SARS CoV2 Studies in the Microbial Pathogenesis Section/LPD

微生物发病机制部分的 SARS CoV2 研究/LPD

• 批准号: 10692239
• 负责人: Stephen Leppla
• 金额: $ 5.14万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10692239
• 关键词: 2019-nCoV; Alkaline Phosphatase; Antibodies; Antibody Specificity; Area; Bacillus anthracis; Biological Assay; C-terminal; COVID detection; Chimeric Proteins; Chinese Hamster Ovary Cell; Complex; Crystallization; DNA; DNA Probes; Detection; Development; Diagnostic Procedure; Diagnostic Reagent Kits; Dot Immunoblotting; Embryo; Enzyme-Linked Immunosorbent Assay; Enzymes; Equipment; Fab Immunoglobulins; Future; Genetic; Human Papillomavirus; Hybrids; Immunoglobulins; Lateral; Lead; Licensing; Messenger RNA; Methods; Modeling; Modification; Molecular; Monoclonal Antibodies; Mus; National Institute of Diabetes and Digestive and Kidney Diseases; Nucleic Acids; Pathogenesis; Pathologic; Peptidyltransferase; Preparation; Production; Property; Protein phosphatase; Proteins; Publications; RNA; Reagent; Recombinant Proteins; Recombinants; Reporter; Research Personnel; Resource-limited setting; Resources; SARS coronavirus; Single-Stranded DNA; Stress; Structure; Surface; Suspension Culture; Swab; System; Testing; Therapeutic Agents; Therapeutic Monoclonal Antibodies; Toxoplasma gondii; United States National Institutes of Health; Vagina; Variant; Viral; Work; antigen binding; base; coronavirus disease; cost; detection method; diagnostic assay; diagnostic platform; diagnostic tool; dimer; improved; interest; lateral flow assay; microbial; milligram; monomer; nanoGold; pandemic coronavirus; pathogen; point-of-care diagnostics; polyclonal antibody; sortase; success; tool; viral DNA; viral RNA; viral detection

The monoclonal S9.6 antibody has the unique property of recognizing DNA/RNA hybrids in a sequence-independent manner. Previously, we popularized this antibody and made it available to hundreds of researchers. We worked with NIH tech transfer staff to license this antibody to several leading reagent supply companies. S9.6 has become a key tool for the study of R-loops, which are natural structures, but ones that become excessively abundant in certain pathological situations. A polyclonal antibody recognizing DNA/RNA hybrids is the basis of hybrid capture ELISA-type commercial diagnostic assays for human papilloma virus (HPV) DNA in vaginal swabs. It follows that the S9.6 antibody offers a generic approach to detection and quantitation of any nucleic acid of known sequence. Development of this diagnostic platform could lead to methods for detection of any pathogen of interest. During FY2022 we continued to improve methods for production of the S9.6 immunoglobulin (IgG) antibody in other formats including single-chain variable fragment (scFv), fragment antigen-binding region (Fab), and molecularly well-defined Fab-reporter conjugates. Thus, we prepared the S9.6 Fab as a recombinant protein equipped with a C-terminal sequence recognized by the sortase transpeptidase. After also producing secreted embryonic alkaline phosphatase (SEAP) with a Gly3 N-terminus, the two proteins were conjugated by sortase in excellent yield to produce a purified and fully defined S9.6 Fab-SEAP conjugate. Once this conjugate proved useful in various diagnostic assay formats (described below), we extended this work to produce the S9.6 Fab (heavy chain)-SEAP protein as a genetic fusion. Because SEAP exists only as a dimer, this genetic fusion protein is effectively bivalent, with an S9.6 Fab attached to each of the SEAP monomers in the dimer. All these S9.6 proteins were produced in Chinese hamster ovary (CHO) cells grown in suspension culture, an efficient system that can yield > 50 milligrams protein per liter. The purified S9.6 antibody variants and conjugates were employed during FY2022 in a variety of assay formats for detection of RNA of Bacillus anthracis and Toxoplasma gondii, as models for future application to COVID RNA detection. In one successful format, hybrids produced from poly-biotinylated single-stranded DNA probes and complementary B. anthracis mRNA were captured on a polystreptavidin-coated assay plate and detected using S9.6 and HRP-conjugated anti-mouse IgG. We also established assays like the HPV ELISA assay, where hybrids are captured on surface-bound S9.6 IgG and then detected with an S9.6 Fab-SEAP conjugate. To make S9.6-based assays more convenient for low-resource areas, we initiated work to implement a lateral flow assay. Our collaborator Marco Biamonte (DDTD, CA) developed an S9.6 IgG-gold nanoparticle conjugate, which we employed to identify SARS-CoV-2 specific DNA:RNA hybrids on a lateral flow strip. Further optimization of this format is continuing. As previously described, work with S9.6 as a diagnostic tool would be aided by understanding the basis for the specificity of the antibody. Our collaborators Charles Bou-Nader and Jinwei Zhang of NIDDK had successfully produced crystals of the S9.6 Fab in complex with a small DNA/RNA hybrid. This work has now been completed by publication of the structure of the complex. Having a structure will facilitate rational approaches to optimizing the selectivity of the antibody.

