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Particle concentration measurement of virus samples using electrospray differential mobility analysis and quantitative amino acid analysis
Journal of Chromatography A, 1216 (2009) 5715–5722
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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Particle concentration measurement of virus samples using electrospray differential mobility analysis and quantitative amino acid analysis Kenneth D. Cole a,∗ , Leonard F. Pease III b , De-Hao Tsai a , Tania Singh a , Scott Lute c , Kurt A. Brorson c , Lili Wang a a b c
Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA Department of Chemical Engineering, University of Utah, Salt Lake City, UT 84112, USA Division of Monoclonal Antibodies, CDER/FDA, 10903 New Hampshire Blvd., Silver Spring, MD 20903, USA
a r t i c l e
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Article history: Received 17 March 2009 Received in revised form 22 May 2009 Accepted 27 May 2009 Available online 6 June 2009 Keywords: Electrospray Differential mobility analysis Amino acid analysis Virus concentration
a b s t r a c t Virus reference materials are needed to develop and calibrate detection devices and instruments. We used electrospray differential mobility analysis (ES-DMA) and quantitative amino acid analysis (AAA) to determine the particle concentration of three small model viruses (bacteriophages MS2, PP7, and X174). The biological activity, purity, and aggregation of the virus samples were measured using plaque assays, denaturing gel electrophoresis, and size-exclusion chromatography. ES-DMA was developed to count the virus particles using gold nanoparticles as internal standards. ES-DMA additionally provides quantitative measurement of the size and extent of aggregation in the virus samples. Quantitative AAA was also used to determine the mass of the viral proteins in the pure virus samples. The samples were hydrolyzed and the masses of the well-recovered amino acids were used to calculate the equivalent concentration of viral particles in the samples. The concentration of the virus samples determined by ES-DMA was in good agreement with the concentration predicted by AAA for these purified samples. The advantages and limitations of ES-DMA and AAA to characterize virus reference materials are discussed. Published by Elsevier B.V.
1. Introduction Determining the concentration of viral particles in solution is essential to many applications, including qualifying filters for viral clearance, calculating the dose of novel gene therapy vectors, and improving environmental sampling methods for biodefense purposes. Most commonly, the virus sample is characterized by measuring the concentration of infective particles using host cells in culture (e.g. plaque assays, focus forming unit assays, tissue culture infection dose 50% assay (TCID50 )). However, not all intact virus particles will be infective and defective viral particles will be present to varying extents in virtually all virus preparations. In addition, host infection and virus replication result from a series of intricate biochemical interactions, which further lower the efficiency of detection by conventional infectivity assays. Biochemical methods can be used to measure the virus concentration based on detection of specific biochemical targets present in or on the virus. For instance, immunoassays detect surface antigens on the virus, and nucleic acid based assays measure the amount of
∗ Corresponding author at: NIST, Mailstop 8312, 100 Bureau Drive, Gaithersburg, MD 20899, USA. Tel.: +1 301 975 2169; fax: +1 301 330 3447. E-mail address: [email protected] (K.D. Cole). 0021-9673/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.chroma.2009.05.083
either DNA or RNA present in the samples [1–4]. Immunoassays and nucleic acid based assays measure their respective targets in the samples regardless of whether the virus particles are intact and infective or otherwise. Some viral capsids do not contain nucleic acid, and free viral nucleic acids in viral preparations can cause false signals, leading to under or overestimated virus titers in TCID50 and PCR-based assays, respectively. Thus, calibration of these assays requires either the use of purified targets (in many cases these are not available) or reference materials, such as well-characterized virus samples and nucleic acid templates. Some efforts to standardize virus preparations used in biotechnology process validation studies have been undertaken [5], but improved methods for the physical and chemical characterization of these preparations remain essential. The total virus concentration can also be measured by counting using microscopy. Electron microscopy (EM) requires sample capture, fixation, treatments to increase contrast, and extensive analysis of the images [6]. Additionally, heterogeneity in the spatial distribution of virus particles on the substrate and sample interpretation (i.e. distinguishing true virions from microscopy artifacts and extraneous impurity complexes) can frustrate conversion to solution concentration. Inclusion of an internal standard of similar size can provide a measure of the heterogeneity and, thus, increase the reliability of counting virus particles by EM [7].
