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Detection of Pathogenic Bacteria Using a Homogeneous Immunoassay Based on Shear Alignment of Virus Particles and Linear Dichroism Raul Pacheco-Gomez,† Julia Kraemer,‡ Susan Stokoe,† Hannah J. England,† Charles W. Penn,† Emma Stanley,|| Alison Rodger,§ John Ward,|| Matthew R. Hicks,†,§ and Timothy R. Dafforn*,† †
School of Biosciences, University of Birmingham, Edgbaston, Birmingham, West Midlands, B15 2TT, U.K. TU Dresden, Institut f€ur Lebensmittel- und Bioverfahrenstechnik (Institute of Food Technology and Bioprocess Engineering) 01062 Dresden § Department of Chemistry, University of Warwick, Coventry, Warwickshire, CV4 7AL, U.K. Department of Biochemical Engineering, University College London, London, WC1E 6BT, U.K.
)
‡
ABSTRACT: Biomolecular detection has for a long time depended on a relatively small number of established methodologies. Many of these depend on the detection of a ligandantibody complex using some kind of optical technique, e.g., chemiluminescence. Before this measurement can be made, the ligandantibody complex generally has to be separated from bulk contaminants. This process involves the implementation of a heterogeneous assay format involving immobilization of the antibody onto a solid support to enable washing of the unbound ligand. This approach has a number of inherent issues including being slow and complex and requiring the use of expensive reagents. In this paper, we demonstrate how we have harnessed a biologically inspired nanoparticle to provide the basis for a homogeneous assay which requires no immobilization. The method relies on using fluid shear flow to align a fiber-like nanoparticle formed from a filamentous virus, M13, combined with a ligandspecific antibody. This renders the particle visible to linear dichroic spectroscopy, which provides an easily interpretable signal. The addition of the target ligand (in this case Escherichia coli O157) leads to the formation of a nanoparticleligand particle that is unable to align, leading to the perturbation of the linear dichroism signal.
ratio. Such systems include DNA5 protein fibers,68 photosynthetic pigmentprotein complexes,9 and hybrid DNA/protein fibers, RecA.10 These experiments have, in general, concentrated on understanding the fundamentals of biomolecular structure organization or the molecular mechanisms that underlie the formation of these assemblies. Our own work has also shown that the magnitude of the LD signal is very sensitive to changes in the shape and hence alignment of molecules. Previously we have utilized this property and used it as the basis of a clinical test that used the exquisite sensitivity of the LD signal to distortions in DNA duplex to detect base pair mismatches in DNA.11 In this study we have used the sensitivity of LD to detect alterations in alignment in order to develop a detection method for a much wider range of biomolecules (see Figure 1). The system relies on the use of highly alignable scaffolds with inherently high LD signals, in this case a bionanoparticle based on bacteriophage M13 that can be functionalized to bind to target ligands (Figure 1). The interaction between this scaffold and the target ligand is then detected as a perturbation in the LD signal resulting
O
ne of the challenges that face biomolecular diagnostics is to develop simple rapid low cost assays that can be used by untrained personnel. Such tests address a common need to carry out detection close to need rather than relying on the delivery of samples to centralized laboratories, resulting in significant delays between sample and result acquisition. The requirement for speed and simplicity places a great challenge on technology development and in many cases requires a completely new approach. In this paper we describe, for the first time, an assay method based on a novel bioreagent, bacteriophage-M13 and a variant of polarized spectroscopy, linear dichroism. Linear dichroism (LD) is a spectroscopic method that provides information on the presence and orientation of chromophores within systems that are aligned at a molecular level (for a review see ref 1). Linear dichroism is simply defined as the differential absorbance by a sample of two light beams with orthogonal polarizations. Data from LD studies provides information on the orientation of chromophores within a system2 as well as the degree of alignment of a system. In most cases, biologically interesting chromophores are aligned hydrodynamically by placing the molecule in a shear flow system such as a Couette cell.3,4 We and others have developed LD as a technique for studying the structure of biomolecular assemblies that have a high aspect r 2011 American Chemical Society
