HIV type 1 (HIV-1) RNA, capsid protein (p24 antigen), and anti-HIV antibodies are the major viral markers that have been used for detecting HIV-1 infection, screening blood donors, monitoring disease progression, and evaluating HIV-1 therapy.1,2 With the advent of sophisticated molecular biological techniques, HIV-1 nucleic acid amplification testing (NAT) assays have been widely used since 1999 and, since the time of NAT assay licensure in 2003, have replaced p24 antigen assay in blood-donor screening in the United States, mainly owing to the high sensitivity and early detection of HIV-1 infection that NAT provides.3 However, NAT assays generally require complex instrumentation for target amplification and detection, and are sensitive to contamination, which ultimately limits their utility in point-of-care venues, especially in resource-limited countries where simple, inexpensive, rapid, and accurate detection formats are needed. In addition, considering the high genetic diversity of HIV-1, the sensitivity of NAT assays might be impaired by the sequence diversity of HIV-1. A possible alternative to testing for HIV-1 RNA is HIV-1 p24 antigen, which is usually detected by enzyme-linked immunosorbent assay (ELISA). During the past decade, HIV-1 p24 antigen assays have been significantly improved by implementing immune complex disruption methods,4,5 using more effective lysis buffer,6 and incorporating a tyramide-mediated boosted assay.7 It has been reported that the boosted ELISA can decrease the lower limit of detection (LOD) of p24 antigen detection to about 1 pg/mL.4,8-10 Another sensitive method called real-time immuno-polymerase chain reaction (IPCR) assay can detect 1000 HIV-1 RNA copies or 40 attograms of HIV-1 p24 antigen per reaction.11 By using these improved p24 antigen assays, it has been found that HIV-1 p24 may be a useful marker for predicting CD4+ T-cell decline and disease progression.12 HIV-1 p24 may also be useful for early detection of HIV-1 infection, resulting in earlier diagnosis and improved patient management. Other potential applications are in testing the blood supply in regions of the world where HIV-1 RNA testing is not available or practical.
Over the past decade, several nanotechnology-based techniques have been widely evaluated in medical testing and could provide new tools for clinical diagnosis due to their potential for high degrees of sensitivity, high specificity, multiplexing capabilities, and ability to operate without enzymes.13,14 In particular, an ultrasensitive nanoparticle (NP)-based assay, called the biobarcode amplification (BCA) assay, was originally introduced by Mirkin and colleagues for the detection of both protein and nucleic acid in the absence of enzymatic reactions.15,16 The BCA assay uses oligonucleotides (the biobarcodes) as surrogates for the indirect amplification of the disease markers and a microarray-based chip detection method that utilizes NP probes and a silver amplification process for signal enhancement and optical readout. The assay, in certain settings, allows one to detect proteins such as prostate-specific antigen (PSA) with an LOD of 30 attomolar (aM)15,17-21 or nucleic acid targets at the level of 500 zeptomolar (zM).16,22,23 In principle, this ultrasensitive NP-based testing system, when applied to HIV-1 p24, could provide a more sensitive alternative to the ELISA-based systems, provided it performs well in the relevant sample types. Herein we report a BCA assay for sensitive and early detection of HIV-1 p24 antigen.
MATERIAL AND METHODS
HIV-1-negative normal serum samples by serology were obtained from a Food and Drug Administration (FDA) HIV laboratory safety routine collection in Bethesda, MD, and kindly provided by Dr. Owen Wood. Twenty-three HIV-1 seroconversion panel samples from 12 donors were originally obtained from ZeptoMetrix (Buffalo, NY) and kindly provided by Dr. Philip Norris under code. Samples from different time points (2 weeks before and up to a month after the first positive HIV-1 PCR test) that had previously shown to be HIV-1 p24 and RNA positive or negative on prior HIV-1 p24 assays or viral load assays were specifically chosen to evaluate the ability of the assay to detect HIV-1 p24 in the early phase of infection. According to the records, 15 samples were HIV-1 RNA positive by PCR (group 1). The median viral load was 4.81 (2.00 to about 6.45) log10 RNA copies/mL. Among them, 13 samples were also HIV-1 p24 positive; one was anti-HIV positive; and one was both HIV-1 p24 and anti-HIV negative. Another 8 samples were HIV-1 RNA, HIV-1 p24, and anti-HIV negative. Ten AIDS patient plasma samples (group 2) with high viral load were selected from patients treated on research protocols in the HIV and AIDS Malignancy Branch of the National Cancer Institute, part of the National Institutes of Health (NIH) in Bethesda, MD. The protocols were approved by the NCI Institutional Review Board, and all patients gave informed consent. Their average CD4 accounts and viral load were 93 ± 33/mL and 5.46 (4.96 to about 5.83) log10 RNA copies/mL, respectively. In addition, 20 blood donor samples identified as positive by NAT (group 3), from HIV-1 seroconverters, were provided by Dr. Susan Stramer. All the samples were anti-HIV negative. Viral load was 3.15 (2.00 to about 5.88) log10 RNA copies/mL. All of the samples were coded and tested in a blinded fashion.
