TY - JOUR
AU - Donath,, Edwin
AB - Abstract Background: Suspension array technology has surpassed ELISA for automated, simultaneous detection and quantification of soluble biomarkers such as virus-specific antibodies. We describe assays in which antigens are attached to a lipid bilayer surrounding color-coded particles. Methods: We used layer-by-layer technology to establish a multiplex suspension array with distinguishable microbeads coated with authentic viral surfaces to catch and quantify virus-specific antibodies in a flow cytometric analysis. Antigenic surfaces were generated by chimeric and wild-type baculoviruses plus 2 different influenza A virus subtypes fused to a lipid bilayer surrounding distinctly colored particles. Specificity of binding of chosen antibodies and sera was detected by immunofluorescence. Results of multiplex analysis were compared with results of ELISA. Results: Titrations of virus-specific antibodies in the multiplex suspension array demonstrated specific binding to the viral surface proteins. The multiplex suspension array gave positive results for up to log 5–diluted primary antibodies with an ∼5- to 10-fold reduced dynamic range compared with the respective ELISA. Conclusions: The bead-based multiplex suspension array is customizable and easy to establish. By displaying native influenza A virus surfaces and recombinant HIV-1 epitopes, the new assay provides a tool for the detection of major viral infections in humans. Suspension-based bead arrays are an attractive alternative to ELISA and other enzyme immunoassays for detection and quantification of soluble biomolecules (1). Bead arrays have great setup flexibility, require only small sample volumes (2), and can be used to analyze multiple components simultaneously (3). Multiplexing is achieved by linking capture molecules to distinctly colored sets of carrier beads. By measuring the fluorescence intensity of the beads and the analyte-specific signal with 2 separate detectors, it is possible to differentiate among the analytes and to quantify them in one experiment. A customizable system (4) allows the simultaneous quantification of up to 100 different biomarkers. Capture molecules can be attached individually to the activated surface of microbeads by use of carboxyl groups for covalent coupling or biotinylated species that can be immobilized on avidin-coated carriers. Several suppliers offer detection reagents, e.g., for quantification of human chemokines (5), which can be readily used with standard flow cytometric devices. Immunosorbent assays (6) and suspension-based arrays (7)(8) can be used to detect and quantify antibodies. Most immunologic tests cleared by the US Food and Drug Administration for virus-specific antibodies are ELISAs. Crude cell lysates of infected cells (9), recombinant viral proteins (10), or synthetic peptides(11) are immobilized on the surface of microtiter plates to capture and quantify the antibodies. Because of heterogeneity of the applied lysates, results may be inadequate unless expensive purified proteins are used, and coupling of the antigens often leads to inhomogeneous structure and surface orientation of the molecules and subsequent loss of activity. Finally, complex procedures for simultaneous detection of various virus-specific antibodies require further extension of the analytical setup. We adapted novel virus-coated layer-by-layer (LbL) colloids (12)(13) to produce a customizable and reliable suspension array for the detection and quantification of virus-specific antibodies. The authentic coating of lipid bilayer-surrounded microparticles with different viruses was demonstrated by Fischlechner et al. (14)(15). We fused 2 different baculoviruses (wild-type and recombinant) plus 2 influenza A viruses with color-coded microparticles and performed multiplexed fluorescence-activated cell-sorting (FACS) 1 titrations with specific antibodies and sera, and compared the results with results from ELISA. Materials and Methods viruses We propagated Autographa californica nuclear polyhedrosis virus (AcNPV; ATCC); its derivative AcCOPSNo10 (16), which displays a 17–amino acid epitope of HIV-1 gp120 N-terminally fused with a second copy of the major envelope protein gp64; and the construct AcZZVSVgTM-EGFP (17) in Sf9 (Spodoptera frugiperda; ATCC) with standard methods (18). We used modified IPL-41(16) from Sigma-Aldrich as medium. Viruses were prepared by ultracentrifugation [100 000g for 75 min at 20 °C with a sucrose cushion (250 g/L)] of cell culture supernatant 4 days after infection. The pellet was resuspended in phosphate-buffered saline (PBS; 8 g/L NaCl, 0.2 g/L KCl, 1.44 g/L Na2HPO4, 0.24 g/L KH2PO4; pH 7.4) containing 1 g/L