HIV-1 RNA, anti-HIV antibodies, and HIV-1 capsid protein (p24 antigen) are the main viral markers used to detect HIV infection and to monitor disease progression. HIV-1 p24 tests are used in combination with anti-HIV antibody testing for early detection of HIV-1 infection.1,2 Anti-HIV antibody/p24 antigen combination assays can reduce the seroconversion “window period” to an average of 17 days postinfection and aid in the identification of acute infection.2 Assays for HIV-1 p24 are also useful for diagnosing HIV infection in infants because HIV antibody tests can yield false positives because of maternal HIV antibodies. Although nucleic acid testing is the gold standard for pediatric samples, nucleic acid testing assays require complex instrumentation and are sensitive to contamination, which ultimately limits their use in resource-limited settings.
Most commercial HIV-1 p24 assays use a standard enzyme-linked immunosorbent assay (ELISA) format for the capture and detection of p24 antigen. A crucial disadvantage shared by these commercially available ELISAs is that they are capable of detecting only 5–25 pg/mL of HIV-1 p24 antigen in the absence of signal amplification.3 At this level of sensitivity, p24 antigen cannot be detected consistently in HIV-positive subjects, particularly in those with asymptomatic infections. In fact, only about 50%–60% of AIDS patients, 30%–40% of AIDS-related complex patients, and 10% of asymptomatic patients will have p24 antigenemia that is detectable through standard ELISA.4 One of the reasons for poor sensitivity of p24 antigen tests in HIV-infected persons is that free p24 antigen in serum is complexed with p24 antibody. A conventional ELISA test cannot detect complexed antigens, unless an immune complex disruption (ICD) step is used. Thus, free p24 is measured using a non-ICD ELISA procedure, whereas the detection of bound p24 antigen requires pretreatment with acid or heat to dissociate the complex. When an ICD procedure is used, the analytical sensitivity of the assay may actually decrease because of the dilution of sample with the ICD reagent. For example, in PerkinElmer HIV-1 p24 assay, manufacturer claims that the sensitivity of detection is 3.5 pg/mL without the acid-mediated ICD treatment and 26 pg/mL with acid-mediated ICD treatment.5 Thus, limited analytical sensitivity of ICD ELISA tests is also an impediment to clinical implementation of p24 antigen tests.
In recent decades, several signal amplification systems have been developed to increase the analytical sensitivity of ELISA.6–10 Each of these approaches has its advantages and limitations. In this study, we present a photochemical signal amplification method, PSAM, which is based on an autocatalytic photochemical reaction of a commonly used colorimetric substrate for ELISA assays.11 In brief, ELISA + PSAM consists of 2 steps. The first step is a conventional ELISA based on the use of one of the most sensitive chromogenic detection systems: the combination of horseradish peroxidase enzyme and its substrate, ortho-phenylenediamine (OPD). Within this step, horseradish peroxidase catalyzes oxidation of OPD, yielding a yellow solution containing the product of this reaction, the dye 2,3-diaminophenazine (DAP) [Fig. 1, reaction (1.1)]. In the second step, the PSAM system takes advantage of the fact that DAP is a good photosensitizer and the small amount of DAP produced during the enzymatic reaction acts as a catalyst for a subsequent autocatalytic photosensitized amplification reaction [Fig. 1, reaction (1.2)]. The possible mechanism of the photosensitized reaction (1.2) is discussed by Bystryak and Santockyte, and Mekler and Bystryak.11,12
Here we show that ELISA + PSAM allows one to increase the analytical sensitivity and dynamic range of ELISA-based p24 antigen tests by a factor of 40. Because ELISA + PSAM is compatible with most widely used colorimetric ELISA format and requires only an inexpensive illumination device, PSAM can become a valuable tool in the field of medical diagnostics.
MATERIALS AND METHODS
Alliance HIV-1 p24 ELISA kit for detection and quantification of the major structural core component of HIV-1 virus, p24 antigen, was purchased from PerkinElmer Life Sciences (Boston, MA). Pathogen-free human serum and OPD substrate tablets were obtained from Sigma (St Louis, MO). Standard 96-well clear microplates were obtained from R&D Systems (Minneapolis, MN). A Labsystems Multiscan MCC/340 microplate reader was used for optical density measurements.
