Single-molecule immunosorbent assay as a tool for human immunodeficiency virus-1 antigen detection
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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.
KeywordsSingle-molecule immunosorbent assay (SMISA) Early diagnosis HIV-1 Fluorescence detection
We thank Dr. Mary Jo Schmerr and Dr. Gufeng Wang for valuable discussions. E.S.Y. thanks the Robert Allen Wright Endowment for Excellence for support. The Ames Laboratory is operated for the U.S. Department of Energy by Iowa State University under contract no. DE-AC02-07CH11358. This work was supported by the Director of Science, Office of Basic Energy Science, Division of Chemical Sciences.
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