Core A: Automated Cell/Biochemical Assays Core

核心 A：自动化细胞/生化检测核心

• 批准号: 10307760
• 负责人: Pamela J Bjorkman
• 金额: $ 41.84万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-02-10 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10307760
• 关键词: Address; Affinity; Animal Model; Animals; Antibodies; Antibody Affinity; Antibody Response; Antigens; Automation; Binding; Biochemical; Biological Assay; Biomedical Research; Calibration; Cells; Custom; Data Analyses; Ensure; Enzyme-Linked Immunosorbent Assay; Freedom; Gene Expression; Grant; HIV; HIV-1; Hand; Harvest; Human Resources; Immunization; Immunize; Immunoglobulin G; In Vitro; Infection; Instruction; Kinetics; Libraries; Liquid substance; Luciferases; Maintenance; Measurement; Measures; Methods; Mus; Postdoctoral Fellow; Preparation; Production; Proteins; Protocols documentation; Reagent; Regimen; Reporter Genes; Reproducibility; Research; Research Assistant; Research Personnel; Robot; Sampling; Scheme; Series; Serum; Specialist; Specificity; Structure; Students; Surface; Surface Plasmon Resonance; System; Testing; Time; Training; Transfection; Vaccinated; Vaccination; Validation; Yeasts; base; design; env Gene Products; experience; experimental study; improved; in vivo; inhibitor; instrument; method development; miniaturize; neutralizing antibody; next generation; protein expression; repaired; response; screening; success; vaccine trial

The ability to elicit potent and broadly neutralizing antibodies (bNAbs) against HIV-1 through vaccination is one of the prime challenges of biomedical research in our time. The proposed HIVRAD project aims to address this challenge. Our approach is based on the concept that immunogens that bind to inferred germline Abs (iGLs) that are precursors to known mature bNAbs can initiate an Ab response that can be guided to produce bNAbs using a series of sequential immunogens. Therefore the focus of this grant is to evaluate immunization regimens in animal models (Project 1) using immunogens selected by structure-based design and library screening (Project 2). Key requirements for the success of this project include the ability to evaluate thousands of serum samples from immunized animals to determine whether they are producing specific and productive responses, producing and screening thousands of potential immunogens and Abs, and assessing binding and neutralization potency and breadth. Here we describe a scientific core dedicated to using liquid handling robots for automated expression of small quantities of many different proteins and high-throughput automated assays. The Cell/Biochemical Assay Automation Core will design and carry out automated cell-based and biochemical assays to express proteins in high-throughput, characterize the binding affinity and specificity, and evaluate the potency of HIV-neutralizing antibodies. Core A personnel will validate, refine, and trouble-shoot protocols for the automated protein expression, binding, and in vitro neutralization assays and perform assays for evaluating antibodies and Env proteins for Drs. Nussenzweig and Bjorkman. The core will train and assist students, postdoctoral fellows, research assistants, and investigators in the analysis and interpretation of data from automated assays. The Automated Cell/Biochemical Assays Core will maintain two custom-equipped Evo Freedom Liquid handling stations and a surface plasmon resonance instrument with high- throughput capabilities for the automated assays proposed in this project.

通过以下途径引发针对HIV-1的有效和广泛中和抗体（bNAb）的能力： 疫苗接种是我们这个时代生物医学研究的主要挑战之一。拟议 HIVRAD项目旨在应对这一挑战。我们的方法是基于这样一个概念， 结合推断的生殖系Ab（iGL）的免疫原，所述生殖系Ab是已知成熟的免疫原的前体。 bNAb可以启动Ab应答，该应答可以使用一系列的免疫抑制剂来引导以产生bNAb。 连续免疫原。因此，这笔赠款的重点是评估免疫方案 在动物模型（项目1）中使用通过基于结构的设计和文库选择的免疫原 筛选（项目2）。该项目成功的关键要求包括： 评估来自免疫动物的数千份血清样品，以确定它们是否 产生具体和富有成效的反应，产生和筛选成千上万的潜在 免疫原和Ab，并评估结合和中和效力和宽度。这里我们 描述了一个致力于使用液体处理机器人自动表达的科学核心， 少量许多不同的蛋白质和高通量自动化测定。的 细胞/生化分析自动化核心将设计和执行自动化细胞为基础， 生物化学测定以高通量表达蛋白质，表征结合亲和力， 特异性，并评估HIV中和抗体的效力。核心A人员将 验证，完善和故障排除协议的自动化蛋白质表达，结合， 体外中和测定和进行测定以评估抗体和Env蛋白， Drs. Nussenzweig和Bjorkman核心将培训和协助学生，博士后研究员， 研究助理和研究人员在分析和解释数据的自动化 分析。自动化细胞/生化分析核心将保留两个定制装备的Evo Freedom液体处理站和表面等离子体共振仪器， 本项目中提出的自动化检测的吞吐能力。