sodium azide. Wild-type influenza A viruses A/Puerto Rico/8/34 (PR8, H1N1; laboratory strain) and A/Singapore/1/57 (Sg, H2N2; ATCC) were propagated in embryonated chicken eggs. The allantoic fluid was collected 4 days after infection and ultracentrifuged [100 000g for 120 min at 4 °C with a sucrose cushion (250 g/L)]; the viruses were resuspended in PBS (pH 7.4) containing 1 g/L sodium azide and inactivated by ultraviolet irradiation (30 min). Concentrated baculovirus was stored at 4 °C, and influenza A virus preparations were kept at −80 °C. The total protein concentration was determined with the Bio-Rad Protein Assay. fabrication of virus-coated LbL colloids Silica particles with a mean (SD) diameter of 3.03 (0.17) μm (microparticles GmbH) were used as template for the stepwise addition of 13 polyelectrolyte layers. Poly(allylamine hydrochloride) (PAH; Mr 70 000; 1 g/L in 0.5 mol/L NaCl) or rhodamine isothiocyanate (RITC)-labeled PAH (1:4.5 molar ratio; 1 g/L in 0.5 mol/L NaCl) was applied for the odd layers, and poly(sodium 4-styrenesulfonate) (Mr 70 000; 1 g/L in 0.5 mol/L NaCl) was applied for the even layers; the polyelectrolytes were obtained from Sigma-Aldrich. PAH-RITC was prepared as described by Reibetanz et al. (19). For each adsorption step, an equal volume of polyelectrolyte solution was added to a 50 mL/L (in 0.5 mol/L NaCl) solution of template beads and incubated for 10 min on a rocking shaker (100 rpm) at 20 °C followed by 3 washing steps with 0.1 mol/L NaCl and subsequent centrifugation (2000g for 2 min at room temperature). Addition of the lipid bilayer was performed by incubation of the polyelectrolyte-coated beads [last layer PAH (positively charged) in 0.1 mol/L NaCl] with an equal volume of unilamellar liposomes in 0.1 mol/L NaCl [lipid composition: 7.5 g/L l-α-phosphatidylserine (brain, porcine; sodium salt), 2.5 g/L l-α-phosphatidylcholine (tissue-derived, egg, 10 g/L) (14)] for 30 min (37 °C and 100 rpm). After 3 washing steps, the pellet was resuspended in 0.2 mol/L phosphate–0.1 mol/L citrate buffer (pH 4.5), and concentrated virus [total protein, 0.75 μg/μL of bead suspension (50 mL/L)] was added and incubated for 10 min (room temperature and 100 rpm). Finally, the virus-coated LbL colloids were washed 3 times with PBS and stored at 4 °C. antibodies The mouse-derived monoclonal antibody (mAb) B12D5 (20), which recognizes the major envelope fusion protein gp64 of AcNPV, was kindly provided by L. Volkman (University of California, Berkeley, CA). The mAb ARP360 (4A7C6, mouse) (21), which is specific for the peptide PQEVVLVNVT from HIV-1 gp120, was obtained from the National Institute for Biological Standards and Control (Potters Bar, UK). The polyclonal rabbit sera anti-PR8 and anti-Singapore were kindly provided by J. Romanova (Institute of Applied Microbiology, Vienna, Austria). Secondary antibodies, all obtained from Sigma-Aldrich, were as follows: fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG (γ-chain specific), peroxidase-conjugated anti-mouse IgG (γ-chain specific), FITC-conjugated anti-rabbit IgG (whole molecule); and peroxidase-conjugated anti-rabbit IgG (whole molecule). sds-page and western blotting Electrophoretic separation of proteins was performed with NuPAGE™ 4%–12% Bis-Tris Gels and NuPAGE MES SDS Running Buffer from Invitrogen. Loading buffer and prestained protein molecular markers were obtained from Fermentas Life Sciences. Samples were boiled for 10 min in loading buffer before analysis. After separation of proteins, the gels were either silver-stained or semidry-blotted on nitrocellulose membrane according to standard methods (22). Western blots were developed with 1/1000 dilutions of primary antibodies and subsequent incubation with the corresponding peroxidase conjugates (1/2000) in PBS containing 1 mL/L Tween 20 and 10 g/L bovine serum albumin fraction V (AppliChem). Detection was by the ECL Plus™ Western Blotting detection system (Amersham Biosciences), and membranes were exposed on a Lumi-Imager F1 (Boehringer Mannheim). confocal laser scanning microscopy Fluorescence imaging (confocal laser scanning microscopy) of influenza virus-coated LbL colloids was performed on a Leica TCS SP2 with Leica Confocal Software (LCS); 3-μm colloids were coated with influenza A PR8 and mixed with the same amount of empty beads. Staining was performed with anti-PR8 [1/500 dilution in PBS containing 50 mL/L fetal calf serum (FCS)] and FITC-conjugated anti-rabbit IgG (1/500 dilution). elisa We performed ELISA with MaxiSorb ELISA plates from Nalge-Nunc, according to previously described standard protocols (22). Concentrated virus diluted in citrate coating buffer (1 mg/L; 100 μL/well) was incubated overnight