JIM-12019; No of Pages 7 Journal of Immunological Methods xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim
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Milan Gunasekera, Mohanlall Narine, Matthew Ashton, Javan Esfandiari ⁎, M. Gunasekera 1, M. Narine 1
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Chembio Diagnostics Systems Incorporated, 3661 Horseblock Road, Medford, NY 11763, United States
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Article history: Received 22 February 2015 Received in revised form 24 April 2015 Accepted 24 April 2015 Available online xxxx
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Keywords: Candida albicans Candidiasis Hyphal Immunodiagnostics Point-of-care Lateral flow
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1. Introduction
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Candida species are diploid fungi that commonly reside in mucosal surfaces of the gastrointestinal and genitourinary tracts (Zucchi et al., 2010). Normally living as a harmless commensal, Candida overgrowth can commonly result in infections such as Oral Candidiasis (OC, occurs in up to approx. 75% of population) and Vulvovaginal Candidiasis (VVC, present in approx. three-quarters of women in their lifetime) (Mayer et al., 2013). An opportunistic pathogen, Candida spp. can enter the systemic circulation through epithelial tissue, a condition known as invasive candidiasis (IC). Therefore, Candida is the fourth most frequent cause of nosocomial blood-stream infections (BSIs) in the United States, accounting for 8–10% nationwide (Pfaller et al., 1998; Yapar, 2014). Attaining a BSI through Candida spp., known as Candidaemia, globally has a mortality rate with a median of 38% (Yapar, 2014). C. albicans accounts for more than 53% of all international Candidaemia cases (Pfaller et al., 1998) and thus we place a strong emphasis on C. albicans detection. In the global diagnostic market however, point-of-care (POC) devices are often employed due to their low cost and user-friendliness. Therefore, we describe herein the development of a rapid POC assay for the detection of C. albicans.
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Candida albicans is an opportunistic pathogen which can lead to Candidiasis and blood-stream infections, resulting in a mortality rate near 40%. Given its high fatality and emerging pathogenicity, there is a strong need for the development of a rapid C. albicans diagnostic assay. Point-of-care devices, specifically lateral flow assays, are an attractive and often employed diagnostic modality for C. albicans detection. However, they lack the required performance characteristics needed for accurate pathogen detection and subsequent treatment options. Thus, we describe herein the utility of the Dual Path Platform (DPP®) device as an immunochromatographic Point-of-care assay for C. albicans. The limit of detection for hyphal and budding C. albicans in DPP® tests are reported to be as low as 7.94 × 105 whole cells/mL in human serum. C. albicans cells were detected with up to a 3.9 fold increase in sensitivity on DPP® when compared to conventional lateral flow modalities. © 2015 Published by Elsevier B.V.
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Development of a Dual Path Platform (DPP®) immunoassay for rapid detection of Candida albicans in human whole blood and serum
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Abbreviations: DPP®, Dual Path Platform; LoD, limit of detection; BSI, blood stream infections; POC, point-of-care; LF, lateral flow. ⁎ Corresponding author. Tel.: +1 631 924 1135x112; fax: +1 631 924 2065. E-mail address: [email protected] (J. Esfandiari). 1 Contributed equally to this work.
Current methods for diagnosis of C. albicans infections have serious limitations in the POC environment, where simplicity, timeliness, and cost efficiency are sought. Microscopy, for example, has approximately 50% sensitivity (Kojic and Darouiche, 2004). ELISA and PCR, the goldstandard methods, have high accuracy but are time-consuming, expensive, and require extensive sample preparation (Kostiala and Kostiala, 1981; Hua et al., 2010). Lateral flow (LF) assays are a common staple in POC testing due to their low cost and rapid results. However, the methodology of the immunoassay – running the sample matrix and the detector probe along the same sorbent – results in sensitivity issues due to less efficient binding of analyte to the testing region (Chembio Diagnostics Systems, 2014). False-positive diagnoses are also common occurrences in LF rapid tests and may result in patient misdiagnosis and erroneous treatment (Iregbu et al., 2011). In an effort to improve the POC diagnostic tools available for C. albicans diagnosis, we demonstrate the development of a Dual Path Platform (DPP®) device for C. albicans detection along with its noticeable improvement in performance characteristics over conventional LF.
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1.1. Dual Path Platform technology
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Briefly, the DPP® technology, patented by Chembio Diagnostic Systems, Inc., is distinct from conventional LF because of its ability to independently deliver analytes and detection probes along different sorbents. The analyte of interest migrates along the first sorbent, binding to the capture agents immobilized on the test region. Then, a buffer solution allows for marked-ligands (probe) to travel along the second sorbent and bind onto the captured analyte in the test region (Fig. 1).
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http://dx.doi.org/10.1016/j.jim.2015.04.014 0022-1759/© 2015 Published by Elsevier B.V.
Please cite this article as: Gunasekera, M., et al., Development of a Dual Path Platform (DPP®) immunoassay for rapid detection of Candida albicans in human whole blood and serum, J. Immunol. Methods (2015), http://dx.doi.org/10.1016/j.jim.2015.04.014
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2.1. Samples, strains, and media
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The yeast strains used in the experiments were Candida albicans SC5314 (Wild-Type) isolated from a clinical sample, Saccharomyces cerevisiae (Wild-Type), Candida lusitiniae (Wild-Type), and Candida tropicalis (Wild-Type) (Gomez-Raja et al., 2008). Strains were plated on YPD (Yeast Peptone Dextrose), and were cultured in YPD media. S. cerevisiae, which divides more slowly than Candida in YPD alone, was cultured in YPD and Uridine-containing media to allow faster growth. Yeast strains and Media were provided by the Konopka lab from the Department of Molecular Genetics & Microbiology at Stony Brook University (Stony Brook, NY).
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2.2. Antibodies
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Multiple monoclonal and polyclonal antibodies for C. albicans were acquired from Virostat (Catalog #: 6411; Portland, ME), Thermo Scientific (Catalog #: MA1-7009; Rockford, IL), Meridian (Catalog #: C86341M, B65411R; Cincinnati, OH), HyTest (Catalog #: 3CA4; Turku, Finland) and Fitzgerald (Catalog #: 10C-CR2155M1; Acton, MA).
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2.3. Growth of yeast strains
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The goal was to create aliquots of equal cell number (measured by optical density) which could be stored for later experimental use. A single colony of each strain was inoculated in YPD media obtained from streaked YPD plates and was grown overnight at 30 °C shaking at 200 rpm in a Delong Flask. Cell density (cell number/mL) was calculated the next day using a spectrophotometer (Beckman) at 660 nm
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2.4. Primary antibody screen by Multi-Antigen Print Immuno-Assay (MAPIA) 122
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MAPIA was used as the primary antibody screening test to select the most immunoreactive reagents to Candida, and lowest cross-reactivity (Lyashchenko et al., 2000). After a lysis step, membrane proteins of the acquired Candida strains as well as the S. cerevisiae sample were solubilized in Urea with Tris and striped across a nitrocellulose membrane (Protran®, Whatman) using Camag's Linomat IV. After printing, the membrane was cut into 3 mm strips perpendicular to the print lines; the number of anti-Candida antibodies stipulates the amount of strips required. The strips were blocked in 1% non-fat milk (Sigma-Aldrich) for 30 min at 25 °C before the addition of the primary antibody. After blocking, each anti-Candida antibody was incubated for 1 hour at 25 °C. Goat anti-Rabbit antibody Alkaline Phosphatase conjugate (GAR) and Goat anti-Mouse antibody Alkaline Phosphatase conjugate (GAM) were used to detect the polyclonal and monoclonal antibodies respectively by incubation for 1 hour at 25 °C. Finally, an alkaline phosphatase substrate was added to each well. Reactivity was evaluated visually, with a visible band of any intensity being considered a positive result.
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2.5. DPP® assay development
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2.5.1. Secondary screen in DPP® The three most immunoreactive antibodies to Candida were chosen to move forward with integration into the DPP® device. Capture antibodies were striped onto a nitrocellulose membrane (mdi Membrane Technologies) whereas the detector antibodies were conjugated to a colloidal gold particle and sprayed onto a conjugate pad. Components were assembled into a total of nine combinations and tested in DPP®.
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2.5.2. Printing of the test and control line to the nitrocellulose membrane for DPP® The concentrations of all the C. albicans antibodies were adjusted to 1 mg/mL in PBS. Depending on whether the conjugated gold antibody was of rabbit or mouse origin, the control line was chosen to be antirabbit or anti-mouse, respectively. The test and control line were printed using the Flatbed Imagene Printer at a print rate of 0.5 μL/cm on to the nitrocellulose membrane used in the DPP® assay.
