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Detection of HIV-1 Specific Monoclonal Antibodies Using Enhancement of Dye-Labeled Antigenic Peptides Kim E. Sapsford,† Juan B. Blanco-Canosa,‡ Philip E. Dawson,‡ and Igor L. Medintz*,§ Division of Biology, Office of Science and Engineering Laboratories, FDA, Silver Spring, Maryland 20993, Departments of Cell Biology and Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, and Center for Bio/Molecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory, Washington, DC 20375. Received August 19, 2009; Revised Manuscript Received December 3, 2009
A simple bifunctional colorimetric/fluorescent sensing assay is demonstrated for the detection of HIV-1 specific antibodies. This assay makes use of a short peptide sequence coupled to an environmentally sensitive dye that absorbs and emits in the visible portion of the spectrum. The core peptide sequence is derived from the highly antigenic six-residue epitope of the HIV-1 p17 protein and is situated adjacent to a terminal cysteine residue which enables site-specific fluorescent labeling with Cy3 cyanine dye. Interaction of the Cy3-labeled p17 peptide with monoclonal anti-p17 antibody resulted in an up to 4-fold increase in dye absorption and greater than 5-fold increase in fluorescent emission, yielding a limit of detection as low as 73 pM for the target antibody. This initial study demonstrates both proof-of-concept for this approach and suggests that the resulting sensor could potentially be used as a rapid screening method for HIV-1 infection while requiring minimal equipment and reagents. The potential for utilizing this assay in simple field-portable point-of-care and diagnostic devices is discussed.
INTRODUCTION Halting the spread of human immunodeficiency virus or HIV (along with many other virulent diseases) is a continuing international health priority. In developing countries where healthcare funding, facilities, and treatment are limited, this is of particular concern as infection rates continue to grow. Reducing infection and providing adequate treatment in the third world will be predicated on the development of rapid diagnostics for HIV screening that are sensitive, cheap, robust, and simple to use, have long-term stability, and require minimal equipment and limited-to-no sample preparation. In the most simplistic terms, these diagnostics must be suitable for use in extreme resource-limited environments (UAIDS/WHO report (2007) AIDS epidemic update http://data.unaids.org/pub/EPISlides/ 2007/2007_epiupdate_en.pdf). Simultaneous developments in two related technologies will help achieve this goal, namely, simple point-of-care (POC) devices combined with robust diagnostic assays. The last two decades have seen tremendous progress in microfabricated POC devices to the point where transition to the field and deployment around the world is now a growing reality (WHO report (2004) Performance evaluations of HIV test kits http://www.who.int/diagnostics_laboratory/ publications/evaluations/en/index.html) (1-4). Although PCRbased detection of conserved HIV-1 DNA sequences has been proposed (1, 5), testing for the presence of serological antibodies against HIV is still among the most common initial screening techniques (1). These tests often target the viral envelope glycoprotein antigens from HIV type 1 or type 2 (HIV-1, HIV2), while others screen for the presence of core viral antigen proteins (1). Among the latter, the HIV-1 p17 protein has been found to play an active role in viral infection, and antibodies reactivity to a number of the gag-proteins including p17 have also been shown to significantly decrease at the later stages of * Corresponding author.
[email protected]. † FDA. ‡ The Scripps Research Institute. § U.S. Naval Research Laboratory.