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Staining viruses with fluorescent dyes allows detection by flow cytometry and counting using epifluorescence microscopy. The conditions of sample preparation and flow cytometry techniques are important to optimize for each sample [8]. Epifluorescence microscopy also requires care in sample preparation [9]. Small viruses are a challenge for both of these techniques. It is important to explore addition approaches and methods to rapidly and accurately determine the concentration of all viral particles. A recent study considered field flow fractionation combined with multiangle light scattering (FFF-MALS) to characterize influenza virus samples [7]. The authors compared several methods and found FFFMALS to be a valuable technique for measuring the concentration and the aggregation state of the influenza virus [7]. In this study we used three model viruses, the bacteriophages MS2, PP7, and X174. Bacteriophages are viruses that infect bacteria. For example, MS2 is hosted by Escherichia coli bacteria and PP7 infects Pseudomonas aeruginosa. Both MS2 and PP7 are members of the family Leviviridae and are single-stranded RNA viruses [10], while X174 is a member of the family Microviridae [11] and has a circular single-stranded DNA of 5386 nucleotides [12]. Model viruses like these are often used to simulate the transport, capture, clearance (removal), and inactivation of more dangerous viruses. For instance, MS2 serves as a model virus for biodefense applications [3,13], environmental fate in ground and waste water [4,14], and disinfection studies in water [15], for hands [16] and on food [17]. PP7, X174, and other model viruses have been used to test the effectiveness of gloves [18] and virus removal filters [19–25]. Here we introduce two additional techniques to determine the total particle concentration. First, electrospray differential mobility analysis (ES-DMA) is a technique, historically used to size environmental aerosols such as soot, that has shown recent promise for the characterization of nanoparticles [26], proteins [27,28] and large macromolecular assemblies [29]. ES-DMA has recently been used to characterize small viruses including MS2 and larger ones including adenovirus (used for gene therapy) [30–34]. In ES-DMA, the samples are electrosprayed into a gas stream, where they undergo evaporation, charge neutralization, size separation in the differential mobility analyzer, and finally detection using a condensation particle counter (CPC). This detector counts the individual particles emerging from the differential mobility analyzer. To develop a reliable and rapid method for measuring viral concentration, we report the use of internal standards added to each sample to determine sample recovery. The second approach is to use amino acid analysis (AAA) to measure the mass of the viral proteins and from this calculate the equivalent concentration of virus particles. AAA is a mature technique that has proved invaluable in the characterization of proteins and peptides [35]. Modern improvements in the instrumentation, use of standards, and refinements to hydrolysis methods have significantly improved the technique for determination of protein concentration [36–38]. AAA can be used to measure the concentration of any purified protein or macromolecular assembly with the provisions that the amino acid sequence of the proteins and the protein composition of the macromolecular assemblies must be known. Our objective in this study is to develop ES-DMA and AAA as reliable measurement methods to more fully characterize virus samples that can be used as reference materials.
2. Experimental 2.1. Chemicals and reagents Chemicals and amino acid standards were obtained from Sigma Chemical Company (St. Louis, MO, USA). Gold particles (15 nm) were obtained from Ted Pella (Redding, CA USA). The initial concentration was 1.4 × 1012 particles/mL (based on supplier’s absorbance
measurements). One milliliter of gold suspension was concentrated 20-fold by centrifugation (20 min at 16,000 × g) and removal of 0.95 mL of visually clear supernatant. To prevent aggregation of the gold particles, the ammonium acetate buffer concentration remained below 4 mmol/L [39]. MS2 RNA (#10165948001) was obtained from Roche Diagnostics Corporation (Indianapolis, IN, USA). 2.2. MS2, PP7 and X174 virus samples and biological assays MS2 (#15597-B1 from American Type Culture Collection, ATCC, Manassas, VA, USA) and its E. coli host (ATCC #15597) were propagated and purified by the methods described for the bacteriophage lambda [40]. Large-scale cultures (1 L) were used to purify the virus followed by clarification, precipitation by polyethylene glycol (8000 molecular weight), and two cycles of CsCl gradient purification [40]. The virus band was collected and dialyzed for several days at 4 ◦ C against 10 mmol/L Tris, 100 mmol/L NaCl, and 1 mmol/L MgCl2 at pH 8 (TSM buffer). MS2 samples in TSM were stored at 4 ◦ C or frozen at −80 ◦ C. Phage PP7 and its host P. aeruginosa were obtained from the ATCC (Manassas, VA, USA; accession numbers 15692-B4 and 15692). Coliphage X174 and host E. coli strain C were obtained from the Félix d’Hérelle Reference Center for Bacterial Viruses (Université Laval, Québec, Canada). Stocks were prepared by CsCl gradient ultracentrifugation method as described elsewhere [41,42]. Plaque assays were done using the respective hosts in top agar. Dilutions of the virus stocks were prepared and added to the plates containing the host in the top agar. Plaques were counted to determine the biological activity of the virus stocks. 2.3. Amino acid analysis AAA was performed as previously described [43]. Good results with AAA were obtained using samples containing approximately 1012 total viral particles per sample (for MS2). Dilute virus samples could be concentrated by lypholyzation, filtration or other techniques to increase sensitivity. Briefly, samples were prepared in triplicate (in 6 mm by 50 mm tubes culture tubes) in 6 N HCl containing liquefied phenol (4%, v/v). The samples were evacuated, flushed with nitrogen for three cycles, evacuated, sealed, and placed in an oven at 110 ◦ C for 22 h. The samples were reduced to dryness using a vacuum centrifuge at 45 ◦ C for 90 min, and the dried samples were suspended in 20 mmol/L HCl. The samples were then analyzed in a Hitachi L-8800 amino acid analyzer using ninhydrin detection. This concentration for each amino acid is divided by the number of respective amino acid per virus particle to determine the molar concentration of the virus. Multiplying by Avogadro’s constant gives the concentration in particles per milliliter. The number of amino acids per virus particle was determined as follows. Amino acid sequences for MS2 were obtained from Swiss Prot (http://ca.expasy.org/sprot/) primary accession numbers P3612 and P03610 for the coat and assembly proteins, respectively. Similar sequences for the PP7-08 virus major coat protein (13.9 kDa, Swiss Prot accession number P03630)) and the maturation protein (50.8 kDa, Swiss Prot accession number Q38061) were also obtained. Total amino acids were calculated with the assumption that a MS2 or PP7 viral particle is composed of 180 copies of the coat protein and 1 copy of the assembly protein. Amino acid sequences for X174 were obtained from Swiss Prot (http://ca.expasy.org/sprot/) primary accession numbers of P03641, P03643, P69592, and P03646, for the F, G, J, and H proteins, respectively. Total amino acids were calculated with the assumption that a viral particle is composed of 60 copies each of the F, G, and J proteins and 12 copies of the H protein. The assumed protein ratios were based on known structural properties of Leviviridae and Microviridae [44].