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2.5.3. Production of antibody nano-gold conjugates (creation of detector antibody conjugate) Gold nanoparticles were prepared through Chembio's proprietary methods using gold chloride, sodium ascorbate and sodium citrate as reducing agents. Colloidal gold particles were verified to be ~40 nm in
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(Feldman and Grenier, 2012). These yeast strains were then added 119 into 500 μL 10.0 mM Phosphate Buffer Solution (PBS, Sigma-Aldrich), 120 refrigerated at 4 °C for future experimental use. 121
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The complex formed is known as a Sandwich assay, which enhances analyte binding and visualization (Esfandiari, 2010). The total time to complete the diagnosis is 20 min, where it can either be visually or quantitatively analyzed (Fig. 1). DPP® technology has performed 10–50× higher in sensitivity tests when compared to conventional LF assays (Chembio Diagnostics Systems, 2010). Additionally, the DPP device is capable of testing various sample matrices such as blood, serum, saliva, feces, oral fluids, urine, and vaginal swabs. DPP® technology has high multiplex potential, with the ability to detect multiple antigens without loss of specificity (Chembio Diagnostics Systems, 2010). This capability allows independent flow and binding of multiple analytes to capture agents at high sensitivity without disturbing specificity due to cross-reactivity (Chembio Diagnostics Systems, 2010). Hence, with its benefits over LF, we incorporate the DPP® technology in developing an improved POC C. albicans assay.
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Fig. 1. DPP® Cassette. (a) DPP® cassette and internals. (b–c) Schematic of Chembio's DPP® Technology. Test line and Control line antibodies are immobilized on a solid phase (Strip 2; S2 Strip) (Esfandiari, 2010). (b) The sample containing the analyte of interest (i.e. C. albicans antigen) is introduced via a secondary strip (Strip 1) and migration is facilitated through a running buffer. This type of sample segregation allows for a pre-incubation of the antigen of interest with the specific antibody striped on the Test Line region in Strip 2. (c) If antigen of interest is present in the sample, binding will occur with the Test Line immobilizing antibody. Running buffer is then added to Strip 2 to allow for the release of nanogold labeled antibodies on the conjugate pad. Labeled antibodies will then bind to the antigen–antibody complex on the Test Line region. This event will produce a colored reaction and thus provide a qualitative means to determine sample reactivity. In addition, through sample segregation, this technology allows for increased device sensitivity for any given analyte(s). Strip1 also acts as a filter for non-homogeneous specimens thereby increasing the robustness of the assay (Esfandiari, 2010).
Please cite this article as: Gunasekera, M., et al., Development of a Dual Path Platform (DPP®) immunoassay for rapid detection of Candida albicans in human whole blood and serum, J. Immunol. Methods (2015), http://dx.doi.org/10.1016/j.jim.2015.04.014
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2.6. Sample preparation and performance characteristics
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2.6.1. Cell lysis procedure A focus was made to determine if lysis of the cells was necessary or aided in the detection of Candida. Cells were harvested at a concentration of 1.0 × 108 cells/mL and the following lysis procedures were performed:
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2.6.2. Bead bashing In order to lyse the cells, 100 μL of glass beads was added to 1.5 mL Eppendorf tubes. Lysis buffer, either with Triton (Cyr-1 Paper, 50 mM Mepes, 40 mM NaCl, 5 mM MgCl2, 1% Triton; pH 7.3) or No Triton (Cyr-1 Paper, 50 mM Mepes, 40 mM NaCl, 5 mM MgCl2; pH 7.28). HaltTM Protease Inhibitor 100 × (Thermo Scientific) was added to the lysis buffer solution. The cells were vortexed at 6000 g for 1 min, and then placed in ice.
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2.7. DPP® sample preparation
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2.7.1. DPP® testing protocol Testing procedure for the DPP® C. albicans assay is as follows: prepare 1:25 dilution of the sample to be tested in the sample migration buffer. Add 60 μL of the mixture into the S1 port of the DPP® device and allow for a 5 minute incubation period. Following, add 150 μL of migration buffer into the S2 port of the DPP® device. Allow 15 min to pass and read device visually and with use of the custom ESEQuant reader for quantification of test and control line results.
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2.7.1.1. Cells in PBS. Candida and S. cerevisiae cells were serially diluted to a concentration of 1:128 with PBS in 1.5 mL Eppendorf tubes to test for reactivity in the DPP® C. albicans assay. Following, samples were further diluted at a 1:25 dilution using Chembio's sample migration buffer as the diluent. 60 μL of the sample and buffer mixture solution was then added to the S1 port of the DPP® assay as per the DPP® C. albicans testing protocol described above.
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In-house LF tests were made by removing the sample strip (S1 Strip; Fig. 2) from the DPP® devices. Sample mixture was then added directly into the S2 well housing the detector particles. The sample and buffer solution then migrated onto the assaying region containing the capture antibodies selected for incorporation into DPP®. Migration of the analytes therefore occurred on the unilateral strip as in conventional
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2.6.3. Enzymatic lysis Zymolase (Zymo Research, 5 units/μL) was used to lyse the cells. 2 μL of zymolase was added to each yeast strain. Cells were incubated at 37 °C for 60 min and then taken out and put on ice to cease the reaction.
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2.8. Lateral flow comparison
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2.5.4. Testing for best capture/detector antibodies In order to determine the best pair of antibodies, a series of DPP® test trials were carried out on the most efficient capture antibodies determined by the MAPIA primary screening process. The assays were prepared according to Chembio proprietary assembly standards and run as follows. First, 60 μL of the diluted C. albicans analyte was added to the S1 port (Fig. 1a). After 5 min of incubation, 150 μL of buffer was added to the S2 port (Fig. 1a), followed by a test reading, both visually and by a semi-automated reader fifteen minutes later (ESEQuant, Qiagen).
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2.7.1.4. Hyphal induction of C. albicans in blood and serum. The virulence of C. albicans is highly dependent on its morphology. The nonfilamentous budding form is less virulent than the pseudohyphal and hyphal forms, the latter being able to create filamentous elongations that penetrate through epithelial tissue and subsequently enter the bloodstream. To induce hyphal growth, spiked specimens negative blood and serum were spiked with C. albicans 6.35 × 108 cells/mL) in a 1:1 dilution) were incubated at 37 °C for 90 min on a rotator set to 200 rpm. After incubation, samples are serially diluted up to 1:128 in both negative blood and serum for use in the DPP® C. albicans assay.
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negative blood sample was found it was spiked with C. albicans 217 (1.0 × 106 cells/mL) at a 1:1 to 1:128 dilution. 218
diameter via dynamic light scattering instrument (Malvern). The pH of the nanogold solution was adjusted near the Isoelectric Point (pI) of each antibody to facilitate a successful and efficient conjugation. Concentration of the conjugated nanogold was then adjusted to 50 O.D. (peak O.D. observed at 520–530 nm) and sprayed onto a fiber composite conjugate pad (Ahlstrom) using a Flatbed Imagene Printer at a spray rate of 5 μL/cm.
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2.7.1.2. Cells in serum. Serological tests were conducted to prescreen for non-reactivity to Candida on the DPP® assay. Unknown patient serum samples (ZeptoMetrix) were spiked with C. albicans (1.0 × 106 cells/mL) at a 1:1 to 1:128 dilution. 2.7.1.3. Cells in human whole blood. Whole blood samples (ZeptoMetrix) were also prescreened for non-reactivity to Candida. When a
Fig. 2. Multi-antigen print immunoassay for varying C. albicans antibodies. 1–20: 1–4) S. cerevisiae lysate protein, 5) whole S. cerevisiae cells, 6–9) C. tropicalis lysate proteins, 10) whole C. tropicalis cells, 11–14) C. lusitaniae lysate proteins, 15) whole C. lusitaniae cells, 16–19) C. albicans lysate proteins, 20) whole C. albicans cells, 21) Protein A. A–F: A) Mouse monoclonal anti-C. albicans antibodies (HyTest Ltd.) at 1:50, B) mouse monoclonal anti-C. albicans antibodies (HyTest Ltd.) at 1:100, C) rabbit polyclonal anti-C. albicans (Virostat, Inc.) at 1:100, D) rabbit polyclonal anti-C. albicans (Virostat, Inc.) at 1:500, E) rabbit polyclonal anti-C. albicans (Meridian Life Science, Inc.) at 1:100, F) rabbit polyclonal anti-C. albicans (Meridian Life Science, Inc.) at 1:500. Printed antibodies were tested against both surface (from whole cells) and intracellular lysate proteins of C. albicans, C. lusitaniae, C. tropicalis, and the negative control S. cerevisiae. Cells were lysed using bead bashing, triton detergent, or Zymolase enzyme. Bands with faint to dark purple intensities are indicative of a reaction with the printed antigens/protein and C. albicans antibodies.