at 4 °C. After 3 washing steps, the serially diluted primary antibody (50 μL) was added and incubated for 1 h. A 1/1000 dilution of the secondary antibody (peroxidase conjugate) was then added (100 μL). Detection was with 1,2-o-phenylenediamine dihydrochloride, and the absorbance at 492 nm was measured with a Sunrise™ plate reader (Tecan) and the supplied Magellan™ software. multiplex suspension array LbL colloids comprising 1, 2, 4, and 7 layers of PAH-RITC were coated with AcCOPSNo10, A/PR8, AcNPV, and A/Singapore, respectively. Before pooling, the virus-coated LbL colloids were treated with 20 mL/L paraformaldehyde (Sigma-Aldrich) on ice for 10 min. Particles were washed and counted with a FACSCalibur (BD Biosciences), and equal amounts of all 4 populations were merged. Approximately 104 beads were stained for 30 min with the indicated antibody in a total volume of 50 μL of PBS containing 25 mL/L heat-inactivated FCS (HyClone). The beads were harvested by centrifugation (2000g for 2 min) and washed once with PBS. Subsequently, the colloids were incubated with the corresponding FITC-conjugated antibody (1/100 dilution in 20 μL of PBS containing 50 mL/L FCS) for 30 min. Finally, 300 μL PBS was added, and the sample was analyzed with the FACSCalibur. For each cytometric analysis, 103 single particles were counted (i.e., ∼250 of each subset). Data analysis was performed with WinMDI2.8 software (TSRI). Results After decomposition of the viral proteins by boiling in a reducing environment, we found that the microparticles were properly coated with the 4 selected viruses (Fig. 11 ). SDS-PAGE of concentrated viruses (Fig. 11 , lane 2 in each gel) and virus-coated colloids (Fig. 11 , lane 5 in each gel) revealed identical protein patterns, showing that the viruses were available on the colloidal composites. Bare LbL-colloid/lipid bilayer controls (Fig. 11 , lane 3 in each gel) and used washing buffer (Fig. 11 , lane 4 in each gel) lacked proteins completely. Estimation of the amount of colloid-bound viral proteins indicated that approximately one third of the applied viruses fused with the polyelectrolyte-supported lipid bilayers, a finding consistent with the amount of fusion reported with unilamellar liposomes (23). Western blot analysis of these gels (Fig. 11 , insets) stained with suitable mAbs or polyclonal sera verified the proper coating of our microparticles with viruses. We identified the most prominent viral surface proteins, hemagglutinin for influenza A virus and gp64 for baculovirus (or the recombinant gp64 (gp64-fus). The confocal laser-scanning micrograph in Fig. 22 shows the localization and distribution of influenza A virus surface proteins on our composites, as visualized by specific binding of anti-PR8 serum. Standard ELISA with serially diluted viruses confirmed specific binding of the antibodies and sera. As expected, the mAb B12D5, which is directed toward an epitope of the major envelope fusion protein gp64 of AcNPV, bound both the wild-type and recombinant baculovirus (Fig. 3A3 ). Moreover, because of its second recombinant copy of the gp64 surface protein, the signal for the AcCOPSNo10-coated composite exceeded that for the wild type (24). The mAb ARP360 specifically bound to its epitope sequence displayed on the recombinant baculovirus AcCOPSNo10 (Fig. 3B3 ), whereas the polyclonal influenza A virus-antisera showed extensive cross-reactivity between the 2 viruses A/PR8 and A/Singapore. The latter finding is not unexpected because the viruses are closely related and have structural similarities recognized by the polyclonal antibody population. Nevertheless, both antisera bound their specific antigen with higher affinity. Apart from these cross-reactions, we did not observe nonspecific binding of the antibodies against the 4 chosen viruses. To compare these results obtained with the presented novel multiplex suspension array, we performed a titration with a flow cytometric device (FACSCalibur). On cytometric analysis, colloids fabricated with 1, 2, 4, and 7 layers of fluorescently labeled polyelectrolytes displayed a lognormal distribution and peak separation of each set in the FL2-H channel (Fig. 44 ). The beads were subsequently coated with specific viruses, and equal numbers of each set were pooled. Before merging, the subpopulations were treated with paraformaldehyde to cross-link viral fusion proteins, a step that prevents undesired exchange of fusion-proficient viral particles between the carrier beads and reliably inactivates all viruses, enabling safe handling of the system. All antigenic virus surfaces on the beads were detected specifically by antibody staining, as shown in Fig. 55 . We performed the titration