Journal of Virological Methods 188 (2013) 153–160 Contents lists available at SciVerse ScienceDirect Journal of Virological Methods jou rnal homepage: www.elsevier.com/locate/jviromet Simple diffusion-constrained immunoassay for p24 protein with the sensitivity of nucleic acid amplification for detecting acute HIV infection Lei Chang, Linan Song, David R. Fournier, Cheuk W. Kan, Purvish P. Patel, Evan P. Ferrell, Brian A. Pink, ∗ Kaitlin A. Minnehan, David W. Hanlon, David C. Duffy, David H. Wilson Quanterix Corp, 113 Hartwell Ave, Lexington, MA 02421, USA a b s t r a c t Article history: Nucleic acid amplification techniques have become the mainstay for ultimate sensitivity for detecting Received 8 May 2012 low levels of virus, including human immunodeficiency virus (HIV). As a sophisticated technology with Received in revised form 22 August 2012 relative expensive reagents and instrumentation, adoption of nucleic acid testing (NAT) can be cost Accepted 29 August 2012 inhibited in settings in which access to extreme sensitivity could be clinically advantageous for detection Available online 2 October 2012 of acute infection. A simple low cost digital immunoassay was developed for the p24 capsid protein of HIV based on trapping enzyme-labeled immunocomplexes in high-density arrays of femtoliter microwells Keywords: and constraining the diffusion of the enzyme–substrate reaction. The digital immunoassay was evalu- Digital ELISA Immunoassay ated for analytical sensitivity for HIV capsid protein p24, and compared with commercially available NAT methods and immunoassays for p24, including 4th-generation antibody/antigen combo assays, for early Single molecule array p24 detection of HIV in infected individuals. The digital immunoassay was found to exhibit 2000–3000-fold HIV greater analytical sensitivity than conventional immunoassays reactive for p24, and comparable sensitiv- ity to NAT methods. Assaying serial samples from 10 HIV-infected individuals, the digital immunoassay detected acute HIV infection as early as NAT methods, and 7–10 days earlier than conventional immunoas- says. Comparison of assay results between the digital immunoassay and a quantitative NAT method 2 from HIV infected serum exhibited a linear correlation R > 0.99. The data indicate that by constraining diffusion of the signal generation step of a simple sandwich immunoassay and enabling the digital count- ing of immunocomplexes, dramatic improvements in sensitivity to virus can be obtained to match the sensitivity of NAT at a fraction of the cost. © 2012 Elsevier B.V. All rights reserved. 1. Introduction sensitivity detection of acute HIV infection. NAT has significantly shortened the window between initial infection and its detection Since the development of the polymerase chain reaction in through amplification of viral nucleic acid rather than detecting the 1980s (Mullis et al., 1986), amplification of specific nucleic the presence of antibody following seroconversion (Busch, 2007). acid sequences for genetic identification has become an indis- Although NAT has become the mainstay for detection of viruses, pensible tool in medical research and diagnostics, including the the technology is relatively complex and high cost (Westreich et al., detection and diagnosis of infectious disease. With its imple- 2008), and can be cost inhibitory or prohibitive in settings where mentation for blood screening in the US in 1999 (Busch et al., access to high sensitivity could be clinically advantageous. These 2000), nucleic acid amplification testing (NAT) for detection of scenarios include blood donor screening in lower-resource settings viral pathogens has helped safeguard blood for transfusion by and clinical screening in higher incidence areas, where a significant providing the most sensitive, economically feasible detection of number of cases of acute infection can be missed by less sensi- infected blood donations possible. For HIV detection, NAT meth- tive immunoassay tests. Rapid detection and reporting of acute HIV ods in use for blood screening are capable of analytical sensitivities infection represents a key opportunity to control the march of the of approximately 60 HIV RNA copies/mL (30 viruses/mL) with indi- disease because viral transmission is 10 times more likely during vidual donors (Assal et al., 2009; Proceix ULTRIO Product Insert, the acute phase than in the chronic phase (Wawer et al., 2005). 