青霉素结合蛋白2a单克隆抗体的研制和初步应用

目的研制青霉素结合蛋白2a(PBP2a)单克隆抗体(Mc Ab),为建立耐甲氧西林金黄色葡萄球菌(MRSA)免疫层析检测方法提供检测用抗体。方法以基因工程抗原r PBP2a免疫BALb/c小鼠,通过常规小鼠B淋巴细胞杂交瘤技术制备单克隆抗体,采用免疫印迹技术(Western blotting)分析单克隆抗体特异性。选取敏感、特异的单克隆抗体进行金标记和硝酸纤维膜包被,建立胶体金免疫层析检测方法。结果共获得11株分泌抗r PBP2a的杂交瘤细胞,其中6株分泌的单克隆抗体能够与天然PBP2a呈阳性反应。用其中2株单克隆抗体建立的PBP2a胶体金免疫层析方法,可在5~20 min内完成检测。结论获得了特异性针对PBP2a蛋白的单克隆抗体,并初步建立了检测PBP2a蛋白的胶体金免疫层析检测方法,为临床快速、简便检测产生PBP2a的细菌提供了检测方法。     

消减免疫法制备克伦特罗单克隆抗体

目的：制备克伦特罗（CL）单克隆抗体。方法：重氮化法制备克伦特罗完全抗原,消减免疫法免疫BALB／c小鼠,常规杂交瘤技术制备抗克伦特罗单克隆抗体。结果：消减免疫法使小鼠获得了对BSA的耐受,单抗筛选过程中获得了高达8．2％的阳性率。最后得到了针对CL的特异性抗体。结论：用消减免疫法制备针对小分子污染物的单克隆抗体,可减少在制备单克隆抗体时筛选的工作量,可增加获得预期抗体的机会。     

抗K物质单克隆抗体的制备

速激肽族肽,包括K物质、P物质、神经素B等都具有神经递质的功能,它们越来越引起人们的关注。免疫学方法诸如:放射免疫测定法、免疫组织化学法等对神经肽的分布、定位和生物学功能等方面的研究提供了极大的方便。我们以K物质-甲状腺球蛋白偶联物免疫动物——Wistar大鼠和Balb/c小鼠。Wistar大鼠对K物质-甲状腺球蛋白的免疫应答能力显著高于Balb/c小鼠。通过Wistar大鼠脾细胞与小鼠骨髓瘤细胞进行异种细胞融合,获得了一株稳定地分泌抗K物质的单克隆抗体的细胞系(SE-1)。用RIA检测该单克隆抗体的特异性,除与P物质有一些交叉外,与其余我们所用的脑肽均不反应。     

开发测定空中花粉的单克隆抗体

兴和和山形研究所卫生研究所开发用单克隆抗体测定室中浮游的杉花粉和丝柏花粉的方法,在10月18日京都召开的日本过敏症学会和国际过敏症学会上发表。花粉症近年来正迅速蔓延。现有方法还停留在用肉眼和显微镜观测在涂抹凡士林的载片上收集到的花粉。因此,必须很有经验,而且由于雨天花粉变形、切断的花粉等原因而难以估测。人们可采用单克隆抗体花粉测定法,测定过敏源,有可能更准确的预报花粉症。该公司用从杉花粉精制的主要过敏源、Cryj Ⅰ免疫小鼠,生产单克隆抗体。已获得只与杉花粉反应的     

单克隆抗体在内分泌学中的应用

把单克隆抗体的技术应用于内分泌学研究仅仅开始,但已显示出巨大的潜力。单克隆抗体可提高激素免疫测定法的敏感性、特异性及标准化的程度,并可降低测定的成本。它可提纯某些激素,或使提纯步骤简化。利用标记的单克隆抗体可能对内分泌细胞膜上或者胞质中的成份进行动态的研究。把单克隆抗体作为分子功能解剖刀,可研究激素或其受体的结构和功能的关系,有利于从分子水平来了解激素与受体的相互作用。     

