Anal. Chem. 2005, 77, 7673-7678
Multiplexed Flow Cytometric Immunoassay for Influenza Virus Detection and Differentiation Xiaomei Yan,*,†,‡ Wenwan Zhong,§ Aijun Tang,§ Erika G. Schielke,†,| Wei Hang,‡,§ and John P. Nolan†,⊥
Bioscience Division and Chemistry Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, and La Jolla Bioengineering Institute, 505 Coast Boulevard South Suite 405, La Jolla, California 92037
Microsphere-based immunoassay by flow cytometry has gained popularity lately in protein detection and infectious disease diagnosis due to its capacity for multiplexed analysis and simple assay format. Here, we demonstrated the power of microsphere-based immunoassay for highsensitivity detection and accurate differentiation of influenza viruses. The effects of sample volume and bead number on the assay sensitivity of viral antigen detection were studied. Compared to enzyme-linked immunosorbent assays, flow-based bead assays provided ∼10-fold lower detection limit for viral particle detection and performed similarly for recombinant viral hemagglutinin protein detection. A four-plexed assay for influenza virus typing and influenza B virus sublineage characterization was developed to demonstrate the potential for multiplexed viral antigen detection and differentiation. Accurate and early disease diagnosis is essential for prompt therapeutic treatment and infection control.1 A multiplexed differentiation tool that can rapidly and accurately discriminate the presence of pathogen species and types causing similar clinical syndromes is critically needed for both public health improvement and biothreat reduction purposes.2 Antibody-based immunoassays are of enormous importance in disease diagnosis because they offer unique detection specificity and sensitivity with relative simple and economical assay format. An array of detection probes is usually employed for detection of a large number of antigens simultaneously.3 Antibody arrays are most often constructed in a planar format by spotting antibodies onto a slide, membrane, well, or other planar surface at different locations.4-6 A suspension array * Corresponding author. E-mail:
[email protected]. † Bioscience Division, Los Alamos National Laboratory. ‡ Present address: Department of Chemistry, and The Key Laboratory of Analytical Sciences of the Ministry of Education, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China. § Chemistry Division, Los Alamos National Laboratory. | Current address: Department of Ecology & Evolutionary Biology, Yale University, 165 Prospect St., New Haven, CT 06520. ⊥ La Jolla Bioengineering Institute. (1) Hinman, A. R. Clin. Chem. 1992, 38, 1532-1538. (2) Jani, I. V.; Janossy, G.; Brown, D. W.; Mandy, F. Lancet Infect. Dis. 2002, 2, 243-250. (3) Joos, T. O.; Stoll, D.; Templin, M. F. Curr Opin Chem Biol 2002, 6, 76-80. (4) Stevens, P. W.; Wang, C. H.; Kelso, D. M. Anal. Chem. 2003, 75, 11411146. (5) Knecht, B. G.; Strasser, A.; Dietrich, R.; Martlbauer, E.; Niessner, R.; Weller, M. G. Anal. Chem. 2004, 76, 646-654. (6) Delehanty, J. B.; Ligler, F. S. Anal. Chem. 2002, 74, 5681-5687. 10.1021/ac0508797 CCC: $30.25 Published on Web 11/02/2005
© 2005 American Chemical Society
created by immobilizing capture reagents on the surfaces of encoded microspheres in conjunction with flow cytometric analysis provides an alternative way of producing antibody arrays for immunodiagnostics.2,7-12 The array elements, the microspheres, are classified by their distinct optical properties, such as light scatter or fluorescence from an internal dye.9 Compared to planar array, suspension array offers greater flexibility, reduced antibody denaturation occurrence, and faster reaction kinetics.8 Also, the high binding capacity of three-dimensional microspheres makes the suspension array a very sensitive platform for immunoassays.13,14 The current generation of suspension arrays contains between 12 and 100 discrete array elements, but an array