系鉴定检测和定量检测的方法。实验对加热条件和时间对检测转基因成分的影响作了探讨 ,并检测了部分市售食品和饲料。检测结果发现 ,加热时间温度越高、时间越长 ,对转基因成分定量检测的影响越大 ;在所检测的样品中可以检测出转基因玉米Bt1 1成分 ,有些样品还同时检出其他转基因成分。本研究实验建立的方法 ,可以用于加工产品中转基因成分的定量检测 ,也可以用于定性检测 ,或作为常规PCR定性检测后的确证实验方法。     

TF-1细胞增殖法测定神经生长因子生物学活性的建立与应用

目的：建立并优化用于检测神经生长因子（NGF）生物学活性的TF-1细胞增殖法,并应用于重组人NGF和鼠NGF制品的活性检测。方法：将梯度稀释的人NGF国际参考品与TF-1细胞相作用,通过MTT法测定细胞增殖情况,建立测定NGF活性的TF-1细胞增殖法;从粒细胞巨噬细胞集落刺激因子（CM-CSF）残留、NGF加样稀释率、TF-1细胞接种浓度、培养时间等多个环节对实验条件进行优化;将建立的TF-1细胞增殖法应用于重组人NGF和鼠NGF的活性检测。结果：建立了用于NGF活性检测的TF-1细胞增殖法;实验条件优化结果表明,在无GM-CSF残留的情况下,将稀释至1／4的NGF样品与4×10^5／mL的细胞相互作用,培养48h,可以得到更为典型的的四参数曲线;利用条件优化后建立的检测方法对重组入NGF和鼠NGF各2批样品分别进行4次测定,结果平均值分别为10．01×10^5、10．81×10^5和3．55×10^5、3．30×10^5U／mg,变异系数分别为7．5％、7．2％和7．2％、9．1％,检测结果稳定均一,表明检测方法具有很好的重复性。结论：建立并优化了用于NGF活性检测的TF-1细胞增殖法,该方法操作简便、定量准确、重复性好、稳定可靠,可有效应用于人NGF和鼠NGF制品的活性检测。     

齿肋赤藓中半定量RT-PCR方法的探讨

目的:建立一种半定量RT-PCR方法,用于齿肋赤藓(Syntrichia caninervis)基因表达研究。方法:比较常用的几种内参照基因actin、tubulin及18S rRNA在齿肋赤藓中的表达情况,以确定表达稳定的内参基因;分析PCR扩增动力学,确定内参和靶基因的PCR体系。结果:琼脂糖凝胶电泳检测分析表明:18S rRNA作为齿肋赤藓的内参基因更稳定;同管扩增试验表明:18SrRNA的引物滞后6个循环加入PCR扩增体系中,至第26个循环,二者同时处于指数扩增期,且靶基因RT-A的扩增效率不受影响。结论:18S rRNA与RT-A可以同管扩增,18S rRNA可以作为半定量RT-PCR的内参照,为齿肋赤藓基因表达研究奠定基础。     

Spoilage Bacteria in Canned Foods: II. Sulfide Spoilage Bacteria in Canned Mushrooms and a Versatile Medium for the Enumeration of Clostridium nigrificans

Clostridium nigrificans was found to be a spoilage organism of canned mushrooms in Taiwan. A modified beef extract tryptone iron medium, both in broth and agar form, was designed for the detection and recovery of the organisms. A procedure of simple plate counting method of C. nigrificans was established.     

A novel colorimetric assay of β‐D‐glucans in basidiomycete strains by alcian blue dye in a 96‐well microtiter plate