1组曲霉单克隆抗体的制备与鉴定

目的制备并鉴定一组抗曲霉不同抗原的单克隆抗体。方法采用烟曲霉细胞壁抗原成分、分泌抗原和灭活分生孢子,分别免疫BALB／c小鼠,制备单克隆抗体,免疫荧光法鉴定单克隆抗体与曲霉属和念珠菌属抗原的交叉反应。结果获得29株稳定分泌抗曲霉单抗的杂交瘤细胞株,其中用烟曲霉细胞壁抗原成分免疫获得11株,用分泌抗原免疫获得13株,用孢子免疫获得5株;Ig亚类鉴定,11个克隆株为IgG1亚类,3个克隆株为IgG3,15个克隆株为IgM。免疫荧光法鉴定29株单抗特异性识别烟曲霉细胞壁抗原,与其他曲霉抗原有交叉反应。结论29株单克隆抗体,对于建立侵袭性曲霉感染早期诊断方法、筛选曲霉保护性抗体以及研究抗体保护机制奠定了实验基础。     

蛋白质芯片技术应用于高通量单克隆抗体制备研究

针对在传统的单克隆抗体制备过程中进行特异性筛选时大量的人力消耗,建立了一种联合应用蛋白质芯片进行单克隆抗体制备的方法。用8种重组蛋白分别免疫BALB/c小鼠,在传统的细胞融合的基础上,将8种抗原免疫的杂交瘤阳性细胞混合后进行克隆化、蛋白质芯片筛选,阳性细胞有限稀释克隆化制备相关抗体。实验结果：混合克隆化共得到单克隆细胞175孔,经蛋白质芯片筛选出阳性孔119孔,选择针对单一抗原阳性的细胞连续2轮克隆化,8种重组蛋白各获得单克隆抗体细胞株1株。与经典的单克隆抗体制备相比,蛋白质芯片筛选与混合克隆化技术联合应用于单克隆抗体制备,1个筛选周期获得了8种重组蛋白的单克隆抗体细胞株,提高了单克隆抗体的制备效率,节省了在筛选中的抗原用量,提供了一种经济、快速、简便的方法。     

单克隆抗体在内分泌学中的应用

把单克隆抗体的技术应用于内分泌学研究仅仅开始,但已显示出巨大的潜力.单克隆抗体可提高激素免疫测定法的敏感性、特异性及标准化的程度,并可降低测定的成本.它可提纯某些激素,或使提纯步骤简化.利用标记的单克隆抗体可能对内分泌细胞膜上或者胞质中的成份进行动态的研究.把单克隆抗体作为分子功能解剖刀,可研究激素或其受体的结构和功能的关系,有利于从分子水平来了解激素与受体的相互作用.     

蛋白质芯片技术应用于高通量单克隆抗体制备研究

针对在传统的单克隆抗体制备过程中进行特异性筛选时大量的人力消耗,建立了一种联合应用蛋白质芯片进行单克隆抗体制备的方法。用8种重组蛋白分别免疫BALB/c小鼠,在传统的细胞融合的基础上,将8种抗原免疫的杂交瘤阳性细胞混合后进行克隆化、蛋白质芯片筛选,阳性细胞有限稀释克隆化制备相关抗体。实验结果:混合克隆化共得到单克隆细胞175孔,经蛋白质芯片筛选出阳性孔119孔,选择针对单一抗原阳性的细胞连续2轮克隆化,8种重组蛋白各获得单克隆抗体细胞株1株。与经典的单克隆抗体制备相比,蛋白质芯片筛选与混合克隆化技术联合应用于单克隆抗体制备,1个筛选周期获得了8种重组蛋白的单克隆抗体细胞株,提高了单克隆抗体的制备效率,节省了在筛选中的抗原用量,提供了一种经济、快速、简便的方法。     