infection (6-9). According to a recent UNAIDS/WHO report, 33 million people (∼0.5% of the world population) are living with HIV with the majority of cases being directly attributable to infection with the HIV-1 strain. This establishes the p17 protein as a prime target antigen for HIV detection that can also be more specifically reflective of the initial stages of HIV-1 infection (5). Immunoassay-based techniques such as Western blotting and enzyme-linked immunosorbent assays (ELISAs) are considered the gold standard for the detection of p17 exposure. Various other methods have also been developed that utilize surfaceimmobilized p17 protein as a capture reagent for detection of HIV-1 p17 specific antibodies (10, 11). However, all techniques including PCR commonly require the use of dedicated laboratory equipment for analysis as compared to control samples and standards. Furthermore, the use of the full p17 protein as a capture reagent can be problematic from a field-portable perspective in that functionalized surfaces need to be prepared and will require storage in a buffered environment and/or refrigeration. Clearly, simple and robust immunoassays for HIV detection that can function in complement with POC devices are still needed. Plaxco’s group have been developing peptide-based beacons, which function in a manner somewhat analogous to DNA molecular beacons, and initial attempts to utilize them for detecting HIV-1 specific p17 antibodies have yielded rather promising results (12, 13). In their approach, a short peptide sequence comprising a highly antigenic six-residue epitope of the HIV-1 p17 protein was labeled with both a long-lived ruthenium(II) bisbipyridine-phenanthroline fluorophore exhibiting a ∼800 ns lifetime at one terminus and the electronaccepting contact-quencher methyl viologen at the other terminus. Significant quenching of the fluorophore was observed in the unstructured polypeptide while in solution until addition and subsequent binding of the target, anti-p17 antibody forced the peptide into a rigid extended conformation, separating the fluorophore and quencher and generating a 6-fold increase in fluorophore emission (13). The resulting optical biosensor
10.1021/bc9003712 2010 American Chemical Society Published on Web 01/08/2010
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demonstrated a limit of detection (LOD) for p17 HIV-1 antibody of 250 pM and was functional in both serum and saliva samples. Although a sensing configuration utilizing this particular fluorophore combination may not yet be directly transferrable to the simplest field-portable devices, it does highlight several inherent advantages. The use of an extremely short peptidyl sequence as opposed to the full p17-protein is preferred, as it can be readily dried down and stored lyophilized for long periods of time prior to reconstitution in an assay. Additionally, monitoring of antibody binding to labeled peptide simplifies the overall assay procedure significantly. Here, we combine a p17 HIV-1 antibody-specific short peptide sequence with a fluorescent Cy3 dye to create a simplified sensor specific for anti-p17 antibodies that is equally functional in both colorimetric and fluorescent assays. This proof-of-concept study demonstrates a significantly lower LOD than previously described sensors and highlights the simplicity of this approach for generalized screening purposes.
Sapsford et al. Scheme 1. Cy3-p17-Peptide Interactions with Monoclonal r-p17 Antibodya
EXPERIMENTAL SECTION Materials. All chemicals were reagent grade and used as received from the manufacturer. Phosphate buffered saline (10 mM phosphate, 137 mM NaCl, 2.7 mM KCl, pH 7.4, PBS), N-[2-hydroxyethyl]piperazine-N′[2-ethane sulfonic acid] (HEPES), bovine serum albumin (BSA), imidazole, dimethyl sulfoxide (DMSO), HPLC-grade acetonitrile and Corning 96-well white polystyrene nonbinding surface (NBS) microtiter plates were obtained from Sigma-Aldrich. Chicken immunoglobulin G protein (IgG) was obtained from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA). Murine monoclonal antibody specific for HIV-1 p17 protein (R-p17, Clone 32/ 1.24.89 IgG1 isotype that reacts to p17 residues 17-22 EKIRLR) was purchased from Zeptometrix Corporation (Buffalo, NY). Cy3-maleimide monoreactive dye (λmax absorption 550 nm, λmax emission 570 nm,) was purchased from GE Healthcare (Piscataway, NJ). Nickel-nitroloacetic acid (Ni-NTA) agarose media was purchased from Qiagen (Valencia, CA). Oligonucleotide purification cartridges (OPC) and triethylamine acetate buffer (TEAA) were obtained from Applied Biosystems (Foster City, CA). Peptide Synthesis. The antigenic peptide recognized by the monoclonal R-p17 antibody (p17-peptide) was prepared using standard in situ neutralization