size of 1000 is possible to be achieved by improving the microsphere encoding approach.8,15 Similar to a microplate-based enzyme-linked immunosorbent assay (ELISA), antigen detection-oriented microsphere-based immunoassays by flow cytometry are mostly sandwich immunoassays involving two different antibodies that target to two different and spatially separated epitopes of the antigen. Analytes are first captured by the antibodies chemically coupled to the surface of microspheres, and then the fluorochrome-conjugated reporter antibodies are bound onto the analytes.3 While the microspheres are interrogated sequentially by the flow cytometer, optical signals from every single microsphere itself and the reporter antibodies are measured to reveal the identity and the quantity of the captured antigens, respectively.2 The microspherebased immunoassays have been used as an efficient tool in multiplexed cytokine secretion measurement,16-18 immunoglobulin quantification,19 and studies of infectious diseases.2,7,11,13 Simulta(7) Nolan, J. P.; Mandy, F. F. Cell. Mol. Biol. 2001, 47, 1241-1256. (8) Nolan, J. P.; Sklar, L. A. Trends Biotechnol. 2002, 20, 9-12. (9) Edwards, B. S.; Oprea, T.; Prossnitz, E. R.; Sklar, L. A. Curr. Opin. Chem. Biol. 2004, 8, 392-398. (10) Iannone, M. A. Clin. Lab. Med. 2001, 21, 731-742. (11) Kellar, K. L. J. Clin. Ligand Assay 2003, 26, 76-86. (12) Vignali, D. A. J. Immunol. Methods 2000, 243, 243-255. (13) Kellar, K. L.; Iannone, M. A. Exp. Hematol. 2002, 30, 1227-1237. (14) McBride, M. T.; Masquelier, D.; Hindson, B. J.; Makarewicz, A. J.; Brown, S.; Burris, K.; Metz, T.; Langlois, R. G.; Tsang, K. W.; Bryan, R.; Anderson, D. A.; Venkateswaran, K. S.; Milanovich, F. P.; Colston, B. W., Jr. Anal. Chem. 2003, 75, 5293-5299. (15) Kettman, J. R.; Davies, T.; Chandler, D.; Oliver, K. G.; Fulton, R. J. Cytometry 1998, 33, 234-243. (16) Chen, R.; Lowe, L.; Wilson, J. D.; Crowther, E.; Tzeggai, K.; Bishop, J. E.; Varro, R. Clin. Chem. 1999, 45, 1693-1694. (17) Carson, R. T.; Vignali, D. A. J. Immunol. Methods 1999, 227, 41-52. (18) Kellar, K. L.; Kalwar, R. R.; Dubois, K. A.; Crouse, D.; Chafin, W. D.; Kane, B. E. Cytometry 2001, 45, 27-36.
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Table 1. Selected Features of Influenza A Viral Proteins protein ID P3 (PB2) P1 (PB1) P2(PA) HA NP NA MP
function
MW (×103 Da)
minor internal protein with NP form ”polymerase complex“ hemagglutinin, major surface protein nucleoprotein, major internal protein neuraminidase, surface protein matrix protein, major internal protein
neous detection of multiple simulants of biological warfare agents has also been demonstrated.14 We reported the first application of microsphere-based immunoassay in respiratory viral antigen detection, particularly influenza virus detection and type differentiation.20 Influenza is a highly contagious acute respiratory viral disease of global importance.21 On the basis of antigenic differences in nucleoprotein (NP) and matrix protein (MP), influenza viruses caused the majority of clinically significant diseases and can be differentiated as A and B types. Selected features of influenza A viral proteins are listed in Table 1.22 Influenza type A viruses are further divided into subtypes based on the antigenic differences in two viral surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). H1N1 and H3N2 are the current circulating influenza A subtypes in humans. Over the past 20 years, influenza B viruses have caused frequent epidemics in humans, as have the H1 and H3 subtypes of influenza A viruses. Although influenza B viruses are generally considered less virulent than influenza A viruses, recent studies have demonstrated a substantial clinical impact attributable to influenza B viruses that were shown to cause severe respiratory symptoms as well as central nervous system diseases.23-25 While there are also no antigenically distinguishable subtypes for influenza B viruses based