PerkinElmer HIV-1 p24 ELISA Procedure
Conventional non-ICD ELISA was performed in accordance with the manufacturer's instructions.5 HIV-positive control provided by the manufacturer was diluted in pathogen-free human serum. The conventional ELISA calibration curves for quantification of HIV-1 p24 antigen were both provided by the manufacturer and prepared by us.
Heat-mediated ICD ELISA was performed using the modified version of the manufacturer's Ultrasensitive HIV-1 p24 assay protocol (PerkinElmer), as described by Jennings et al,13 and using a virus disruption buffer (VDB) [30 mM Tris–HCl (pH 7.2), 450 mM NaCl, 1.5% Triton X-100, 1.5% deoxycholic acid sodium salt, 0.3% sodium dodecyl sulfate, 10 mM EDTA] as described by Schupbach et al.14 Briefly, 50 μL of serum was mixed with 25 μL of VDB and incubated at room temperature for 10 minutes. These samples were then treated with 225 μL of pre-diluted kit complex disruption buffer and heat denatured as described in the manufacturer's instructions. We also excluded the tyramide signal amplification (TSA) step15 that requires the use of PerkinElmer ELAST p24 ELISA amplification system.
All core ELISA steps were performed as described in the manufacturer's instructions. Analytical sensitivity of the conventional ELISA tests was determined through the least squares fit to the standard curve at an absorbance equal to the cutoff defined by the manufacturer (ie, mean negative control optical density (OD) + 0.050).
PerkinElmer HIV-1 p24 ELISA + PSAM Procedure
Minor adjustments to the p24 ELISA reaction conditions were made so as to minimize background in the p24 ELISA + PSAM system. This was necessitated because the PSAM reactions performed in conjunction with the conventional p24 ELISA were rather “noisy,” that is, the conventional p24 ELISA was characterized by a high level of nonspecific binding of reagents, which caused amplification of both signal and background. In the non-ICD p24 ELISA + PSAM assays, the steps were the same as for the conventional assay procedure described above, except that we used Sigma OPD tablets instead of the tablets provided by PerkinElmer and decreased incubation time with OPD substrate solution (10 minutes instead of 30 minutes in conventional assay). The OPD substrate solution contained OPD and H2O2 in concentrations of 6.0 × 10−3 M and 4 × 10−3 M, respectively, in 0.05 M phosphate–citrate buffer, pH 5.0. After 10 minutes of incubation with the OPD substrate solution, samples were transferred to the clean microtiter plate (R&D Systems). Thirty microliters of the PSAM reagent solution (250 μM of ascorbic acid in phosphate–citrate buffer)11 was added to 100 μL of OPD substrate solution, and the samples were irradiated in 2-minute increments for the first 4 minutes and in 1-minute increments thereafter for a total of 12 minutes using an in-house made illumination device configured for even illumination of 96-well microtiter plates (see Text, Supplemental Digital Content 1, http://links.lww.com/QAI/A702, which provides the description of the illumination device). The samples were illuminated at an average power density of 0.044 W/cm2. Calibration curves for determination of p24 antigen were prepared by double dilution of positive control in negative control (normal human serum).
In the heat-mediated ICD p24 ELISA + PSAM assays, the steps were the same as in the modified version of Ultrasensitive HIV-1 p24 assay protocol (PerkinElmer), described in refs,13,15 except that 2 changes were made: (1) the TSA step was omitted and (2) the Sigma OPD tablets were used instead of the tablets provided by PerkinElmer. After a 30-minute enzymatic step of OPD oxidation, samples were transferred to R&D Systems microtiter plate and 30 μL of the PSAM reagent solution was added. The samples were irradiated in 2-minute increments for the first 4 minutes and in 1-minute increments thereafter for a total of 9 minutes as described above.
Differences in ELISA procedures used for conventional PerkinElmer ELISA and ELISA + PSAM assays are displayed in Table 1.