Received: June 17, 2011 Accepted: October 23, 2011 Published: October 24, 2011 91
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Growth and Purification of Wild-Type M13 (wtM13). Infection of Escherichiae coli by bacteriophage M13 requires an intact F pilus. Tetracycline was added to the growth media to ensure that bacteria used for propagating bacteriophage M13 maintain the F0 plasmid encoding the pilus structure. Using this selection eliminates the need to use minimal media. Wild type M13 was purified as described below, and it was concentrated by precipitating with polyethylene glycol (PEG) in the presence of high salt. A volume of 50 mL of autoclaved NB2 (Oxoid) medium in a 250 mL flask containing tetracycline to a final concentration of 5 μg/mL was inoculated with 50 μL of One Shot Top10F0 (Invitrogen). The culture was then incubated overnight at 37 °C and 200 rpm. A volume of 40 mL of the overnight E. coli TOP10F0 culture were then transferred into 400 mL of autoclaved NB2 medium in a 2 L flask (no extra tetracycline is required at this step) and allowed to prewarm at 37 °C. M13 bacteriophage was then inoculated into the culture flasks (1 mL of 1 1013 pfu/mL or as low as 1 mL of 1 106 pfu/mL can be used without affecting the final titer of progeny of M13), and the sample was incubated overnight at 37 °C and 200 rpm. The samples were centrifuged at 8000g, and the supernatant was mixed with 60 mL of 25% w/v PEG6000, 2.5 M NaCl. The precipitated bacteriophage particles were then recovered by centrifugation of the samples at 8000g for 20 min. The supernatant was then removed, and the pellet was resuspended in 50 mM phosphate buffer, pH 8.0. Growth and Purification of FBM13 Bacteriophage. The phagemid was transformed into One Shot Top10F0 chemically competent cells (Invitrogen) and incubated on ice for 30 min. The mixture was then incubated in a water bath for 90 s at 42 °C and transferred to ice. A volume of 250 mL of sterile, prewarmed Luria Broth was then added to each vial, and the transformed cells were incubated at 37 °C for 30 min at 250 rpm in a shaking incubator. A volume of 100 μL from each vial was then spread in a Luria Broth agar plate (containing a final concentration of 100 μg/mL ampicillin) and incubated overnight at 37 °C. Propagation of Phagemid Phage Stocks from Bacteria Containing Phagemid DNA. A volume of 50 mL of autoclaved NB2 medium containing ampicillin to a final concentration of 100 μg/mL in a 250 mL flask was inoculated with 1 mL of an overnight phagemid bacterial stock and incubated at 37 °C, 200 rpm. A volume of 40 mL of culture was then removed at OD600 of around 0.5 to which 40 μL of M13 K07 helper phage (stock 1 1013 pfu/ml) was added. The culture was then incubated for 30 min at 37 °C, static, and transferred afterward to 400 mL of NB2 in a 2 L flask containing ampicillin to a final concentration of 100 μg/mL. The culture was incubated overnight at 200 rpm at 37 °C, and the samples were centrifuged at 4 °C for 15 min at 8000g. The supernatant was then collected and 30 mL of 25% w/v PEG6000, 2.5 M NaCl were added per 200 mL of phage supernatant. The samples were centrifuged at 4 °C for 15 min at 8000g, and the pellets were resuspended in 2 mL of 50 mM phosphate buffer, pH 8.0 per 200 mL of centrifuged PEG/NaCl/phage. The purified FBM13 bacteriophage was then dialyzed overnight against 50 mM phosphate buffer, pH 8.0 with 2 changes of 1 L each. Purification. Purification of FBM13 bacteriophage utilized the affinity of the FB domain for antibodies. An IgG-Sepharose prepacked column was first washed with at least 5 bed volumes of Tris-saline Tween 20 (TST) buffer (50 mM Tris buffer, pH 7.6, 150 mM NaCl and 0.05% v/v Tween 20) to remove any
Figure 1. Diagrammatic representation of the rationale behind an LD/ bacteriophage based assay. (Left) The bacteriophage are genetically engineered to bind to a chosen target via a Y-shaped function (representing an antibody attached to an FB domain engineered into the bacteriophage coat) on the end of the bacteriophage. In Couette flow, the bacteriophage align and produce an LD signal, whereas small or low aspect ratio impurities remain unaligned. In the presence of the target (a bacterial pathogen in this case), the bacteriophage (right) binds disrupting the ordered alignment of the bacteriophage and hence the LD signal.