on the surface antigens, two genetically and antigenically distinct lineages of influenza B viruses, represented by the reference strains B/Victoria/2/87 (Victoria) and B/Yamagata/16/1988 (Yamagata), have co-circulated in humans since at least 1983.26-28 Recent advances in antiviral chemotherapy and the ever-present potential of pandemic influenza emphasize the importance of accurate and timely diagnostic techniques.29-31 (19) Dasso, J.; Lee, J.; Bach, H.; Mage, R. G. J. Immunol. Methods 2002, 263, 23-33. (20) Yan, X.; Schielke, E. G.; Grace, K. M.; Hassell, C.; Marrone, B. L.; Nolan, J. P. J. Immunol. Methods 2004, 284, 27-38. (21) Cox, N. J.; Subbarao, K. Lancet 1999, 354, 1277-1282. (22) Schild, G. C. In Topley and Wilson’s Principles of Bacteriology, Virology and Immunity; Brown, F. W., G., Ed.; Williams & Wilkins: Baltimore, 1984; Vol. IV, pp 315-344. (23) Fujimoto, S.; Kobayashi, M.; Uemura, O.; Iwasa, M.; Ando, T.; Katoh, T.; Nakamura, C.; Maki, N.; Togari, H.; Wada, Y. Lancet 1998, 352, 873-875. (24) Chan, C. H.; Wu, M. C.; Huang, C. T.; Wu, K. G.; Liu, W. T. J. Med. Virol. 1999, 59, 208-214. (25) McCullers, J. A.; Facchini, S.; Chesney, P. J.; Webster, R. G. Clin. Infect. Dis. 1999, 28, 898-900. (26) Nerome, R.; Hiromoto, Y.; Sugita, S.; Tanabe, N.; Ishida, M.; Matsumoto, M.; Lindstrom, S. E.; Takahashi, T.; Nerome, K. Arch. Virol. 1998, 143, 1569-1583. (27) McCullers, J. A.; Wang, G. C.; He, S.; Webster, R. G. J. Virol. 1999, 73, 7343-7348. (28) Nakagawa, N.; Maeda, A.; Kase, T.; Kubota, R.; Okuno, Y. J. Virol. Methods 1999, 79, 113-120. (29) Playford, E. G.; Dwyer, D. E. Pathology 2002, 34, 115-125.
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approx copy no./virus
80
50
97 86 72 55 45 27
50 50 1000 1000 100-200 (150) 3000
total weight (×103 Da)
percentage
4000
1.76
4850 4300 72000 55000 6750 81000
2.13 1.89 31.59 24.13 2.96 35.54
Moreover, influenza resembles many other respiratory diseases (such as severe acute respiratory syndrome, SARS) and infections caused by many biothreat agents (such as Bacillus anthracis, Yersinia pestis, Botulinum toxin, etc.) in initial symptoms (sudden onset of high fever, headache, muscle pain, severe malaise, etc.). Therefore, the development of a method for influenza virus detection and characterization has immediate applications in therapeutic treatment, infection control, epidemiological surveillance, vaccine development, and biothreat reduction. In the present study, performance of microsphere-based immunoassay was compared with ELISA for both intact viral particles and recombinant viral protein detection. A four-plexed assay was developed with the capability to simultaneously detect and differentiate influenza virus types A and B and lineages B/Yamagata and B/Victoria to demonstrate the power of the liquid array assay format in multiplexed analyte detection. EXPERIMENTAL SECTION Reagents and Chemicals. Monoclonal antibodies (MAbs) against influenza virus A nucleoprotein (IVF8), A subtype H3 hemagglutinin (12/5), B hemagglutinin (4H7) were obtained from Biodesign International (Saco, ME). Influenza virus B nucleoprotein-specific MAb B114 was purchased from DakoCytomation California (Carpinteria, CA). Influenza virus B Victoria lineagespecific MAb 10B8 was a generous gift from Dr. N. Nakagawa at the Division of Bacteriology, Department of Public Health, Osaka Prefectural Institute of Public Health (Osaka, Japan). Inactivated influenza viruses were purchased from Research Diagnostics (Flanders, NJ) (A/Beijing/262/95, A/New Caledonia/20/99, and A/Taiwan/1/86), Advanced ImmunoChemical (Long Beach, CA) (A/Leningrad/325/88, B/Hong Kong/330/2001, B/Leningrad/ 86/93), and Biodesign International (A/Kiev/301/94, A/Panama/ 2007/99, A/Shandong/9/93, B/Qingdao/102/91, B/Tokio/53/ 99, B/Victoria/504/2000). Viral protein concentrations were provided in the manufacturer’s data sheet. Recombinant hemagglutinin protein of A/Panama2007/99 strain was purchased from