cycles for Boc-solid-phase-peptide synthesis (Boc-SPPS) as described (14, 15) and consisted of the sequence CEKIRLRSGLGAibAAAWGGHHHHHH-NH2 (Mw of 2652, Aib is the non-natural amino acid R-aminoisobutyric acid, and NH2 is an amide group blocking the C-terminal carboxyl in this sequence). Following synthesis and deprotection, the peptide was purified and isolated by HPLC and both the purity (>99%) and the sequence confirmed by mass-spectral analysis (see Supporting Information for data). The modular design of this peptide (16), as highlighted by the different colors in Scheme 1, includes a unique N-terminal cysteine allowing site-specific dye-labeling, the six-residue EKIRLR antigenic epitope recognized by the R-p17, an AibAAA R-helical spacer which is disrupted by flanking G residues, W allowing UV absorption monitoring if needed, and a C-terminal hexahistidine sequence (His)6 which provides an affinity handle for purification via metal-affinity chromatography. Fluorescent Dye Labeling of p17-Peptide. The peptide was labeled and purified in a manner similar to the method described in detail in ref 17. Briefly, ∼1 mg of the purified p17-peptide was dissolved in 1 mL 10× PBS pH 7.4 and combined with a large excess of Cy3-maleimide monoreactive dye to ensure complete labeling. The reaction mixture was incubated overnight at 4 °C, and unreacted Cy3 dye was removed using three consecutive 0.5 mL Ni-NTA-agarose columns. The reaction
a (Not to scale). The peptide contains several functional modules including a (His)6 affinity sequence for purification over Ni-NTA media (blue), an Aib-alanine containing helical spacer sequence that is disrupted by flanking glycine residues (green), the antibody recognition element (orange), and a unique terminal cysteine (red) for site-specific fluorophore labeling. R-p17 antibody binding to the Cy3-p17-peptide alters the peptide structure and/or dye environment significantly enhancing both dye absorption and emission and thus transducing signal.
mixture was loaded onto Ni-NTA-agarose media as the Cterminal (His)6 sequence provides a specific affinity handle for this type of purification. Bound peptide was then washed with 10 mL 1× PBS to remove unreacted free dye, and the Cy3labeled p17-peptide (abbreviated as Cy3-p17-peptide) was eluted using 300 mM imidazole in 1× PBS. A reverse-phase media containing OPC was then used to remove the imidazole and desalt the Cy3-p17-peptide. The OPC cartridge was first equilibrated with 3 mL acetonitrile followed by 3 mL 2 M TEAA before the Cy3-p17-peptide was loaded in. The cartridge was then washed with 50 mL 0.02 M TEAA before the Cy3p17-peptide was eluted with ∼1 mL 70% acetonitrile in H2O. The OPC media were regenerated for further rounds of Cy3p17-peptide purification, as needed, by washing with 3 mL acetonitrile followed by 3 mL 2 M TEAA until the original Ni-NTA-imidazole elutant became clear. Desalted Cy3-p17peptide was quantitated using UV-vis spectroscopy by measuring the Cy3 absorption (150,000 M-1 cm-1 at 550 nm) before being aliquoted, dried down, and stored at -20 °C in a desiccator until required. Assays Monitoring Binding of Anti-p17 Antibody to Cy3p17-Peptide. Interactions of Cy3-p17-peptide with murine R-p17 antibody were monitored by exposing increasing concentrations of antibody to dye-labeled peptide dissolved in buffer. Cy3p17-peptide was resuspended by first dissolving in DMSO (∼5% of the final volume) followed by 20 mM HEPES buffer pH 8.0. Samples consisted of either 300 or 600 nM (30 or 60 pmol/ well in 100 uL final volume) Cy3-p17-peptide exposed to increasing concentrations of the R-p17, control chicken IgG, or BSA ranging from 0 to 10 nM in HEPES buffer. These concentrations were chosen by initially testing the peptide against the antibody for optimal concentrations that allowed sensitive fluorescent and absorption detection prior to the assay (data not shown). The solutions were incubated for 1 h at room temperature before being transferred to 96-well microtiter plates and fluorescence measurements (spectra and intensities) were measured using 530 nm excitation on a Tecan Safire Dual Monochromator Multifunctional Plate Reader (Tecan, Research Triangle Park, NC). Intensities were an average of 9 readings
Technical Notes
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Figure 1. UV-vis absorbance data. Data from a representative experiment that monitored changes in the absorbance spectra of Cy3-p17-peptide (600 nM) exposed to (A) the indicated increasing concentrations of R-p17 antibody or (B) nonspecific control chicken IgG protein. (C) Summary of the corresponding signal-over-noise (S/N) ratio monitored at the Cy3 absorbance maxima (555 nm) versus antibody concentration for both the R-p17 antibody and control chicken IgG.