from an alteration in the hydrodynamic shape of the scaffold once in complex with the target ligand. A key element in the development of an LD-based assay is identifying a suitable alignable scaffold. Ideally such a scaffold should take the form of a long rigid rod that aligns under shear flow. In addition it should contain a large number of ordered chromophores required to produce an LD signal. Finally it should also be stable and easily functionalized to attach moieties that can provide the specificity for the ligand of choice. Over a period of years, we have studied a number of potential scaffolds leading us to choose a virus, bacteriophage M13. M13 is easily aligned in shear flow to a very high degree. This capability was first observed by Clack et al.12 who used LD to determine the structural arrangements of DNA and protein in the bacteriophage. The high alignment is of M13 is the result of the particle’s high aspect ratio with a diameter of 67 nm and a length of approximately 900 nm13 combined with its remarkable rigidity (M13 bacteriophage has a persistence length in excess of 1200 nm14). The second reason for choosing a bacteriophage as the alignable scaffold for an assay is its genetic simplicity and biochemical flexibility. The bacteriophage particle is made up of only five different proteins (g3p, g6p, g7p, g8p, and g9p) which together form a capsid that encapsulates the single stranded circular DNA genome of the bacteriophage. Researchers have manipulated the bacteriophage genome for many years and are able to fuse exogenous proteins to those in the capsid of the virus. Perhaps the most notable of these studies has been the use of bacteriophage with antibody fragments fused to the g3p protein to develop new antibody fragments for use in the biopharmaceutic industry. In this study, a fusion between g3p and an antibody binding domain (FB) derived from protein A15 is used to provide an alignable scaffold with functionality for binding to target ligands. Finally the bacteriophage shows a very high degree of stability compared to other protein-based assemblies: the particle has evolved to survive on surfaces and is resistant to desiccation as well as to extremes of temperature.
’ MATERIALS AND METHODS Materials. The FB-bacteriophage phagemid was a generous gift from Professor R. C. Willson, Department of Biochemical and Biophysical Sciences, University of Houston. 92
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traces of ethanol storage solution. The column was then equilibrated with 3 bed volumes each of acetic acid (HAc, 0.5 M pH 3.4), TST buffer. The sample was then applied to the column in phosphate buffer, (50 mM pH 8.0). A total of 10 bed volumes of TST were then applied to the column followed by 2 bed volumes of ammonium acetate, (5 mM pH 5.0) to remove bacteriophage that either did not contain the FB domain or that contained nonfunctional FB domain. M13 bacteriophage containing the FB domain was eluted with HAc, (0.5 M, pH 3.4) and dialyzed in phosphate buffer (50 mM, pH 8.0). The eluted sample was then concentrated using a 30 000 molecular weight cutoff (MWCO) Vivaspin 15R centrifugal concentrator. Linear Dichroism. Linear dichroism is the difference in absorbance of two orthogonally oriented linearly polarized light beams by a sample. In order to obtain an LD signal, the biomolecules need to be aligned with respect to a known orientation axis. For long molecules, this can be achieved using Couette flow which is observed in systems that trap a liquid sample between two concentric cylinders one of which is rotating at a constant velocity. This induces a velocity gradient in the sample aligning long molecules by the viscous drag created by the system. The method aligns only biomolecules with appropriate hydrodynamic properties which in this case means molecules with a high aspect ratio, e.g., that are long and thin. In this study we use a micro-Couette system that uses only 50 μL of sample.3 The sample is placed into the micro-Couette cell which is housed in a JASCO J-715 spectrapolarimeter (JASCO Japan) which has been modified to measure linear dichroism. Measurement of LD Spectra. Unless otherwise stated, data were collected using either a spectrum measurement program (for single scans) or an interval scan measurement program (for multiple scans in which one of the variables is changing) both of which are implemented with the JASCO spectrum manager software suite. Unless otherwise stated, LD spectral data were recorded at room temperature over a wavelength range from 350190 nm at a speed of 100 nm min1, with a 0.2 nm data pitch, 2.0 nm bandwidth, and 1 s response time. For each experiment, a baseline (non-rotating capillary) was collected and then subtracted and the signal was zeroed at 350 nm. Measurement of Number of E. coli O157. The total cell count of E. coli O157 cells in solution was obtained using a 0.1 mm depth improved Neubauer hemocytometer and an optical microscope. The cell suspension was placed in the area between the glass coverslip and the microscopic slide (counting chamber), and the number of cells over a defined area were then counted. The volume was given by the depth and the area, and thus the number of cells in that volume could be calculated. Growth of E. coli O157. The Sakai strain of E. coli O157 was previously isolated by Hayashi and co-workers16 before being modified to disable both the STX toxin genes, in order to downgrade it to containment category 2.16 Cultures of the new strain of E. coli O157 were then grown using the methods detailed by Lee and co-workers.17 Measurement of M13 Concentration. Absorption spectra to determine phage concentrations for LD experiments were measured using a JASCO V-550 spectrophotometer. Unless otherwise stated, UV absorption spectra were measured from 700 to 200 nm in a 0.1 cm path length quartz cuvette (Starna, U.K.) at room temperature using 0.5 nm data pitch, 100 nm min1 scanning speed, and 1.0 nm bandwidth. Spectra of 50 mM phosphate buffer were also collected and used as the baseline for each experiment. Diluted samples were used where appropriate to
obtain data below 240 nm in a 0.1 cm path length cell avoiding absorbance values above 1.5.
