Micro Analytical Immunochemistry

子项目#1 组织样品的MALDI成像以鉴定新蛋白质 MALDI-TOF成像是一种新兴的无标记测量多肽、蛋白质、脂质、药物和代谢物的工具。 组织可以直接通过MALDI-TOF成像，并与其他模式相结合，以研究组织样本的分子谱以及空间位置。 目前，大多数组织样本通过MALDI成像，并与组织学染色的组织进行比较。 与NIAID合作，我们正在开发组织成像协议，然后将其与共聚焦显微镜/组织细胞术成像切片进行比较。共聚焦显微镜成像提供细胞水平的分辨率和空间映射，而MALDI-IMS提供更多的高通量蛋白质组学和分子映射，可以识别与组织炎症和其他过程相关的生物分子。我们的目标是使用这些技术对感染艾滋病毒的淋巴结进行成像，希望能够识别新的生物标志物并提供它们的空间分辨率。 在与LDRR的合作中，我们正在开发协议，以成像已被超声辐照的小鼠组织。 我们正在对治疗前后的组织进行成像，以确定是否有任何蛋白质、肽或脂质不受该过程的调节，并在它们不受调节的情况下进行识别。 目的是确定小鼠是否对手术表现出炎症反应。 子项目#2 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： - 氧化锰纳米颗粒中锰的量和在酸性溶液中释放的量的分析 - 分析多模态造影剂以定量铁和钆的量 本 - 分析新型药物洗脱珠以定量存在的硼的量 - 分析聚（丙烯酸）凝胶以定量钠、钙 钾和氯的存在。 - 分析从诊断患有丙型肝炎的患者收集的唾液以定量钠和 钾含量 - 小鼠口干综合征模型唾液分析 次级项目3 通过基质辅助激光解吸/电离（MALDI）-飞行时间（TOF）-质谱（MS）对IgG糖基化进行高通量分析。 NIAID免疫调节实验室的研究重点是阐明健康和疾病中人体免疫反应调节的细胞和分子机制。这些研究工作的一个主要组成部分是了解HIV感染中B细胞的免疫发病机制。B细胞免疫学单位的Susan Moir博士最近描述了IgG 3在临床研究方案02-I-0202中登记的HIV感染个体中调节B细胞的作用。研究结果表明，IgG 3和可能的其他血清蛋白的糖基化在研究中描述的调节功能中发挥了作用;研究结果来自对100多名参与者分离的标本的分析。血清蛋白中糖基化模式的评价必须使用与从相对少量的材料处理相对大量的样品相容的方法进行。将对一些阳性和阴性对照品进行初步测定，然后对来自开始具有阳性IgG 3特征然后随时间推移而丧失或随时间推移获得特征的个体的一些纵向样本进行测定。 次级项目#4 血清学调查以确定SARS-COV-2蛋白抗体的存在 NIBIB的免疫工程科已经开展了基于ELISA的血清学调查，以检测SARS-COVID 2 SPIKE和RBD蛋白的IgG、IgM和伊加抗体的存在。 使用该血清调查的研究包括： - 在时间点0收集9，300个健康血液样品，以确定 社区内SARS-COVID-2的社区传播。 - 大约7000个健康的血液样本从最初的个人收集的时间 点六个月，以确定社区传播的程度和疫苗接种状况。 - 从最初的个体中收集了大约5000份健康血液样本， 点一年，以确定社区传播的程度和疫苗接种状况。 - 2，542份创伤患者样本用于确定SARS-COV-2血清阳性 - 疑似SARS-COV-2死亡患者的尸检样本，以确定 SARS-CoV-2 - 大约15份样本来自于在治疗后发生反弹感染的患者， 给药百忧解以确定血清阳性 - 大约75名接受过加强剂的卫生保健工作者样本， 血清阳性 - 约30份患者样本入组IL-7试验，以确定血清阳性 - 大约4000个具有选择性免疫缺陷和免疫缺陷的个体样本 与健康志愿者相比， 响应 - 大约300个线粒体疾病患者的样本， 血清阳性

Sub-project #1 MALDI imaging of tissue samples to identify novel proteins MALDI-TOF Imaging is an emerging tool for the label free measurement of peptides, proteins, lipids, drugs and metabolites. The tissue can be imaged directly be the MALDI-TOF and combined with other modalities to study the molecular profiles as well as spatial placement of the tissue samples. Currently most tissue samples are imaged by MALDI and compared to histological stained tissues. In collaboration with NIAID, we are developing protocols to image tissues and then compare them to confocal microscopy/histo-cytometry imaged sections. The confocal microscopy imaging provides cellular level of resolution and spatial mapping whereas MALDI-IMS provides more high-throughput proteomics and molecular mapping that can identify biomolecules associated with tissue inflammation and other processes. The goal is to use these techniques to image lymph nodes infected with HIV in hopes of identifying new biomarkers and providing spatial resolution of them. In collaboration with LDRR, we are developing protocols to image mice tissue that has been irradiated with ultrasound. We are imaging tissues pre and post treatment to determine if any proteins, peptides or lipids are unregulated by this procedure and identify them if they are. The goal of is to determine if the mice exhibit an inflammatory response to the procedure. Sub-Project #2 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 manganese oxide nanoparticles for the amount of manganese in the particle and released in acidic sloutions - Analysis of multimodal contrast agents to quantify the amount of iron and gadolinium present - Analysis of novel drug eluting beads to quantify