Antigen, Antibody, and DNA
HIV-1 p24 antigen standard with known concentration was kindly provided by Dr. Robert J. Gorelick (AIDS Vaccine Program, SAIC-Frederick, Frederick, MD) or obtained from HIV-1 p24 ELISA kits (PerkinElmer, Boston, MA). The anti-HIV monoclonal antip24 antibody #353724 and antip24 antibody #652125 were obtained through the NIH AIDS Research and Reference Reagent Program in Germantown, MD. Biotinylation of the secondary anti-HIV antibodies was performed using an EZ-Link Sulfo-NHS-LC-Biotin kit (Pierce, Rockford, IL). Biotinylated biobarcode DNA LT68, amine-terminated biobarcode capture DNA, and thiol-functionized dT20 probe DNA were ordered from Integrated DNA Technologies (Coralville, IA).
The detailed method of performing an ELISA has been described previously.24,26 Nunc Immuno Maxisorp strips (Fisher Scientific, Pittsburgh, PA), coated with monoclonal antip24 antibody (#3537) at 2.5 μg/μL, were incubated at 37°C for 1 hour with 100 μL of a series of standard p24 dilutions in 1 × phosphate-buffered saline (PBS)/0.025% Tween 20/0.1% BSA (phosphate-buffered saline/Tween [PBS-T]) and appropriate dilutions of samples (anti-HIV-positive samples were acid disrupted and neutralized before testing). After washing 5 times with PBS-T, 100 μL of biotinylated antip24 antibody (#6521) in appropriate dilutions in PBS-T was added and incubated 30 minutes at 37°C, followed by further washing and incubation with diluted peroxidase-streptavidin conjugate (Pierce, Rockford, IL) for 30 minutes at 37°C. Finally, O-phenylenediamine (OPD) substrate (Pierce) was added and incubated for 30 minutes at room temperature, followed by quantification with a microtiter plate reader (Molecular Devices, Sunnyvale, CA). The cutoff value was the sum of the means of the absorbance of 8 negative controls plus 3 standard deviations (SD). Samples with signal-to-cutoff (S/CO) ratios ≥1.00 were considered positive for HIV-1 p24 antigen.
The capture of HIV-1 p24 antigen and binding to the biotinylated antip24 antibody were done as in the ELISA described above. After washing, 100 μL of assay buffer containing 6.6 μg/μL tRNA and 1010 streptavidin-coated 15-nm gold NPs (British Biocell, Cardiff, UK) were added and incubated at 37°C for 30 minutes, followed by further washing and incubation with 1 nM biotinylated biobarcode DNA LT68 for 30 minutes at 37°C. Finally, the strips were washed 5 times with PBS-T and rinsed in 50 μL of 98% formamide and 10 mM ethylenediamine tetraacetic acid (EDTA, pH 8.0). The biobarcode DNA LT68 was released by incubation at 80°C for 5 minutes and detected by a scanometric assay.27
CodeLink Activated slides (Amersham Biosciences, Piscataway, NJ) were printed with biobarcode capture oligonucleotides (complementary to the specific 25mer biobarcode LT68 sequence) and control sequences (noncomplementary to the LT68 sequence). Microarray hybridization reactions were carried out by loading 50-μL barcodes in hybridization buffer (40% formamide, 3× saline-sodium citrate (SSC), and 0.02% Tween 20) per array and incubating at 40°C for 90 minutes. After removing the hybridization solution, 40 μL of the dT20 gold NP chip-probe solution (1 nM gold NP, 10% formamide, 3× SSC, and 0.02% Tween 20) was added and allowed to hybridize for 30 minutes at 40°C. Then the slides were washed once with 0.5 N NaNO3 and once with 0.1 N NaNO3. The detection of the biobarcode signal was done by pouring Signal Enhancement mixture (Nanosphere, Northbrook, IL) over slides and incubating at 20°C for 5 minutes and 30 seconds. Reactions were stopped by rinsing in pure H2O. Slides were then scanned and quantified on the Verigene ID image analyzer (Version 1.1.6, Nanosphere). The cutoff value was the average signal intensity of negative controls plus 3 times the SD. Samples with S/CO values ≥1.00 were considered positive for HIV-1 p24 antigen.