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2.4. Size-exclusion chromatography
2.7. Preparation of virus samples for ES-DMA
Size-exclusion chromatography was done at ambient temperature (21 ◦ C) using one column (Biosep SEC-S 2000 column, 7.8 mm diameter and 600 mm long) or with a second column in series (Biosep SEC-S 4000, 7.8 mm diameter and 300 mm long, Phenomenex, Torrance, CA, USA). The mobile phase comprised 100 mmol/L sodium phosphate buffer at pH 7.2 with a flow rate of 0.75 mL/min. Samples (0.02 mL) were injected and monitored at 280 nm. Samples for chromatography were diluted in the sodium phosphate buffer. In some cases MS2 virus samples containing 0.45% (mass fraction) sodium dodecyl sulfate (SDS) were heated at 75 ◦ C for 7 min before injection (0.020 mL) to dissociate the protein coat from the RNA.
To reduce the concentration of nonvolatile salts that may encrust the surface of the aerosolized virus particles, the samples were dialyzed through a 10-kDa membrane into ammonium acetate. The X174 and PP7 were dialyzed into 10 mmol/L ammonium acetate for 2 days; the MS2 was dialyzed into 2 mmol/L ammonium acetate for 3 days. The volume of the sample before and after dialysis, which varied from sample to sample, was recorded to determine the dilution and to allow reporting of pre-dialysis concentrations. This dialysis step was found to not significantly diminish the infectivity of bacteriophages (data not shown). ES-DMA needs a concentration of about 1011 viral particles/mL, but a small volume is sufficient (approximately 0.05 mL) using our conditions. Virus samples spiked with gold were prepared as follows. The dialyzed MS2-03 and MS2-07 samples (0.004 mL) were mixed with 0.020 mL of the concentrated gold, and 0.176 mL of 2.12 mmol/L ammonium acetate buffer (pH 8.5). The dialyzed MS2-06 sample (0.04 mL) was mixed with 0.020 mL of the concentrated gold, and 0.140 mL of 2.12 mmol/L ammonium acetate. The dialyzed X174 sample (0.008 mL) was mixed with 0.020 mL of the concentrated gold, 0.010 mL of 20 mmol/L ammonium acetate (pH 8.5), and 0.162 mL of water. The dialyzed PP7 sample (0.001 mL) was mixed with 0.005 mL of the concentrated gold, 0.0025–0.0050 mL of 20 mmol/L ammonium acetate (pH 8.5), and 0.039 mL of water. Samples were run in triplicate.
2.5. Gel electrophoresis RNA gel electrophoresis proceeded as follows. The MS2 RNA and the phage samples were mixed with 10 mmol/L 3-(N-Morpholino)propanesulfonic acid (MOPS) pH 7.0 buffer, 0.25% (mass fraction) sodium dodecyl sulfate (SDS), 1.5% (mass fraction) Ficoll 400, and a trace of bromophenol blue and then heated at 75 ◦ C for 10 min to release the RNA from the virus [45,46]. Gel electrophoresis was then performed in a 1% (mass fraction) agarose gel in 10 mmol/L MOPS pH 7.0 at 50 ◦ C for 55 min at 3 V/cm. The gel was stained in ethidium bromide (1 g/mL) for 1 h and then twice rinsed for 10 min with water, before imaging using a UV transilluminator. Precast denaturing gels (1.5 mm thick 15% polyacrylamide, bis tris (Invitrogen, Carlsbad, CA, USA)) were run using Tris glycine buffer at 150 V for 90 min. Samples were denatured and reduced using the sodium dodecyl sulfate (SDS) sample buffer containing 50 mmol/L dithiothreitol by heating at 70 ◦ C for 10 min. The gels were microwaved (high setting) in 100 mL of water for 1 min and gently rocked for 1 min; this washing step was repeated twice for a total of three washes to remove SDS and buffer. Gels were then placed in 100 mL of Coomassie brilliant blue G-250 (0.075 mg/mL in 8 mM HCl) [47], microwaved for 1 min, gently rocked for 1–18 h, and then washed in water to destain. 2.6. MS2 immunoassay using a suspension array An immunoassay for MS2 was developed using the suspension array format (Luminex 100, Luminex Corp., Austin TX, USA). Polyclonal antibodies against MS2 were obtained from Tetracore (rabbit polyclonal, Rockville, MD, USA). The antibody preparations were coupled to the Luminex beads using the recommended procedures (Luminex Corp.). Another sample of the same antibody preparation was biotinylated using a commercial kit (Biotin-XX Protein Labeling Kit, Invitrogen, Molecular Probes, Carlsbad, CA, USA). The immunoassay for MS2 virus was done as follows. In subdued light, diluted samples of MS2 samples (0.02 mL) were mixed with the microspheres immobilized with the antibody (1000 spheres in 0.014 mL) in a 96-well plate. After 30 min, 0.03 mL of the biotinylated-antibody preparation (13 g/mL) was added to each sample. After an additional 30 min, 0.01 mL of a streptavidin–phycoerythrin dye complex (0.1 mg/mL, Invitrogen, Molecular Probes, Carlsbad, CA, USA) was added to each sample. After each addition, the samples were mixed by pipetting up and down a few times. After 10 min the plate was placed in the suspension assay instrument, and the samples were analyzed by interrogating 100 microspheres per well. Samples, including blanks, were run in triplicate. All reagents and sample dilutions were done using blocking buffer (0.01 mol/L phosphate, 0.138 mol/L sodium chloride, 0.0027 mol/L KCl, 10 mg/mL bovine serum albumin, pH 7.4).