Comparison of the Non-ICD ELISA + PSAM and Conventional Non-ICD ELISA
The calibration curves for non-ICD detection of HIV-1 p24 antigen prepared using ELISA + PSAM are shown in Figures 2A, B as compared with conventional non-ICD ELISA (Fig. 2B, closed squares). All data are plotted on log 2 scales to show the best fitted lines for the signal-amplified ELISA. The regression line for the conventional ELISA was obtained after adding stop solution (signal readout at 492 nm) and has the best fit curve if data are plotted on the linear x scale (y = 0.0135x + 0.1023, the best fit curve is not shown). For ELISA + PSAM, the calibration curve obtained after 12 minutes of illumination (Fig. 2A, closed circles) represents the curve yielding the best sensitivity of the assay. Indeed, the lower detection limit was calculated as the analyte concentration corresponding to twice the value of the background signal. Using a regression analysis for the p24 antigen ELISA + PSAM (12-minute illumination procedure), we calculated the lower limit of detection as 0.08 pg/mL. The detection limits of the conventional non-ICD HIV-1 p24 ELISA (PerkinElmer) provided by the manufacturer and according to our calculations are 3.55 and 3.3 pg/mL, respectively. Thus, the analytical sensitivity of the ELISA + PSAM is more than 40-fold higher than that for the conventional assay.
It should be noted that HIV-1 p24 ELISA + PSAM results can be read in both kinetic (0 and 4 minutes of illumination) and endpoint modes (12 minutes of illumination). Curves represented by open and closed triangles (Fig. 2B) correspond to signal-amplified ELISA at 0 and 4 minutes of illumination and are suitable for determination of high HIV-1 p24 antigen concentrations. Indeed, the first OD reading is taken before illumination (Fig. 2B, open triangles). In the ELISA + PSAM procedure, stop solution is not added before the photo-amplification step because the addition of the stop solution inhibits the photochemical amplification reaction. Samples are incubated with OPD for only 10 minutes before amplification and measurement (Table 1). The shortened period of incubation with OPD causes the slope of the “before illumination” calibration curve (ie, the PSAM assay at 0 minutes of illumination) to be lower than that for the corresponding curve prepared according to the manufacturer's instructions (Fig. 2B, closed squares). Although this causes the PSAM assay to be less sensitive before amplification, the net effect is to extend the dynamic range of the assay. In this configuration, the estimated dynamic range of the ELISA + PSAM assay at 0 minutes of illumination is between 80 and 2560 pg/mL (upper detection limit not shown). Here we did not attempt to get the best fitting curve for data points acquired before illumination. Data points obtained during the second OD reading at 4 minutes of illumination (Fig. 2B, closed triangles) were used to construct a calibration curve for measuring p24 antigen concentrations in the range between 5.0 and 500 pg/mL [y = 0.4111 log (x) + 0.0433, where x is the antigen concentration and y is OD]. The final calibration curve (endpoint data) obtained after 12 minutes of illumination (Fig. 2A, closed circles) is used for calculation of p24 antigen concentration in the range between 0.08 and 5.00 pg/mL [y = 0.5663 log (x) + 0.8685]. If all the 3 calibration curves (lines obtained at 0, 4, and 12 minutes of illumination) are combined, the estimated total dynamic range of the assay boosted with PSAM is more than 4 logs, between 0.08 and 2506 pg/mL. Data acquired during kinetic and endpoint readings can easily be pooled using QuantiKin software.16 Thus, the dynamic range of this configuration of the non-ICD ELISA + PSAM is 2 orders of magnitude larger than that of the conventional PerkinElmer non-ICD ELISA (3.5–100 pg/mL).5 It should be emphasized, however, that the dynamic range of the conventional ELISA could be increased in 2 ways: (1) samples with high p24 concentrations can be diluted in such a way that analyte concentrations fall within the dynamic range of the conventional PerkinElmer non-ICD ELISA and (2) using a kinetic mode in which the signal is measured at different times of the substrate solution incubation. Thus, the same upper limit of detection could be achieved using both the conventional and the ELISA + PSAM assays. Because the lower limit of detection of the ELISA + PSAM is increased approximately 40-fold as compared with that for the conventional assay, the dynamic range of ELISA + PSAM is increased at least 40-fold.