Please cite this article as: Gunasekera, M., et al., Development of a Dual Path Platform (DPP®) immunoassay for rapid detection of Candida albicans in human whole blood and serum, J. Immunol. Methods (2015), http://dx.doi.org/10.1016/j.jim.2015.04.014
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A1 = Monoclonal Ab from HyTest (CAT # 3CA4)
A1/A1 A1/A2 A1/A3 A2/A1 A2/A2 A2/A3 A3/A1 A3/A2 A3/A3
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A3 = Polyclonal Ab from Virostat (CAT # 6411)
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3. Results & discussion
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To investigate the feasibility of developing a C. albicans antigen detection assay utilizing the DPP® technology, anti-C. albicans antibodies were first screened on MAPIA for reactivity against members of the Candida genus and the S. cerevisiae negative control (Fig. 2). Following this primary screen, various capture and detector antibody combinations were then introduced into the DPP® platform and further tested for reactivity (Table 1).
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3.1. MAPIA screen
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Fig. 2 illustrates the immunoreactivity for all commercially purchased anti-C. albicans antibodies, as well as their affinity for lysate epitopes. Monoclonal anti-C. albicans antibody was nonreactive to other members of the Candida genus (Candida lusitaniae, and C. tropicalis) in
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To establish the negative threshold for the DPP® C. albicans assay, the S. cerevisiae control was tested in triplicate at varying concentrations in 10 mM PBS. For all testing, no test line was made visible, thereby indicating a negative result, and the highest reader output was 26 mV in intensity. Therefore, to account for non-trending occurrences, a
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3.3. Negative threshold
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A secondary screening assessed various combinations of antiC. albicans capture and detector antibodies in the DPP® platform for signal intensity (Table 1). Monoclonal antibody combinations resulted in signals of low intensity. Polyclonal exclusive anti-C. albicans antibody combinations (Virostat and Meridian) created high signal intensity, indicating binding of the analyte at the test line. Subsequently, these combinations were selected as the best capture/detector agent for incorporation into the DPP® C. albicans assay. Upon the selection of capture and detector antibodies for the DPP® C. albicans assay, experiments were conducted to determine device sensitivity, specificity, cross-reactivity, and limit of detection (Figs. 4–8), as well as comparison with in-house LF assays In addition, a comparative study was performed to illustrate how the DPP® technology fares against LF (Fig. 8). All results were plotted against an established negative threshold for the DPP® C. albicans assay (Fig. 3).
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3.2. DPP® screening test
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LF assays. After a 20 minute wait period, device results were interpreted using the same reader instrument utilized in DPP® quantification. The in-house LF assay was semi-optimized with 2 M urea into the buffer solution. Previous empirical data has shown that LF generates a high degree of false positive results due to non-specific binding of the analyte–detector complex (Chembio Diagnostics Systems, 2014). Hence, a more accurate representation of the sensitivities between the two prototypes was achieved by addition of the urea denaturant.
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A2 = Polyclonal Ab from Meridian (CAT# B65411AR)
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addition to the negative control, S. cerevisiae (Fig. 2: lane A and B). Faint purple bands were observed for printed C. albicans whole cells and printed C. albicans cell lysate proteins (mix of intracellular and surface/ membrane proteins), indicating weak reactivity of the monoclonal antibody to epitopes on Candida cells. Polyclonal anti-C. albicans antibodies (Fig. 2: lanes C–F) were reactive to C. albicans, C. lusitaniae, C. tropicalis, and S. cerevisiae. In particular, darker bands were visible for printed whole cells of C. albicans, C. lusitaniae and C. tropicalis whereas, faint bands were visible for their printed intracellular lysate protein counterparts and S. cerevisiae. Hence, all polyclonal antibodies display a higher affinity for extracellular proteins of the various yeast organisms, thereby, eliminating the need for a sample pre-treatment (lysis of whole cells) step in the DPP® C. albicans assay. As such, all further testing was conducted using whole Candida and S. cerevisiae cells.
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Table 1 Anti-C. albicans antibody pairs tested in the DPP® format. Combinations with Meridian and Virostat antibodies as capture and detector agents elicited a strong signal.
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Fig. 3. Establishment of negative threshold for DPP® C. albicans Assay using S. cerevisiae as negative control. Negative threshold for DPP® C. albicans assay. S. cerevisiae cells (unlysed) were serially diluted to 1:4096 from neat (6.35 × 108 cells/mL) in 10 mM PBS. Average taken from samples tested in triplicate using DPP® C. albicans' assay. Error bars indicate standard deviation. Negative threshold established to be 30 mV.
Please cite this article as: Gunasekera, M., et al., Development of a Dual Path Platform (DPP®) immunoassay for rapid detection of Candida albicans in human whole blood and serum, J. Immunol. Methods (2015), http://dx.doi.org/10.1016/j.jim.2015.04.014
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In order to assess the POC assay's and performance characteristics, cultured whole C. albicans cells (both hyphal and yeast budding forms of strain SC5314), were spiked into negative human blood and serum. Following serial dilutions were made and tested so as to represent varying concentrations of the C. albicans analyte in human blood and serum medium. Testing was performed in quintuplicate and nonreactive results were characterized with a reader output of ≤30 mV in conjunction with the absence of a colored test line. The DPP® C. albicans assay
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was unsuccessful at detecting C. albicans (in its yeast budding form) analytes at a 1:32 and 1:64 dilution in human blood and serum respectively whereas, detection of C. albicans in its hyphal form ceased at a concentration of 1:16 and 1:32 in the DPP® C. albicans assay in human blood and serum, respectively (Fig. 4). The limit of detection (LoD) was calculated for hyphal C. albicans in blood and serum as 1.59 × 106 and 7.94 × 105 cells/mL (95,300 and 47,600 whole cells), respectively. In turn, the LoD for budding yeast form of C. albicans in blood and serum are 7.94 × 105 and 3.97 × 105 cells/mL (47,600 and 23,800 whole cells), correspondingly (Fig. 5). These numbers are comparable to present PCR diagnostic modalities that are able to detect C. albicans at a magnitude of 105 whole cells in human blood (Maaroufi et al., 2003).
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Cross reactant tests of pathogens inherent of immunocompromised 321 patients, as well as non-Candida yeast samples were performed to 322
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reader value of 30 mV was chosen to be the negative threshold for the DPP® C. albicans assay (Fig. 3). Reader values were measured using a customized ESEQuant reader (Qiagen) outfitted for DPP® devices. Using a series of imaging algorithms, the ESEQuant reader is able to quantify the color reaction (or lack of) observed on both the Test and Control lines on the DPP® C. albicans assay.
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Fig. 4. Average DPP® C. albicans test line intensity of hyphal and yeast Candida albicans in negative human serum and blood at varying concentrations. Sensitivity profile of DPP® C. albicans assay using spiked negative human blood (Zeptometrix Corp.) and serum (BioreclamationIVT). Blood and serum were spiked with both hyphal and yeast C. albicans' strain SC5314 to a concentration of 6.35 × 108 cells/mL. Samples were then serially diluted in either negative human blood or serum concentration down to 1:128. Averages were taken from samples tested in quintuplicate using DPP® C. albicans assay. Negative threshold set at 30 mV. Error bars indicate standard deviation.. Hyphal C. albicans detection in human blood and serum ceases at 1:16 and 1:32, respectively, whereas yeast C. albicans detection ceases at 1:32 dilution and 1:64 dilution in human blood and serum, respectively.
Fig. 5. Limit of detection of hyphal C. albicans and yeast C. albicans in human whole blood and serum in DPP® assay. Limit of detection for the DPP® C. albicans assay. C. albicans cells (both yeast budding and hyphal forms) were spiked into negative human blood and serum (Zeptometrix Corp.; BioreclamationIVT) at varying concentrations until no positive signal was elicited by the assay. Limit of detection (LoD) for hyphal C. albicans in blood and serum is 1.59 × 106 and 7.94 × 105 cells/mL, respectively. LoD for budding yeast form of C. albicans in blood and serum is 7.94 × 105 and 3.97 × 105 cells/mL, respectively.