2.8. ES-DMA operation The electrospray was operated with gas flow rates of 1.0 L/min of house air and 0.2 L/min of CO2 . A vial containing the Au colloids (>100 L) was placed under pressure, which drove the sample solution to the nozzle through a capillary (25 m in diameter, 24 cm in length) at approximately 66 nL/min. Voltages between 1.0 kV and 3.5 kV were applied as needed until the meniscus at the exit of the electrospray capillary formed into a Taylor cone and the measured current remained stable. Electrospray currents varied substantially based on the conductivity of the solution and the protein concentration, ranging from 29 nA to 182 nA. Achieving a stable Taylor cone as described in the Supporting information of a previous study [26] is critical because other meniscus regimes at higher or lower voltages failed to produce the narrow distribution of small droplet sizes necessary to achieve high quality, reproducible results. Highly charged droplets formed at the tip of the Taylor cone passed into a bipolar charger where the charge distribution on the droplets, and consequently the virus particles (or components thereof) remaining as the droplet evaporates, achieves a steady-state distribution dominated by +1, 0, −1 as described by Wiedensohler [48]. The aerosolized, charged, and essentially dry particles passed into the differential mobility analyzer. The DMA was run with a sheath-tosample flow rate ratio of 30:1.2 and voltages ranging from 0.0 kV to −11 kV were applied to the central electrode to classify the particles. The flow rate exiting the DMA was 1.0 L/min which was supplemented by 0.5 L/min of HEPA filtered air as it entered the condensation particle counter (CPC). Data points were collected at a rate of three per minute with a step interval of 0.2 nm. 2.9. Data reduction for ES-DMA samples Because a negative bias is applied to the DMA electrode, only positively charged particles are counted by the CPC. The raw CPC count is divided by the fraction of positively charged particles described by Wiedensohler [48] to obtain the count for all particles regardless of their charge state when entering the DMA. The DMA voltage is converted into a spherically equivalent size as described elsewhere [26,49] to obtain a size distribution. Because the DMA
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Table 1 Compiled measurements of the values biological assays, amino acid analysis, and ES-DMA for the virus samples. Values are means and one standard deviation (in parenthesis) of at least three experiments. Values without standard deviations are the means of two measurements. Sample
Biological activity (plaques/mL)
Predicted by amino acid (particles/mL)
ES-DMA (particles/mL)
MS2-03 (stored at 4 ◦ C) MS2-06 MS2-07 PP7-08 X174-07
4.5 (±0.3) × 1011 a 1.2 (±0.7) × 1012 4.3 (±2.4) × 1012 8 × 1012 3 × 1012
6.22 (±0.37) × 1014 6.99 (±0.13) × 1013 3.40 (±0.67) × 1014 1.69 (±0.02) × 1014 7.7 (±1.3) × 1013
5.9 (±1.4) × 1014 2.7 (±0.4) × 1013 3.9 (±0.2) × 1014 1.93 (±0.76) × 1014 4.1 (±0.2) × 1013 b 8.8 (±0.3) × 1013 c
a b c
Initial biological activity (measured in 2003) was 1.3 (±0.3) × 1013 . Based on monomer peak alone. Based on monomer, dimer, trimer, and tetramer peaks.
may bin particles of the same size at two neighboring voltages, Pease et al. [50] recently developed a correction to account for this overlap, which is essential to determining the true concentration. This correction is given for the operating conditions here as foverlap = 10.94/d − 29.94/d2 . The raw count divided by the fraction positively charged and multiplied by foverlap is reported for each size versus a spherically equivalent diameter, d, termed the mobility diameter. The portion of the spectrum attributable to each type of peak (e.g. gold, virus, etc.) is determined and the sum of all corrected counts within that range of diameters is determined. The concentration reported is the product of the known concentration of the gold particle and the summed count due for the virus all divided by the summed count for the gold particles. This concentration is multiplied by the dilution factor as necessary to determine the concentration prior to dilution. Where significant aggregates of the virus were observed, concentrations for both the individual virus particles and all virus particles including small aggregates of the virus were reported. The summed count was multiplied by two for the dimer peak, three for the trimer peak, etc. Because free protein, capsomer or capsomer assembly have a distinctly smaller size, they do not overlap with either the gold or intact virus peaks, and thus their presence does not bias the concentration reported by ES-DMA.
measurement. This sample was limited in amount and not further characterized. SDS PAGE was used to examine the protein composition of the virus samples. The samples gave the expected bands. The purities of the virus samples were 95% or greater, based on the stained intensity of the bands (results not shown). SEC was also used to assess the heterogeneity of the samples under native conditions (in phosphate buffer at pH 7.2). The MS2 and PP7 samples eluted as single peaks in Fig. 1A, while the X174 sample gave two peaks: one with retention times similar to MS2 and PP7, and the other peak eluting earlier at the exclusion volume of the columns. This latter peak indicates aggregation of X174 virus sample.