Comparison of the Heat-Mediated ICD ELISA Without Signal Amplification, ICD ELISA + PSAM, and Conventional Ultrasensitive p24 ELISA
Figure 3 shows calibration curves for heat-mediated ICD ELISA + PSAM (Figs. 3A, B) as compared with the conventional heat-mediated ICD ELISA (Fig. 3B, closed squares). The regression line for the conventional ICD ELISA has the best fit curve if data are plotted on the linear x scale (y = 0.0064x + 0.0817, the best fit curve is not shown). Heat-mediated ICD ELISA + PSAM assay was more sensitive after 9 minutes of illumination (Fig. 3A, closed circles) with a lower limit of detection of approximately 0.1 pg/mL estimated as twice the value of the background signal as described above. In contrast, the lower detection limit for ICD ELISA without signal amplification is equal to 4.0 pg/mL. Thus, the ELISA + PSAM has 40-fold higher analytical sensitivity as compared with the conventional ELISA. It is worth noting that the documented lower detection limit of the PerkinElmer Ultrasensitive HIV-1 p24 ELISA, which uses a rather complex TSA, is from 0.5 to 1 pg/mL.15,17–24 Therefore, the analytical sensitivity of the ELISA + PSAM is 5- to 10-fold higher than that of the conventional Ultrasensitive assay, which uses the labor-intensive and expensive ELAST amplification system.
Heat-mediated HIV-1 p24 ELISA + PSAM results can be read in both kinetic and endpoint modes the same way as described for non-ICD ELISA. The first OD reading is taken before illumination (Fig. 3B, open triangles). The estimated dynamic range of this variant of the ELISA + PSAM assay at 0 minutes of illumination is between 128 and 8196 pg/mL (upper detection limit not shown), whereas the linear range of conventional heat-mediated ICD ELISA is between 4.0 and 256 pg/mL. Data points obtained during the second OD reading at 4 minutes of illumination (Fig. 3B, closed triangles) were used to construct a calibration curve for measuring p24 antigen concentrations in the range between 8.0 and 512 pg/mL [y = 0.4102 log (x) − 0.093, where x is the antigen concentration and y is OD]. The final calibration curve obtained after 9 minutes of illumination (Fig. 3A, closed circles) is used for calculation of p24 antigen concentration in the range between 0.1 and 8.0 pg/mL [y = 0.4215 log (x) + 0.5425]. All 3 calibration curves (lines obtained at 0, 4, and 9 minutes of illumination) can be combined to get an estimated total dynamic range of the assay between 0.1 and 8196 pg/mL, which is more than 4 logs in magnitude.
It is worth noting that the conventional heat-mediated ICD ELISA has a 2 log dynamic range. Thus, ELISA + PSAM has almost 2 orders of magnitude larger dynamic range than the conventional assay. The Ultrasensitive ICD ELISA has a 4 log dynamic range if sample optical density is measured in both kinetic and endpoint modes. This range is comparable with the estimated dynamic range of the ICD ELISA + PSAM.
Our results obtained using the p24 ELISA system confirm that ELISA + PSAM could dramatically increase the detection sensitivity and extend the dynamic range of a commercially available assay. Given the clinical challenges involved in the detection and treatment of AIDS, PSAM technology holds the potential to significantly improve the diagnostic utility of p24 immunoassays. Indeed, our experiments show that the analytical sensitivity of the p24 antigen assays and, consequently, their dynamic range can potentially be increased approximately 40-fold as compared with the conventional tests. PSAM technology increases analytical sensitivity of conventional ELISA and requires only one additional short step of illumination (9–12 minutes) of the microtiter plate using an inexpensive illumination device. Amplification occurs as a result of the photochemical amplification of DAP produced within enzymatic reaction of the regular colorimetric ELISA.11
It is worth noting that in all instances, the error (coefficient of variation) of the measurements using the PSAM system did not exceed 15%, which is acceptable for immunoassays (Figs. 2, 3).