Please cite this article as: Gunasekera, M., et al., Development of a Dual Path Platform (DPP®) immunoassay for rapid detection of Candida albicans in human whole blood and serum, J. Immunol. Methods (2015), http://dx.doi.org/10.1016/j.jim.2015.04.014
M. Gunasekera et al. / Journal of Immunological Methods xxx (2015) xxx–xxx
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Fig. 6. Average test line signal intensity for C. albicans, S. cerevisiae, C. lusitaniae, & C. tropicalis in human serum. Specificity testing of the DPP® C. albicans assay. C. albicans, S. cerevisiae, C. lusitaniae, and C. tropicalis were individually spiked in negative human serum (BioreclamationIVT). Averages taken from triplicate testing of each sample in DPP® C. albicans assay. Negative threshold set at 30 mV. Error bars indicate standard deviation.
3.6. DPP® vs. LF technology
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The DPP® assay was tested against an in-house LF device (semioptimized with 2 M urea in buffer solution) counterpart for evaluation
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The results generated indicate the feasibility of producing a DPP® assay for detection of C. albicans cells in human blood and serum and its marked improvement over the conventional LF assay. Experiments have demonstrated a limit of detection (LoD) for hyphal C. albicans in blood and serum as 1.59 × 106 and 7.94 × 105 cells/mL, respectively whereas the LoD for budding yeast form of C. albicans in blood and serum are 7.94 × 105 and 3.97 × 105 cells/mL, respectively. The assay's reactivity with HIV-2 specimens, as well as with C. lusitaniae and
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4. Conclusion
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between the two technologies. Negative human serum was spiked with varying concentrations of C. albicans cells. In every instance, the DPP® assay was able to generate intensity results of at minimum 2.5× higher than LF based assay for samples that contained cell concentration within the DPP® C. albicans' LoD (Fig. 8).
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assess the specificity of DPP®. Tests were done in quintuplicate and were evaluated as positive if the average signal intensities were above 30 mV. Reactants tested include antigens specific to: HIV-1, HIV-2, HSV-1, HSV-2, Rubella, and Hepatitis C. Additionally, select members of the Candida genus along with the yeast S. cerevisiae were assessed. Of all tested reactants, the DPP® C. albicans assay generated a reactive result to HIV-2 antigens whereas all tested Candida genus' elicited low positive results for the C. albicans test line and S. cerevisiae resulted in a nonreactive status (Figs. 6 and 7). Note that other Candida spp.– C. glabrata, and C. parapsilosis, were not utilized for testing due to unavailability.
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Fig. 7. Cross-reactivity profile of the DPP® C. albicans assay. HIV1 1:240: strong HIV1 positive sample. HIV1 1:1800: low HIV1 positive sample. HIV2 1:32: strong HIV2 positive sample. HIV2 1:128: low HIV2 positive sample. PTH201-06: low HSV1 positive sample. PTH201-05: low HSV1 positive sample. PTH201-04: strong HSV 1/2 positive sample. PTR201-13: strong Rubella positive sample. PTR201-01: low positive Rubella sample. PHV207-08: strong Hepatitis C positive sample. PHV207-02: low Hepatitis C positive sample. HIV samples provided by Zeptometrix Corporation. All other samples acquired from SeraCare Life Sciences. Averages taken from quintuplicate testing of each sample in DPP® C. albicans assay. Negative threshold set at 30 mV. Error bars indicate standard deviation. HIV2 1:32 results are above the negative threshold and thus yields a positive result.
Please cite this article as: Gunasekera, M., et al., Development of a Dual Path Platform (DPP®) immunoassay for rapid detection of Candida albicans in human whole blood and serum, J. Immunol. Methods (2015), http://dx.doi.org/10.1016/j.jim.2015.04.014
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We would like to thank Dr. James Konopka, as well as his lab members (Molecular Genetics and Microbiology Department at Stony Brook) for providing strains, support, and the lab space for MG to carry out initial experiments on C. albicans.
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Chembio Diagnostics Systems, I.M., NY, US), 2010. Dual Path Platform vol. 2015. Chembio Diagnostics Systems, I.M., NY, US), 2014. Comparison of Lateral Flow to Dual Path Platform in Rubella Antigen detection assay.
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Esfandiari, J.S.B., NY, US), 2010. Dual path immunoassay device vol. 7682801. Chembio Diagnostic Systems, Inc., (Medford, NY, US), United States. Feldman, M., Grenier, D., 2012. Cranberry proanthocyanidins act in synergy with licochalcone A to reduce Porphyromonas gingivalis growth and virulence properties, and to suppress cytokine secretion by macrophages. J. Appl. Microbiol. 113, 438. Gomez-Raja, J., Andaluz, E., Magee, B., Calderone, R., Larriba, G., 2008. A single SNP, G929T (Gly310Val), determines the presence of a functional and a non-functional allele of HIS4 in Candida albicans SC5314: detection of the non-functional allele in laboratory strains. Fungal Genet. Biol. 45 (4), 527. Hua, Z., Rouse, J.L., Eckhardt, A.E., Srinivasan, V., Pamula, V.K., Schell, W.A., Benton, J.L., Mitchell, T.G., Pollack, M.G., 2010. Multiplexed real-time polymerase chain reaction on a digital microfluidic platform. Anal. Chem. 82, 2310. Iregbu, K.C., Esfandiari, J., Nnorom, J., Sonibare, S.A., Uwaezuoke, S.N., Eze, S.O., Abdullahi, N., Lawal, A.O., Durogbola, B.S., 2011. Dual Path Platform HIV 1/2 assay: evaluation of a novel rapid test using oral fluids for HIV screening at the National Hospital in Abuja, Nigeria. Diagn. Microbiol. Infect. Dis. 69, 405. Kojic, E.M., Darouiche, R.O., 2004. Candida infections of medical devices. Clin. Microbiol. Rev. 17, 255. Kostiala, A.A., Kostiala, I., 1981. Enzyme-linked immunosorbent assay (ELISA) for IgM, IgG and IgA class antibodies against Candida albicans antigens: development and comparison with other methods. Sabouraudia 19, 123. Lyashchenko, K.P., Singh, M., Colangeli, R., Gennaro, M.L., 2000. A multi-antigen print immunoassay for the development of serological diagnosis of infectious diseases. J. Immunol. Methods 242, 91. Maaroufi, Y., Heymans, C., De Bruyne, J.M., Duchateau, V., Rodriguez-Villalobos, H., Aoun, M., Crokaert, F., 2003. Rapid detection of Candida albicans in clinical blood samples by using a TaqMan-based PCR assay. J. Clin. Microbiol. 41, 3293. Mayer, F.L., Wilson, D., Hube, B., 2013. Candida albicans pathogenicity mechanisms. Virulence 4, 119. Pfaller, M.A., Jones, R.N., Messer, S.A., Edmond, M.B., Wenzel, R.P., 1998. National surveillance of nosocomial blood stream infection due to species of Candida other than Candida albicans: frequency of occurrence and antifungal susceptibility in the SCOPE Program. SCOPE Participant Group. Surveillance and Control of Pathogens of Epidemiologic. Diagn. Microbiol. Infect. Dis. 30, 121. Yapar, N., 2014. Epidemiology and risk factors for invasive candidiasis. Ther. Clin. Risk Manag. 10, 95. Zucchi, P.C., Davis, T.R., Kumamoto, C.A., 2010. A Candida albicans cell wall-linked protein promotes invasive filamentation into semi-solid medium. Mol. Microbiol. 76, 733.
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C. tropicalis cells, indicates a need for improvement of the assay's specificity performance for clinical trials. The polyclonal nature of antiC. albicans capture and detector antibodies may be the causative agent responsible for the observed false-positive results; due to the lack of viable commercially available monoclonal anti-C. albicans antibodies for immunochromatographic tests, the DPP® C. albicans assay is restrained to the antibody's characteristics. Although cross-reactivity and specificity issues are of concern within the DPP® C. albicans assay, it is important to note that the device can effectively detect C. albicans cells at varying concentration at a level much higher than its LF counterpart while still preserving the current device specificity. Furthermore, the positive signal achieved with the DPP® C. albicans assay is enhanced by 2.5–3.9× when compared to LF-based detection for samples within the device's LoD. Hence, the DPP® technology is considered superior in sensitivity and specificity over LF technology. Albeit the experiments within have been conducted with cultured cells, future work is expected to be well-translated in the clinical setting. Thus, impending research will focus on the clinical efficacy of the DPP® C. albicans assay over its lateral flow counterpart.
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Fig. 8. Comparison of DPP® versus lateral flow (LF) technology. C. albicans spiked in negative human serum at varying dilutions and tested in triplicate in DPP® C. albicans assay. Averages plotted. Negative threshold set at 30 mV. Error bars indicate standard deviation. At maximum, DPP® is 3.9× greater in sensitivity than LF.