ProteinSciences (Meriden, CT). Goat anti-influenza virus A/H1N1, A/H3N2, and B polyclonal antibodies (PAbs) were purchased from Cortex Biochem (San Leandro, CA). SPHERO carboxyl polystyrene particles of 5.4-µm diameter were purchased from Spherotech (Libertyville, IL). Luminex 100 (30) Li, K. S.; Guan, Y.; Wang, J.; Smith, G. J.; Xu, K. M.; Duan, L.; Rahardjo, A. P.; Puthavathana, P.; Buranathai, C.; Nguyen, T. D.; Estoepangestie, A. T.; Chaisingh, A.; Auewarakul, P.; Long, H. T.; Hanh, N. T.; Webby, R. J.; Poon, L. L.; Chen, H.; Shortridge, K. F.; Yuen, K. Y.; Webster, R. G.; Peiris, J. S. Nature 2004, 430, 209-213. (31) Ellis, J. S.; Zambon, M. C. Rev. Med. Virol. 2002, 12, 375-389.
LabMAP carboxylated microspheres were purchased from Luminex (Austin, TX). R-PE goat anti-mouse IgG (H+L) and rabbit anti-goat IgG (H+L) horseradish peroxidase conjugate were purchased from Molecular Probes (Eugene, OR). The Phycolink R-phycoerythrin (R-PE) Conjugation Kit was obtained from Prozyme (San Leandro, CA). Reporter antibodies were labeled with R-phycoerythrin as a 1:1:1 mixture of goat anti-influenza virus A/H1N1, A/H3N2, and B polyclonal antibodies. Siliconized microcentrifuge tubes and Maxisorp Nunc-Immuno Plates were purchased from Fisher Scientific (Pittsburgh, PA). Dulbecco’s phosphate-buffered saline (PBS, 1×, pH 7.4, without calcium and magnesium) was purchased from Invitrogen (Carlsbad, CA). Tween 20, bovine serum albumin (BSA), sodium azide, sodium acetate trihydrate, and 3,3′,5,5′-tetramethylbenzidine (TMB) Liquid Substrate System for ELISA were purchased from Sigma (St. Louis, MO). N-Hydroxysuccinimide (NHS) and 1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride (EDC) were obtained from Pierce Biotechnology (Rockford, IL). Incubation buffer (PBS/1% BSA/0.2% Tween 20) was used as the dilution buffer for virus, recombinant protein, and reporter antibodies in the microsphere-based assays. Less Tween 20 (0.05%) was used in the incubation buffer for ELISA. Covalent Coupling of Monoclonal Antibodies to Microspheres. A two-step coupling procedure optimized in our laboratory was used to covalently attach the capture MAbs to the carboxylated microspheres. Briefly, 1 × 107 microspheres were pipetted into 500 µL of activation buffer (10 mM sodium acetate, pH 5.0), to which 10 and 33 µL of freshly made NHS (50 mg/ mL) and EDC (100 mg/mL) solutions in the activation buffer were added, respectively. After 20 min of mixture incubation at room temperature, excess EDC/NHS was removed by microsphere pelleting (centrifugation at 10000g for 2 min) and supernatant aspiration. After washing once with 500 µL of the coupling buffer (10 mM sodium acetate, pH 5.0), the microspheres were resuspended in 500 µL of the coupling buffer, to which 50 µg of MAb was added. After incubating the microsphere and MAb mixture on a shaker at room temperature for 3.5 h, the MAb-bearing microspheres were washed twice with 500 µL of washing buffer (PBS/0.05% Tween 20), and stored at 4 °C in blocking/storage buffer (PBS/1% BSA/0.05% sodium azide) at a concentration of 2 × 104 microspheres/µL. Although same buffer (10 mM sodium acetate, pH 5.0) was used as both the activation and coupling buffer, removing excess EDC/NHS after bead activation was found to be critical in avoiding MAb cross-linking and in maintaining its antigen binding capacity (unpublished data). The relative efficiency of antibody coupling was estimated by incubating 2 × 104 MAb-coupled microspheres with 50 µL of 10 µg/mL R-PE conjugated goat anti-mouse IgG at 37 °C for 30 min. After washing once with 500 µL of washing buffer, the microspheres were resuspended in 300 µL of PBS and analyzed on a flow cytometer. Immunoassay Procedure for Microsphere-Based Assay. A total of 1000-50 000 MAb-coupled microspheres of each bead class or their mixture were added to 100 µL of inactivated influenza virus or recombinant HA protein diluted in incubation buffer. Following incubation at 37 °C for 45 min, two washes were carried out to remove the unbound viral antigens. Then 50 µL of 50 µg/ mL R-phycoerythrin-conjugated reporter antibody was added to resuspend the microsphere pellet. Following a 30-min incubation
