Micro Analytical Immunochemistry

子项目#1 生物样品的免疫亲和毛细管电泳法分析 毛细管电泳联用激光诱导荧光(LIF)检测是一种成熟的生物样品分析方法。样品体积小，分析时间短，试剂使用量少，比高效液相色谱法等更传统的方法更具优势。带有抗体的毛细管衍生化允许从复杂的生物样品中特定地捕获和检测单个或多个分析物。ICE提高了CE的检测限，而不需要对样品进行前处理。此外，该技术既可以在传统的CE机器上使用，也可以在微芯片CE上使用。 ICE已经被用于处理来自NIH校园内几个合作者的样本。研究包括以下内容： -对人体血浆样本进行肾脏疾病指示的细胞因子分析 -来自阿尔茨海默病疾病模型研究的小鼠和大鼠的组织分析 -对给予细胞因子抑制药物的大鼠的组织样本进行分析 子项目#2 细胞分泌物的多重酶联免疫吸附分析 多重酶联免疫吸附试验是对传统的酶联免疫吸附试验模式的改进。多重技术不是在单个样本中检测单个抗原，而是允许从单个样本中检测多达16个抗原。这减少了与ELISA相关的总体时间和成本，并需要更少的样本量来产生更多的信息。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多肽。 使用多重酶联免疫吸附试验的研究包括： -分析参与社会失败模型的小鼠的血浆样本 -对参与睡眠刺激试验的受试者的血浆样本进行分析 -分析受紫外线和寒冷刺激的Hela细胞的细胞分泌产物。 子项目#3 蛋白质的MALDI质谱学 MALDI质谱仪是一种用来精确确定分析物质量的技术，范围从几百个质量单位到几十个千达单位。这项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。这项技术使研究人员能够肯定地识别孤立的未知数，并确定元素组成。 使用MALDI的研究包括： -从恒河猴中分离出的IgA分析是否存在J链成分 -分析荧光蛋白EGFP和M-Ruby-2，以获得准确的切割分子量 产品 子项目#4 比亚科雷 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光对表面等离子体的激发。其中一个相互作用物(配体)固定在传感器芯片表面，而另一个(分析物)以连续流动的方式在表面上注入。如果发生相互作用，分析物的表面浓度会增加，从而导致RI变化。SPR技术实现了对生物分子事件的无标记实时检测和监测，并提供了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。实验设计具有很高的灵活性，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： 分析HER蛋白的Kd与DNA适配子的相互作用 子项目#5 SELEX毛细管电泳法 SELEX是一种用来筛选DNA适体文库以寻找与特定蛋白质具有亲和力的独特DNA序列的技术。然后收集并通过PCR扩增适配子，并重复选择，直到获得显示与蛋白质有亲和力的富集库。这一过程耗费了时间和物质。毛细管电泳法已被用来提高这一过程的灵敏度，并减少这一过程所需的时间。 使用SELEX_CE的研究包括 -筛选与HER-2蛋白有亲和力的适体DNA文库 -筛选与MIG蛋白有亲和力的适体DNA文库

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. 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 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 affinity for Her-2 protein - Screening DNA libraries for aptamers that show an affinitiy for MIG protein