Micro Analytical Immunochemistry

子项目#1 使用免疫亲和毛细管电泳（ICE）分析生物样品 毛细管电泳-激光诱导荧光（LIF）检测是一种成熟的生物样品分析方法。 小样品体积、短分析时间和低试剂用量优于更传统的方法，如HPLC。 毛细管与抗体的衍生化允许从复杂的生物样品中特异性捕获和检测单个或多个分析物。ICE提高了CE的检测限，而无需预处理样品。 此外，该技术可以用于传统的CE机器以及微芯片CE。 ICE已被用于处理NIH校园内几个合作者的样本。 研究包括以下内容： - 分析人血浆样品中用于肾脏疾病的细胞因子 - 从阿尔茨海默病的疾病模型研究中对小鼠和大鼠进行组织分析 - 分析给予细胞因子抑制药物的大鼠的组织样品 次级项目2 细胞分泌物的多重ELISA分析 多重ELISA改进了传统的ELISA形式。 多重技术不再检测单个样本中的单个抗原，而是允许从单个样本中检测多达16种抗原。 这减少了与ELISA相关的总时间和成本，并且需要更少的样品体积来产生更多的信息。 多重格式是可定制的，使研究人员能够研究一系列独特的蛋白质和肽。 使用多重ELISA的研究包括： - 来自参与社交失败模型的小鼠的小鼠血浆样品的分析 - 分析来自参与睡眠刺激试验的受试者的人血浆样品 - 分析由UV光和冷刺激的Hela细胞的细胞分泌产物。 - 分析来自被照射的人支气管上皮细胞的细胞分泌产物 x射线 子项目3 蛋白质的MALDI质谱 MALDI质谱法是一种用于精确测定分析物质量的技术，范围从几百个质量单位到几十个KDa单位。 该技术是对我们分离技术的补充，使我们能够精确定位从复杂样品中分离出的未知分析物的质量。 这种技术使研究人员能够积极识别孤立的未知物质并确定元素组成。 使用MALDI的研究包括： - 分析从恒河猴分离的伊加中J链组分的存在 - 分析荧光蛋白EGFP和M-Ruby-2的精确分子量， 切割产物 次级项目#4 Biacore Biacore 3000检测系统使用表面等离子体共振（SPR）来监测分子在传感器芯片表面相互作用时的折射率（RI）变化。表面等离子体激元是由光激发的表面等离子体激元。一种相互作用物（配体）固定在传感器芯片表面上，而另一种（分析物）则以连续流动的方式注入表面。如果发生相互作用，则分析物的表面浓度增加，导致RI变化。SPR技术能够无标记地实时检测和监测生物分子事件，并提供关于两个分子之间结合的特异性、亲和力和相互作用动力学的定性和定量信息。实验设计高度灵活，该技术可应用于蛋白质与其他蛋白质、核酸、脂质、小分子的相互作用。 使用Biacore的研究包括： - Her蛋白与DNA适体的Kd相互作用的分析 - 分析RasGRP 1和RasGRP 3与脂质的膜相互作用，以确定 不同结构域在结合相互作用中的作用 子项目#5 毛细管电泳法测定SELEX SELEX是一种筛选DNA适体文库的技术，用于筛选对某种蛋白质显示亲和力的独特DNA序列。 然后收集适体并通过PCR扩增，重复选择，直到获得显示对蛋白质的亲和力的适体的富集池。 这个过程是费时耗料的。 毛细管电泳已被用于增加该过程的灵敏度并减少该过程所需的时间。 使用SELEX_CE的研究包括 - 从DNA文库中筛选与α蛋白有亲和力的适体 子项目#6 电感耦合等离子体（ICP）痕量金属分析 具有UV/维斯检测的ICP分析可用于分析样品中痕量金属的存在。 将样品引入等离子体火焰中，然后通过连续波长分光光度计检测，以允许对感兴趣的金属进行定量测量。该技术允许研究人员确定反应的产率，测量金属化合物的细胞摄取或确定造影剂实验中组织中的金属水平。 使用ICP-OES的研究包括： - 磁等离子体Janus囊泡与金纳米粒子的结合分析 - 分析组织样品中硼纳米颗粒的存在

Sub-project #1 Analysis of biological samples using immunoaffinity capillary electrophoresis (ICE) Capillary electrophoresis combined with lased induced fluorescent (LIF) detection is an established method for analyzing biological samples. The small sample volumes, short analysis times and low reagent use are advantageous over more traditional methods such as HPLC. Derivatization of the capillary with antibodies allows for specific capture and detection of single or multiple analytes from a complex biological sample. ICE increases the limits of detection for CE without the need for pre-processing samples. Additionally this technique can be used on both a traditional CE machine as well as a micro-chip CE. ICE has been used to process samples from several collaborators on the NIH campus. Studies include the following: -Analysis of human plasma samples for cytokines indicated in kidney diseases -Tissue analysis from mice and rats from disease model studies looking at Alzheimer's Disease -Analysis of tissue samples from rats given cytokine inhibition drugs Sub-project #2 Multiplex ELISA analysis of cell secretions Multiplex ELISA improves upon the traditional ELISA format. Rather than detecting a single antigen in a single sample, multiplex technology allows for the detection of up to 16 antigens from a single sample. This reduces the overall time and cost associated with ELISA and requires far less sample volume to produce more information. The multiplex format is customizable to allow researchers to investigate a unique array of proteins and peptides. Studies using multiplex ELISA include: -Analysis of mouse plasma samples from mice involved in social defeat models -Analysis of human plasma samples from subjects involved in sleep