Micro Analytical Immunochemistry

子项目#1 组织样品的MALDI成像以识别新的蛋白质 MALDI-TOF成像是一种新兴的无标记测量多肽、蛋白质、脂类、药物和代谢物的工具。组织可以直接用MALDI-TOF成像，并结合其他方式来研究组织样品的分子轮廓以及空间位置。目前，大多数组织样本都是通过MALDI成像，并与组织学染色的组织进行比较。 在与NIAID的合作下，我们正在开发对组织成像的方案，然后将它们与共焦显微镜/组织细胞成像切片进行比较。共聚焦显微镜成像提供了细胞水平的分辨率和空间图谱，而MALDI-IMS提供了更高通量的蛋白质组学和分子图谱，可以识别与组织炎症和其他过程相关的生物分子。目标是使用这些技术对感染艾滋病毒的淋巴进行成像，希望识别新的生物标记物并提供它们的空间分辨率。 在与LDRR的合作中，我们正在开发对受超声波辐射的小鼠组织进行成像的协议。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、多肽或脂类不受这一程序的调节，并确定它们是否存在。其目的是确定小鼠是否对该程序表现出炎症反应。 子项目#2 电感耦合等离子体分析痕量金属 采用UV/Vis检测的电感耦合等离子体分析可用于分析样品中痕量金属的存在。样品被引入等离子体火焰中，然后由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。这项技术允许研究人员在造影剂实验中确定反应的产率，测量细胞对金属化合物的摄取，或确定组织中的金属水平。 使用电感耦合等离子体发射光谱仪的研究包括： -分析与金纳米颗粒结合的磁等离子体Janus囊泡 -分析组织样本中是否存在纳米硼颗粒 -分析多模式造影剂以量化铁和格拉量 现在时 -分析新型药物洗脱微珠，以量化存在的硼的量 -分析聚丙烯酸凝胶，以量化钠、钙、 有钾和氯存在。 -分析从诊断为丙型肝炎的患者收集的唾液，以量化钠和 钾含量 --口干综合征模型小鼠唾液分析 子项目#3 基质辅助激光解吸/电离(MALDI)-飞行时间(TOF)-质谱仪(MS)高通量分析免疫球蛋白糖基化 NIAID免疫调节实验室的研究重点是阐明人体免疫反应在健康和疾病中的调节细胞和分子机制。这些研究工作的一个主要组成部分涉及了解B细胞在艾滋病毒感染中的免疫病理机制。B细胞免疫学单位的苏珊·莫尔博士最近描述了IgG3在登记在临床研究方案02-I-0202中的HIV感染者中调节B细胞的作用。研究结果表明，IgG3的糖基化和可能的其他血清蛋白在研究中描述的调节功能中发挥了作用；这一发现是通过对100多名参与者的样本进行分析得出的。对血清蛋白糖基化模式的评估必须使用一种方法，该方法与从相对少量的材料中处理相对大量的样本相兼容。初步化验将对几个阳性和阴性对照进行，然后从一些人的纵向样本中进行，这些人要么一开始有阳性的IgG3图谱，然后随着时间的推移失去它，要么随着时间的推移获得图谱。 子项目#4 血清调查确定SARS-COV-2蛋白抗体的存在 NIBIB免疫工程科开发了一种新的检测方法，用于检测SARS-CoVID2刺突蛋白和RBD蛋白的免疫球蛋白、免疫球蛋白M和免疫球蛋白A抗体的存在。 使用此血清调查的研究包括： -在时间点零点采集了9,300份健康血液样本，以确定 SARS-CoVID-2在社区内的社区传播。 -当时从最初的个人中采集了大约7000个健康的血液样本 点六个月来确定社区传播的程度和疫苗接种情况。 -当时从最初的个人中采集了大约5000份健康血液样本 每一年确定社区传播程度和疫苗接种状况。 -从创伤患者中采集2542个样本，以确定SARS-COV-2的血清阳性率 -疑似SARS-COV-2死亡患者的尸检样本，以确定血清阳性率 SARS-COV-2 -大约15个样本来自在被感染后经历反弹感染的患者 注射巴昔洛韦以确定血清阳性率 -大约75名医护人员样本接受了免疫接种，以确定 血清阳性 -大约30个参加IL-7试验的患者样本，以确定血清阳性率 -大约4000个有特定免疫缺陷和免疫缺陷的个人样本 与健康志愿者比较评估疫苗接种前和疫苗后免疫功能障碍 回应 -大约300个线粒体疾病患者的样本，以确定 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 magneto-plasmonic Janus vesicles integrated with Gold nanoparticles - Analysis of tissue samples for the presence of Boron nanoparticles - 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