title: Single-molecule immunosorbent assay as a tool for human immunodeficiency virus-1 antigen detection.
abstract: Ultrasensitive detection and quantification of viral antigen with a novel single-molecule immunosorbent assay (SMISA) was achieved. Antigen from human immunodeficiency virus type 1 (HIV-1), the major etiological agent of acquired immune deficiency syndrome, served as the screening target in this study. The target molecule was sandwiched between a polyclonal capture antibody and a monoclonal detector antibody. The capture antibody was covalently immobilized on (3-glycidoxypropyl) trimethoxy silane-modified glass slides. The detector antibody was conjugated with fluorescent Alexa Fluor 532 labeled secondary antibody prior to being used as a probe for the antigen. Imaging was performed with a total internal reflection fluorescence single-molecule detection system. This technique is demonstrated for detecting HIV-1 p24 antigen down to 0.1 pg/mL with a dynamic range of over four orders of magnitude. A Langmuir isotherm fits the molecule count dependence on the target concentration. The target antigen was further tested in 20% human serum, and the results showed that neither sensitivity nor dynamic range was affected by the biological matrix. SMISA is therefore a promising approach for the early diagnosis of viral induced diseases.
authors.affiliation: Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, IA, 50011, USA.
authors.forename: Jiangwei
authors.initials: J
authors.lastname: Li
authors.forename: Wenjun
authors.initials: W
authors.lastname: Xie
authors.forename: Ning
authors.initials: N
authors.lastname: Fang
authors.forename: Edward S
authors.initials: ES
authors.lastname: Yeung
mesh_terms: D000956:Antigens, Viral / Q000032:analysis* / Q000737:chemistry; D015497:HIV-1 / Q000276:immunology* / Q000302:isolation & purification*; D007163:Immunosorbent Techniques / Q000295:instrumentation*; D015394:Molecular Structure
chemical_list: D000956:Antigens, Viral