Basidiomycete strains synthesize several types of β‐d ‐glucans, which play a major role in the medicinal properties of mushrooms. Therefore, the specific quantification of these β‐d ‐glucans in mushroom strains is of great biochemical importance. Because published assay methods for these β‐d ‐glucans present some disadvantages, a novel colorimetric assay method for β‐d ‐glucan with alcian blue dye was developed. The complex formation was detected by following the decrease in absorbance in the range of 620 nm and by hypsochromic shift from 620 to 606 nm (～14 nm) in UV‐Vis spectrophotometer. Analysis of variance was used for optimization of the slope of the calibration curve by using the assay mixture containing 0.017% (w/v) alcian blue in 2% (v/v) acetic acid at pH 3.0. The high‐throughput colorimetric assay method on microtiter plates was used for quantification of β‐d ‐glucans in the range of 0–0.8 μg, with a slope of 44.15 × 10^?2 and a limit of detection of 0.017 μg/well. Recovery experiments were carried out by using a sample of Hericium erinaceus, which exhibited a recovery of 95.8% for β‐1,3‐d ‐glucan. The present assay method exhibited a 10‐fold higher sensitivity and a 59‐fold lower limit of detection compared with the published method with congo red. β‐d ‐glucans of several mushrooms strains were isolated from fruiting bodies and mycelia, and they were quantified by this assay method. This assay method is fast, specific, simple, and it can be used to quantify β‐d ‐glucans from other biological sources. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1526–1535, 2015     

Enzyme-linked immunosorbent assay for the determination of p21-activated kinase activity

An enzyme-linked immunosorbent assay (ELISA) for the measurement of p21-activated kinase (PAK) activity is described. The development of this method takes advantage of the fact that a phospho-epitope-specific antibody against the regulatory autophosphorylation site sequence of PAK was successfully produced, and after being phosphorylated by PAK, a cross-linked peptide containing the autophosphorylation site of PAK could be recognized on immunoblot by this antibody. This procedure involves coating the cross-linked peptide on microtiter plates, phosphorylating the cross-linked peptide by adding active PAK plus ATP.Mg(2+), and detecting peptide phosphorylation using the phospho-epitope-specific antibody and secondary antibody conjugated with alkaline phosphatase followed by reaction with p-nitrophenyl phosphate (for colorimetric detection) or fluorescein diphosphate (for fluorimetric detection). The PAK activity detected by this method was linearly proportional to the amount of kinase used in the reaction and to the duration of the kinase reaction. Furthermore, fluorimetric detection proved more sensitive than colorimetric detection in terms of both detection limit and signal magnitude. Kinase inhibitor assay revealed that the IC(50) value of staurosporine obtained by this ELISA was very close to that obtained in radioassay. Besides staurosporine, the inhibitory activity of several kinase inhibitors was also tested by the PAK ELISA. The results taken together demonstrate the feasibility and efficacy of this solid phase method for the measurement of PAK activity in a non-radioactive way. Development of this method can be helpful in further high-throughput screening of potential inhibitors of this kinase.     

A convenient microscale colorimetric method for terminal galactose on immunoglobulins.

A new approach for quantitative determination of terminal galactose (Gal) residues of immunoglobulins was developed by combining exoglycosidase digestion with the classical colorimetric estimation of reducing sugars. The ferricyanide colorimetric method was modified to increase the stability of the chromophore (Prussian blue) and adapted to determine the amount of terminal Gal residues present in immunoglobulins. The method involves the release of covalently bound Gal from immunoglobulins by Diplococcus pneumoniae beta-D-galactosidase (specific for beta(1,4) linked galactose), removal of the glycoprotein and enzyme from the reaction mixture by heat denaturation or ethanol precipitation, followed by colorimetric measurement of the released sugar using the ferricyanide assay. The ferricyanide method was modified to enhance the solubility and stability of the chromophore by increasing the concentration of aqueous sulfuric acid and sodium dodecyl sulfate (SDS). The linear range of the modified method was from approximately 11 to 111 microM Gal. Typical variation in assay results was on the order of 5%. Using the modified method, the terminal Gal content of a recombinant chimeric monoclonal antibody (anti-CD20, rIgG) expressed in Chinese hamster ovary (CHO) cells was determined and evaluated for batch-to-batch consistency. The method was used to optimize pH, time, temperature, and enzyme concentration for beta-galactosidase digestion for maximal release of terminal Gal residues from rIgG.     

基于纳米金的比色分析法在核酸检测中的应用

随着纳米技术的发展,运用纳米粒子检测核酸成为研究的热点.在众多检测方法中,基于纳米金的比色分析法操作较为简便,只需普通光学仪器甚至肉眼即可观察结果,从而表现出广阔的市场及临床应用前景.基于纳米金的比色分析法有多种,不同检测原理的方法在灵敏度和实用性上存在差异.根据纳米金是否经寡核苷酸探针修饰可将其分为基于功能化纳米金的比色分析法和基于未功能化纳米金的比色分析法,前者又分为利用纳米金颜色变化的聚集反应体系以及利用纳米金特殊氧化-还原能力的银染增强体系.     