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Please cite this article as: Gunasekera, M., et al., Development of a Dual Path Platform (DPP®) immunoassay for rapid detection of Candida albicans in human whole blood and serum, J. Immunol. Methods (2015), http://dx.doi.org/10.1016/j.jim.2015.04.014
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Contents lists available at ScienceDirect
Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim
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Milan Gunasekera, Mohanlall Narine, Matthew Ashton, Javan Esfandiari ⁎, M. Gunasekera 1, M. Narine 1
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Chembio Diagnostics Systems Incorporated, 3661 Horseblock Road, Medford, NY 11763, United States
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Article history: Received 22 February 2015 Received in revised form 24 April 2015 Accepted 24 April 2015 Available online xxxx
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Keywords: Candida albicans Candidiasis Hyphal Immunodiagnostics Point-of-care Lateral flow
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Candida species are diploid fungi that commonly reside in mucosal surfaces of the gastrointestinal and genitourinary tracts (Zucchi et al., 2010). Normally living as a harmless commensal, Candida overgrowth can commonly result in infections such as Oral Candidiasis (OC, occurs in up to approx. 75% of population) and Vulvovaginal Candidiasis (VVC, present in approx. three-quarters of women in their lifetime) (Mayer et al., 2013). An opportunistic pathogen, Candida spp. can enter the systemic circulation through epithelial tissue, a condition known as invasive candidiasis (IC). Therefore, Candida is the fourth most frequent cause of nosocomial blood-stream infections (BSIs) in the United States, accounting for 8–10% nationwide (Pfaller et al., 1998; Yapar, 2014). Attaining a BSI through Candida spp., known as Candidaemia, globally has a mortality rate with a median of 38% (Yapar, 2014). C. albicans accounts for more than 53% of all international Candidaemia cases (Pfaller et al., 1998) and thus we place a strong emphasis on C. albicans detection. In the global diagnostic market however, point-of-care (POC) devices are often employed due to their low cost and user-friendliness. Therefore, we describe herein the development of a rapid POC assay for the detection of C. albicans.
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Candida albicans is an opportunistic pathogen which can lead to Candidiasis and blood-stream infections, resulting in a mortality rate near 40%. Given its high fatality and emerging pathogenicity, there is a strong need for the development of a rapid C. albicans diagnostic assay. Point-of-care devices, specifically lateral flow assays, are an attractive and often employed diagnostic modality for C. albicans detection. However, they lack the required performance characteristics needed for accurate pathogen detection and subsequent treatment options. Thus, we describe herein the utility of the Dual Path Platform (DPP®) device as an immunochromatographic Point-of-care assay for C. albicans. The limit of detection for hyphal and budding C. albicans in DPP® tests are reported to be as low as 7.94 × 105 whole cells/mL in human serum. C. albicans cells were detected with up to a 3.9 fold increase in sensitivity on DPP® when compared to conventional lateral flow modalities. © 2015 Published by Elsevier B.V.
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Development of a Dual Path Platform (DPP®) immunoassay for rapid detection of Candida albicans in human whole blood and serum
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Abbreviations: DPP®, Dual Path Platform; LoD, limit of detection; BSI, blood stream infections; POC, point-of-care; LF, lateral flow. ⁎ Corresponding author. Tel.: +1 631 924 1135x112; fax: +1 631 924 2065. E-mail address: [email protected] (J. Esfandiari). 1 Contributed equally to this work.
Current methods for diagnosis of C. albicans infections have serious limitations in the POC environment, where simplicity, timeliness, and cost efficiency are sought. Microscopy, for example, has approximately 50% sensitivity (Kojic and Darouiche, 2004). ELISA and PCR, the goldstandard methods, have high accuracy but are time-consuming, expensive, and require extensive sample preparation (Kostiala and Kostiala, 1981; Hua et al., 2010). Lateral flow (LF) assays are a common staple in POC testing due to their low cost and rapid results. However, the methodology of the immunoassay – running the sample matrix and the detector probe along the same sorbent – results in sensitivity issues due to less efficient binding of analyte to the testing region (Chembio Diagnostics Systems, 2014). False-positive diagnoses are also common occurrences in LF rapid tests and may result in patient misdiagnosis and erroneous treatment (Iregbu et al., 2011). In an effort to improve the POC diagnostic tools available for C. albicans diagnosis, we demonstrate the development of a Dual Path Platform (DPP®) device for C. albicans detection along with its noticeable improvement in performance characteristics over conventional LF.
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1.1. Dual Path Platform technology
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Briefly, the DPP® technology, patented by Chembio Diagnostic Systems, Inc., is distinct from conventional LF because of its ability to independently deliver analytes and detection probes along different sorbents. The analyte of interest migrates along the first sorbent, binding to the capture agents immobilized on the test region. Then, a buffer solution allows for marked-ligands (probe) to travel along the second sorbent and bind onto the captured analyte in the test region (Fig. 1).
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http://dx.doi.org/10.1016/j.jim.2015.04.014 0022-1759/© 2015 Published by Elsevier B.V.
Please cite this article as: Gunasekera, M., et al., Development of a Dual Path Platform (DPP®) immunoassay for rapid detection of Candida albicans in human whole blood and serum, J. Immunol. Methods (2015), http://dx.doi.org/10.1016/j.jim.2015.04.014
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2.1. Samples, strains, and media
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The yeast strains used in the experiments were Candida albicans SC5314 (Wild-Type) isolated from a clinical sample, Saccharomyces cerevisiae (Wild-Type), Candida lusitiniae (Wild-Type), and Candida tropicalis (Wild-Type) (Gomez-Raja et al., 2008). Strains were plated on YPD (Yeast Peptone Dextrose), and were cultured in YPD media. S. cerevisiae, which divides more slowly than Candida in YPD alone, was cultured in YPD and Uridine-containing media to allow faster growth. Yeast strains and Media were provided by the Konopka lab from the Department of Molecular Genetics & Microbiology at Stony Brook University (Stony Brook, NY).
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Multiple monoclonal and polyclonal antibodies for C. albicans were acquired from Virostat (Catalog #: 6411; Portland, ME), Thermo Scientific (Catalog #: MA1-7009; Rockford, IL), Meridian (Catalog #: C86341M, B65411R; Cincinnati, OH), HyTest (Catalog #: 3CA4; Turku, Finland) and Fitzgerald (Catalog #: 10C-CR2155M1; Acton, MA).
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The goal was to create aliquots of equal cell number (measured by optical density) which could be stored for later experimental use. A single colony of each strain was inoculated in YPD media obtained from streaked YPD plates and was grown overnight at 30 °C shaking at 200 rpm in a Delong Flask. Cell density (cell number/mL) was calculated the next day using a spectrophotometer (Beckman) at 660 nm
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MAPIA was used as the primary antibody screening test to select the most immunoreactive reagents to Candida, and lowest cross-reactivity (Lyashchenko et al., 2000). After a lysis step, membrane proteins of the acquired Candida strains as well as the S. cerevisiae sample were solubilized in Urea with Tris and striped across a nitrocellulose membrane (Protran®, Whatman) using Camag's Linomat IV. After printing, the membrane was cut into 3 mm strips perpendicular to the print lines; the number of anti-Candida antibodies stipulates the amount of strips required. The strips were blocked in 1% non-fat milk (Sigma-Aldrich) for 30 min at 25 °C before the addition of the primary antibody. After blocking, each anti-Candida antibody was incubated for 1 hour at 25 °C. Goat anti-Rabbit antibody Alkaline Phosphatase conjugate (GAR) and Goat anti-Mouse antibody Alkaline Phosphatase conjugate (GAM) were used to detect the polyclonal and monoclonal antibodies respectively by incubation for 1 hour at 25 °C. Finally, an alkaline phosphatase substrate was added to each well. Reactivity was evaluated visually, with a visible band of any intensity being considered a positive result.
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2.5.1. Secondary screen in DPP® The three most immunoreactive antibodies to Candida were chosen to move forward with integration into the DPP® device. Capture antibodies were striped onto a nitrocellulose membrane (mdi Membrane Technologies) whereas the detector antibodies were conjugated to a colloidal gold particle and sprayed onto a conjugate pad. Components were assembled into a total of nine combinations and tested in DPP®.
