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 - 来自用生长因子刺激的星形胶质细胞的细胞分泌产物的分析 - 分析由UV光和冷刺激的Hela细胞的细胞分泌产物。 次级项目3 表面等离子体共振 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。配体被固定在传感器芯片表面上，而分析物以连续流注入表面。如果发生相互作用，则分析物的表面浓度增加，这导致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。 使用表面等离子体共振的研究包括： - TPRV 1与毒素K1、K2和K1-K2的相互作用。 - 蛋白激酶C1与脂质表面的相互作用。 - HIV-1 gp 120与生物素化v2肽的动力学和热力学相互作用的分析。 - 功能性大麻素受体CB 2的研究。

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 astrocytes stimulated with growth factors -Analysis of cell secretion products from Hela cells stimulated by UV light and cold. Sub-project #3 Surface Plasmon Resonance The Biacore 3000 detection system uses Surface Plasmon Resonance (SPR) to monitor refractive index (RI) change as molecules interact at the sensor chip surface. SPR is the excitation of surface plasmons by light. The ligand is immobilized on the sensor chip surface, while the analyte is injected in continuous flow over the surface. If an interaction takes place, the surface concentration of the analyte is increased which results in an 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 surface Plasmon resonance include: -Interaction of TPRV1 with toxins K1,K2 and K1-K2. -Interaction of protein kinase C1 with lipid surfaces. -Analysis of kinetic and thermodynamic interactions of HIV-1 gp120 with biotinylated v2 peptide. -A study of functional cannabinoid receptor CB2.