3. Results and discussion 3.1. Biological activity, gel electrophoresis, and SEC to characterize sample purity Table 1 shows the five lots of virus samples that were characterized by ES-DMA and quantitative AAA. These samples were evaluated as prototype virus reference materials in this study. The samples are named by the year the lot was produced. The MS2 samples included three lots prepared in three different years. The lots are identified by their years of production, for instance MS2-03, MS2-06, and MS2-07 were produced in years 2003, 2006, and 2007, respectively. We first ascertained the biological activity, purity, and aggregation state of the samples using plaque assays, gel electrophoresis, and size-exclusion chromatography (SEC). The biological activities of the samples were measured by plaque assays using their respective host bacteria (Table 1). MS2-03 (prepared in 2003) used for the ES-DMA and amino acid analysis was stored at 4 ◦ C maintaining its initial activity for approximately two years (results not shown), but when the activity was again measured after over four years of storage, the activity decreased by approximately 30-fold (Table 1). This sample thus represented an old sample that had degraded biological activity and was used as comparison to results using the new lots. A small amount of the MS2-03 lot was stored at −80 ◦ C for the entire period, in contrast, retained the initial biological activity when thawed but once. This sample is referred to as MS2-03 (−80 ◦ C) was only used to confirm the effect of storage conditions on the biological activity
Fig. 1. Size-exclusion chromatography. (A) Virus samples analyzed on a SEC 2000 (6 mm × 600 mm) column and a SEC 4000 (6 mm × 300 mm) column. (B) MS2 virus and MS2 RNA analyzed on a SEC 2000 (6 mm × 600 mm) column. In some cases, MS2 virus samples were heated at 75 ◦ C for 7 min in the presence of SDS (0.4%) to strip the coat proteins from the RNA. The insert has the absorbance (y-axis) multiplied by 10 to show details. Flow rate was 0.75 mL/min using 0.1 M sodium phosphate pH 7.2.
K.D. Cole et al. / J. Chromatogr. A 1216 (2009) 5715–5722 Table 2 Amino acid compositions of MS2, PP7, and X174. Amino acid
MS2 a
Aspartic acid Threonine Serine Glutamic acida Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Tryptophan Arginine Proline Total
2,550 1,647 2,376 2,015 1,648 2,555 363 2,550 367 1,456 1,296 736 736 1,094 5 372 749 1,097 23,612
PP7 2,016 2,369 2,195 2,374 1,114 1,645 367 3,094 5 735 2,746 555 202 1,280 191 189 1,662 569 23,308
PhiX174 4,620 3,288 3,060 3,396 3,192 2,952 480 2,736 1,104 1,992 3,288 1,800 2,196 1,956 1,056 516 2,196 2,508 42,336
a The aspartic acid numbers include asparagines and the glutamic acid numbers include glutamines because the amides are converted to their respective amino acids during the hydrolysis conditions. The source of the viral protein amino acid sequences is described in Section 2.
SEC was also used to examine the integrity of the virus particles in the samples at gross level (large structural changes in the virus). If the virus particles had degraded by dissociation of the coat proteins, then there would be a shift of the peaks to lower molecular weights, indicated by longer retention times on SEC column. Heating the MS2 virus in SDS detergent was a way to cause the complete disaggregation of the virus structure and this served as a control for detection of virus disaggregation. SEC was thus used to detect the presence of significant amounts free protein not associated with the intact virus particle. Fig. 1B shows that MS2-03 (stored at 4 ◦ C)
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and MS2-07 are predominantly intact virus particles and do not contain significant amounts of dissociated proteins. In contrast, the chromatogram of the MS2-07 sample, which had been heated in SDS to dissociate the capsid shell into smaller free proteins, did display a peak corresponding to the dissociated proteins (seen in the insert of Fig. 1B with expanded scale). The MS2 samples that were dissociated with SDS detergent contain the RNA peak that elutes near the excluded volume of the column and strongly absorbs at the monitored wavelength (280 nm) due to the broad absorbance spectrum of RNA. This is in addition to the proteins that do not absorb as strongly (Fig. 1B). This can be seen from the MS2 RNA sample (Fig. 1B). Thus, the SEC data indicated that the virus samples did not contain detectable amounts of virus protein that was not associated into larger macromolecular complexes. This is an important result because the presence of any free protein could have lead to an overestimate of the concentration of virus particles when measured by AAA.
3.2. Electrophoresis of MS2 RNA The quality of the RNA in the MS2 virus samples was also analyzed using agarose gel electrophoresis. The MS2 virus and MS2 RNA (as an internal standard) samples were heated in the presence of SDS detergent before loading onto the gel (to strip the viral coat proteins and to help denature any RNA secondary structure). The agarose gel was also heated to 50 ◦ C to reduce interference from the secondary structure of the MS2 RNA [45]. The MS2-03 that had been stored at 4 ◦ C for almost four years, showed considerable degradation as indicated by the smearing of low molecular weight components (results not shown). This degradation explains in part the decrease in MS2-03’s infectivity (see Table 1). However, samples of the MS2-07 appeared very similar to the MS2 RNA commercial preparation when analyzed in this manner.
Fig. 2. ES-DMA of (A) MS2, (B) PP7, (C) X174, and (D) size distribution showing both gold particles and MS2 shortly after mixing (circle symbols) and stored for 10 days at ambient temperature later (× symbols).