Psychoactive tryptamines from basidiomycetes

The review lists natural sources,i.e. strains and species of fungi producing predominantly psychoactive tryptamines (indolealkylamines), their chemical structure and properties, toxic effects on the man and psychic symptoms of intoxication. It describes the biosynthesis and production of some tryptamines by the mycelial culture ofPsilocybe bohemica Šebek, a survey of methods for their analysis and isolation. It evaluates the worldwide use and abuse of psychoactive fungi as sources of drugs in general and in the Czechia in particular during the last two and a half decades. Dedicated to the memory of Dr.Marta Semerdžieva     

G-quadruplex DNAzyme-based Hg2+ and cysteine sensors utilizing Hg2+-mediated oligonucleotide switching

A simple and sensitive colorimetric Hg(2+) detection method is reported, based on the Hg(2+)-mediated structural switch of an unlabeled oligonucleotide strand. In the absence of Hg(2+), the oligonucleotide strand forms a stem-loop. A G-rich sequence in the strand is partially caged in the stem-loop structure and cannot fold into a G-quadruplex. In the presence of Hg(2+), T-Hg(2+)-T coordination chemistry leads to the formation of another stem-loop structure and the release of the G-rich sequence. The released sequence folds into a G-quadruplex, which binds hemin to form catalytically active G-quadruplex DNAzymes. This is detected as an absorbance increase in a H(2)O(2)-2,2'-azinobis(3-ethylbenzothiozoline)-6-sulfonic acid (ABTS) reaction system using UV-vis absorption spectroscopy. This simple colorimetric sensor can detect aqueous Hg(2+) at concentrations as low as 9.2 nM with high selectivity. Based on the strong binding interaction between Hg(2+) and the sulfur-containing amino acid cysteine (Cys), and the competition between Cys and a oligonucleotide for Hg(2+), the proposed Hg(2+)-sensing system can be further exploited as a Cys-sensing method. The method has a detection limit for Cys of 19 nM.     

Colorimetric assay method for determination of the tannin acyl hydrolase (EC 3.1.1.20) activity

A new colorimetric method of tannase (tannin acyl hydrolase, EC 3.1.1.20) assay has been developed using its specific substrate tannic acid. It is based on the changes in optical density of substrate tannic acid after enzymatic reaction at 530 nm. The residual tannic acid was measured by a modified BSA precipitation method. This assay is very simple, reproducible, and very convenient, and with it tannase activity can be measured in relation to the growth of the organism.     

Rapid and convenient determination of oxalic acid employing a novel oxalate biosensor based on oxalate oxidase and SIRE technology

A new method for rapid determination of oxalic acid was developed using oxalate oxidase and a biosensor based on SIRE (sensors based on injection of the recognition element) technology. The method was selective, simple, fast, and cheap compared with other present detection systems for oxalate. The total analysis time for each assay was 2-9 min. A linear range was observed between 0 and 5 mM when the reaction conditions were 30 degrees C and 60 s. The linear range and upper limit for concentration determination could be increased to 25 mM by shortening the reaction time. The lower limit of detection in standard solutions, 20 microM, could be achieved by means of modification of the reaction conditions, namely increasing the temperature and the reaction time. The biosensor method was compared with a conventional commercially available colorimetric method with respect to the determination of oxalic acid in urine samples. The urine oxalic acid concentrations determined with the biosensor method correlated well (R=0.952) with the colorimetric method.     

碳青霉烯耐药的肠杆菌科细菌诊断进展

碳青霉烯耐药的肠杆菌科细菌(Carbapenem-resistant Enterobacteriaceae,CRE)在全球快速上升,已出现了不同的基因型,为临床诊治提出新的挑战。CRE分为具有不同特点和耐药基因的三大类五大家族;诊断方法包括Kirby-Bauer法初筛试验,碳青霉烯类药物的协同试验(EDTA与美罗培南、苯硼酸与美罗培南的双纸片抑制法)、改良的Hodge试验、显色培养基检测等CRE筛选试验,而Carba NP比色微管试验、PCR及测序等为CRE确认试验。上述方法均各有其优缺点,可根据当地的主要流行CRE型别和实验条件选择应用。