Figure 2. Fluorescence data. Representative changes in the fluorescence spectra of Cy3-p17-peptide (600 nM) exposed to (A) the indicated increasing concentrations of R-p17 antibody or (B) the control chicken IgG protein. (C) Summary of the fluorescent S/N ratio monitored at the Cy3 emission maxima (570 nm) with samples consisting of either 300 nM or 600 nM Cy3-p17-peptide versus increasing R-p17 antibody, chicken IgG, and BSA protein concentrations. (n ) 4 or 6, duplicate or triplicate wells measured twice for the data shown).
per well. UV-vis spectra of the solutions were recorded using an Agilent Technologies 8453 UV-vis spectrophotometer (Santa Clara, CA). Data from the assays were analyzed and plotted using Microsoft Excel (Microsoft, Redmond, WA) and SigmaPlot (Systat Software Inc., San Jose, CA), respectively.
RESULTS AND DISCUSSION A schematic overview of the R-p17 sensing assay discussed here is shown in Scheme 1. The freely diffusing Cy3-p17peptide in solution has a nominal Cy3-fluorescent emission. Addition of R-p17 to the sensing solution results in specific antibody recognition and binding to the antigenic EKIRLR epitope which is proximal to the dye attachment site. This binding event alters the peptide structure and/or dye environment resulting in a significant enhancement of the Cy3 fluorescent dye photophysical properties which serves as the overall mechanism of signal transduction. We evaluated the R-p17 induced changes in both Cy3 absorbance and emission properties following binding to the labeled peptide. Figure 1A,B shows representative UV-vis spectra collected from samples consisting of 600 nM Cy3-p17-peptide samples consecutively exposed to increasing concentrations of either R-p17 antibody or a nonspecific chicken IgG control, respectively. The data from this UV-vis analysis is summarized and directly compared in Figure 1C, which plots the signal-overnoise (S/N) ratio at 550 nm (Cy3 absorbance maximum) versus the R-p17 and IgG concentrations. The data clearly show a significant increase in the intensity of the Cy3 absorbance spectra as a function of the R-p17 antibody concentration added. For R-p17 antibody binding, the effect appears to reach an asymptotic plateau at around a 4-fold increase, and this plateau is
observed starting at a minimal concentration of ∼1.25 nM. Importantly, almost no significant changes in Cy3 absorption were noted when the Cy3-p17-peptide was exposed to the same concentrations of control chicken IgG; a protein with a similar size and conserved structure as the IgG class of antibody proteins. Using just these changes in Cy3 absorption, an overall LOD (defined as a S/N ratio that is greater than 1 plus 3 times the standard deviation of the blank) of ∼0.156 nM could be derived for R-p17 binding to the Cy3-p17-peptide. The corresponding fluorescent emission spectra for the same samples prepared in the same manner were also recorded, and representative data are shown in Figure 2. As with the UV-vis data, the fluorescence intensity of 600 nM Cy3-p17-peptide increased as a direct function of the R-p17 antibody concentration added (Figure 2A). The fluorescent enhancement appears to also reach a maximum effect beginning at around 1.25 nM of R-p17 which corresponds to the same concentration noted for the plateau in the absorption data (Vide supra). Although the data from Cy3-p17-peptide exposed to equivalent concentrations of chicken IgG do show some changes in Cy3 emission, these appear to consist of both increases and decreases in the emission and do not seem to become significant (S/N of 1.1 ( 0.1 for 2.5 nM chicken IgG) until a concentration of 5 nM, which is five times the minimal amount of R-p17 antibody needed for the maximal enhancement effect (Figure 2B). The data from the fluorescence analysis are summarized and compared in Figure 2C, which plots the S/N ratio at 570 nm (Cy3 emission maximum) versus the IgG/R-p17 concentration for sample sets consisting of either 300 nM or 600 nM Cy3p17-peptide substrate. Exposure of R-p17 antibody to the lower concentration Cy3-p17-peptide was found to generate an up to
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Figure 3. Photographic image of a typical assay performed in a 96-well white microtiter plate where each well contains 600 nM Cy3-p17-peptide. The concentration of the R-p17 antibody in the top two rows (A,B) and the chicken IgG control in the bottom two rows (C,D) is steadily increased (0-10 nM) from right to left.