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3.3. Amino acid analysis of the virus samples The total virus concentration can be determined from AAA. The total number of each amino acid present in a virus particle was calculated from available amino acid sequences of the viral proteins and the number of each protein present in a mature virus particle. Table 2 represents the expected amino acid composition for a theoretical virus particle. To decompose the viruses into their individual proteins, the samples were hydrolyzed. An internal standard (norvaline, which is not naturally found in the native species) was added to the samples prior to hydrolysis to quantify the recovery of the hydrolyzed samples. The recovery of nor-valine exceeded 95%, indicating that hydrolysis, sample preparation, and amino acid analysis sustained minimal sample loss. The amino acid concentrations were compared to the expected virus composition to calculate the molar concentration of virus particles in the samples. Of the amino acids shown in Table 2, only those with the highest recoveries were used to determine the concentration [37]. These include aspartic acid, glutamic acid, glycine, alanine, leucine, and arginine. Aspartic acid includes asparagine, and glutamic acid includes glutamine residues, because the amides are converted to their respective acids during hydrolysis. The use of six analytes improves the reliability of the measurement because the concentrations determined from all six amino acids were aggregated to obtain an average and standard deviation for each sample. If the amino acid compositions deviate significantly from the predicted values, this would have indicated a sample purity problem. 3.4. ES-DMA of the virus samples The total virus concentration was also determined from ESDMA. Prior to analysis by ES-DMA, the samples were dialyzed into a low ionic strength, volatile buffer (ammonium acetate). This was done to avoid the formation of a salt layer on the dry particles and provide an acceptable conductivity for the electrospray. Samples were analyzed at several dilutions, until consistent results were obtained to ensure that any aggregation was present in the dialyzed sample and not an artifact of the measurement technique [27]. MS2 and PP7 viruses gave predominantly single peaks when analyzed by ES-DMA (Fig. 2). For our system the detection limit is somewhat less than 10 kDa as particles as small as 3 nm are also detectible [28]. The smaller peaks seen in the MS2 sample (Fig. 2A) are probably due to amounts of either impurities or small amounts of protein or salt remaining in the sample. A small amount of larger sized material was observed in the PP7 sample (Fig. 2 B) that may be due to a small amount of aggregation. In contrast, the X174 sample was more heterogeneous with additional peaks in the spectrum (Fig. 2C). The presence of additional peaks is not unexpected, as X174 is generally more difficult to purify than PP7, leading to the choice of the latter for virus filter standardization [19]. Peaks smaller than the primary peaks (i.e. <25 nm) represent capsomer, free protein, or other contaminants. Indeed, Fig. 2 shows the presence of several peaks suggesting that there is a broad range of partially disintegrated viral structures present in the sample. These structures are similar to the degradation products observed by Pease et al., for the larger virus PR772 [50]. The figure also shows several peaks larger than the primary peak. These peaks represent aggregates of X174 and the figure shows peaks attributable to dimers, trimers and tetramers. While the free protein may contribute to the protein content modestly (less than approximately 10%), the aggregate peaks represent at least as many virus particles as the primary peak, and thus can substantially influence the concentration reported. It should also be noted that phage aggregates, while possibly infectious could lead to titer underestimates because several phage in one aggre-
Fig. 3. Calibration curves for an immunoassay of MS2 virus using a suspension array. The calculated concentrations of the indicated samples of MS2 were determined by amino acid analysis (A) and by biological assay (B). The samples were run in triplicate and the results averaged (the bars show the standard deviation of the mean).
gate are likely to form one plaque due to spatial and infectivity constraints. To establish ES-DMA as a tool to measure the total concentration of virus particles, a solution of nominally 15 nm gold nanoparticles was added to each virus sample before analysis to serve as an internal standard with which to quantify the overall recovery. Table 1 shows the concentrations determined for the three viruses. The concentrations measured by ES-DMA are in good agreement with those measured by amino acid analysis, mutually confirming the analyses. For X174, the presence of aggregates (i.e. dimers, trimers, and tetramers) affects the concentration reported. Table 1 reports two concentrations for X174: one derived from the count of individual particles and a second that includes the individual particles and the aggregates. The difference of a factor of two shows the importance of accounting for the aggregation state of the virus when using ES-DMA to determine the viral load. It is important to confirm that the gold nanoparticles did not form a complex with the virus samples. Fig. 2D shows that the gold nanoparticles did not react with the MS2 virus even under prolonged storage (i.e. 10 days), while the PP7 samples had to be analyzed shortly after the gold particle were added to prevent the formation of complexes. We note that ES-DMA is also a rapid means of measuring the size of particles. We find the measured sizes for MS2, PP7, and X174 to be 23.4 nm, 24.0 nm, and 27.0 m, respectively, consistent with previous measurements [19].
K.D. Cole et al. / J. Chromatogr. A 1216 (2009) 5715–5722 Table 3 Ratio of biological activity to total viral particle concentration as measured by amino acid analysis and ES-DMA. Sample
Percentage infectivity to concentration (amino acid analysis)
Percentage infectivity to concentration (ES-DMA)
MS2-03 MS2-06 MS2-07 PP7-08 X14-07
2.0a (0.072b ) 1.7 1.3 4.7 3.9
2.2a (0.076b ) 4.4 1.1 4.1 3.4c
a b c
Calculated using initial biological activity shown in Table 1. Calculated using biological activity at time of analysis (Table 1). Calculated values for X174 included aggregated forms.
