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Background:  The nanoparticle-based bio-bar code amplification (BCA) assay is a simple, rapid, homogeneous assay for the detection and quantification of target analytes. Here we developed a nanoparticle-based BCA assay for quantitative measurements of HIV p24^CA and p7^NC antigens in serum or plasma at point-of-care.

Methods:  The BCA assay employs 100-nm magnetic microparticles functionalized with antibodies against the HIV p24^CA or p7^NC protein and 30-nm gold nanoparticles functionalized with both oligonucleotide barcodes and antibodies against a non-overlapping region of the cognate viral protein. In the presence of HIV p24^CA or p7^NC, the magnetic microparticles and the gold nanoparticles form sandwich structures that are magnetically separated from plasma. After dehybridization of the DNA barcodes on the gold nanoparticle, the released barcodes are quickly identified by a nanoparticle-based scanometric detection. We analyzed plasma samples that were confirmed to be positive (n = 16) or negative (n = 10) for HIV by antibody and nucleic acid amplification testing. All study participants were men enrolled in the Chicago component of the MACS. For the HIV-infected men, levels of HIV-1 RNA measured by quantitative RT-PCR ranged between < 50 to 77,000 copies/mL of plasma.

Results:  All men infected with HIV had > 5 pg of p24^CA/mL of plasma; a quantity below the limit of detection by the commercial HIV p24^CA assay. Of 16 plasma samples from men with proven HIV infection, all 16 were positive for immune complex dissociated p24^CA and p7^NC by the BCA assay. Each positive value for antigens exceeded the threshold signal intensity of the nanoparticle-based scanometric detection method by at least 3 orders of magnitude. Of 10 uninfected men, all 10 were negative. Thus, the tests with plasma samples identified the status of HIV infection correctly for all 26 men.

Conclusions:  These data show that the BCA assay coupled with a nanoparticle-based colorimetric detection method offers an innovative, rapid approach to HIV detection and quantification. Demonstration of measurable amounts of HIV p24^CA in plasma obtained from men with < 50 copies of RNA per mL of plasma (25 fg of p24^CA/mL) illustrates that the results from the test can exceed the limit of detection of conventional ELISA-based immunoassays by more than 1000-fold. The BCA assay is robust, sensitive, specific, and easy-to-use for measurement of protein levels; attributes that make it a suitable diagnostic for resource-limited settings.

Keywords: Nanoparticle based bio-barcode amplification assay; HIV detection; p24 CA and P7 NC