SARS CoV2 Studies in the Microbial Pathogenesis Section/LPD

微生物发病机制部分的 SARS CoV2 研究/LPD

基本信息

批准号：
10272279
负责人：
Stephen Leppla
金额：
$ 7.8万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
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/10272279
关键词：
2019-nCoV Antibodies Area Biological Biological Assay Cells Chemistry Complex Coronavirus DNA DNA Probes Detection Diagnostic Diagnostic Procedure Dot Immunoblotting Enzyme-Linked Immunosorbent Assay Enzymes Eukaryotic Cell Fluorescent Dyes Human Papillomavirus Hybrids Impairment Lateral Membrane Methods Modeling Modification Monoclonal Antibodies Nucleic Acids Pathogenesis Plasmids Preparation Production Property RNA Reagent Recombinant Proteins Recombinants Reporter Resources SARS coronavirus Sampling Sensitivity and Specificity Signal Transduction Site Stress System Testing Therapeutic Agents Therapeutic Monoclonal Antibodies Transfection Viral Work cost diagnostic assay enzyme activity improved microbial pandemic disease point-of-care diagnostics polyclonal antibody scale up sortase success viral DNA viral RNA viral detection

项目摘要

Using model nucleic acids, we established methods for membrane and plate assays (i.e., dot blots and ELISAs) that show good sensitivity and specificity for detection of DNA/RNA hybrids. Many controls have been used to demonstrate that the assay detects DNA/RNA hybrids rather than other types of nucleic acids that might be present in a complex biological sample. In parallel, we have created plasmids for expression of a recombinant S9.6 Fab antibody in eukaryotic cells. Transient transfection of these plasmids into expiCHO cells has the potential to produce the Fab at levels of 1 gm per liter culture. Initial tests have confirmed production of the Fab and scaled-up production is proceeding. The Fab version now being made is equipped with a sortase signal so that site-specific conjugation can be done to attach various materials, such as fluorescent dyes and reporter enzymes. This approach has the potential to improve the diagnostic assays over what can be done with S9.6 that is conjugated by random bifunctional chemistries that might impair either the antibody or the reporter enzyme activities.

使用模型核酸，我们建立了膜和板测定方法（即斑点印迹和 ELISA），这些方法在检测 DNA/RNA 杂交体时显示出良好的灵敏度和特异性。许多对照已被用来证明该测定检测的是 DNA/RNA 杂交体，而不是复杂生物样品中可能存在的其他类型的核酸。与此同时，我们创建了用于在真核细胞中表达重组 S9.6 Fab 抗体的质粒。将这些质粒瞬时转染至 expiCHO 细胞中，有可能产生每升培养物 1 gm 水平的 Fab。初步测试已确认该工厂已投产，并且扩大生产正在进行中。目前正在制造的 Fab 版本配备了分选酶信号，以便可以进行位点特异性缀合以附着各种材料，例如荧光染料和报告酶。与 S9.6 相比，这种方法有可能改进诊断测定，S9.6 是通过随机双功能化学物质缀合的，可能会损害抗体或报告酶的活性。