Ultrasensitive Bispecific Antibody Assay for SARS Virus

描述(由申请人提供)：i.广泛的、长期的目标：目前的目标是开发使用针对SARS抗原的新的单克隆的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.具体目的：(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)的高亲和力单抗，用于同源抗原(如全病毒中的重复表位)或异源抗原(如在脱落单体抗原中)；(B)开发针对NP(或SG)和HRPO的双特异性抗体；(C)优化在微板上对SARS病毒全病毒进行超灵敏的同种或间接免疫印迹分析的方法；(D)建立用于筛查从咽喉或痰中采集的样本的免疫学试纸或试纸。研究设计：各种SARS抗原将通过皮下注射、腹腔注射和静脉注射的短期和长期方案用于免疫小鼠。打针。将免疫的脾细胞与骨髓瘤细胞融合，获得抗原特异性杂交瘤细胞。来自不同克隆的杂交瘤上清液将用重组抗原检测同种和异种形式的抗原的能力。我们还将开发四倍体来产生双功能单抗(BsMAb)，其中一个对偶能够与SARS抗原结合，另一个能够与过氧化物酶等酶标记物结合。我们将探索不同的检测全病毒、NP或其他脱落抗原的ELISA法。免疫拭子检测将通过使用带有柔性铝轴的商用鼻咽拭子进行优化，用于使用bsMAbs直接检测SARS病毒或其脱落抗原。或者，我们还将测试我们的单抗与碰撞金标记的效用，以开发一种可能用于快速筛查可疑个体的免疫波分析原型。拟议的免疫拭子检测的简单之处在于它在机场和高危社区和卫生保健工作者的筛查中的实用性。

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.