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Detection of HIV-1 p24 at Attomole Level by Ultrasensitive ELISA with Thio-NAD Cycling.

Nakatsuma A, Kaneda M, Kodama H, Morikawa M, Watabe S, Nakaishi K, Yamashita M, Yoshimura T, Miura T, Ninomiya M, Ito E - PLoS ONE (2015)

Bottom Line: 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.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.An additional recovery test using blood supported the reliability of our ultrasensitive ELISA.

View Article: PubMed Central - PubMed

Affiliation: Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Sanuki, Japan.

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. 10(3) copies of the HIV-1 RNA/assay. That is, our ultrasensitive ELISA is chasing the detection limit (10(2) 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.

No MeSH data available.


Related in: MedlinePlus

Linear calibration curves for HIV-1 p24 antigen obtained by an ultrasensitive ELISA coupled with a thio-NAD cycling.The absorbance of thio-NADH was measured at a cycling reaction of 90 min. Three data measured in different plates are presented as A, B and C. LOD is short for the limit of detection.
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pone.0131319.g002: Linear calibration curves for HIV-1 p24 antigen obtained by an ultrasensitive ELISA coupled with a thio-NAD cycling.The absorbance of thio-NADH was measured at a cycling reaction of 90 min. Three data measured in different plates are presented as A, B and C. LOD is short for the limit of detection.

Mentions: 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 ELISA coupled with a thio-NAD cycling (Fig 2). The curves were obtained from the absorbance of thio-NADH at a cycling reaction of 90 min. The data were measured in different plates. One linear calibration curve is expressed as y = 0.27x + 0.019, R2 = 0.99 (Fig 2A). The limit of detection of p24 was 0.0055 IU/assay (i.e., ca. 2 x 10−18 moles/assay). We thus claim that the ultrasensitive ELISA coupled with a thio-NAD cycling succeeds in detecting p24 at the attomole level. The minimum limit of determination of p24 was 0.0275 IU/assay (i.e., ca. 1 x 10−17 moles/assay).


Detection of HIV-1 p24 at Attomole Level by Ultrasensitive ELISA with Thio-NAD Cycling.

Nakatsuma A, Kaneda M, Kodama H, Morikawa M, Watabe S, Nakaishi K, Yamashita M, Yoshimura T, Miura T, Ninomiya M, Ito E - PLoS ONE (2015)

Linear calibration curves for HIV-1 p24 antigen obtained by an ultrasensitive ELISA coupled with a thio-NAD cycling.The absorbance of thio-NADH was measured at a cycling reaction of 90 min. Three data measured in different plates are presented as A, B and C. LOD is short for the limit of detection.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4476629&req=5

pone.0131319.g002: Linear calibration curves for HIV-1 p24 antigen obtained by an ultrasensitive ELISA coupled with a thio-NAD cycling.The absorbance of thio-NADH was measured at a cycling reaction of 90 min. Three data measured in different plates are presented as A, B and C. LOD is short for the limit of detection.
Mentions: 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 ELISA coupled with a thio-NAD cycling (Fig 2). The curves were obtained from the absorbance of thio-NADH at a cycling reaction of 90 min. The data were measured in different plates. One linear calibration curve is expressed as y = 0.27x + 0.019, R2 = 0.99 (Fig 2A). The limit of detection of p24 was 0.0055 IU/assay (i.e., ca. 2 x 10−18 moles/assay). We thus claim that the ultrasensitive ELISA coupled with a thio-NAD cycling succeeds in detecting p24 at the attomole level. The minimum limit of determination of p24 was 0.0275 IU/assay (i.e., ca. 1 x 10−17 moles/assay).

Bottom Line: 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.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.An additional recovery test using blood supported the reliability of our ultrasensitive ELISA.

View Article: PubMed Central - PubMed

Affiliation: Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Sanuki, Japan.

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. 10(3) copies of the HIV-1 RNA/assay. That is, our ultrasensitive ELISA is chasing the detection limit (10(2) 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.

No MeSH data available.


Related in: MedlinePlus