of the multiplex bead array with primary antibody dilutions from 10−2 to 10−6. Titration data acquired for each of the 4 antibodies are shown in Fig. 66 : the geometric mean fluorescence intensities (geo MFI) in the FL1-H channel are shown as a function of dilution of the primary antibody. The 4 virus-coated subpopulations were differentiated by their fluorescence in the FL2-H channel. All antigens could be detected with the suspension array, and nonspecific background signals were constantly low (geo MFI <7.5). Comparison of these results with the ELISA data showed similar sigmoidal regressions. ELISA displayed a sensitivity up to 10−5 or 10−6 dilutions of the primary antibodies and dynamic ranges of 3 to 4 logs, whereas the dynamic ranges were 1 log lower for the multiplexed FACS analysis, which led to ∼5- to 10-fold lower sensitivity depending on the assay (Figs. 33 and 66 ). Because of its dense protein load and enzymatic signal amplification, the applied ELISA outperformed the bead array in dynamic range and sensitivity. Furthermore, the bead array with the B12D5 antibody reached higher geo MFI values with the wild-type AcNPV-coated microspheres than with the recombinant AcCOPSNo10. This lower sensitivity might be a result of the recombinant fraction of gp64 on the latter construct interfering with proper membrane fusion, which could lead to less dense distribution on the colloids. However, the curve progressions within one array cannot be compared directly among themselves; therefore, a preliminary calibration for each subpopulation has to be performed and each antigen quantified individually on this basis. To estimate the stability of the virus-coated colloids, we stored a paraformaldehyde-treated array at 4 °C and analyzed samples as a function of time with anti-PR8 (Fig. 77 ). Peak separations of the different bead subpopulations in the FL2-H channel remained completely stable. The geo MFI values of the different components in the FL1-H channel decreased slightly (∼30% in 38 days), but the results indicated that, if calibrators are run with the assays, the array can be used for at least 5 weeks after preparation. We obtained similar results when we tested for the other viral surface proteins. The signal in the FL2 channel itself is rather stable. Even for arrays stored for more than 1 year, we did not observe a noticeable decrease in resolution (data not shown). Using a recombinant baculovirus that displays IgG-binding domains (ZZ) of protein A linked to a vesicular stomatitis virus (VSV) anchor domain on its outer membrane (AcZZVSVgTM-EGFP), we were able to immobilize immunoglobulins on the surface of the composites, as demonstrated by the specific coating of virus-coated beads with FITC-conjugated anti-mouse IgG shown in Fig. 88 . Discussion We used an interesting virus species, influenza A virus, for virus-coated LbL-colloid/lipid bilayer composites, enlarging the range of bead-based applications. This easily customizable method can be used for basic research on viral functions, for biological interaction studies, and for potential viral gene delivery or vaccination tasks. Furthermore, the use of recombinant AcNPV displaying a gp64-fusion protein allowed us not only to mimic other viruses, e.g., HIV-1, but also to demonstrate that any chosen surface-modified baculovirus can be used for this purpose. Diverse functions such as affinity tags (e.g., StrepTag) (25), receptor-binding domains for pseudotyping, and surface libraries (26) have already been established in the baculovirus system and can be readily introduced into our system. Moreover, by fusing 2 different baculoviral clones with 1 population of lipid-coated beads, we have shown that several functions can be engineered simultaneously on the colloidal particles (15), combining their features. We used these new composites to measure virus-specific antibodies in a refined multiplex suspension array. Fabrication of distinct subpopulations of beads was achieved by incorporation of fluorescently labeled polyelectrolytes. Immunostaining demonstrated authentic presentation of viral surfaces on our beads, and comparison with a commonly used ELISA showed a good correlation. Background signals were rather low because the surrounding lipid bilayer ensured very low nonspecific adsorption. A multiplex influenza-coated suspension array could be used to detect and quantify relevant virus-specific antibody titers in patient blood and to subtype influenza virus infections. Moreover, fusing existing recombinant baculoviral HIV-1 gp120 surface libraries (16)(27) to our composite could facilitate a more precise and reliable determination of early-phase HIV infections. The molecular principle of the