细胞工程

<正> 853037抗神经元γ-γ烯醇化酶专一性单克隆抗体免疫测定法的性质和应用[英]/Ki-mura,S.…//Biochem.Biophys.ActaG.-1984,799(3).-252～259[译自DBA,1984,3(18),84-08812]本文报告了抗人和牛γ-γ烯醇化酶单克隆抗体的生产与特性。用纯化的人或牛γ-γ烯醇化酶皮下注射免疫6～8周龄的BALB/c小鼠,重复免疫7次,每周1次。最后以腹     

重金属离子的免疫检测研究进展

农畜产品中残留的重金属离子已对人类安全构成严重威胁,急需快速、高效的重金属残留检测方法。重金属离子免疫检测是一种新型的检测方法,与传统检测方法相比,具有省时、省力、费用低廉、便于携带、易于操作等优点。除了化学螯合剂之外,植物螯合肽和金属硫蛋白也可用来制备重金属免疫原。重金属离子的免疫检测可分为多克隆抗体免疫检测和单克隆抗体免疫检测,前者包括荧光偏振免疫检测,后者包括间接竞争性ELISA、一步法竞争性免疫检测和KinExA免疫检测。作为一种辅助方法,胶体金快速免疫层析法可初步检测样品中的重金属离子浓度。     

Microfluidic heavy metal immunoassay based on absorbance measurement

A simple and rapid flow-based multioperation immunoassay for heavy metals using a microfluidic device was developed. The antigen-immobilized microparticles in a sub-channel were introduced as the solid phase into a main channel structures through a channel flow mechanism and packed into a detection area enclosed by dam-like structures in the microfluidic device. A mixture of a heavy metal and a gold nanoparticle-labeled antibody was made to flow toward the corresponding metal through the main channel and make brief contact with the solid phase. A small portion of the free antibody was captured and accumulated on the packed solid phase. The measured absorbance of the gold label was proportional to the free antibody portion and, thus, to the metal concentration. Each of the monoclonal antibodies specific for cadmium-EDTA, chromium-EDTA, or lead-DTPA was applied to the single-channel microfluidic device. Under optimized conditions of flow rate, volume, and antibody concentration, the theoretical (antibody K(d)-limited) detection levels of the three heavy metal species were achieved within only 7 min. The dynamic range for cadmium, chromium, and lead was 0.57-60.06 ppb, 0.03-0.97 ppb, and 0.04-5.28 ppb, respectively. An integrated microchannel device for simultaneous multiflow was also successfully developed and evaluated. The multiplex cadmium immunoassay of four samples was completed within 8 min for a dynamic range of 0.42-37.48 ppb. Present microfluidic heavy metal immunoassays satisfied the Japanese environmental standard for cadmium, chromium and, lead, which provided in the soil contamination countermeasures act.     

Structural transitions in bovine factor X associated with metal binding and zymogen activation. Studies using conformation-specific antibodies

Conformation-specific antibodies against distinct regions of Factor X were employed to locate antigenic determinants which are altered during zymogen activation or by metal binding. Anti-Factor X antibodies, raised in rabbits against Factor X, were purified by affinity chromatography using Factor X covalently bound to Sepharose. Quantitative equilibrium and kinetic measurements of precipitation of Factor X and Factor Xa by antibodies indicated differences in the antigenic structure of the zymogen and the enzyme form of factor X. The factor X antibodies were further fractionated by sequential immunoabsorption using fragments of Factor X and Factor Xa. With conformation-specific antibodies directed against the heavy chain and the light chain of Factor X, zymogen activation was shown to involve a structural transition in the heavy chain but not the light chain. Antibodies directed against the activation peptide domain 1-51 of the heavy chain, the trypsin-like region of the heavy chain 52-290, and the substrate-binding site suggest a generalized conformational transition in the heavy chain. Antibodies were isolated which are specific for the Factor X:Ca(II) complex and bind to Factor X only in the presence of metal ions. Subfractions were directed against either the heavy chain or the light chain, indicating that both the heavy chain and the light chain of Factor X undergo a metal-induced conformational transition. Half-maximal antibody-factor X interaction was observed at 0.13 mM CaCl2 for the light chain and 0.7 mM CaCl2 for the heavy chain. These results indicate that zymogen activation is limited to structural changes in the heavy chain, but metal binding is associated with changes in the structure of both the heavy and light chains. Metal-dependent binding of Factor X to the platelet Factor Xa receptor after activation may involve surfaces of the heavy as well as the light chains.     

