Simple diffusion-constrained immunoassay for p24 protein with the sensitivity of nucleic acid amplification for detecting acute HIV infection
| Autor: | Kaitlin A. Minnehan, Cheuk W. Kan, Brian A. Pink, David Hanlon, David Fournier, David C. Duffy, David H. Wilson, Lei Chang, Purvish P. Patel, Linan Song, Evan P. Ferrell |
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| Rok vydání: | 2012 |
| Předmět: |
Immunoassay
P24 capsid protein biology medicine.diagnostic_test Clinical Laboratory Techniques HIV Core Protein p24 HIV HIV Infections Nucleic acid amplification technique p24 Virology Molecular biology Sensitivity and Specificity Virus Single molecule array Antigen Digital ELISA Nat biology.protein medicine Nucleic acid Humans Antibody |
| Zdroj: | Journal of virological methods. 188(1-2) |
| ISSN: | 1879-0984 |
| Popis: | Nucleic acid amplification techniques have become the mainstay for ultimate sensitivity for detecting low levels of virus, including human immunodeficiency virus (HIV). As a sophisticated technology with relative expensive reagents and instrumentation, adoption of nucleic acid testing (NAT) can be cost inhibited in settings in which access to extreme sensitivity could be clinically advantageous for detection of acute infection. A simple low cost digital immunoassay was developed for the p24 capsid protein of HIV based on trapping enzyme-labeled immunocomplexes in high-density arrays of femtoliter microwells and constraining the diffusion of the enzyme–substrate reaction. The digital immunoassay was evaluated for analytical sensitivity for HIV capsid protein p24, and compared with commercially available NAT methods and immunoassays for p24, including 4th-generation antibody/antigen combo assays, for early detection of HIV in infected individuals. The digital immunoassay was found to exhibit 2000–3000-fold greater analytical sensitivity than conventional immunoassays reactive for p24, and comparable sensitivity to NAT methods. Assaying serial samples from 10 HIV-infected individuals, the digital immunoassay detected acute HIV infection as early as NAT methods, and 7–10 days earlier than conventional immunoassays. Comparison of assay results between the digital immunoassay and a quantitative NAT method from HIV infected serum exhibited a linear correlation R2>0.99. The data indicate that by constraining diffusion of the signal generation step of a simple sandwich immunoassay and enabling the digital counting of immunocomplexes, dramatic improvements in sensitivity to virus can be obtained to match the sensitivity of NAT at a fraction of the cost. |
| Databáze: | OpenAIRE |
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