All of the statistical analyses were performed with GraphPad Prism software (Version 4.0, GraphPadSoftware, San Diego, CA). The unpaired t test was used to compare the differences between sample means of different groups. The correlation analysis was performed by nonparametric Pearson rank to test the relationship between the concentration of HIV-1 p24 and p24 S/CO ratios.
BCA Assay Scheme and Sensitivity in Detecting HIV-1 p24 Antigen
As shown in Figure 1, the BCA assay used in these studies adapts a sandwich immunoassay format to capture its target. Typically, HIV-1 p24 antigen is captured by an antip24 antibody coated on the surface of microtiter plate wells and further complexed with a secondary antip24 antibody labeled with biotin molecules. In the assay developed here, the sandwiched complex is then coupled to streptavidin-coated gold NPs through biotin-streptavidin interaction. The biotinylated biobarcode DNAs are bound to the free streptavidin molecules at the surface of the NPs. After extensive washing between steps with detergent solution to remove unbound or nonspecifically bound conjugates, the so-formed Ab-Ag-Ab-NPs complex is heated to 80°C to release the biobarcode DNA into the supernatant when the DNA is detected by the scanometric method.27 For detection of biobarcode DNA, it is first captured by the probes immobilized on the surface of slides that are complementary to half of the biobarcode DNAs, followed by a second hybridization of another half of the biobarcode DNAs with oligonucleotides that functionalize the surface of the NPs. With the addition of silver amplification solution (hydroquinone and Ag+), silver ions are reduced to silver metal, which grows on the NPs. This process significantly increases the ability of NPs to scatter light. The sensitivity of this scanometric detection system can be 100 times greater than that of the analogous, conventional PCR-less fluorescence-based assays.27 We used the NP-based BCA assay and conventional colorimetric in-house ELISA to establish a calibration curve for the positive HIV-1 p24 antigen control with known concentration (Fig. 2). The BCA assay exhibited a detection range from 0.1 to 500 pg/mL, whereas the lower LOD for ELISA was about 15 pg/mL. A good correlation between HIV-1 p24 concentration and BCA signal intensity (S/CO) was demonstrated (Pearson correlation coefficient r = 0.9357; R2 = 0.8756; P < 0.0001) and characterized as a linear dose-dependent model (Fig. 2). These results demonstrate that the HIV-1 p24 BCA assay offers 150-fold improvement in the lower detection limit over the traditional colorimetric ELISA under the current conditions.
Sensitivity and Specificity of BCA Assay in Detecting HIV-1 p24
Serum samples from 30 HIV-1-negative adults and 45 HIV-1-positive plasma samples from 3 groups were evaluated by BCA assay. As shown in Figure 3, the distribution of the S/CO ratios for HIV-1-positive samples was very different from that for HIV-1-negative human control samples. The S/CO values for HIV-1-negative samples were considerably <1.00, and the average signal intensity was 0.57 ± 0.2. In contrast, the S/CO values for all HIV-1-positive samples were >1.00, although a small fraction (5 of 45, 11.1%) of the samples exhibited S/CO values of <1.5. The average signal intensity for HIV-1-positive samples was 5.9 ± 6.0. The difference of the mean signal intensity between HIV-1-positive and HIV-1-negative samples was statistically significant (unpaired t test, P < 0.0001). The average p24 concentration (log10 fg/mL) was 2.9 ± 0.5, 3.3 ± 0.5, and 3.0 ± 0.5 for group 1, 2, and 3 of HIV-1-positive samples, respectively. In addition, the correlation between HIV-1 viral load (log10 RNA copies/mL) and p24 antigen (log10 fg/mL) was demonstrated to be significant (r = 0.4434, P < 0.01). Taken together, our data indicated that our BCA assay generated no false positive results with 30 HIV-1-negative samples, and it found all 45 HIV-1 RNA positive samples to be HIV-1 p24 positive. The HIV-1 p24 antigen BCA assay correlated well with the HIV-1 RNA viral load assay.