重金属残留的快速检测

环境及农畜产品中的重金属残留已对人类安全构成严重威胁,急需快速、高效的重金属残留检测方法。该文总结了国内外有关采用生物、化学传感器、免疫学方法等快速检测环境及农畜产品中重金属残留的研究进展,并对其发展前景及市场化作了预测及展望。     

Phytochelatins and heavy metal tolerance

The induction and heavy metal binding properties of phytochelatins in heavy metal tolerant (Silene vulgaris) and sensitive (tomato) cell cultures, in water cultures of these plants and in Silene vulgaris grown on a medieval copper mining dump were investigated. Application of heavy metals to cell suspension cultures and whole plants of Silene vulgaris and tomato induces the formation of heavy metal–phytochelatin-complexes with Cu and Cd and the binding of Zn and Pb to lower molecular weight substances. The binding of heavy metal ions to phytochelatins seems to play only a transient role in the heavy metal detoxification, because the Cd- and Cu-complexes disappear in the roots of water cultures of Silene vulgaris between 7 and 14 days after heavy metal exposition. Free heavy metal ions were not detectable in the extracts of all investigated plants and cell cultures. Silene vulgaris plants grown under natural conditions on a mining dump synthesize low molecular weight heavy metal binding compounds only and show no complexation of heavy metal ions to phytochelatins. The induction of phytochelatins is a general answer of higher plants to heavy metal exposition, but only some of the heavy metal ions are able to form stable complexes with phytochelatins. The investigation of tolerant plants from the copper mining dump shows that phytochelatins are not responsible for the development of the heavy metal tolerant phenotypes.     

A novel screening method for microorganisms that efficiently remove heavy metals from aqueous solution

A novel bioassay system for estimating concentrations of several heavy metal ions was carried out with yeast mutants which are highly sensitive to heavy metal ions. The method does not need an atomic adsorption spectrometer or other special equipment. It is suitable for screening of microorganisms that efficiently remove heavy metal ions from aqueous solution.     

Studies on thermoprotection induced by heavy metal ions in spring barley seedlings

Barley seedlings (Hordeum vulgare L., cv. Fatran) were pretreated with various concentrations of five heavy metal ions (Zn, Cu, Fe, Mg, Mn) for 3 d. When the subsequent heat shock was administered for 2 h, the heavy metal ions had thermoprotective effect against the sub-lethal (40 °C) and lethal (45 °C) temperature stresses, which were otherwise lethal to control (water grown) seedlings. The effectiveness of each of the heavy metal ions was different, the most effective being Cu. The level of protection provided by these heavy metal ions was dependent on both the time and the concentration that plants were exposed to them. The greatest differences were recorded in the thermotolerance mediated by applied metal ions in the shoot and root cells. Thermotolerance was exhibited by both the shoot and root of pretreated seedlings, even though the heavy metal stresses were applied solely to the roots.     