5-fold increase in the fluorescence emission at 570 nm, with a corresponding LOD of 0.073 nM R-p17 antibody. Overall R-p17 LOD’s, determined across multiple studies (n ) 5), were 0.098 ( 0.012 and 0.073 ( 0.060 nM for the 600 and 300 nM Cy3p17-peptide samples, respectively. The 73 pM LOD represents a 3.4-fold improvement over that determined by Oh and coworkers using their contact-quenching peptide beacon design (13). We did not note any dye enhancement when R-p17 antibody was exposed to a similarly labeled generic control peptide lacking the antigenic epitope (see Supporting Information Figure 3). Beyond chicken IgG, we also tested 600 nM Cy3p17-peptide interactions with BSA at a similar concentration range. In this case, BSA represents a nonspecific protein commonly found in serum samples and also often utilized as a blocking agent. The BSA elicits an almost identical response to the nonspecific chicken IgG at the same concentration ranges; see Figure 2C. We also noted that concentrations of BSA g 1000 nM, which is g100 times that of the highest R-p17 antibody concentration used, approached a S/N of ∼3 with some sensor configurations (different amounts of Cy3-p17-peptide were tested, data not shown) which is still below the upper S/N limit detected with R-p17 antibody. The enhancement of Cy3 absorbance and fluorescence properties observed upon R-p17 binding to the labeled-peptide sequence is likely a result of several putative changes in the dye’s localized environment. These may include a decrease in polarity or solvation around the dye in the presence of the antibody as well as a reduction in the conformational/rotational mobility of the dye, as suggested by Gruber and co-workers who investigated anomalous fluorescence enhancement of Cy3 upon covalent attachment to IgG and noncovalent binding to avidin (18). Similar changes in Cy3 properties have been previously exploited in conjunction with the conformationally coupled bacterial periplasmic maltose binding protein (MBP) to prototype a number of maltose sensing modalities. For these, reduction of Cy3-labeled MBP emission upon maltose addition was either monitored directly (19) or in conjunction with semiconductor quantum dot donors in steady-state, excited-state lifetime, single-molecule, and multiphoton fluorescence resonance energy transfer sensing configurations (20-22). The overall concept based on pairing of fluorogenic dyes with myriad cognate protein or nucleic acid partners such that each has limited fluorescence independently but a strong emission upon binding to each other during biosensing has recently been termed “fluoromodules” (23). Indeed, several cyanine dyes structurally analogous to Cy3 have been tested against various single-chain antibodies (scFV) for this type of activity and suggested the exciting possibility of a range of such fluoromodules that can span most of the visible to near-IR spectrum (23).
As our overall goal was to evaluate this biosensing approach for the most basic screening purposes, we focused last on demonstrating assay utility with minimal to no electronic instrumentation. Figure 3 shows a digital color image taken of duplicate samples consisting of Cy3-p17peptide in buffer exposed to increasing concentrations of either R-p17 antibody and the chicken IgG control, top and bottom two rows, respectively. The enhancement in Cy3 dye color intensity upon R-p17 binding, as compared to the IgG control, is most significant for the six antibody concentrations starting at 0.31 nM and higher. This colorimetric approach alone suggests that a rapid assessment of positive binding can easily be made by visual comparison of the samples versus controls against a white background in a microtiter well plate or even a test tube. Should observation of fluorescence also be desired, samples prepared in a similar fashion could be investigated using some form of LED or equivalent light source combined with CCD detection, both powered by batteries in the simplest configuration (24, 25). The enhancement of Cy3 absorption or emission is far more desirable for signal transduction and assay monitoring than observing losses in these properties as the latter can also be caused by a variety of nonspecific or ancillary processes, for example, pH changes, ion presence, or chemical denaturation. We have also found that the Cy3-p17-peptide can be stored lyophilized for long periods of time (6 months to 1 year) and still remain fully functional. This is another desirable property, as it can allow reagents to be prepared beforehand, dried, shipped, and stored until use on an as-needed basis.