3.5. Immunoassay application of MS2 Common methods used to detect viruses and to measure titers are immunoassays. Immunoassays need to be calibrated with ether well-characterized virus reference materials or purified antigens of known concentrations. We included data on the immunoassay of MS2 to further emphasize the importance and value of measuring the total particle concentration. The three MS2 samples (03, 06, and 07 lots) were used in an immunoassay implemented in a suspension array format. To calibrate the assay, the virus concentration is correlated with the fluorescence intensity, and the slope of these curves varies substantially depending on the concentration metric selected. For example, fluorescence intensity in Fig. 3A of 10,000 corresponds to a narrow range of concentration around 1.6 × 1010 virus/mL, whereas the same intensity in panel B corresponds to a broad range of concentration ranging from just 0.2 × 108 virus/mL to 2.2 × 108 virus/mL. This is due in part to the 30-fold decrease in biological activity of the MS2-03 sample stored for four years at 4 ◦ C. Yet when the AAA derived concentration of the three samples was used to calibrate the assay (values shown for the x-axis in Fig. 3A) the samples gave very similar slopes, in contrast to the results obtained if the biological activity was used to calibrate the assays (Fig. 3B). 4. Conclusions ES-DMA and AAA gave similar results in most cases, and the mean concentration values were within one or two standard deviations of their means. The difference between the concentration of biologically active particles and the total particle concentration is very large. Table 3 shows that only 1–5% of the total particle concentration leads to a plaque in the infectivity assay. Studies on a related RNA virus (phage R17) revealed heterogeneity in the functional characteristics of the virus samples [51]. They observed that approximately 80% of the total particles did not efficiently bind to the F pilli of their host bacteria and consequently did not transfer the RNA to the host cell. Another class of R17 virus lacked the A protein, which is essential to viral assembly, and these virus particles were not infective [51]. It was also shown that the RNA in defective particles lacking the assembly protein was susceptible to degradation by RNase [51]. An important observation is that even though the MS2-03 sample stored at 4 ◦ C lost the majority of its biological activity, the gross structural integrity of the virus remained intact as evidenced by the SEC and ES-DMA data showed that the virus proteins did not dissociate significantly. Minor changes to the structure of the virus particle may not be detected by either ES-DMA or SEC. This indicates that either sizing technique did not resolve live from dead virus particles. Minor structural changes could result in compromising the biological activity. ES-DMA and AAA techniques both require sample preparation. Samples for ES-DMA must be relatively free of salt and this can
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be accomplished by dilution (if samples are concentrated) or by exchange to a low ionic strength using dialysis, membranes concentrators or size-exclusion chromatography. A concentration of about 1011 viral particles/mL was used for ES-DMA using our conditions. ES-DMA can count millions of particles per hour, providing a lot of data for analysis in a relatively short time. The samples for AAA need to be hydrolyzed to their amino acids. Sample preparation for AAA requires overnight hydrolysis, removal of the acid, and suspension of the amino acids in a buffer. This is best accomplished by batching a number of samples together for processing. The amino acid samples are stable and can be analyzed later. The chromatographic separation of the amino acids is done in less than an hour. Advances in the AAA instruments have increased the reliability and sensitivity of the analysis, and automation of sample injection increases measurement productivity. We have demonstrated that protein content determined by AAA and ES-DMA are two methods to measure the particle concentration of viral samples that are independent of each other, and provide complimentary information. ES-DMA is a counting method that relies upon a particle standard (in this case gold nanoparticles), whose concentration has been determined independently by the manufacturer. ES-DMA also provides information on the aggregation state of the sample that AAA does not provide. AAA is a technique that is used to measure the total mass of the proteins of the samples. The standard used to calibrate the AAA instrument is a standard whose mass concentrations (of the individual amino acids) were determined by the manufacturer. Our results indicate that the two methods provide complementary information on virus reference samples that are pure and not aggregated, such as the MS2 and PP7 samples. Acknowledgements We wish to thank Dr. Bert Coursey (U.S. Department of Homeland Security) for support of this project. Reference to commercial equipment, supplies, or software neither implies its endorsement by the National Institute of Standards and Technology (NIST) nor that it is necessarily best suited for this purpose. Views expressed in this article reflect those of the authors and do not constitute official positions of the Food and Drug Administration or the U.S. Government. Inclusion or exclusion of any product in this study does not constitute an endorsement by the Food and Drug Administration or the U.S. Government. References [1] J.D. Callahan, S.J. Wu, A. Dion-Schultz, B.E. Mangold, L.F. Peruski, D.M. Watts, K.R. Porter, G.R. Murphy, W. Suharyono, C.C. King, C.G. Hayes, J.J. Temenak, J. Clin. Microbiol. 39 (2001) 4119. [2] M.A. Espy, J.R. Uhl, L.M. Sloan, S.P. Buckwalter, M.F. Jones, E.A. Vetter, F.R. Cockerill III, T.F. Smith, Clin. Microbiol. Rev. 19 (2006) 165. [3] K.P. O’Connell, J.R. Bucher, P.E. Anderson, C.J. Cao, A.S. Khan, M.V. Gostomski, J.J. Valdes, Appl. Environ. Microbiol. 72 (2006) 478. [4] M.D. Sobsey, D.A. Battigelli, G.-A. Shin, S. Newland, Water Sci. Technol. 38 (1998) 91. [5] PDA Virus Preparation Standardization Task Force, Technical Report: Preparation of Virus Spikes used for Virus Clearance Studies, PDA (Bethesda, MD) in preparation. [6] E.K. Wagner, M.J. Hewlet, Basic Virology, Blackwell Publisher, 2004. [7] Z. Wei, M. Mcevoy, V. Razinkiv, A. Polozova, E. Li, J. Casas-Finet, G.I. Tous, P. Balu, A.A. Pan, H. Mehta, M.A. Schenerman, J. Virol. Methods 144 (2007) 122. [8] C.P.D. Brussaard, Appl. Environ. Microbiol. 70 (2004) 1506. [9] K. Wen, A.C. Ortmann, C.A. Suttle, Appl. Environ. Microbiol. 70 (2004) 3862. [10] J. van Duin, N. Tsareva, in: R. Calendar (Ed.), The Bacteriophages, second ed., Oxford University Press, Oxford, 2006, p. 175. [11] B.A. Fane, K.L. Brentlinger, A.D. Burch, M. Chen, S. Hafenstein, E. Moore, C.R. Novak, A. Uchiyama, in: R. Calendar (Ed.), The Bacteriophages, second ed., Oxford University Press, Oxford, 2006, p. 129. [12] F. Sanger, A.R. Coulson, T. Friedmann, G.M. Air, B.G. Barrell, N.L. Brown, J.C. Fiddes, C.A.I. Hutchison, P.M. Slocombe, M. Smith, J. Mol. Biol. 125 (1978) 225. [13] M.T. McBride, S. Gammon, M. Pitesky, T.W. O’Brien, T. Smith, J. Aldrich, R.G. Langlois, B. Colston, K.S. Venkateswaran, Anal. Chem. 75 (2003) 1924.