Early Detection by BCA Assay of HIV-1 p24 in Seroconversion Samples
To test the capacity of the HIV-1 p24 BCA assay for early diagnosis of HIV-1 infection, 23 samples (representing different time points after infection) from 12 donors were analyzed for HIV-1 p24 in parallel by the BCA assay and in-house ELISA. The day of the first HIV-1 qualitative PCR positive was arbitrarily defined as day 0 of the timescale for each donor. The BCA assay could detect HIV-1 p24 in 15 specimens that were also HIV-1 RNA positive, whereas the conventional HIV-1 p24 ELISA detected only 8 of them (data not shown). As shown in Figure 4, the BCA assay could detect HIV-1 p24 at 7 days after the first PCR positive test in 4 donors (no. 1, 6, 7, and 10) and as early as the same day of positive PCR in 1 donor (no. 2). In contrast, the earliest date of positive HIV-1 p24 by ELISA was 11 days after the first PCR positive test in donor 10. The average number of days at which HIV-1 p24 was first detected by BCA and ELISA were 12 and 15 days after detection of HIV-1 RNA, respectively. These results indicated that the BCA assay was more sensitive than the conventional ELISA and could detect HIV-1 p24 antigen around 3 days earlier than ELISA in this panel of samples.
Unlike nucleic acid molecules, which can be amplified directly by enzyme-mediated molecular techniques such as PCR to significantly increase their detection sensitivity, protein molecules per se cannot yet be duplicated in in vitro testing assays. The current signal booster methods for protein assays rely on the amplification of surrogate markers. For example, IPCR uses oligonucleotides as a surrogate marker and PCR for amplification. The BCA assay increases its detection sensitivity mainly through 2 steps: 1) the amplification process that occurs as a result of the large number of biobarcode DNA strands that bind to the NPs and are released from Ag-Ab-Ag-NPs complexes in each antigen recognition and binding event15,16 and 2) a highly sensitive biobarcode detection process that involves NP-based silver enhancement and a microarray method.27,28 BCA techniques have been successfully used for sensitive detection of PSA;15,17,20 human chorionic gonadotropin (HCG) and α-fetoprotein (AFP);20 amyloid-β-derived diffusible ligand (ADDL), a possible pathogenic Alzheimer disease marker;18 and interleukin-2 (IL-2).19 We have now utilized a novel BCA technique for HIV-1 p24 antigen detection. Our results are consistent with other reports and confirm that the BCA technique provides higher sensitivity than the conventional ELISA and can reach PCR-like sensitivity in the absence of enzymatic reactions.15,17-20
Theoretically, the ultrasensitive BCA assay could enable the detection of as few as hundreds of target molecules.17,18 Because each HIV-1 virion contains around 3000 copies of p24 molecules29 or at least 1200 to 1500 copies of p24 molecules,30,31 a lower LOD would be expected with further optimization. It should be noted that the current NAT assays can routinely detect 400 to 500 HIV-1 RNA copies/mL, with reported LODs as low as 50 HIV-1 RNA copies/mL.32-34 Our preliminary results indicate that the lower LOD of our BCA assay is 100 fg/mL (equal to around 2000 RNA copies/mL) under current assay conditions, which is slightly higher than PCR. One explanation is that like other immunoassays, the ultrasensitive nature of the BCA assay is strongly influenced by the efficiency of target capture, which in turn relies on the binding affinity and specificity of antibody pairs used in the assay system.35 Large LOD differences have been observed for immunoassays that use different antibody pairs, indicating that antibody selection is critical for assay sensitivity.36 In our study, we have only screened several antibodies obtained from the NIH AIDS Research and Reference Reagent Program (http://www.aidsreagent.org; accessed July 18, 2007), and the results we obtained with these antibodies are encouraging. This leaves great potential for further improvement of the BCA assay for sensitive detection of HIV-1 p24 antigen.