’ RESULTS The aim of this study was to use M13 bacteriophage as an alignment scaffold in a linear dichroism-based assay. This relies on the ability of the M13 bacteriophage to perform two tasks. First the M13 bacteriophage has to bind to the antibody that will provide the target specific binding capacity for the system. Second, the M13 bacteriophage needs to be able to align in fluid flow in order to provide the LD signal that will respond to M13 bacteriophagepathogen complex formation. To provide both of these characteristics, M13 bacteriophage that contained the antibody binding FB domain from protein A was expressed and purified. Production and Characterization of FB-M13. M13 bacteriophage with an FB domain fused to g3p (FB-M13) was produced by inoculation of the FB-M13 phagemid into E. coli in the presence of M13 helper bacteriophage. The resulting FB-M13 bacteriophage was then purified using standard methods. The success of the culture and purification process was confirmed by sequencing of DNA purified from the cultured bacteriophage, which showed the presence of the FB-g3p fusion. Infection of phagemid-carrying E. coli with the M13 helper phage (M13KO7) results in packaging of the SpA-FB (fragment B) of protein A phagemid into bacteriophage particles. The FB-g3p fusion is displayed on the surface of the phage, along with wild-type g3p which is encoded in the genome of the helper phage (data not shown). The functionality of the FB-g3p fusion was then assessed using an assay based on a modified Western blot protocol. The phage proteins were separated using SDS-PAGE and the proteins transferred onto nitrocellulose. The proteins where then probed with an antibody fused to horseradish peroxidase. The presence of a functional FB-g3p fusion in the capsid of the bacteriophage should lead to a complex between the probe antibody and the fusion protein. This should then be able to be detected by conventional enhanced chemiluminescence methods. Figure 3A shows a band at approximately 43 kDa on the SDS PAGE that resulted from this experiment. This indicates that the modified purified bacteriophage contains intact FB fused g3p protein that is able to bind to antibodies and confirms that this bacteriophage construct should be appropriate for use in an LD-based immunoassay. LD Spectrum of the FB Bacteriophage M13. Previous work by Clack et al.12 showed that M13 bacteriophage can be aligned effectively in extension flow and an LD spectrum of the bacteriophage measured. The experiments in our study differ from the earlier studies in two aspects. First, the extension flow system used in the study by Clack et al. has been replaced with a Couette flow system, and second the bacteriophage strain used in the study is not wild type but a genetically modified version containing an extra protein subunit. It was therefore important to establish that this new system could still produce an LD signal that would form the basis for the assay. The Couette flow alignment system used in the current study offers advantages over the system used by Clack et al.12 as it is able to align significantly smaller samples. Sample requirement is a very important parameter that has to be addressed if the system proposed here were to be used as a bioassay system. The system used by Clack et al. required 30 mL of sample for each experiment; by contrast, bioassay systems ideally use 3 orders of magnitude 93