单克隆S9.6抗体具有独特的独特特性，即以序列无关的方式识别DNA/RNA杂种。以前，我们普及了这种抗体，并将其提供给数百名研究人员。我们与NIH技术转移人员合作，将该抗体许可给几家领先的试剂供应公司。 S9.6已成为研究自然结构的R-loops研究的关键工具，但在某些病理情况下会过多。 识别DNA/RNA杂种的多克隆抗体是阴道拭子中人乳头状瘤病毒（HPV）DNA的杂种捕获ELISA型商业诊断测定的基础。因此，S9.6抗体提供了一种通用方法来检测和定量已知序列的任何核酸。这种诊断平台的开发可能导致检测任何感兴趣的病原体的方法。 在2022年期间，我们继续改善其他格式中S9.6免疫球蛋白（IgG）抗体的生产方法，包括单链可变片段（SCFV），片段抗原结合区域（FAB）和分子定义明确的Fab-Reporter-Reporter-Reporter-Reporter-Reporter-Reporter-Reporter-Reporter。因此，我们准备了S9.6 FAB作为一种重组蛋白，该蛋白具有由分子酶转肽酶识别的C末端序列。在与Gly3 n末端产生了分泌的胚胎碱性磷酸酶（SEAP）之后，通过排序酶以出色的产量结合了这两种蛋白质，以产生纯化的纯化且完全定义的S9.6 Fab-Seap-Seap-seap-conjugate。一旦这种共轭被证明在各种诊断测定格式中有用（如下所述），我们将这项工作扩展到生产S9.6 Fab（重链） - SEAP蛋白作为遗传融合。由于SEAP仅作为二聚体存在，因此该遗传融合蛋白有效地二价，S9.6 Fab附着在二聚体中的每个SEAP单体上。所有这些S9.6蛋白都是在悬浮培养物中生长的中国仓鼠卵巢（CHO）细胞中产生的，这是一种有效的系统，每升可产生> 50毫克的蛋白质。 纯化的S9.6抗体变体和偶联物在2022年期间采用多种测定格式使用，用于检测炭疽芽孢杆菌和弓形虫弓形虫的RNA，作为未来用于共证RNA检测的模型。在一种成功的格式中，通过在多蛋白蛋白糖蛋白涂层的测定板上捕获了由多生物素化的单链DNA探针产生的杂种和互补的B.炭疽杆菌mRNA，并使用S9.6和HRP结合的抗小鼠IgG检测。我们还建立了HPV ELISA测定法，该测定法在杂种中捕获在表面结合的S9.6 IgG上，然后用S9.6 s.6 fab-eap-eap conjugate检测。 为了使基于S9.6的测定对于低资源区域更方便，我们开始了实施横向流量测定法的工作。我们的合作者Marco Biamonte（DDTD，CA）开发了S9.6 IgG-gold纳米颗粒结合物，我们采用了该SARS-COV-2特异性DNA：侧向流动带上的RNA混合体。这种格式的进一步优化仍在继续。 如前所述，通过了解抗体的特异性的基础，将使用S9.6作为诊断工具的工作。我们的合作者Charles Bou-Nader和Niddk的Jinwei Zhang成功地生产了S9.6 Fab的晶体，并具有小型DNA/RNA混合动力。这项工作现已通过公布综合体的结构来完成。具有结构将有助于优化抗体选择性的有理方法。