at 37 °C, the beads were washed twice. In the end, the beads were resuspended in 300 or 50 µL of PBS buffer for analysis on the FACSCalibur or Luminex 100 system, respectively. Flow Cytometric Analysis. A FACSCalibur system (equipped with 488-nm air-cooled argon ion laser, Becton Dickinson, San Diego, CA) and a Luminex 100 system (equipped with 532-nm solid-state laser and 635-nm red diode laser, Luminex Corp.) were used in this study for single-plexed and multiplexed analysis, respectively. On the FACSCalibur flow cytometer, the microspheres were gated on forward light scatter and side (90°) light scatter, and the R-PE fluorescence signals were measured on the FL2 channel (564-606 nm). Median fluorescence intensity of 500 gated beads was reported by CellQuest software (Beckton Dickson). On the Luminex 100 system, the red laser excites the dye molecules inside the optically encoded bead and classifies the bead to its unique bead set through two classification channels. The green laser excites the fluorochromes bound to the bead and quantifies the assay on the bead surface. Median fluorescence intensity was reported by analyzing 100 gated beads for each bead set. Immunoassay Procedure for ELISA. The ELISA protocol described here has been optimized in our laboratory specifically for influenza viral antigen detection. Capture MAb was diluted in PBS to 1 µg/mL, and 100 µL was added to each well of an ELISA plate (Nunc Maxisorb). Plates were incubated overnight at 4 °C and then blocked with 200 µL of 1% BSA in PBS for 1 h at 37 °C. After washing twice with 200 µL of washing buffer, inactivated influenza virus or recombinant HA protein at variable concentrations in a 100-µL volume was added. After 37 °C incubation for 2 h, the plates were washed four times with 200 µL of washing buffer. A 100-µL aliquot of unconjugated reporter antibody cocktail (15 µg/mL total, 5 µg/mL for each of the three goat anti-influenza PAb) diluted in incubation buffer was added to each well. Plates were incubated at 37 °C for 1 h. After five washes, 100 µL of rabbit anti-goat horseradish peroxidase conjugate (1 mg/mL) at 1:1000 dilution was added to each well and incubated at 37 °C for 1 h. Following another five stringent washes, 100 µL of horseradish peroxidase substrate TMB was added and the reaction was allowed at room temperature for 5 min before being stopped by addition of 100 µL of 2 N H2SO4. The absorption was measured on a Microplate reader (SPECTRAFluor Plus, Tecan Group Ltd., Maennedorf, Switzerland) at 450 nm. It is worth noting that vigorous tapping of the plate against absorbent paper after each wash is important to reduce the background signal. RESULTS AND DISCUSSION Preliminary Investigation. Optimization of the assay procedure is possible to improve the performance of the assay. In the present study, we investigated the effects of the number of beads and sample volume used in the assay on the sensitivity. In the suspension array, each set of encoded microspheres represents one array element, and the flow cytometric measurement of every microsphere is actually one replicate analysis of the array element.8 Readings from 100 microspheres of the same bead set can provide adequate statistics for the behavior of that specific array element.8 However, in practice, a higher density of beads is employed. For example, 20 000 beads per reaction have been used in our studies to allow faster counting rate and clearer visual inspection of the bead pellet after microcentrifuging and during aspiration of Analytical Chemistry, Vol. 77, No. 23, December 1, 2005
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Figure 1. Bead number effect on the detection sensitivity of microsphere-based immunoassay.