assembly, together with the possibility of genetically modifying viruses, provides the opportunity to reverse the detection method: The use of recombinant AcZZVSVgTM-EGFP in our system allows the beads to be equipped with antibodies, enabling array-based quantification of antigens and opening new approaches in the field of antibody purification and detection. The presented system is easy to establish and offers customizable and individual setup. The beads can be used for single analyte quantification or for multiplex arrays, and assays can be performed in <2 h. Large quantities of distinctly colored microspheres with virus-like surfaces can be manufactured at a very low cost. All components for the array can be deposited for months and can be assembled in a short time. The prepared multiplex bead array can be stored in a refrigerator and is stable for several weeks. Furthermore, arrays can be enlarged by encoding the bead subpopulations with templates of different diameters and by introducing additional fluorescence markers within the polyelectrolyte layers (e.g., Cy5). The sensitivity of the array could be further increased, e.g., by use of phycoerythrin-labeled secondary antibodies with a superior signal-to-noise ratio. Because the signal intensities increase with decreasing bead numbers at a given amount of antibodies, the performance of the system can be further optimized by adjustment for a proper particle-to-antibody ratio, thus ensuring use of most of the virus epitopes. The described novel multiplex suspension array features various properties that make it competitive with ELISA for fast and easy measurement of virus-specific antibodies. The introduction of influenza A virus particles and baculovirus-HIV chimers takes advantage of the combination of authentic virus-like surfaces and processable multifunctional carriers that can be individually configured. Figure 1. Open in new tabDownload slide SDS-PAGE and Western blots of virus-coated LbL-colloid/lipid bilayer constructs. For each gel, lane 1 contains a mixture of prestained molecular markers, lane 2 contains 0.5 μg of concentrated virus, lane 3 contains 10 μL of a 50 g/L solution of liposome-coated LbL colloids, lane 4 contains 10 μL of supernatant of last washing step after virus fusion, and lane 5 contains 2 μL of a 50 g/L solution of virus-coated LbL-colloid/lipid bilayer constructs. The insets demonstrate specific binding of the chosen antibodies in the Western blot analyses. (A), wild-type baculovirus AcNPV (antibody: B12D5); (B), recombinant baculovirus AcCOPSNo10 (antibody: ARP360); (C), wild-type influenza virus viruses A/PR8/34 (H1N1; antibody: anti-PR8); (D), wild-type influenza virus A/Singapore/1/57 (H2N2; antibody: anti-Sg). Arrows indicate prominent viral surface proteins: gp64, major envelope fusion protein of AcNPV; gp64-fus, recombinant gp64 harboring 17–amino acid epitope of HIV-1 gp120; HA, major envelope fusion protein hemagglutinin of influenza A virus. The values at the left of each gel are the molecular masses of the markers (× 1000). Figure 1. Open in new tabDownload slide SDS-PAGE and Western blots of virus-coated LbL-colloid/lipid bilayer constructs. For each gel, lane 1 contains a mixture of prestained molecular markers, lane 2 contains 0.5 μg of concentrated virus, lane 3 contains 10 μL of a 50 g/L solution of liposome-coated LbL colloids, lane 4 contains 10 μL of supernatant of last washing step after virus fusion, and lane 5 contains 2 μL of a 50 g/L solution of virus-coated LbL-colloid/lipid bilayer constructs. The insets demonstrate specific binding of the chosen antibodies in the Western blot analyses. (A), wild-type baculovirus AcNPV (antibody: B12D5); (B), recombinant baculovirus AcCOPSNo10 (antibody: ARP360); (C), wild-type influenza virus viruses A/PR8/34 (H1N1; antibody: anti-PR8); (D), wild-type influenza virus A/Singapore/1/57 (H2N2; antibody: anti-Sg). Arrows indicate prominent viral surface proteins: gp64, major envelope fusion protein of AcNPV; gp64-fus, recombinant gp64 harboring 17–amino acid epitope of HIV-1 gp120; HA, major envelope fusion protein hemagglutinin of influenza A virus. The values at the left of each gel are the molecular masses of the markers (× 1000). Figure 2. Open in new tabDownload slide Confocal laser scanning micrograph of bare and influenza A virus-coated 3-μm LbL-colloid/lipid bilayer microparticles stained with anti-PR8 and FITC-conjugated anti-rabbit IgG. (Left image), transmission light micrograph showing two 3-μm colloids; (right image), fluorescence detection of a single scan with the ArKr laser (λ = 488 nm). Compared with the transmission light micrograph (left), in the fluorescence image (right), the particle