CONCLUSIONS We have shown here a proof-of-concept design for a simple colorimetric and/or fluorescent sensing platform that uses a highly specific peptide coupled with a cyanine dye for the sensitive detection of R-p17 antibodies with a demonstrated LOD of 73 pM. This represents both a first use of a monoclonal antibody with such a fluorescent peptide in this type of sensing strategy and the detection of a clinically relevant and important target with both absorption and fluorescent emission signal transduction modalities. Although encouraging, these initial results also suggest that much research and engineering remains before transitioning to field studies. The overall enhancement mechanism is not yet been fully elucidated, and a greater understanding of the process is still warranted to answer a variety of complex mechanistic questions. For example, the extremely low concentration of antibody utilized to elicit a maximal enhancement effect (1 antibody per ∼4000 Cy3-p17peptides for the 300 nM peptide sample in Figure 2) strongly suggests that only a very small fraction of the dyes are appropriately responsive perhaps reflecting the antibodies bind-
Technical Notes
ing affinity/constant for the epitope in this context. The influence of Cy3 dye spacing/position relative to the epitope sequence on the peptide is clearly important and may be amenable to improvement to allow even more signal enhancement, while placement too far from the epitope may disrupt productive sensing. Iterative testing of such placement along with fluorescent polarization and anisotropy studies may help address this aspect. Testing a variety of other environmentally sensitive dyes in the sensor, including both cyanine-based and other structures, may provide better LODs, larger observed enhancements, and wider sensing ranges with the caveat that signal transduction in the visible portion of the spectrum is preferred for simplicity. Optimization of the dye attachment site in the peptide structure may also contribute similar benefits. Although similar concentrations of nonspecific antibody or dye-labeled generic peptide lacking the epitope did not trigger any significant sensor changes, we did note that excess concentrations of BSA (>100 times that of the antibody) were able to elicit a sensor response. Therefore, while this platform has demonstrated potential for the sensitive detection of R-p17 antibodies, it is likely that some sample dilution and/or purification may be required before analysis where complex matrices such as blood are used. Significant dilution of serum samples or utilizing an integrated sample cleanup and concentration module are generally accepted sample-pretreatment strategies commonly utilized prior to POC device analysis and can readily help accomplish this (1, 2, 4, 26, 27). Testing of relevant spiked controls and clinical samples will help answer and optimize the LOD and sample dilution/pretreatment issues (using approved BSL III facilities). These results in combination with other studies (12, 13, 23) strongly suggest the possibility of developing similar fluoromodule-based assays that are specifically targeted to sensing a variety of other antibodies or antigens. In principle, the method should be applicable to most antibody-epitope interactions regardless of peptide substrate length, as dyepositioning is the determinant. The various fluoromodules could be arrived at by screening combinatorially synthesized peptide libraries against targets of choice in parallel formats. Following on this, it is not hard to envision assays where several different dye-labeled peptides, each expressing different specific epitopes, are utilized in parallel-simultaneous formats to detect the presence of many different antibodies. This could allow the detection of other antibodies associated with different HIV infection stages, secondary infections, or even different strains of HIV. In conclusion, this report adds to the growing body of work which suggests that simplified bioassay formats, such as those exemplified by the fluoromodule-based assays described here, may have much to offer for challenging global health and biosecurity concerns especially in resource-poor environments (28).
ACKNOWLEDGMENT The authors thank Dr. J. Golden (NRL/CBMSE) for the digital photograph shown in Figure 3. This work was supported in part by CDRH/OSEL/Division of Biology funds. The mention of commercial products, their sources, or their use in connection with material reported herein is not to be construed as either an actual or implied endorsement of such products by the Department of Health and Human Services. IM acknowledges the CB Directorate/Physical S&T Division DTRA/ARO, ONR, NRL, and the NRL-NSI for financial support. J.B.B-C. gratefully acknowledges the Marie Curie Foundation for a postdoctoral fellowship. Supporting Information Available: The peptide purification protocol, along with HPLC and mass spectroscopy characteriza-
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tion, and a control study involving exposure of a generic Cy3 peptide to the p17 antibody. This material is available free of charge via the Internet at http://pubs.acs.org.
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