Abstract
To reduce the window period between HIV-1 infection and the ability to diagnose it, a fourth-generation immunoassay including the detection of HIV-1 p24 antigen has been developed. However, because the commercially available systems for this assay use special, high-cost instruments to measure, for example, chemiluminescence, it is performed only by diagnostics companies and hub hospitals. To overcome this limitation, we applied an ultrasensitive ELISA coupled with a thio-NAD cycling, which is based on a usual enzyme immunoassay without special instruments, to detect HIV-1 p24. The p24 detection limit by our ultrasensitive ELISA was 0.0065 IU/assay (i.e., ca. 10-18 moles/assay). Because HIV-1 p24 antigen is thought to be present in the virion in much greater numbers than viral RNA copies, the value of 10-18 moles of the p24/assay corresponds to ca. 103 copies of the HIV-1 RNA/assay. That is, our ultrasensitive ELISA is chasing the detection limit (102 copies/assay) obtained by PCR-based nucleic acid testing (NAT) with a margin of only one different order. Further, the detection limit by our ultrasensitive ELISA is less than that mandated for a CE-marked HIV antigen/antibody assay. An additional recovery test using blood supported the reliability of our ultrasensitive ELISA.
Highlights
During the window period between infection with human immunodeficiency virus type 1 (HIV-1) and the appearance of detectable antibodies to HIV-1, the infection cannot be diagnosed
We obtained three linear calibration curves for HIV-1 p24 antigen in the range of 0.1 - 1.0 IU/mL that were provided with the ultrasensitive enzyme-linked immunosorbent assay (ELISA) coupled with a thio-NAD cycling (Fig 2)
We claim that the ultrasensitive ELISA coupled with a thio-NAD cycling succeeds in detecting p24 at the attomole level
Summary
During the window period between infection with human immunodeficiency virus type 1 (HIV-1) and the appearance of detectable antibodies to HIV-1, the infection cannot be diagnosed. Attempts to shorten this period have been made using a fourth-generation immunoassay that detects both HIV-1/2 IgG/M and HIV-1 p24 antigens [1, 2]. Most of the commercially available detection systems for fourth-generation immunoassays use chemiluminescent measurement and need special, high-cost, automated measurement instruments. For this reason, fourth-generation immunoassays are performed only at diagnostics companies and hub hospitals. To overcome this limitation and to test many samples simultaneously, it is PLOS ONE | DOI:10.1371/journal.pone.0131319 June 22, 2015
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