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bands are probably dominant. As these bands lie either side of the long axis of tryptophan, this suggests the average tryptophan is tilted toward the fiber axis. The negative LD between 240 and 260 nm will be dominated by the absorbance of the bases of the ssDNA. The sign of this signal suggests that, on average, the base pairs of the ssDNA (with absorption centered at 260 nm) are more perpendicular than parallel to the long axis of the bacteriophage. So the ladder of DNA base pairs lies along the long axis of the bacteriophage. The LD signals in the far UV region (below 240 nm) arise mainly from the peptide chromophore transitions. The LD spectrum in this region shows two positive bands below 240 nm. The one at 222226 nm will be due to the long axis 230 nm band of the tyrosine phenol groups and the approximately long axis 220 nm band of the tryptophans in the coat protein. As in the 280 nm region of the spectrum, tryptophan will dominate. The sign of this band supports the tilting of the tryptophan proposed above. The negative band at approximately 216 nm, which is apparent as a minimum between the neighboring large positive bands, corresponds to the n f π* transition of the peptides. The long wavelength component of the lowest energy π f π* peptide transition of any residues located in helices is apparent as the positive signal at ∼206 nm (only peptides in helices have an absorbance maximum near 208 nm). The short wavelength component of this transition is visible as the negative LD signal is below 190 nm. The sign and amplitude of these spectral features agree well with the previous investigation. In a Couette flow cell, the alignment of the sample is related to the shear field in the sample which is in turn related to the rotation speed of the Couette cell itself. To use this system as part of an assay, it is important to produce the largest LD signal possible to provide the largest dynamic range for the assay. To determine the maximum alignment attainable, the rotation speed of the Couette cell was therefore increased while the LD signal was monitored. These data (Figure 2B) show that the LD signal increases quickly and remains relatively invariant until the maximum rating for the motor in the LD cell is reached (3840 rpm). This suggests that the bacteriophage aligns exceptionally efficiently in shear flow. Comparison of these data with those recorded by Clack et al. show that the LD signal produced using Couette alignment in our experiment is of the same order of magnitude as that obtained by extension flow, 0.036 ΔOD (4.8 mg/mL) at 250 nm compared to that of approximately 0.016 ΔOD (1.86 mg/mL) by Clack et al. This suggests that in this application, Couette flow is adequate for aligning the bacteriophage, and that once aligned changes in the shear field over a relatively large range will not effect the alignment and hence LD signal of the bacteriophage. Thus, the higher shear rates of Clack and Gray’s system provide no added advantage. In fact because stability of alignment is essential for any applications of LD as an assay system, Couette flow is superior. Formation of a Complex between FB-M13 Bacteriophage, IgG, and a Pathogen. The basis of the detection system proposed in this study is the formation of a complex between the FB-M13 bacteriophage and the pathogen. This interaction is mediated by a third molecule, an antibody that is able to bind to both the FB domain and the pathogen. In this study an antibody is used that is specific for a protein found only on the outer surface of the pathogenic bacterium E. coli O157. To ensure that a complex can be formed between the FBM13antibodyE. coli O157 prior to any investigation using LD,
Figure 2. (A) The LD spectrum of wild type bacteriophage (0.7 μM) aligned in a Couette flow cell rotating at 3000 rpm. (B) The relationship between bacteriophage LD signal and Couette rotation speed at a range of bacteriophage concentrations (b, 0.56 μM; 2, 0.28 μM; [, 0.07 μM; 9, 5.6 nM; /, 4.4 nM,). The LD units are Δ optical density.