Figure 2. Sample volume effect on the detection sensitivity of microsphere-based immunoassay.
washing buffer. As suggested, higher signal intensity can be expected by reducing the number of beads,12 which would increase the concentration of virus on the surface of each microsphere. A total of 1000-5000 beads per cytokine class have been reported in a multiplexed assay for human cytokine quantitation in serum and culture supernatants.18 In Figure 1, 1000, 2000, 5000, 10 000, 20 000, and 50 000 MAb IVF8-coupled beads were used to capture 500 and 100 ng/mL inactivated A/New Caledonia/20/99 virus in 100 µL of sample volume. Blanks (no viral antigen in the sample) were run for each tested bead number. Median fluorescence intensity with arbitrary units is shown on the Y-axis. As we can see, assay fluorescence intensity kept increasing as bead number decreased, while the fluorescence background for the blank controls seemed quite stable. When the bead number was reduced from 20 000 to 5000, the signal increased ∼30% for both 500 and 100 ng/mL viral protein concentrations. These results suggest that fewer beads can be used to enhance the detection sensitivity if it does not slow the analysis rate dramatically or incur bead handling problems. To be conservative, 5000 beads of each bead class were adopted in the later experiments. A total of 5000 beads per well has been reported in human serum immunoglobulin analysis using a 96well filter plate and vacuum manifold for sample preparation.19 To develop a more standardized assay format, sample volume needs to be compatible with clinical settings and preferably also with a 96-well plate format. For the detection of respiratory viruses, nasopharyngeal aspirates and nasal wash specimens are usually the specimens of choice, but nasal and throat swabs are also often used because of their greater ease of collection. In general, aspirate or wash specimens generate sample volumes larger than 0.5 mL, while swabs can contain the specimen in a small volume if not diluted in viral transport media. Upon viral antigen extraction using a specific buffer, the resulting sample volume can be as little as 100 µL or as large as several milliliters. Besides the specific diagnostic test, specimens may also need to be saved for other purposes or archived for chain of custody. Therefore, the available sample volume for respiratory viral antigen detection and characterization may vary from tens of microliters to several milliliters. The sample volume effect on the detection sensitivity of the microsphere-based immunoassay was investigated by incubating various volumes of 100 and 500 ng/mL inactivated A/New
Caledonia/20/99 virus with 5000 MAb-IVF8 coupled beads. Blanks were run for each tested volume. As shown in Figure 2, for both viral protein concentrations tested, the fluorescence signal kept increasing with increasing sample volume whereas the background signal was quite steady over all the tested volumes. This implies that if higher detection sensitivity is desired and if there is ample sample supply, larger sample volume can be used to enhance the assay sensitivity. Of course, standards need to be analyzed at the same volume to construct the calibration curves for quantitative measurement of viral antigen concentrations in the sample. To standardize the assay platform and for a fair comparison with the ELISA format, 100 µL of sample volume was used in our further tests. Assay Performance Comparison with ELISA. The dynamic range, reproducibility, and limit of detection (LOD) of the microsphere-based flow cytometric immunoassay were compared to results obtained using ELISA, the default standard of immunoassay. Titrations were conducted via 2-fold serial dilutions for both the inactivated influenza A/Panama/2007/99 virus and the recombinant HA protein of the same strain. MAb 12/5, which has demonstrated highly specific binding to the HA protein of A/Panama/2007/99 strain (data not shown), was used as the capture antibody. The same capture and reporter antibody pair was used in both the microsphere-based assay and ELISA. The titration results are displayed as log-log plots in Figure 3 with each data point and error bar representing the average and standard deviation of three replicates. The antigen concentration was examined over 6 orders of magnitude. LOD is taken as the antigen concentration at which the net median fluorescence intensity or absorbance (blank subtracted) is three times the standard deviation of the blank samples. As shown in Figure 3, the titration curves of the microspherebased assay are obviously shifted to the left of the ELISA curves for both inactivated virus and recombinant HA protein detection. Specifically, in comparison to ELISA, the microsphere-based immunoassay has ∼10-fold lower LOD for viral particle detection and a comparable LOD for recombinant HA protein. Wider dynamic ranges were obtained on microsphere-based assay for both the viral particle and recombinant protein detection. The sensitivity improvement in viral particle detection could be ascribed to the homogeneous liquidlike interaction in the micro-
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Figure 3. Performance comparison between microsphere-based immunoassay and ELISA on (a) viral particle and (b) recombinant hemagglutinin protein detection.
