Ultrasensitive Bispecific Antibody Assay for SARS Virus

概述（由申请人提供）：1、广泛、长期目标：目前的目标是使用新的针对SARS抗原的单克隆药物开发廉价的护理点诊断方法。同样的抗体也可能具有治疗作用。2。(a)开发针对SARS相关包膜(E)、膜糖蛋白(M)、核衣壳蛋白（NP）或刺突蛋白（SP）的高亲和力单克隆抗体，这些单克隆抗体可用于同型夹心（重复表位，如在整个病毒中）或异型夹心（如在脱落的单体抗原中）测定；(b)制备对NP（或SG）和HRPO具有特异性的双特异性抗体；(c)优化在微孔板上对整个SARS病毒进行超灵敏尼龙搭扣均匀夹心测定的方法(d)为从喉咙或痰中收集的样本开发免疫拭子或试纸测定方法，以进行筛选应用。3。研究设计：各种SARS抗原将通过短期和长期方案，通过s.c、i.p.和静脉注射对小鼠进行免疫。免疫后的脾细胞和骨髓瘤细胞融合形成抗原特异性杂交瘤。不同克隆的杂交瘤上清液将用重组抗原检测同型和异型两种格式的抗原。我们还将开发quadromas以产生双特异性单克隆抗体（bsMAb），其中一个假面能够与SARS抗原结合，另一个假面能够与酶标记物如过氧化物酶结合。我们将探索不同的ELISA格式来检测整个病毒、NP或其他脱落抗原。免疫拭子试验将使用市售的带有柔性铝轴的鼻咽拭子进行优化，用于使用bsMAbs直接检测SARS病毒或其脱落抗原。另外，我们还将测试用碰撞金标记的单克隆抗体的效用，以开发一种原型免疫拭子试验，可能用于快速筛选可疑个体。所提出的免疫拭子检测方法的简单性是它在机场、高风险社区和卫生保健工作者筛查中的实用性。

DESCRIPTION (provided by applicant): I. BROAD,LONG-TERM OBJECTIVES: The current objective is to develop inexpensive point of care diagnostics using new monoclonals against SARS antigens. The same antibodies may have therapeutic utility as well. II. SPECIFIC AIMS: (a)To develop high affinity MAbs against SARS associated envelope (E), membrane glycoprotein (M), the nucleocapsid protein (NP) or spike proteins (SP) useful in homosandwich(repeat epitopes as in whole virus) or heterosandwich (as in shed monomeric antigens) assay; (b) To develop bispecific antibodies with specificity to NP (or SG) and HRPO; (c) To optimize an ultrasensitive molecular velcro homosandwich assay of the whole SARS virus on microplates(d) To develop an immunoswab or dipstick assay for samples collected from throat or sputum for screening applications. III. RESEARCH DESIGN: The various SARS antigens will be used to immunize mice by short-term and long-term protocols with s.c., i.p.,and i.v. injections. Immunized splenocytes and myeloma cells will be fused to obtain antigen specific hybridomas. The hybridoma supernatants from different clones will be tested for their ability to detect antigens in homosandwich and heterosandwich formats with recombinant antigen. We will also develop quadromas to generate bispecific monoclonal antibodies (bsMAb) with one paratope capable of binding to SARS antigen and the other to an enzymatic marker like peroxidase. We will explore different ELISA formats for detection of whole virus, for NP or other shed antigens. The immunoswab assay will be optimized using the commercially available nasopharyngeal swab with a flexible aluminum shaft for direct detection of SARS virus or its shed antigen using bsMAbs. Alternatively, we will also test the utility of our Mabs labeled with collidal gold to develop a prototype immunoswab assay that could potentially be used for rapid screening of suspected individuals. The simplicity of the proposed immunoswab assay is its utility in screening at airports and high risk communities and health care workers.