lower volumes of sample (a capacity of only 50 μL per experiment). The extension flow system used by Clack et al.12 does have the advantage of being able to generate much higher shear forces, and hence molecular alignment, than Couette flow. As LD intensity is proportional to the degree of alignment, then it is possible that Couette flow might not induce enough alignment in the bacteriophage to permit measurement of an LD spectrum. To test the new sample chamber configuration, we measured the LD spectrum of wild type M13 bacteriophage in the Couette cell and compared the results to that obtained in the previous study.12 Figure 2 shows the LD spectrum obtained for the M13 bacteriophage aligned in the low volume LD Couette. The spectrum shows all of the features observed previously indicating that Couette flow is sufficient to align the bacteriophage. For simplicity, the LD spectrum of the bacteriophage can be divided in two distinct regions: the near-UV region (from 260 to 350 nm), also described as the aromatic region, and the far-UV region (from 190 to 260 nm), which is dominated by electronic transitions (π f π* and n f π*) of the peptide backbone (for a summary of the absorbance transition moments of proteins and DNA, see ref 1). In the aromatic region, the LD spectrum contains overlapping contributions from the ssDNA and aromatic residues of the major coat protein g8p whose abundance dominates any signals from the other coat proteins. The signals from g8p in this region are the result of 1 tryptophan (λmax 279 nm), 2 tyrosines (λmax 274 nm), and 3 phenylalanine (λmax 258 nm) per protein monomer (approximately 2700 monomers per assembly). The positive LD band between 270 and 310 nm is thus dominated by the long wavelength absorbance of the tyrosine and tryptophan. The sign of these signals indicates that on average these side chains in g8p have absorption transition moments that are more parallel than perpendicular to the long axis of the bacteriophage. Given the structure in the band, the 285 and 265 nm tryptophan 94
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Figure 3A, lane 8, only the antibody is observed in the sample indicating that unexpectedly the ternary complex has not been formed. Instead the FB-M13 bacteriophage remains in solution indicating that the formation of the complex of all three components is in some way blocked. We concluded that this was likely to be due to steric hindrance between the components of the assay blocking simultaneous interaction between the antibody and both the bacteriophage and the E. coli. To alleviate this, a secondary antibody was therefore employed. This antibody interacts with both the FB-domain and the constant region of the pathogen-specific antibody (primary antibody) thereby spatially separating the bacterium from the bacteriophage. A repeat of the previous experiment (Figure 3B, lane 4) in the presence of this fourth component produced a positive result in which all components were cosedimented with the bacteria, indicating the presence of a quaternary complex. To further ensure that the proposed complex was specific for E. coli O157, the experiment was repeated with a nonpathogenic strain of E. coli, BL21 DE3 which does not contain the target epitope found on the E. coli O157 strain. As expected, in these experiments the FB-M13 bacteriophage did not sediment. Taken together, these experiments show that a complex can be formed between the FB-M13 bacteriophage and a pathogen specific antibody via a secondary antibody. This complex can then form a complex with E. coli O157 and not the nonpathogenic strain E. coli, BL21 DE3. Detection of Pathogenic E. coli Using FB-M13 Bacteriophage and Linear Dichroism. The central assumption of the detection system proposed here is that the binding of a large near-spherical bacterium to the rodlike bacteriophage particle will disrupt the ability of the bacteriophage to align in shear flow. This disruption would then lead to a decrease in the LD signal of the bacteriophage. To test this hypothesis, the LD of the complex of FB-M13 bacteriophagesecondary (antimouse) antibody primary (anti-E. coli O157 raised in mouse) antibodyE. coli O157 was compared to that with the E. coli omitted. As can be seen in Figure 4, the hoped for result is observed this time with the LD intensity reducing significantly upon addition of the pathogen. It is possible that this result could be achieved as the result of the bacteriophageE. coli complexes cross reacting and simply precipitating out of the reaction solution. However, such behavior leads to an increase in light scattering by virtue of the large size of such aggregates. Such increases in scattering lead to a scattering signal which adds to the absorbance LD signal. This signal increases with decreasing wavelength leading to an increase in the apparent LD signal at low wavelengths. We observe no such signal suggesting that no such cross-linked aggregates are occurring. To ensure that this effect is not a nonspecific effect of the incubation of E. coli with the FB-M13 bacteriophage, control experiments were carried out (see Figure 4A) with nonpathogenic bacteria (BL21 DE3) in place of the O157 strain. In this case, no significant change in LD was observed. Optimization of Antibody Concentration. To optimize the concentration of antibodies used in the assay, experiments were carried out with each antibody reduced 10-fold or increased by 2-fold to examine the sensitivity of the assay to antibody concentration. These data (Figure 4B) show that a reduction in concentration of the antibody reduces the ability of the assay to detect the target. Increasing the antibody did not significantly enhance the signal change observed upon the addition of the target. Concentration Dependence of LD Signal. In order to examine the concentration dependence of the interaction between
Figure 3. Western blot analysis of the bacteriophage based immunoassay. (A) A Western blot of a 10% SDS PAGE showing the presence and absence of E. coli O157 and an appropriate 1° antibody and WT and FBbacteriophage in a range of experiments. Bands were visualized using enhanced chemiluminescence using an HRP-conjugated antimouse monoclonal. (B) A Western blot showing that FB-M13 can be sedimented only in the presence of E. coli O157 and an appropriate 1° and 2° antibody. Bands were visualized using enhanced chemiluminescence using an HRP-conjugated antimouse monoclonal.