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2.5.2. Printing of the test and control line to the nitrocellulose membrane for DPP® The concentrations of all the C. albicans antibodies were adjusted to 1 mg/mL in PBS. Depending on whether the conjugated gold antibody was of rabbit or mouse origin, the control line was chosen to be antirabbit or anti-mouse, respectively. The test and control line were printed using the Flatbed Imagene Printer at a print rate of 0.5 μL/cm on to the nitrocellulose membrane used in the DPP® assay.
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2.5.3. Production of antibody nano-gold conjugates (creation of detector antibody conjugate) Gold nanoparticles were prepared through Chembio's proprietary methods using gold chloride, sodium ascorbate and sodium citrate as reducing agents. Colloidal gold particles were verified to be ~40 nm in
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(Feldman and Grenier, 2012). These yeast strains were then added 119 into 500 μL 10.0 mM Phosphate Buffer Solution (PBS, Sigma-Aldrich), 120 refrigerated at 4 °C for future experimental use. 121
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The complex formed is known as a Sandwich assay, which enhances analyte binding and visualization (Esfandiari, 2010). The total time to complete the diagnosis is 20 min, where it can either be visually or quantitatively analyzed (Fig. 1). DPP® technology has performed 10–50× higher in sensitivity tests when compared to conventional LF assays (Chembio Diagnostics Systems, 2010). Additionally, the DPP device is capable of testing various sample matrices such as blood, serum, saliva, feces, oral fluids, urine, and vaginal swabs. DPP® technology has high multiplex potential, with the ability to detect multiple antigens without loss of specificity (Chembio Diagnostics Systems, 2010). This capability allows independent flow and binding of multiple analytes to capture agents at high sensitivity without disturbing specificity due to cross-reactivity (Chembio Diagnostics Systems, 2010). Hence, with its benefits over LF, we incorporate the DPP® technology in developing an improved POC C. albicans assay.
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Fig. 1. DPP® Cassette. (a) DPP® cassette and internals. (b–c) Schematic of Chembio's DPP® Technology. Test line and Control line antibodies are immobilized on a solid phase (Strip 2; S2 Strip) (Esfandiari, 2010). (b) The sample containing the analyte of interest (i.e. C. albicans antigen) is introduced via a secondary strip (Strip 1) and migration is facilitated through a running buffer. This type of sample segregation allows for a pre-incubation of the antigen of interest with the specific antibody striped on the Test Line region in Strip 2. (c) If antigen of interest is present in the sample, binding will occur with the Test Line immobilizing antibody. Running buffer is then added to Strip 2 to allow for the release of nanogold labeled antibodies on the conjugate pad. Labeled antibodies will then bind to the antigen–antibody complex on the Test Line region. This event will produce a colored reaction and thus provide a qualitative means to determine sample reactivity. In addition, through sample segregation, this technology allows for increased device sensitivity for any given analyte(s). Strip1 also acts as a filter for non-homogeneous specimens thereby increasing the robustness of the assay (Esfandiari, 2010).
Please cite this article as: Gunasekera, M., et al., Development of a Dual Path Platform (DPP®) immunoassay for rapid detection of Candida albicans in human whole blood and serum, J. Immunol. Methods (2015), http://dx.doi.org/10.1016/j.jim.2015.04.014
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2.6.1. Cell lysis procedure A focus was made to determine if lysis of the cells was necessary or aided in the detection of Candida. Cells were harvested at a concentration of 1.0 × 108 cells/mL and the following lysis procedures were performed:
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2.6.2. Bead bashing In order to lyse the cells, 100 μL of glass beads was added to 1.5 mL Eppendorf tubes. Lysis buffer, either with Triton (Cyr-1 Paper, 50 mM Mepes, 40 mM NaCl, 5 mM MgCl2, 1% Triton; pH 7.3) or No Triton (Cyr-1 Paper, 50 mM Mepes, 40 mM NaCl, 5 mM MgCl2; pH 7.28). HaltTM Protease Inhibitor 100 × (Thermo Scientific) was added to the lysis buffer solution. The cells were vortexed at 6000 g for 1 min, and then placed in ice.
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2.7.1. DPP® testing protocol Testing procedure for the DPP® C. albicans assay is as follows: prepare 1:25 dilution of the sample to be tested in the sample migration buffer. Add 60 μL of the mixture into the S1 port of the DPP® device and allow for a 5 minute incubation period. Following, add 150 μL of migration buffer into the S2 port of the DPP® device. Allow 15 min to pass and read device visually and with use of the custom ESEQuant reader for quantification of test and control line results.
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2.7.1.1. Cells in PBS. Candida and S. cerevisiae cells were serially diluted to a concentration of 1:128 with PBS in 1.5 mL Eppendorf tubes to test for reactivity in the DPP® C. albicans assay. Following, samples were further diluted at a 1:25 dilution using Chembio's sample migration buffer as the diluent. 60 μL of the sample and buffer mixture solution was then added to the S1 port of the DPP® assay as per the DPP® C. albicans testing protocol described above.
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In-house LF tests were made by removing the sample strip (S1 Strip; Fig. 2) from the DPP® devices. Sample mixture was then added directly into the S2 well housing the detector particles. The sample and buffer solution then migrated onto the assaying region containing the capture antibodies selected for incorporation into DPP®. Migration of the analytes therefore occurred on the unilateral strip as in conventional
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2.6.3. Enzymatic lysis Zymolase (Zymo Research, 5 units/μL) was used to lyse the cells. 2 μL of zymolase was added to each yeast strain. Cells were incubated at 37 °C for 60 min and then taken out and put on ice to cease the reaction.
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2.5.4. Testing for best capture/detector antibodies In order to determine the best pair of antibodies, a series of DPP® test trials were carried out on the most efficient capture antibodies determined by the MAPIA primary screening process. The assays were prepared according to Chembio proprietary assembly standards and run as follows. First, 60 μL of the diluted C. albicans analyte was added to the S1 port (Fig. 1a). After 5 min of incubation, 150 μL of buffer was added to the S2 port (Fig. 1a), followed by a test reading, both visually and by a semi-automated reader fifteen minutes later (ESEQuant, Qiagen).
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2.7.1.4. Hyphal induction of C. albicans in blood and serum. The virulence of C. albicans is highly dependent on its morphology. The nonfilamentous budding form is less virulent than the pseudohyphal and hyphal forms, the latter being able to create filamentous elongations that penetrate through epithelial tissue and subsequently enter the bloodstream. To induce hyphal growth, spiked specimens negative blood and serum were spiked with C. albicans 6.35 × 108 cells/mL) in a 1:1 dilution) were incubated at 37 °C for 90 min on a rotator set to 200 rpm. After incubation, samples are serially diluted up to 1:128 in both negative blood and serum for use in the DPP® C. albicans assay.
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diameter via dynamic light scattering instrument (Malvern). The pH of the nanogold solution was adjusted near the Isoelectric Point (pI) of each antibody to facilitate a successful and efficient conjugation. Concentration of the conjugated nanogold was then adjusted to 50 O.D. (peak O.D. observed at 520–530 nm) and sprayed onto a fiber composite conjugate pad (Ahlstrom) using a Flatbed Imagene Printer at a spray rate of 5 μL/cm.
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2.7.1.2. Cells in serum. Serological tests were conducted to prescreen for non-reactivity to Candida on the DPP® assay. Unknown patient serum samples (ZeptoMetrix) were spiked with C. albicans (1.0 × 106 cells/mL) at a 1:1 to 1:128 dilution. 2.7.1.3. Cells in human whole blood. Whole blood samples (ZeptoMetrix) were also prescreened for non-reactivity to Candida. When a
Fig. 2. Multi-antigen print immunoassay for varying C. albicans antibodies. 1–20: 1–4) S. cerevisiae lysate protein, 5) whole S. cerevisiae cells, 6–9) C. tropicalis lysate proteins, 10) whole C. tropicalis cells, 11–14) C. lusitaniae lysate proteins, 15) whole C. lusitaniae cells, 16–19) C. albicans lysate proteins, 20) whole C. albicans cells, 21) Protein A. A–F: A) Mouse monoclonal anti-C. albicans antibodies (HyTest Ltd.) at 1:50, B) mouse monoclonal anti-C. albicans antibodies (HyTest Ltd.) at 1:100, C) rabbit polyclonal anti-C. albicans (Virostat, Inc.) at 1:100, D) rabbit polyclonal anti-C. albicans (Virostat, Inc.) at 1:500, E) rabbit polyclonal anti-C. albicans (Meridian Life Science, Inc.) at 1:100, F) rabbit polyclonal anti-C. albicans (Meridian Life Science, Inc.) at 1:500. Printed antibodies were tested against both surface (from whole cells) and intracellular lysate proteins of C. albicans, C. lusitaniae, C. tropicalis, and the negative control S. cerevisiae. Cells were lysed using bead bashing, triton detergent, or Zymolase enzyme. Bands with faint to dark purple intensities are indicative of a reaction with the printed antigens/protein and C. albicans antibodies.