2011). In the clinical setting, NAT is the gold standard for high Immunoassays, on the other hand, are simpler and lower cost than NAT, and can be deployed in more diverse environ- ments. Conveniently, nature provides its own amplification of ∗ protein molecules from each virus in the form of antibodies to the Corresponding author. E-mail address: [email protected] (D.H. Wilson). virus and the viral proteins. Immunoassays to HIV antibodies are 0166-0934/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2012.08.017 154 L. Chang et al. / Journal of Virological Methods 188 (2013) 153–160 straightforward, but they require the immune response and are genetically engineered Saccharomyces cerevisiae. One amino acid unable to detect acute infection when antibodies are not yet (met) was inserted at the N-terminus to enable expression. present. 3rd generation antibody assays (Constantine et al., 1994) detect the earliest stage of the immune response (immunoglobu- 2.2. Cultured virus lin M), reducing the serological window to approximately 3 weeks (Fiebig et al., 2003). Viral proteins are present at the start of infec- The cultured HIV-1 Group M, Subtype B virus was purchased tion, and each HIV particle produces approximately 2000 copies from Seracare Life Sciences (Part number PN242B, lot 9899P-10, 9 ® of p24 capsid protein (NIAID, 2011), which works in favor of 1.15 × 10 HIV-1 RNA/mL by Roche AMPLICOR HIV-1 MonitorTM immunoassay detection of acute infection. 30 viruses/mL equates Assay, v. 1.5) and diluted in delipidated, defibrinated human to approximately 60,000 p24 molecules per mL, or a concentration serum (Seracare PN: HS-210). This virus is harvested from the of 3 fg/mL. Unfortunately, current, state-of-the-art conventional culture supernatant without additional purification; therefore the immunoassays are not sufficiently sensitive to detect these low standard could contain free p24 in addition to virion-incorporated 7 numbers. Immunoassays for p24 (Kontio, 1991) and 4th genera- p24. The diluted virus, at 1 × 10 copies HIV-1 RNA/mL, was inactiv- tion combo assays that combine reactivity for p24 and anti-HIV ated by the addition of Triton X-100 to 0.4% and incubation of the ◦ antibodies (Weber et al., 1998) can only detect the presence sample for 1 h at 37 C (Ukkonen et al., 1988). of circulating p24 antigen after it has elevated to greater than 11,000–70,000 fg/mL (Marcel et al., 2011). While this can reduce 2.3. Seroconversion panels the detection window another 4 or 5 days relative to serology tests, immunoassays for p24 remain thousands-fold less sensi- Sets of seroconversion serum samples from HIV infected indi- tive for virus than HIV RNA detection, which reduces the window viduals were purchased from SeraCare (PRB956, PRB958, PRB967, an additional week or more relative to antigen detection (Fiebig RB968, PRB969, PRB972) and from Zeptometrix (ZM-HIV9016, ZM- et al., 2003). Thus conventional immunoassays remain blind to HIV9018, ZM-HIV9024, ZM-HIV9031). most of the acute phase window that is detectable by NAT. A simple immunoassay capable of the sensitivity of NAT for HIV could repre- 2.4. Digital immunoassay sent another step forward in broader, more cost effective detection of acute HIV infection, which would help further control the spread Digitization of immunoassay analyte detection using single ® of the disease. molecule arrays (Simoa ) has been described (Rissin et al., 2010, Thermodynamically, antigen–antibody interactions should 2011). In brief, it involves performing a standard paramagnetic enable sufficient sensitivity for detection of fg/mL concentrations microbead-based enzyme-linked immunosorbent assay (ELISA), of antigen with little amplification. The problem for unamplified singulation of individual microbeads in high-density arrays of 50 fL methods has been acquiring sufficient signal of this interaction. microwells, and confinement within the microwells of fluorescent Conventional immunoassays employing optical signal detection product generated by enzymes labeling the immunocomplexes. (such as chemiluminescence) require millions to thousands of Preventing diffusion of the fluorescent product out of the wells per- millions of signal molecule events before signal can be reliably mits rapid buildup of fluorescence that is readily visualized with a discerned by a luminometer or fluorometer. This limitation arises CCD camera. Immunocomplexes are labeled with ␤-galactosidase because the ensemble signal is diluted in a typical reaction vol- (␤G) during the ELISA, and 30 s of conversion of enzyme substrate ume of hundreds of microliters required for measurement. When (resorufin ␤-d-galactopyranoside, RGP) into fluorescent product the signal molecules are constrained from diffusing beyond a small (resorufin) by a single molecule of ␤G provides sufficient signal local volume, then lower numbers of signal molecules would be suf- to differentiate wells containing beads with an enzyme label from ficient for optical detection due to their local high concentration. wells containing beads without an enzyme. At very low