Effect of Heavy Metals (Cd, Cu) on the Gametophytes of Laminaria japonica Aresch

Effects of various concentrations of two heavy metals, namely Cd and Cu, on gametophytes of Laminariajaponica Aresch were determined by recording morphological changes of gametophytes, determining pH values and the heavy metal content of the culture solution, calculating the germination rate of sporophytes, and observing heavy metal (Cd) distribution using a fluorescence microscope. The results showed that heavy metals damaged the gametophytes, and were even lethal, and that the higher the concentration of heavy metal ions, the greater the injury to gametophytes. Gametophytes could not survive in culture solutions containing more than 100 mg/L Cd and 50 mg/L Cu and were only able to survive in culture solution containing a mixture of Cd and Cu up to a concentration of 10 mg/L, which indicates that gametophytes have a higher tolerance to Cd than Cu and that multiple heavy metal ions in solution markedly aggravate the damage to gametophytes compared with individual heavy metal ions. With increases in the concentration of the heavy metal, the burgeoning rate of sporophytes decreased acutely, and solutions containing multiple heavy metal ions caused even more marked harm to sporophytes than solutions containing a single heavy metal ion, because most sporophytes died in mixed solutions. The pH value of the culture medium dropped immediately at the beginning (the first day) of treatment, increased over the following days, and then decreased again. The pH of culture media containing multiple heavy metal ions showed greater variation than media containing a single heavy metal ion, with the extent of the decrease in pH of culture media containing multiple ions being greatest during the last period of the experiment. With increases in the concentration of heavy metals, the capacity of gametophytes to accumulate these ions increased. The blue fluorescent light emitted by the Cd- and Cd-binding protein complex existing in gametophytes in media containing different concentrations of Cd showed clearly the distribution of the ion in gametophytes and the results obtained were consistent with distribution determined using other methods.All results of the present study showed that gametophytes of L. japonica play a remarkable role as heavy metal decontaminators, especially with regard to Cd.     

Resistance to heavy metal toxicity in organisms under chronic exposure

The rate of changes of heavy metal ions concentrations in the organism or biological system will determine the choice of strategy of realization of heavy metal ions effect and, consequently, the biological effect itself. Which of the possible strategies will dominate, depend on the rate of changes of concentration of heavy metal ions in biological systems, functional activity of organism at the moment of metal action and on metals chemical properties. Keeping this view, the present review deals with the concept of time-based alterations of concentration of heavy metal ions (TACMI) in biological systems. On the basis of TACMI concept formation of organism resistance to metal ions action could be explained. In the event of slow increase of its concentration in organism, it produces induction of metallothioneins, other stress-proteins and relative changes in the whole metabolic system. This, first of all, results in formation of new specific epigenotypes, which provide higher resistance (hormesis effect) not only to metal ions that induced this effect, but also to such stress-factors as high temperature (at least, for micro-algae cells).     

Effect of Heavy Metals （Cd, Cu） on the Gametophytes of Laminaria japonica Aresch

Effects of various concentrations of two heavy metals, namely Cd and Cu, on gametophytes of Laminariajaponica Aresch were determined by recording morphological changes of gametophytes, determining pH values and the heavy metal content of the culture solution, calculating the germination rate of sporophytes, and observing heavy metal （Cd） distribution using a fluorescence microscope. The results showed that heavy metals damaged the gametophytes, and were even lethal, and that the higher the concentration of heavy metal ions, the greater the injury to gametophytes. Gametophytes could not survive in culture solutions containing more than 100 mg/L Cd and 50 mg/L Cu and were only able to survive in culture solution containing a mixture of Cd and Cu up to a concentration of 10 mg/L, which indicates that gametophytes have a higher tolerance to Cd than Cu and that multiple heavy metal ions in solution markedly aggravate the damage to gametophytes compared with individual heavy metal ions. With increases in the concentration of the heavy metal, the burgeoning rate of sporophytes decreased acutely, and solutions containing multiple heavy metal ions caused even more marked harm to sporophytes than solutions containing a single heavy metal ion, because most sporophytes died in mixed solutions. The pH value of the culture medium dropped immediately at the beginning （the first day） of treatment, increased over the following days, and then decreased again. The pH of culture media containing multiple heavy metal ions showed greater variation than media containing a single heavy metal ion, with the extent of the decrease in pH of culture media containing multiple ions being greatest during the last period of the experiment. With increases in the concentration of heavy metals, the capacity of gametophytes to accumulate these ions increased. The blue fluorescent light emitted by the Cd-and Cd-binding protein complex existing in gametophytes in media containing different concentrations of Cd showed clearly the distribution of the ion in gametophytes and the results obtained were consistent with distribution determined using other methods. All results of the present study showed that gametophytes of L. japonica play a remarkable role as heavy metal decontaminators, especially with regard to Cd.