on the left (virus-coated) displays influenza A-specific fluorescence, whereas the particle on the right (control) does not show background binding of anti-PR8 serum. Figure 2. Open in new tabDownload slide Confocal laser scanning micrograph of bare and influenza A virus-coated 3-μm LbL-colloid/lipid bilayer microparticles stained with anti-PR8 and FITC-conjugated anti-rabbit IgG. (Left image), transmission light micrograph showing two 3-μm colloids; (right image), fluorescence detection of a single scan with the ArKr laser (λ = 488 nm). Compared with the transmission light micrograph (left), in the fluorescence image (right), the particle on the left (virus-coated) displays influenza A-specific fluorescence, whereas the particle on the right (control) does not show background binding of anti-PR8 serum. Figure 3. Open in new tabDownload slide ELISA titration of virus-specific antibodies. Maxisorb plates were coated with prepared viruses (AcNPV, AcCOPSNo10, PR8, and Sg; all at 1 mg/L) and incubated with serially diluted primary antibodies [B12D5 (A), ARP360 (B), anti-PR8 (C), and anti-Sg (D)] and suitable peroxidase conjugates. Values are depicted as the extinction at 492 nm (y axes) vs dilution of primary antibody (x axes). Figure 3. Open in new tabDownload slide ELISA titration of virus-specific antibodies. Maxisorb plates were coated with prepared viruses (AcNPV, AcCOPSNo10, PR8, and Sg; all at 1 mg/L) and incubated with serially diluted primary antibodies [B12D5 (A), ARP360 (B), anti-PR8 (C), and anti-Sg (D)] and suitable peroxidase conjugates. Values are depicted as the extinction at 492 nm (y axes) vs dilution of primary antibody (x axes). Figure 4. Open in new tabDownload slide FL2-H histogram of a flow cytometric analysis of distinctly colored sets of LbL colloids. Four populations of LbL colloids were fabricated by incorporation of 1 (a), 2 (b), 4 (c), or 7 (d) layers of fluorescent PAH-RITC, respectively. Subpopulations were measured individually, and histograms presenting single particles were overlayed. Figure 4. Open in new tabDownload slide FL2-H histogram of a flow cytometric analysis of distinctly colored sets of LbL colloids. Four populations of LbL colloids were fabricated by incorporation of 1 (a), 2 (b), 4 (c), or 7 (d) layers of fluorescent PAH-RITC, respectively. Subpopulations were measured individually, and histograms presenting single particles were overlayed. Figure 5. Open in new tabDownload slide Multiplex suspension array. Distinctly colored subpopulations of LbL colloids (Fig. 44 ) were covered with lipid bilayers and coated with viruses: AcCOPSNo10 (1 layer of PAH-RITC), A/PR8 (2 layers of PAH-RITC), AcNPV (4 layers of PAH-RITC), and A/Singapore (7 layers of PAH-RITC). After treatment with paraformaldehyde, equal amounts of each set were merged, and 106 beads were reacted with antibody [(A), B12D5; (B), ARP360; (C), anti-PR8; (D), anti-Sg] diluted 1/25 in 100 μL of PBS containing 25 mL/L FCS, followed by suitable FITC conjugates (1/50). Gates were used to differentiate between the different subsets in FL2-H (RITC). FL1-H (FITC) signals of the subpopulations indicate specific binding of antibodies to virus-coated LbL colloids. Each panel is a dot plot showing ∼104 particles of each subset. Figure 5. Open in new tabDownload slide Multiplex suspension array. Distinctly colored subpopulations of LbL colloids (Fig. 44 ) were covered with lipid bilayers and coated with viruses: AcCOPSNo10 (1 layer of PAH-RITC), A/PR8 (2 layers of PAH-RITC), AcNPV (4 layers of PAH-RITC), and A/Singapore (7 layers of PAH-RITC). After treatment with paraformaldehyde, equal amounts of each set were merged, and 106 beads were reacted with antibody [(A), B12D5; (B), ARP360; (C), anti-PR8; (D), anti-Sg] diluted 1/25 in 100 μL of PBS containing 25 mL/L FCS, followed by suitable FITC conjugates (1/50). Gates were used to differentiate between the different subsets in FL2-H (RITC). FL1-H (FITC) signals of the subpopulations indicate specific binding of antibodies to virus-coated LbL colloids. Each panel is a dot plot showing ∼104 particles of each subset. Figure 6. Open in new tabDownload slide Titration of multiplex suspension array (Fig. 55 ). For each reaction, 104 beads coated with the viruses (AcNPV, AcCOPSNo10, PR8, and Sg) were incubated with dilutions of each antibody [(A), B12D5; (B), ARP360; (C), anti-PR8; (D), anti-Sg] and suitable FITC conjugates (1/100). FL1-H (FITC) geo MFI values of the gated subpopulations were measured (y axes) and are displayed vs the dilution factor of the primary antibody (x axes). Figure 6. Open in new tabDownload slide Titration of multiplex suspension array (Fig. 55 ). For each reaction, 104 beads coated