the amount of boron present - Analysis of poly(acrylic acid) gels to quantify the amount of sodium, calcium, potassium and chlorine present. - Analysis of saliva collected from patients diagnosed with Hepatitis C to quantify sodium and potassium content - Analysis of saliva collected from mice modeling dry mouth syndrome Sub-project #3 High-throughput analysis of IgG glycosylation by matrix assisted laser desorption/ionization (MALDI) -time-of-flight (TOF)- mass spectrometry (MS). The Laboratory of Immunoregulation at NIAID focuses its research efforts on the elucidation of cellular and molecular mechanisms of the regulation of the human immune response in health and disease. A major component of these research efforts involves understanding the immunopathogenesis of B cells in HIV infection. Dr. Susan Moir of the B-Cell Immunology Unit has recently described a role for IgG3 in regulating B cells in HIV-infected individuals enrolled in clinical research protocol 02-I-0202. The findings indicated that glycosylation of IgG3 and possibly other serum proteins played a role in the regulatory function described in the study; the findings were obtained from the analysis of specimens isolated from over 100 participants. The evaluation of glycosylation patterns in serum proteins must be performed using an approach that is compatible with processing a relatively large number of samples from a relatively small quantity of material. Preliminary assays will be performed on a few positive and negative controls, followed by a few longitudinal samples from individuals who either begin with a positive IgG3 profile and then lose it over time or acquire the profile over time. Sub-project #4 Serosurvey to determine antibody presence to SARS-COV-2 protein The Section for Immuno-Engineering in NIBIB has undertaken the development of and ELISA based serosurvey to detect the presence of IgG, IgM and IgA antibodies to SARS-COVID2 SPIKE and RBD proteins. Studies using this serosurvey include: - 9,300 healthy blood samples were collected at time point zero to determine the extent of community spread of SARS-COVID-2 within the community. - Approximately 7000 healthy blood samples were collected from the initial individuals at time point six months to determine the extent of community spread and vaccination status. - Approximately 5000 healthy blood samples were collected from the initial individuals at time point one year to determine the extent of community spread and vaccination status. - 2,542 samples from trauma patients to determine seropositivity for SARS-COV-2 - Autopsy samples from patients suspected of SARS-COV-2 death to determine seropositivity for SARS-COV-2 - Approximately 15 samples from patients who experienced rebound infections after being administered paxlovid to determine seropositivity - Approximately 75 health care worker samples who have received boosters to determine seropositivity - Approximately 30 samples of patients enrolled in an IL-7 trial to determine seropositivity - Approximately 4000 samples of individuals with select immunodeficiencies and immune dysregulations compared to healthy volunteers to assess pre- and post- vaccine immune responses - Approximately 300 samples of individuals with Mitochondrial Disease to determine seropositivity