In our study, we modified the BCA assay by using a microtiter plate in place of antibody-coated magnetic particles (MMPs) to capture the p24 target. When compared to a traditional ELISA, the BCA assay uses additional reagents including streptavidin and DNA-coated gold probes, DNA biobarcodes, signal-enhancement solution, and a DNA-coated glass substrate to detect DNA barcodes. These additional reagents add minimal assay cost because only a very small amount of gold probe and barcode DNA is required for each individual assay. Nanosphere's Verigene ID Reader was used to quantify the amount of DNA barcode (and therefore protein) detected in our assay. It was chosen on the basis of providing high-sensitivity nanoparticle detection using relatively simple, low-cost optics (no moving parts for image acquisition)28 compared to other commercially available systems. The advantages of the modified BCA assay are that 1) all the reagents are universal and can be used for different targets; 2) the entire assay does not include any enzymatic reactions for target amplification; 3) target capture is based on an ELISA format widely used in laboratories and clinics; 4) it does not need specific instruments and training; and 5) it is more suitable for high-throughput screening. The current assay format could be finished within 6 hours. We are now further simplifying the assay by using highly fluorescent europium nanoparticles to replace the microarray detection system. This assay could be completed within 2 to 3 hours (Tang et al, manuscript in preparation). Nam and colleagues19 have recently reported a simple colorimetric bio-barcode assay that could reach attomolar sensitivity without using a chip-based microarray detection system. These modifications can further simplify the assay, decrease the reaction time, and potentially reduce cost significantly. We believe that with these modifications, the BCA technology could be useful for diagnostics and blood-donor testing, particularly in resource-limited settings where NAT is not feasible or practical.
In conclusion, the modified BCA assay for HIV-1 p24 antigen detection showed no false positive results using HIV-1-negative, healthy adult serum samples and 100% sensitivity for HIV-1 RNA positive plasma samples from acute or end-stage HIV infection. It is 150-fold more sensitive and detects HIV-1 p24 antigen 3 days earlier than the conventional ELISA. Additionally, there is a good correlation between the HIV-1 p24 BCA and HIV-1 viral load assays. To the best of our knowledge, this is one of the first reports demonstrating the potential applicability of nanotechnology in HIV diagnosis. However, these data should be considered preliminary in that the BCA assay needs to be evaluated with a larger panel of both HIV-1-positive and HIV-1-negative samples, including samples that contain high RNA/DNA loads and other potentially interfering proteins from other viral agents. Studies comparing HIV-1 RNA and p24 in clinical specimens are warranted because HIV RNA levels may not run completely in parallel with p24 titers in HIV-1 patients. In some AIDS patients under long-term antiviral treatment, HIV-1 RNA is negative but HIV-1 p24 is positive.12,37 Considering that HIV-1 p24 is more stable and easier to handle than HIV-1 RNA,38,39 HIV-1 p24 antigen testing based on new approaches such as the BCA technology, which can provide further improvements in assay sensitivity, may be a useful tool to detect and monitor HIV-1 infection in settings where NAT is currently not routinely performed.
We wish to acknowledge the FDA Office of Science and Health Coordination for funding this work. Chad A. Mirkin acknowledges support from the NIH and the National Science Foundation. In addition, he is grateful for an NIH Director's Pioneer Award. We wish to acknowledge Dr. Owen Wood, Kathleen M. Wyvill, and Dr. Robert Gorelick for providing plasma samples and reagents; Dr. C. Shad Thaxton, Dimitra Georganopoulou, Savka Stoeva, and Jae-Seung Lee for technical support; Dr. Steven Wolinsky for helping to initiate the project; and Dr. Carol Weiss, Dr. Wood, and Hira Nakhasi for review of the manuscript. The findings and conclusions in this article have not been formally disseminated by the FDA and should not be construed to represent any agency determination or policy.
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Keywords:© 2007 Lippincott Williams & Wilkins, Inc.
HIV-1; p24 antigen; nanoparticle; biobarcode; detection