It should also be emphasized that the aforementioned calibration curves and calculations of the analytical sensitivity for ELISA and ELISA + PSAM assays are based on the use of “ideal” negative (normal) sera. To determine a real cutoff of the assay, at least several dozen clinical negative samples must be run. An increase in cutoff value of the assay in real clinical samples as compared with that for ideal negative serum is a common phenomenon. For example, the cutoffs for p24 positivity and for p24 negativity for the Ultrasensitive PerkinElmer HIV-1 p24 assay using VDB and Tanzanian plasma samples were calculated as corresponding to 3.5 and 2.3 pg/mL, respectively.25 These values are 2.5- to 7-fold higher than those for the ideal analytical sensitivity (0.5–1 pg/mL) of the same assay. With respect to ELISA + PSAM, preclinical studies including examination of hundreds of clinical negative and positive samples are underway, and the results on the ELISA + PSAM clinical sensitivity and specificity will be presented in the near future elsewhere.26 We should mention here, however, that our preliminary data obtained for 25 negative sera show that real cutoffs for non-ICD and heat-mediated ELISA + PSAM correspond to 0.15 and 0.3 pg/mL, respectively,26 and these values are 2- to 3-fold higher than the corresponding ideal analytical sensitivities. We should also note that the real cutoff for non-ICD ELISA + PSAM is equal cutoff corresponding to its analytical sensitivity when the method is used for determination of recombinant p24 antigen in cell culture supernatants.
Several signal amplification strategies have been applied to p24 ELISA assays, in an effort to address the need for more sensitive detection of p24. One approach is to boost the signal intensity using amplification systems. For example, the aforementioned TSA is the method that has been applied to the p24 antigen test in an attempt to increase its analytical sensitivity.9 It should be noted, however, that the TSA is labor intensive and includes a time-consuming TSA step (Table 1), which introduces a high coefficient of variation. Moreover, the cost of TSA is much higher than that for PSAM: TSA and PSAM reagents add approximately $15 and $1 per sample, respectively, whereas the cost of the projected PSAM illumination device, $2,000, will approach zero in a clinical setting performing tens of thousands of tests.
Another approach, immuno-polymerase chain reaction amplification method, uses an oligonucleotide as a surrogate marker and polymerase chain reaction for its amplification. This method has been applied to the p24 antigen assay and, at present, is claimed to be the most sensitive p24 antigen test with the detection limit of 10–100 HIV-1 p24 molecules per reaction (in ag/mL concentration range) and 4-log dynamic range.27 This test has a potential to detect HIV-1 infection earlier than nucleic acid tests.27 However, this assay is very intricate, extremely sensitive to contamination, has reproducibility issues, and requires complex equipment. Moreover, it is not commercially available, perhaps because of the aforementioned shortcomings.
Several new p24 tests that use different platforms have the potential to offer enhancement in the detection limit over the traditional colorimetric ELISA and have been described in the literature. Gold nanoparticle-based bio-barcode amplification assay claims sensitivity of 0.1 pg/mL.28–30 A novel HIV-1 p24 antigen single-molecule immunosorbent assay has the detection limit of 0.1 pg/mL of p24 antigen, and a linear dynamic range of the assay is over 3 orders of magnitude between 0.1 and 100 pg/mL.31 An Ultrasensitive Microsphere Immunoassay with TSA and the heat-mediated immune complex disruption procedure using commercially available anti-p24 has a sensitivity of 1 pg/mL.32 An immunofluorescent cytometric bead assay is another flow cytometry–based assay that offers a wide dynamic measurement range and allows for the detection of p24 concentrations over 4 orders of magnitude from less than 0.4 up to 20,000 pg/mL.33 These techniques uniformly require complex optical or flow cytometric equipment that complicates their use in the resource-limited settings. It should also be emphasized that all aforementioned techniques are complex, involve numerous incubation and wash steps, and are labor intensive. Consequently, none have yet been implemented in commercially available formats.
Although a number of methods that allow for ultrasensitive detection of protein analytes have been developed, these methods are generally characterized by complex protocols, expensive reagents, or a combination of both. The advantages of the ELISA + PSAM assay over other signal amplification methods include ultrasensitive detection capabilities and extended dynamic range of the assay without the use of the complex and expensive equipment and without significant changes in assay procedure. Unlike other ultrasensitive techniques, the PSAM is simple and straightforward, requiring minimal effort to achieve markedly improved results from commercially available ELISA reagent systems.
The authors wish to acknowledge the financial support of National Institutes of Health. In addition, the authors would also like to thank Dr. Addison D. Ault for assistance in the preparation of the manuscript.
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