Please cite this article as: Gunasekera, M., et al., Development of a Dual Path Platform (DPP®) immunoassay for rapid detection of Candida albicans in human whole blood and serum, J. Immunol. Methods (2015), http://dx.doi.org/10.1016/j.jim.2015.04.014
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A1 = Monoclonal Ab from HyTest (CAT # 3CA4)
A1/A1 A1/A2 A1/A3 A2/A1 A2/A2 A2/A3 A3/A1 A3/A2 A3/A3
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A3 = Polyclonal Ab from Virostat (CAT # 6411)
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To investigate the feasibility of developing a C. albicans antigen detection assay utilizing the DPP® technology, anti-C. albicans antibodies were first screened on MAPIA for reactivity against members of the Candida genus and the S. cerevisiae negative control (Fig. 2). Following this primary screen, various capture and detector antibody combinations were then introduced into the DPP® platform and further tested for reactivity (Table 1).
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Fig. 2 illustrates the immunoreactivity for all commercially purchased anti-C. albicans antibodies, as well as their affinity for lysate epitopes. Monoclonal anti-C. albicans antibody was nonreactive to other members of the Candida genus (Candida lusitaniae, and C. tropicalis) in
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To establish the negative threshold for the DPP® C. albicans assay, the S. cerevisiae control was tested in triplicate at varying concentrations in 10 mM PBS. For all testing, no test line was made visible, thereby indicating a negative result, and the highest reader output was 26 mV in intensity. Therefore, to account for non-trending occurrences, a
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A secondary screening assessed various combinations of antiC. albicans capture and detector antibodies in the DPP® platform for signal intensity (Table 1). Monoclonal antibody combinations resulted in signals of low intensity. Polyclonal exclusive anti-C. albicans antibody combinations (Virostat and Meridian) created high signal intensity, indicating binding of the analyte at the test line. Subsequently, these combinations were selected as the best capture/detector agent for incorporation into the DPP® C. albicans assay. Upon the selection of capture and detector antibodies for the DPP® C. albicans assay, experiments were conducted to determine device sensitivity, specificity, cross-reactivity, and limit of detection (Figs. 4–8), as well as comparison with in-house LF assays In addition, a comparative study was performed to illustrate how the DPP® technology fares against LF (Fig. 8). All results were plotted against an established negative threshold for the DPP® C. albicans assay (Fig. 3).
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LF assays. After a 20 minute wait period, device results were interpreted using the same reader instrument utilized in DPP® quantification. The in-house LF assay was semi-optimized with 2 M urea into the buffer solution. Previous empirical data has shown that LF generates a high degree of false positive results due to non-specific binding of the analyte–detector complex (Chembio Diagnostics Systems, 2014). Hence, a more accurate representation of the sensitivities between the two prototypes was achieved by addition of the urea denaturant.
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addition to the negative control, S. cerevisiae (Fig. 2: lane A and B). Faint purple bands were observed for printed C. albicans whole cells and printed C. albicans cell lysate proteins (mix of intracellular and surface/ membrane proteins), indicating weak reactivity of the monoclonal antibody to epitopes on Candida cells. Polyclonal anti-C. albicans antibodies (Fig. 2: lanes C–F) were reactive to C. albicans, C. lusitaniae, C. tropicalis, and S. cerevisiae. In particular, darker bands were visible for printed whole cells of C. albicans, C. lusitaniae and C. tropicalis whereas, faint bands were visible for their printed intracellular lysate protein counterparts and S. cerevisiae. Hence, all polyclonal antibodies display a higher affinity for extracellular proteins of the various yeast organisms, thereby, eliminating the need for a sample pre-treatment (lysis of whole cells) step in the DPP® C. albicans assay. As such, all further testing was conducted using whole Candida and S. cerevisiae cells.
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Fig. 3. Establishment of negative threshold for DPP® C. albicans Assay using S. cerevisiae as negative control. Negative threshold for DPP® C. albicans assay. S. cerevisiae cells (unlysed) were serially diluted to 1:4096 from neat (6.35 × 108 cells/mL) in 10 mM PBS. Average taken from samples tested in triplicate using DPP® C. albicans' assay. Error bars indicate standard deviation. Negative threshold established to be 30 mV.
Please cite this article as: Gunasekera, M., et al., Development of a Dual Path Platform (DPP®) immunoassay for rapid detection of Candida albicans in human whole blood and serum, J. Immunol. Methods (2015), http://dx.doi.org/10.1016/j.jim.2015.04.014
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In order to assess the POC assay's and performance characteristics, cultured whole C. albicans cells (both hyphal and yeast budding forms of strain SC5314), were spiked into negative human blood and serum. Following serial dilutions were made and tested so as to represent varying concentrations of the C. albicans analyte in human blood and serum medium. Testing was performed in quintuplicate and nonreactive results were characterized with a reader output of ≤30 mV in conjunction with the absence of a colored test line. The DPP® C. albicans assay
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was unsuccessful at detecting C. albicans (in its yeast budding form) analytes at a 1:32 and 1:64 dilution in human blood and serum respectively whereas, detection of C. albicans in its hyphal form ceased at a concentration of 1:16 and 1:32 in the DPP® C. albicans assay in human blood and serum, respectively (Fig. 4). The limit of detection (LoD) was calculated for hyphal C. albicans in blood and serum as 1.59 × 106 and 7.94 × 105 cells/mL (95,300 and 47,600 whole cells), respectively. In turn, the LoD for budding yeast form of C. albicans in blood and serum are 7.94 × 105 and 3.97 × 105 cells/mL (47,600 and 23,800 whole cells), correspondingly (Fig. 5). These numbers are comparable to present PCR diagnostic modalities that are able to detect C. albicans at a magnitude of 105 whole cells in human blood (Maaroufi et al., 2003).
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Cross reactant tests of pathogens inherent of immunocompromised 321 patients, as well as non-Candida yeast samples were performed to 322
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reader value of 30 mV was chosen to be the negative threshold for the DPP® C. albicans assay (Fig. 3). Reader values were measured using a customized ESEQuant reader (Qiagen) outfitted for DPP® devices. Using a series of imaging algorithms, the ESEQuant reader is able to quantify the color reaction (or lack of) observed on both the Test and Control lines on the DPP® C. albicans assay.
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Fig. 4. Average DPP® C. albicans test line intensity of hyphal and yeast Candida albicans in negative human serum and blood at varying concentrations. Sensitivity profile of DPP® C. albicans assay using spiked negative human blood (Zeptometrix Corp.) and serum (BioreclamationIVT). Blood and serum were spiked with both hyphal and yeast C. albicans' strain SC5314 to a concentration of 6.35 × 108 cells/mL. Samples were then serially diluted in either negative human blood or serum concentration down to 1:128. Averages were taken from samples tested in quintuplicate using DPP® C. albicans assay. Negative threshold set at 30 mV. Error bars indicate standard deviation.. Hyphal C. albicans detection in human blood and serum ceases at 1:16 and 1:32, respectively, whereas yeast C. albicans detection ceases at 1:32 dilution and 1:64 dilution in human blood and serum, respectively.
Fig. 5. Limit of detection of hyphal C. albicans and yeast C. albicans in human whole blood and serum in DPP® assay. Limit of detection for the DPP® C. albicans assay. C. albicans cells (both yeast budding and hyphal forms) were spiked into negative human blood and serum (Zeptometrix Corp.; BioreclamationIVT) at varying concentrations until no positive signal was elicited by the assay. Limit of detection (LoD) for hyphal C. albicans in blood and serum is 1.59 × 106 and 7.94 × 105 cells/mL, respectively. LoD for budding yeast form of C. albicans in blood and serum is 7.94 × 105 and 3.97 × 105 cells/mL, respectively.
Please cite this article as: Gunasekera, M., et al., Development of a Dual Path Platform (DPP®) immunoassay for rapid detection of Candida albicans in human whole blood and serum, J. Immunol. Methods (2015), http://dx.doi.org/10.1016/j.jim.2015.04.014
M. Gunasekera et al. / Journal of Immunological Methods xxx (2015) xxx–xxx
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Fig. 6. Average test line signal intensity for C. albicans, S. cerevisiae, C. lusitaniae, & C. tropicalis in human serum. Specificity testing of the DPP® C. albicans assay. C. albicans, S. cerevisiae, C. lusitaniae, and C. tropicalis were individually spiked in negative human serum (BioreclamationIVT). Averages taken from triplicate testing of each sample in DPP® C. albicans assay. Negative threshold set at 30 mV. Error bars indicate standard deviation.