with the viruses (AcNPV, AcCOPSNo10, PR8, and Sg) were incubated with dilutions of each antibody [(A), B12D5; (B), ARP360; (C), anti-PR8; (D), anti-Sg] and suitable FITC conjugates (1/100). FL1-H (FITC) geo MFI values of the gated subpopulations were measured (y axes) and are displayed vs the dilution factor of the primary antibody (x axes). Figure 7. Open in new tabDownload slide Stability of multiplex suspension array. After 1 × 105 microparticles comprising the 4 different subsets were reacted with anti-PR8 (1/1000) for 30 min, the beads were washed once and stained for another 30 min with FITC-conjugated anti-rabbit IgG (1/100). geo MFI values were acquired at days 0, 1, 6, and 38 after fabrication. Figure 7. Open in new tabDownload slide Stability of multiplex suspension array. After 1 × 105 microparticles comprising the 4 different subsets were reacted with anti-PR8 (1/1000) for 30 min, the beads were washed once and stained for another 30 min with FITC-conjugated anti-rabbit IgG (1/100). geo MFI values were acquired at days 0, 1, 6, and 38 after fabrication. Figure 8. Open in new tabDownload slide Reaction of AcZZVSVgTM-EGFP-coated colloids with FITC-conjugated anti-mouse IgG. FACS histogram (FL1-H) overlay of 3-μm lipid bilayer-coated colloids carrying wild-type AcNPV (trace with maximum counts between 100 and 101 in FL1-H) and AcZZVSVgTM-EGFP (trace with maximum counts between 101 and 102 in FL1-H). Approximately 104 colloid particles were incubated for 20 min in 20 μL of a 1/100 dilution of FITC-conjugated anti-mouse IgG (γ-chain specific) in PBS containing 50 mL/L FCS. Particles were washed once and analyzed in a FACSCalibur. The strong fluorescence signal of the AcZZVSVgTM-EGFP-coated particles demonstrates proper adsorption of antibody conjugates to the displayed viral IgG-binding domains. Figure 8. Open in new tabDownload slide Reaction of AcZZVSVgTM-EGFP-coated colloids with FITC-conjugated anti-mouse IgG. FACS histogram (FL1-H) overlay of 3-μm lipid bilayer-coated colloids carrying wild-type AcNPV (trace with maximum counts between 100 and 101 in FL1-H) and AcZZVSVgTM-EGFP (trace with maximum counts between 101 and 102 in FL1-H). Approximately 104 colloid particles were incubated for 20 min in 20 μL of a 1/100 dilution of FITC-conjugated anti-mouse IgG (γ-chain specific) in PBS containing 50 mL/L FCS. Particles were washed once and analyzed in a FACSCalibur. The strong fluorescence signal of the AcZZVSVgTM-EGFP-coated particles demonstrates proper adsorption of antibody conjugates to the displayed viral IgG-binding domains. 1 Nonstandard abbreviations: FACS, fluorescence-activated cell sorting; AcNPV, Autographa californica nuclear polyhedrosis virus; LbL, layer-by-layer; PBS, phosphate-buffered saline; PAH, poly(allylamine hydrochloride); RITC, rhodamine isothiocyanate; mAb, monoclonal antibody; FITC, fluorescein isothiocyanate; FCS, fetal calf serum; and geo MFI, geometric mean fluorescence intensity. We thank Wolfgang Ernst, Alexandra Spenger, and Uta Reibetanz for support and guidance. We also thank Julia Romanova for providing influenza A viruses and antibodies, and Christian Oker-Blom and Kirsi Ojala for supplying the recombinant AcZZVSVgTM-EGFP. We acknowledge Hermann Unger for technical advice. This research was supported by a grant from Volkswagenstiftung within the framework of the program “Complex Materials”. 1 Khan SS, Smith MS, Reda D, Suffredini AF, McCoy JP, Jr. Multiplex bead array assays for detection of soluble cytokines: comparisons of sensitivity and quantitative values among kits from multiple manufacturers. [Erratum published in: Cytometry B Clin Cytom 2005;64:53]. Cytometry B Clin Cytom 2004 ; 61 : 35 -39. 2 Tarnok A, Hambsch J, Chen R, Varro R. Cytometric bead array to measure six cytokines in twenty-five microliters of serum. Clin Chem 2003 ; 49 : 1000 -1002. 3 Hodge G, Hodge S, Haslam R, McPhee A, Sepulveda H, Morgan E, et al. Rapid simultaneous measurement of multiple cytokines using 100 microl sample volumes–association with neonatal sepsis. Clin Exp Immunol 2004 ; 137 : 402 -407. 4 Fulton RJ, McDade RL, Smith PL, Kienker LJ, Kettman JR, Jr. Advanced multiplexed analysis with the FlowMetrix system. Clin Chem 1997 ; 43 : 1749 -1756. 5 Morgan E, Varro R, Sepulveda H, Ember JA, Apgar J, Wilson J, et al. Cytometric bead array: a multiplexed assay platform with applications in various areas of biology [Review]. Clin Immunol 2004 ; 110 : 252 -266. 6 Lin S, Arcangel P, Medina-Selby A, Coit D, Ng P, Nguyen S, et al. Design of novel conformational and genotype-specific antigens for improving sensitivity of immunoassays for hepatitis C virus-specific antibodies. J Clin Microbiol 2005 ; 43 : 3917 -3924. 