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

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.

Urinary adiponectin as a new diagnostic index for chronic kidney disease due to diabetic nephropathy

“…An ultrasensitive ELISA coupled with thionicotinamide-adenine dinucleotide (thio-NAD) cycling was originally developed by Watabe and Ito 11 23–26. Shortly, a sandwich method using a primary and a secondary antibody for antigens is employed in an ELISA.…”

Immunoreactive Insulin in Diabetes Mellitus Patient Sera Detected by Ultrasensitive ELISA with Thio-NAD Cycling

“…By contrast, our colorimetric ultrasensitive ELISA provides several major advantages in terms of cost and simplicity, without requiring a specialized instrument. However, this sensitivity of 10 16 moles/assay does not surpass that for recombinant human insulin (10 19 moles/assay) (19) or that for HIV-1 p24 (10 18 moles/assay) (31), which have also been obtained by our ultrasensitive ELISA. This issue of sensitivity thus seems to depend on the antigen and antibodies used.…”

Advances in biosensing strategies for HIV-1 detection, diagnosis, and therapeutic monitoring

“…, horseradish peroxidase, fluorescent molecules, quantum dots). ELISA tests have had remarkable success with detecting p24 antigens as shown by a recent publication, which used an inexpensive approach to achieve an attomolar limit of detection [105]. ELISA based tests have also been used to detect T cell CD4 proteins collected from a fingerpick blood sample, and have a 97% sensitivity when the CD4 + T lymphocyte count is as low as 350 cells/μL, suggesting that it could be used to quantify response to ART in resource-limited settings.…”