stimulation trials -Analysis of cell secretion products from Hela cells stimulated by UV light and cold. -Analysis of cell secretion products from Human Bronchial Epithelial cells irradiated X-rays Sub- project #3 MALDI mass spectrometry of proteins MALDI mass spectrometry is a technique used to pinpoint the mass of analytes ranging from a few hundred mass units to tens of KDa units. The technique is a complement to our isolation techniques, allowing us to pinpoint the mass of unknown analytes isolated from complex samples. This technique allows researchers to positively identify isolated unknowns and determine elemental composition. Studies using MALDI include: -Analysis of IgA isolated from rhesus macaques for the presence of J-chain component -Analysis of fluorescent proteins EGFP and M-Ruby-2 for precise molecular weights of cleavage products Sub-project #4 Biacore Biacore 3000 detection system uses Surface Plasmon Resonance (SPR) to monitor the refractive index (RI) change as molecules interact at the sensor chip surface. SPR is the excitation of surface plasmons by light. One of the interactants (ligand) is immobilized on the sensor chip surface, while the other (analyte) is injected in continued flow over the surface. If the interaction takes place, the surface concentration of the analyte is increased that results in the RI change. SPR technology enables label-free real-time detection and monitoring of biomolecular events and provides qualitative and quantitative information on specificity of binding between two molecules, affinity, and kinetics of the interaction. Experimental design is highly flexible and the technology can be applied to protein interaction with other proteins, nucleic acids, lipids, small molecules. Studies using Biacore include: -Analysis of the interaction of the Kd of Her- protein with DNA aptamers -Analysis of the membrane interactions of RasGRP1 and RasGRP3 with lipids to determine the role of different domains in binding interactions Sub-Project #5 SELEX by capillary electrophoresis SELEX is technique by which DNA aptamer libraries are screened for unique DNA sequences that show an affinity for a certain protein. The aptamers are then collected and amplified by PCR and the selection is repeated until an enriched pool of aptamers showing affinity for the protein is obtained. This procedure is time and material consumming. Capillary electrophoresis has been used to increase the sensitivity of this process and decrease the amount of time the procdure takes. Studies using SELEX_CE include - Screening DNA libraries for aptamers that show an affinitiy for MIG protein Sub-Project #6 Trace metal analysis by Inductively Coupled Plasma (ICP) ICP analysis with UV/Vis detection can be used to analyze samples for the presence of trace metals. The samples are introduced into a plasma flame and then detected by a continuous wavelength spectrophotometer to allow for quantitative measurements of the metal of interest. This technique allows researchers to determine yields for reactions, measure cellular uptake of metal compounds or determine metal levels in tissues in contrast agent experiments. Studies using ICP-OES include: - Analysis of magneto-plasmonic Janus vesicles integrated with Gold nanoparticles - Analysis of tissue samples for the presence of Boron nanoparticles