7 Seideman J, Peritt D. A novel monoclonal antibody screening method using the Luminex-100 microsphere system. J Immunol Methods 2002 ; 267 : 165 -171. 8 Pickering JW, Martins TB, Schroder MC, Hill HR. Comparison of a multiplex flow cytometric assay with enzyme-linked immunosorbent assay for quantitation of antibodies to tetanus, diphtheria, and Haemophilus influenzae Type b. Clin Diagn Lab Immunol 2002 ; 9 : 872 -876. 9 Zelnik V, Harlin O, Fehler F, Kaspers B, Gobel TW, Nair VK, et al. An enzyme-linked immunosorbent assay (ELISA) for detection of Marek’s disease virus-specific antibodies and its application in an experimental vaccine trial. J Vet Med B Infect Dis Vet Public Health 2004 ; 51 : 61 -67. 10 Carattoli A, Bonito PD, Grasso F, Giorgi C, Blasi F, Niedrig M, et al. Recombinant protein-based ELISA and immuno-cytochemical assay for the diagnosis of SARS. J Med Virol 2005 ; 76 : 137 -142. 11 Hohlich BJ, Wiesmuller KH, Schlapp T, Haas B, Pfaff E, Saalmuller A. Identification of foot-and-mouth disease virus-specific linear B-cell epitopes to differentiate between infected and vaccinated cattle. J Virol 2003 ; 77 : 8633 -8639. 12 Donath E, Sukhorukov GB, Caruso F, Davis SA, Möhwald H. Novel polymer shells via colloid templated assembly of polyelectrolytes. Angew Chem Int Ed Engl 1998 ; 37 : 2201 -2205. 13 Moya S, Donath E, Sukhorukov GB, Auch M, Bäumler H, Lichtenfeld H, et al. Lipid coating on polyelectrolyte surface modified colloidal particles and polyelectrolyte capsules. Macromolecules 2000 ; 33 : 4538 -4544. 14 Fischlechner M, Zschornig O, Hofmann J, Donath E. Engineering virus functionalities on colloidal polyelectrolyte lipid composites. Angew Chem Int Ed Engl 2005 ; 44 : 2892 -2895. 15 Fischlechner M, Toellner L, Messner P, Grabherr R, Donath E. Virus engineered colloidal particles—a novel surface display system. Angew Chem Int Ed Engl 2006 ; 45 : 784 -789. 16 Toellner L. Establishment of a baculovirus surface display library using the major envelope protein gp120 from human immuno deficiency virus 1 as a model. http://edocs.tu-berlin.de/diss/2002/toellner_lars.pdf (accessed March 2006) PhD thesis.. 17 Ojala K, Koski J, Ernst W, Grabherr R, Jones I, Oker-Blom C. Improved display of synthetic IgG-binding domains on the baculovirus surface. Technol Cancer Res Treat 2004 ; 3 : 77 -84. 18 O’Reilly DR, Miller LK, Luckow VA. Baculovirus Expression Vectors: A Laboratory Manual 1992 : 347pp WH Freeman and Company New York. . 19 Reibetanz U, Claus C, Typlt E, Hofmann J, Donath E. Defoliation and plasmid delivery with layer-by-layer coated colloids. Macromol Biosci 2006 ; 6 : 153 -160. 20 Keddie BA, Aponte GW, Volkman LE. The pathway of infection of Autographa californica nuclear polyhedrosis virus in an insect host. Science 1989 ; 243 : 1728 -1730. 21 Moore JP, Sattentau QJ, Yoshiyama H, Thali M, Charles M, Sullivan N, et al. Probing the structure of the V2 domain of human immunodeficiency virus type 1 surface glycoprotein gp120 with a panel of eight monoclonal antibodies: human immune response to the V1 and V2 domains. J Virol 1993 ; 67 : 6136 -6151. 22 Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual, 3rd ed 2001 : 2344pp Cold Spring Harbor Laboratory Press Cold Spring Harbor, NY. . 23 Hoekstra D, de Boer T, Klappe K, Wilschut J. Fluorescence method for measuring the kinetics of fusion between biological membranes. Biochemistry 1984 ; 23 : 5675 -5681. 24 Tani H, Nishijima M, Ushijima H, Miyamura T, Matsuura Y. Characterization of cell-surface determinants important for baculovirus infection. Virology 2001 ; 279 : 343 -353. 25 Ernst WJ, Spenger A, Toellner L, Katinger H, Grabherr RM. Expanding baculovirus surface display. Modification of the native coat protein gp64 of Autographa californica NPV. Eur J Biochem 2000 ; 267 : 4033 -4039. 26 Oker-Blom C, Airenne KJ, Grabherr R. Baculovirus display strategies: Emerging tools for eukaryotic libraries and gene delivery [Review]. Brief Funct Genomic Proteomic 2003 ; 2 : 244 -253. 27 Spenger A, Grabherr R, Tollner L, Katinger H, Ernst W. Altering the surface properties of baculovirus Autographa californica NPV by insertional mutagenesis of the envelope protein gp64. Eur J Biochem 2002 ; 269 : 4458 -4467. © 2006 The American Association for Clinical Chemistry This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Virus-Coated Layer-by-Layer Colloids as a Multiplex Suspension Array for the Detection and Quantification of Virus-Specific Antibodies
JO - Clinical Chemistry
DO - 10.1373/clinchem.2005.065789
DA - 2006-08-01
UR - https://www.deepdyve.com/lp/oxford-university-press/virus-coated-layer-by-layer-colloids-as-a-multiplex-suspension-array-0ahYWUPM23
SP - 1575
VL - 52
IS - 8
DP - DeepDyve
ER -