a simple test was devised using a precipitation-based assay. FBM13 bacteriophage was mixed with the antibody and E. coli O157, and the bacteria were then sedimented by centrifugation. The components that cosedimented were analyzed by Western blot (the bacteriophage and antibody alone do not sediment at the low centrifugal force used to sediment the bacteria). Interactions between the bacteriophage and the antibody could also be established in a similar fashion in which the bacteriophage was first precipitated by the addition of polyethylene glycol. The precipitate was then sedimented, and any interaction between the antibody and bacteriophage was indicated by cosedimentation of the antibody with the bacteriophage. These data show that the antibody interacts with both the bacteriophage and E. coli (Figure 3A, the presence of the appropriate band in lanes 5 and 7, respectively). To test whether the interaction between the FB-bacteriophage and the antibody was specific, the same experiment was carried out using WT bacteriophage in place of the FB derivatized virus. As expected, no antibody was observed in the pellet (Figure 3A, lane 3). To ensure that the bacteriophages do not interact directly with the bacterium, an experiment was carried out with the two components in the absence of the antibody. As expected, no bacteriophage was observed in the pellet when the bacteria was sedimented (Figure 3B, lane 2). The final experiment was to established whether the ternary complex of bacteriophage antibodyE. coli O157 can be formed. As can be seen from 95
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Figure 5. The relationship between the concentration of E. coli O157 (in cells per milliliter of sample) and LD signal at 280 nm. Each point represents an average of three experiments, and standard errors over all three measurements are less than 10% of the total signal. The LD units are Δ optical density.
adequate for aligning bacteriophages to a level required for accurate LD measurements. Comparison of the LD signal with the only previous study of this sort showed that the signal was similar both quantitatively and qualitatively. The observation that the intensities were very similar to the theoretical maximum proposed in the previous study demonstrates for the first time that the alignment produced by the miniaturized Couette cell is of a very similar level to that obtained by the more conventional extension flow system. This has significant implications for the use of LD as an assay methodology. The extension flow system had previously been thought to yield higher degrees of alignment than the available Couette cells. However extension flow is seldom used experimentally for biological systems as inherent in its design is a requirement for a large sample volume. The system used by Clack required 30 mL of sample circulating through the flow cell at between 20 and 300 mL/min. Our system required only a total volume of 5060 μL and produces a similar level of alignment, making it ideal for bioassays. The establishment of bacteriophage alignment and hence an LD signal allowed the use of bacteriophage as a detection system to be assessed. The proposed system has many similarities to a conventional immunoassay system where a ligand is detected by a specific primary antibody which itself is then detected by a secondary antibody that contains some kind of identifiable functionality, e.g., fluorescent tag. In these sorts of assay, the major issue is separating the unbound assay elements (1° and 2° antibody) from those involved in an interaction with the target ligand. Many of the established methods use a heterogeneous assay format that employs some kind of physical separation method involving immobilization of the antibodytarget complex either onto the surface of a reaction vessel or onto magnetic beads that can be separated using a magnet. These separations add an extra level of complexity to an assay, increasing assay time which LD completely avoids. In addition, the reagents and supports required to carry out the existing immunoassay methods add a significant cost to the overall system. For a number of years, the bioassay industry has been on the search for simpler methods that ideally have a homogeneous format (i.e., the assay is self-contained in the liquid phase with no requirement for insoluble supports). However, in the majority of cases it has been difficult to overcome sensitivity
Figure 4. (A) The effect of various protein complexes with FB-M13 on the bacteriophage LD spectrum. Thick dark line, FB-M13 (0.7 μM) + E. coli O157 (107 cells/mL final concentration). Dotted line, FB-M13 (0.7 μM) + 1° antibody (1 mg/mL) + 2° antibody (1 mg/mL) + E. coli BL21 (107 cells/mL final concentration). Thin line, FB-M13 (0.7 μM) + 1° antibody (1 mg/mL) + 2° antibody (1 mg/mL) + E. coli O157 (107 cells/mL final concentration). The LD signal is only disrupted when the target bacterium is present. (B) The effect of varying concentrations of 1° and 2° antibodies on the assay signal. The LD units are Δ optical density.
the bacterium and the detection complex (defined as bacteriophage 2°antibody1°antibody), a set of experiments were carried out where the concentration of bacteria was varied while the concentration of the detection complex was kept constant. Figure 5 shows that the LD signal varies in a linear fashion with respect to bacterial concentration showing that the assay is quantitative. In it also important to note that the LD measurement is made using the near UV feature centered around 280 nm. The use of near UV as opposed to far UV greatly reduces the complexity of the apparatus used to measure the signal and reduces any influences from scattering. Both of these factors will be important in future developments of commercial instrumentation.