sphere-based assay that made the microsphere-captured viral particles more accessible to the binding of reporter antibodies, while in ELISA, the reaction took place only on the flat surface of the microplate. This 0.016 ng/mL detection limit obtained for influenza A/Panama/99/2007 strain is ∼380-fold lower than the detection limit (6.2 ng/mL) reported for the A/Beijing/262/95 strain (anti-NP MAb was used as capture antibody) in our earlier study.32 This significantly reduced detection limit for influenza virus detection could be attributed to the high-affinity binding of MAb12/5 to the surface HA protein of the influenza A virus subtype H3 and the easier protein accessibility of surface protein versus internal protein to the capture antibody. Of course, the optimized assay conditions, such as the improved MAb-microsphere coupling and the reduced bead number, were also helpful. Because hemagglutinin constitutes 29-32% of the total protein of the virus particles,22,33 0.016 ng/mL of the viral protein detection limit corresponds to 4.6-5.1 pg/mL for the hemagglutinin. However, when the recombinant HA protein was tested as the analyte, the detection limit obtained with the microsphere-based immunoassay was 0.45 ng/mL (Figure 3b), which was ∼100-fold higher. This can be explained by the fact that each viral particle provides remarkably more binding sites for the fluorochromeconjugated reporter antibodies than the individual hemagglutinin protein. Meanwhile, comparable reproducibility was obtained with ELISA and the microsphere-based immunoassay for both viral particle and recombinant protein detection. The relatively larger detection variation observed for the recombinant protein detection on both assay formats was mainly due to the hydrophobic interaction between protein and the sample tube or well. Multiplexed Immunoassays for Influenza Virus Detection and Differentiation. It has been reported that the two lineages of influenza B viruses are so different antigenically that vaccination (32) Yan, X. M.; Tang, A. J.; Schielke, E. G.; Hang, W.; Nolan, J. P. International Congress Series, 5th International Conference on Options for the Control of Influenza; October 7-11, 2003; Okinawa, Japan, 2004; Vol. 1263, pp 342345. (33) Oxford, J. S.; Corcoran, T.; Hugentobler, A. L. J. Biol. Stand. 1981, 9, 483491.
with strains from one lineage may not provide good protection against infection with strains from another lineage.34 Since influenza virus vaccines are currently formulated to include only a single strain of influenza B virus, this lack of antigenic cross-reactivity has made the designation of a type B vaccine strain problematic in seasons when viruses of both lineages circulate. Nevertheless, it would be important to examine whether the vaccine influenza B strain matches the lineage of the dominant circulating strain. Therefore, including lineage identification of influenza B viruses into the first steps in influenza virus characterization is necessary for improved surveillance and vaccine formulation. Using four sets of Luminex LabMAP microspheres and the Luminex 100 system, a multiplexed immunoassay was developed to simultaneously differentiate influenza virus types (A vs B) and influenza B virus sublineages (Yamagata vs Victoria). After screening more than 20 monoclonal antibody candidates, MAbIVF8 (anti-A-NP), MAb-114 (anti-B-NP), MAb-4H7 (anti-B-HA), and MAb-10B8 (anti-B-HA) were selected as the capture antibodies for influenza A, B, B/Yamagata, and B/Victoria viruses, respectively. Anti-HA MAb-10B8 has been demonstrated to react specifically with the strains of the B/Victoria group and did not react with any strains of the B/Yamagata group.28 For each of the 12 influenza strains (7 strains of influenza type A and 5 strains of type B) tested, a 100 ng/mL viral protein concentration was used. The trivalent influenza virus vaccine Fluzone (Aventis Pasteur Inc., Swiftwater, PA) was formulated to contain 45 µg of hemagglutinin per 0.5-mL dose, in the recommended ratio of 15 µg of HA each. The 2003-2004 formula contains following three prototype strains: A/New Caledonia/20/99 (H1N1), A/Panama/ 2007/99 (H3N2) (an A/Moscow/10/99-like strain), and B/Hong Kong/1434/2002 (a B/Hong Kong/330-2001-like strain). Before being tested in the microsphere-based immunoassay, this vaccine has been storage at 4 °C for 18 months. As shown in Figure 4, highly sensitive and specific detection and differentiation of influenza viruses were attained for all the (34) Levandowski, R. A.; Regnery, H. L.; Staton, E.; Burgess, B. G.; Williams, M. S.; Groothuis, J. R. Pediatrics 1991, 88, 1031-1036.
