LOWERING THE DETECTION LIMITS OF HIV-1 VIRAL LOAD USING REAL-TIME IMMUNO-PCR FOR HIV-1 P24 ANTIGEN Niel T. Constantine, Ph.D., Daniel Edelman, M.S., Janet Barletta, Ph.D. University of Maryland, Baltimore, School of Medicine, Baltimore, MD Poster #40 HIV Diagnostics Meeting March 2005 Orlando, Florida Objective To develop a new HIV p24 antigen detection method that offers an enhanced sensitivity that can detect HIV infection earlier than current methods and can monitor HIV infection at viral loads below 50 RNA copies/ml. Rationale (see Figure 1) HIV RT-PCR maximum analytical sensitivity=50 copies or 25 virions. p24 antigen molecules (p24Ag) outnumber RNA molecules 1500:1 since 1 HIV virion contains ~3000 p24Ag molecules but only 2 copies RNA. Detection of p24 Ag at 75,000 molecules (~3 fg) should approximate the sensitivity of RT-PCR for HIV RNA. A protein detection method capable of detecting fewer than 75,000 molecules of p24 antigen (<3 fg) will detect HIV infection earlier than can RT- PCR, and may provide information for effective therapeutic monitoring when copy number is low. Figure 1: Marker Kinetics During HIV Infection Early in HIV infection, and when effective therapy is provided, RNA is below RT-PCR’s lower limit of detection. Ultra-sensitive methods for p24 detection have the potential to detect infection earlier and at lower copy number (red line). Methods: I-PCR Principle: see Figure 2 Detection Limits (see Figure below) A standard curve was generate using dilutions of HIV-1 p24 Ag from HIV- 1 infected cell culture supernatants quantified by ELISA. These dilutions included log fold differences from 10 3 to 10 8 HIV-1 p24 molecules Reproducibility replicates from HIV-1 antibody positive patients with known HIV-1 RNA viral loads (determined by the Amplicor HIV-1 Monitor Test) were diluted within groups of three logs (between viral RNA copies), and then analyzed by real-time IPCR. In addition to these samples, 52 samples from HIV-1 infected persons (antibody positive) who had viral loads <50 RNA copies/mL were tested by real-time IPCR. Introduction/Background Currently, nucleic acid tests can detect infection by HIV as soon as 12 days after infection, and are capable of identifying as few as 50 copies RNA/mL. These tests have been instrumental in protecting the blood supply and monitoring response to antiretroviral therapy. More sensitive methods that detect lower levels of viremia could provide a safer blood supply and assist clinicians in better assessing virologic responses to therapy. We developed a real-time Immuno-PCR (IPCR) method that combines the specificity of serologic detection with the exquisite sensitivity of molecular techniques to detect HIV p24 antigen (p24Ag) in serum. We defined the analytical sensitivity of the IPCR method with p24 antigen positive tissue culture fluid, and challenged it using patient samples with known viral RNA copy numbers, including those below 50 RNA copies/mL. Figure 2: Principles of Immuno-PCR A typical sandwich ELISA is performed, followed by the addition of biotinylated reporter DNA, which binds streptavidin conjugated to a biotinylated 2˚Ab. This DNA can then be amplified by real-time PCR, and the product detected by fluorogenic probe binding and hydrolysis. Results When derived from the IPCR standard curve, a dose response was observed by IPCR for samples with known viral loads diluted at , , and 5179 – 6514 copies/mL In addition, IPCR detected 42 % of patient samples which could not be detected by RT-PCR (i.e., <50 RNA copies/mL). The limit of detection for the IPCR was equivalent to 20 viral RNA copies/mL and 0.66 viral RNA copies per reaction; this corresponded to 40 attograms/reaction of p24 Ag and approximately 1000 HIV-1 p24 Ag molecules. Conclusions Immuno-PCR for HIV p24 antigen detection was shown to be more analytically sensitive for the detection of viremia than approved nucleic acid tests. It has the potential to confirm the diagnosis of HIV-1 at an earlier time than current methods, and should be valuable to monitor the response to anti-retroviral treatment when RNA copy number is below 50 copies/mL. Published: Barletta, Edelman, Constantine, Am J Clin Path: 122, 20-27, 2004