’ DISCUSSION The primary aim of this study was to examine whether linear dichroism spectroscopy and M13 bacteriophage could be combined to form the basis of an assay system. To achieve this, a number of individual tasks were undertaken. First the ability for Couette flow to align bacteriophages was established. The data presented in this paper show that Couette flow is more than 96
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issues related to high background signals inherent in such assays. Two systems (CEDIA and EMIT) that have attempted to address these issues utilize reporter enzymes that have their activities up-regulated in the presence of the target ligand.18 The CEDIA system uses a variant of β-galactosidase that requires two peptide chains to be reunited by interactions with the target ligand in order to regain activity. In contrast, EMIT assays are based on the free target ligand competing against a drug enzyme conjugate from a ligand specific antibody. When bound to the antibody, the enzyme activity is diminished but is enhanced upon release. Both systems have shown promise but both have significant issues; for example, EMIT assays suffer from relatively low repeatability as well as lower sensitivities then corresponding radio-immunoassays.18 The system demonstrated provides a completely different solution to the homogeneous assay problem. Linear dichroism is used in a shear aligned solution to probe specific changes in the physical shape of the bacteriophage induced by the presence of the ligand. These data show that such an assay can be carried out and that the assay is quantitative and fast (a single wavelength measurement taking less than 2 min). The sensitivity of the assay is not as high as a number of other methods, but further refinement of the method should lead to sensitivity increases in the future. The system has the advantage over other homogeneous methods in that it requires no enzymatic assay, significantly reducing the potential for issues relating to high background signal levels. The lack of an enzyme means that the system is chemically more stable and less prone to inactivation due to adverse environmental factors. Taken together, these data show that the combination of LD and bacteriophage can form the basis for a homogeneous assay that has the potential to be used to detect a wide range of large (>1 μm) targets including bacteria.
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’ AUTHOR INFORMATION Corresponding Author
*E-mail: t.r.daff
[email protected].
’ ACKNOWLEDGMENT The manuscript was written through contributions of all authors. T.R.D. would like to thank R. C. Willson, Department of Biochemical and Biophysical Sciences, University of Houston, for the kind gift of the FB-M13 bacteriophage. The work was funded by the EPSRC, U.K. (Grants GR/T09224/01 and EP/ G005869/1) and an Advantage West Midlands Advanced Proof of Concept Fund Award. ’ REFERENCES (1) Dafforn, T. R.; Rodger, A. Curr. Opin. Struct. Biol. 2004, 14 (5), 541–546. (2) Bulheller, B. M.; Hicks, M. R.; Dafforn, T. R.; Serpell, L. C.; Marshall, K. E.; Bromley, E. H.; King, P. J.; Channon, K. J.; Woolfson, D. N.; Hirst, J. D. J. Am. Chem. Soc. 2009, 131 (37), 13305–13314. (3) Marrington, R.; Dafforn, T. R.; Halsall, D. J.; MacDonald, J. I.; Hicks, M.; Rodger, A. Analyst 2005, 130 (12), 1608–1616. (4) Rodger, A.; Marrington, R.; Geeves, M. A.; Hicks, M.; de Alwis, L.; Halsall, D. J.; Dafforn, T. R. Phys. Chem. Chem. Phys. 2006, 8 (27), 3161–3171. (5) Hicks, M. R.; Rodger, A.; Thomas, C. M.; Batt, S. M.; Dafforn, T. R. Biochemistry 2006, 45 (29), 8912–8917. (6) Dafforn, T. R.; Rajendra, J.; Halsall, D. J.; Serpell, L. C.; Rodger, A. Biophys. J. 2004, 86 (1 Pt 1), 404–410. 97
dx.doi.org/10.1021/ac201544h |Anal. Chem. 2012, 84, 91–97