3.6. DPP® vs. LF technology
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The DPP® assay was tested against an in-house LF device (semioptimized with 2 M urea in buffer solution) counterpart for evaluation
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The results generated indicate the feasibility of producing a DPP® assay for detection of C. albicans cells in human blood and serum and its marked improvement over the conventional LF assay. Experiments have demonstrated a limit of detection (LoD) for hyphal C. albicans in blood and serum as 1.59 × 106 and 7.94 × 105 cells/mL, respectively whereas the LoD for budding yeast form of C. albicans in blood and serum are 7.94 × 105 and 3.97 × 105 cells/mL, respectively. The assay's reactivity with HIV-2 specimens, as well as with C. lusitaniae and
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between the two technologies. Negative human serum was spiked with varying concentrations of C. albicans cells. In every instance, the DPP® assay was able to generate intensity results of at minimum 2.5× higher than LF based assay for samples that contained cell concentration within the DPP® C. albicans' LoD (Fig. 8).
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assess the specificity of DPP®. Tests were done in quintuplicate and were evaluated as positive if the average signal intensities were above 30 mV. Reactants tested include antigens specific to: HIV-1, HIV-2, HSV-1, HSV-2, Rubella, and Hepatitis C. Additionally, select members of the Candida genus along with the yeast S. cerevisiae were assessed. Of all tested reactants, the DPP® C. albicans assay generated a reactive result to HIV-2 antigens whereas all tested Candida genus' elicited low positive results for the C. albicans test line and S. cerevisiae resulted in a nonreactive status (Figs. 6 and 7). Note that other Candida spp.– C. glabrata, and C. parapsilosis, were not utilized for testing due to unavailability.
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Fig. 7. Cross-reactivity profile of the DPP® C. albicans assay. HIV1 1:240: strong HIV1 positive sample. HIV1 1:1800: low HIV1 positive sample. HIV2 1:32: strong HIV2 positive sample. HIV2 1:128: low HIV2 positive sample. PTH201-06: low HSV1 positive sample. PTH201-05: low HSV1 positive sample. PTH201-04: strong HSV 1/2 positive sample. PTR201-13: strong Rubella positive sample. PTR201-01: low positive Rubella sample. PHV207-08: strong Hepatitis C positive sample. PHV207-02: low Hepatitis C positive sample. HIV samples provided by Zeptometrix Corporation. All other samples acquired from SeraCare Life Sciences. Averages taken from quintuplicate testing of each sample in DPP® C. albicans assay. Negative threshold set at 30 mV. Error bars indicate standard deviation. HIV2 1:32 results are above the negative threshold and thus yields a positive result.
Please cite this article as: Gunasekera, M., et al., Development of a Dual Path Platform (DPP®) immunoassay for rapid detection of Candida albicans in human whole blood and serum, J. Immunol. Methods (2015), http://dx.doi.org/10.1016/j.jim.2015.04.014
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Acknowledgments
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We would like to thank Dr. James Konopka, as well as his lab members (Molecular Genetics and Microbiology Department at Stony Brook) for providing strains, support, and the lab space for MG to carry out initial experiments on C. albicans.
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References
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Chembio Diagnostics Systems, I.M., NY, US), 2010. Dual Path Platform vol. 2015. Chembio Diagnostics Systems, I.M., NY, US), 2014. Comparison of Lateral Flow to Dual Path Platform in Rubella Antigen detection assay.
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Esfandiari, J.S.B., NY, US), 2010. Dual path immunoassay device vol. 7682801. Chembio Diagnostic Systems, Inc., (Medford, NY, US), United States. Feldman, M., Grenier, D., 2012. Cranberry proanthocyanidins act in synergy with licochalcone A to reduce Porphyromonas gingivalis growth and virulence properties, and to suppress cytokine secretion by macrophages. J. Appl. Microbiol. 113, 438. Gomez-Raja, J., Andaluz, E., Magee, B., Calderone, R., Larriba, G., 2008. A single SNP, G929T (Gly310Val), determines the presence of a functional and a non-functional allele of HIS4 in Candida albicans SC5314: detection of the non-functional allele in laboratory strains. Fungal Genet. Biol. 45 (4), 527. Hua, Z., Rouse, J.L., Eckhardt, A.E., Srinivasan, V., Pamula, V.K., Schell, W.A., Benton, J.L., Mitchell, T.G., Pollack, M.G., 2010. Multiplexed real-time polymerase chain reaction on a digital microfluidic platform. Anal. Chem. 82, 2310. Iregbu, K.C., Esfandiari, J., Nnorom, J., Sonibare, S.A., Uwaezuoke, S.N., Eze, S.O., Abdullahi, N., Lawal, A.O., Durogbola, B.S., 2011. Dual Path Platform HIV 1/2 assay: evaluation of a novel rapid test using oral fluids for HIV screening at the National Hospital in Abuja, Nigeria. Diagn. Microbiol. Infect. Dis. 69, 405. Kojic, E.M., Darouiche, R.O., 2004. Candida infections of medical devices. Clin. Microbiol. Rev. 17, 255. Kostiala, A.A., Kostiala, I., 1981. Enzyme-linked immunosorbent assay (ELISA) for IgM, IgG and IgA class antibodies against Candida albicans antigens: development and comparison with other methods. Sabouraudia 19, 123. Lyashchenko, K.P., Singh, M., Colangeli, R., Gennaro, M.L., 2000. A multi-antigen print immunoassay for the development of serological diagnosis of infectious diseases. J. Immunol. Methods 242, 91. Maaroufi, Y., Heymans, C., De Bruyne, J.M., Duchateau, V., Rodriguez-Villalobos, H., Aoun, M., Crokaert, F., 2003. Rapid detection of Candida albicans in clinical blood samples by using a TaqMan-based PCR assay. J. Clin. Microbiol. 41, 3293. Mayer, F.L., Wilson, D., Hube, B., 2013. Candida albicans pathogenicity mechanisms. Virulence 4, 119. Pfaller, M.A., Jones, R.N., Messer, S.A., Edmond, M.B., Wenzel, R.P., 1998. National surveillance of nosocomial blood stream infection due to species of Candida other than Candida albicans: frequency of occurrence and antifungal susceptibility in the SCOPE Program. SCOPE Participant Group. Surveillance and Control of Pathogens of Epidemiologic. Diagn. Microbiol. Infect. Dis. 30, 121. Yapar, N., 2014. Epidemiology and risk factors for invasive candidiasis. Ther. Clin. Risk Manag. 10, 95. Zucchi, P.C., Davis, T.R., Kumamoto, C.A., 2010. A Candida albicans cell wall-linked protein promotes invasive filamentation into semi-solid medium. Mol. Microbiol. 76, 733.
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C. tropicalis cells, indicates a need for improvement of the assay's specificity performance for clinical trials. The polyclonal nature of antiC. albicans capture and detector antibodies may be the causative agent responsible for the observed false-positive results; due to the lack of viable commercially available monoclonal anti-C. albicans antibodies for immunochromatographic tests, the DPP® C. albicans assay is restrained to the antibody's characteristics. Although cross-reactivity and specificity issues are of concern within the DPP® C. albicans assay, it is important to note that the device can effectively detect C. albicans cells at varying concentration at a level much higher than its LF counterpart while still preserving the current device specificity. Furthermore, the positive signal achieved with the DPP® C. albicans assay is enhanced by 2.5–3.9× when compared to LF-based detection for samples within the device's LoD. Hence, the DPP® technology is considered superior in sensitivity and specificity over LF technology. Albeit the experiments within have been conducted with cultured cells, future work is expected to be well-translated in the clinical setting. Thus, impending research will focus on the clinical efficacy of the DPP® C. albicans assay over its lateral flow counterpart.
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Fig. 8. Comparison of DPP® versus lateral flow (LF) technology. C. albicans spiked in negative human serum at varying dilutions and tested in triplicate in DPP® C. albicans assay. Averages plotted. Negative threshold set at 30 mV. Error bars indicate standard deviation. At maximum, DPP® is 3.9× greater in sensitivity than LF.
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Please cite this article as: Gunasekera, M., et al., Development of a Dual Path Platform (DPP®) immunoassay for rapid detection of Candida albicans in human whole blood and serum, J. Immunol. Methods (2015), http://dx.doi.org/10.1016/j.jim.2015.04.014
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