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Figure 4. A four-plexed microsphere-based immunoassay for influenza virus types and influenza B virus sublineage characterization on the Luminex 100 system.
tested samples. Though the nonspecific binding observed on the encoded Luminex beads was higher than that from the nonfluorescent Spherotech beads, the background readings generated by alternate influenza type or sublineages were comparable with that of the negative control (blank). Very low nonspecific binding was detected on the Flu B-specific bead as compared to other bead sets. With samples containing influenza A viruses, signals on the influenza A-specific beads were dominant and distinct compared to the readings on the influenza B-specific or B/sublineage-specific beads. On the other hand, the presence of influenza B virus triggered signals on both the influenza B-specific bead and one of the two sublineage-specific beads. The positive signals on two bead sets not only identified the influenza B sublineage but also confirmed the presence of influenza B virus. For the trivalent influenza virus vaccine of the 2003-2004 season, the presence of both influenza A and B viruses was confirmed by the strong signals on influenza A- and B/Victoria-specific beads even after 500-fold dilution. The marginal fluorescence intensity obtained on the influenza B-specific bead could be due to the vaccine manufacturing process, which was only focused on the preservation of HA protein upon purification. Another apparent feature reflected in Figure 4 was the stronger signal for influenza virus detection when surface protein (such as HA) was used as the target for capture antibody, compared to internal protein (such as NP). This can be ascribed to the greater accessibility of surface protein to the capture probe. Figure 4 demonstrated that with the availability of high-affinity and very specific antibodies, a microsphere-based immunoassay can be developed as a powerful tool in disease diagnosis and infection control. To our knowledge, this is the first reported multiplexed assay for influenza virus antigenic differentiation of types and sublineages of influenza B viruses.
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CONCLUSIONS Influenza virus detection and differentiation was demonstrated using a multiplexed microsphere-based immunoassay. Assay conditions were optimized and standardized by decreasing the number of beads and fixing the sample volume to 100 µL. Compared to ELISA, microsphere-based immunoassay displayed ∼10-fold lower detection limit for influenza viral particle and comparable detection limit for recombinant hemagglutinin protein. Microsphere-based immunoassay also provided a wider dynamic range and significantly less assay time as compared to ELISA. The four-plexed immunoassay developed for the antigenic detection and differentiation of influenza viruses demonstrated that microsphere-based flow cytometric immunoassay is an attractive and practical platform for accurate infectious disease diagnosis. This approach can be used to accurately identify and characterize pathogens causing similar medical syndromes for prompt treatment and infection control. ACKNOWLEDGMENT This work was supported by the Laboratory Directed Research & Development Program of Los Alamos National Laboratory and by the NIH-funded National Flow Cytometry Resource (RR01315). We thank Dr. A. Klimov of the CDC influenza branch, for providing us the WHO Influenza Reagent Kit, and Dr. N. Nakagawa of Osaka Prefectural Institute of Public Health, for his kind gift of monoclonal antibodies against influenza viruses.
Received for review May 19, 2005. Accepted September 29, 2005. AC0508797