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Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Particle concentration measurement of virus samples using electrospray differential mobility analysis and quantitative amino acid analysis Kenneth D. Cole a,∗ , Leonard F. Pease III b , De-Hao Tsai a , Tania Singh a , Scott Lute c , Kurt A. Brorson c , Lili Wang a a b c
Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA Department of Chemical Engineering, University of Utah, Salt Lake City, UT 84112, USA Division of Monoclonal Antibodies, CDER/FDA, 10903 New Hampshire Blvd., Silver Spring, MD 20903, USA
a r t i c l e
i n f o
Article history: Received 17 March 2009 Received in revised form 22 May 2009 Accepted 27 May 2009 Available online 6 June 2009 Keywords: Electrospray Differential mobility analysis Amino acid analysis Virus concentration
a b s t r a c t Virus reference materials are needed to develop and calibrate detection devices and instruments. We used electrospray differential mobility analysis (ES-DMA) and quantitative amino acid analysis (AAA) to determine the particle concentration of three small model viruses (bacteriophages MS2, PP7, and X174). The biological activity, purity, and aggregation of the virus samples were measured using plaque assays, denaturing gel electrophoresis, and size-exclusion chromatography. ES-DMA was developed to count the virus particles using gold nanoparticles as internal standards. ES-DMA additionally provides quantitative measurement of the size and extent of aggregation in the virus samples. Quantitative AAA was also used to determine the mass of the viral proteins in the pure virus samples. The samples were hydrolyzed and the masses of the well-recovered amino acids were used to calculate the equivalent concentration of viral particles in the samples. The concentration of the virus samples determined by ES-DMA was in good agreement with the concentration predicted by AAA for these purified samples. The advantages and limitations of ES-DMA and AAA to characterize virus reference materials are discussed. Published by Elsevier B.V.
1. Introduction Determining the concentration of viral particles in solution is essential to many applications, including qualifying filters for viral clearance, calculating the dose of novel gene therapy vectors, and improving environmental sampling methods for biodefense purposes. Most commonly, the virus sample is characterized by measuring the concentration of infective particles using host cells in culture (e.g. plaque assays, focus forming unit assays, tissue culture infection dose 50% assay (TCID50 )). However, not all intact virus particles will be infective and defective viral particles will be present to varying extents in virtually all virus preparations. In addition, host infection and virus replication result from a series of intricate biochemical interactions, which further lower the efficiency of detection by conventional infectivity assays. Biochemical methods can be used to measure the virus concentration based on detection of specific biochemical targets present in or on the virus. For instance, immunoassays detect surface antigens on the virus, and nucleic acid based assays measure the amount of
∗ Corresponding author at: NIST, Mailstop 8312, 100 Bureau Drive, Gaithersburg, MD 20899, USA. Tel.: +1 301 975 2169; fax: +1 301 330 3447. E-mail address: [email protected] (K.D. Cole). 0021-9673/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.chroma.2009.05.083
either DNA or RNA present in the samples [1–4]. Immunoassays and nucleic acid based assays measure their respective targets in the samples regardless of whether the virus particles are intact and infective or otherwise. Some viral capsids do not contain nucleic acid, and free viral nucleic acids in viral preparations can cause false signals, leading to under or overestimated virus titers in TCID50 and PCR-based assays, respectively. Thus, calibration of these assays requires either the use of purified targets (in many cases these are not available) or reference materials, such as well-characterized virus samples and nucleic acid templates. Some efforts to standardize virus preparations used in biotechnology process validation studies have been undertaken [5], but improved methods for the physical and chemical characterization of these preparations remain essential. The total virus concentration can also be measured by counting using microscopy. Electron microscopy (EM) requires sample capture, fixation, treatments to increase contrast, and extensive analysis of the images [6]. Additionally, heterogeneity in the spatial distribution of virus particles on the substrate and sample interpretation (i.e. distinguishing true virions from microscopy artifacts and extraneous impurity complexes) can frustrate conversion to solution concentration. Inclusion of an internal standard of similar size can provide a measure of the heterogeneity and, thus, increase the reliability of counting virus particles by EM [7].
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Staining viruses with fluorescent dyes allows detection by flow cytometry and counting using epifluorescence microscopy. The conditions of sample preparation and flow cytometry techniques are important to optimize for each sample [8]. Epifluorescence microscopy also requires care in sample preparation [9]. Small viruses are a challenge for both of these techniques. It is important to explore addition approaches and methods to rapidly and accurately determine the concentration of all viral particles. A recent study considered field flow fractionation combined with multiangle light scattering (FFF-MALS) to characterize influenza virus samples [7]. The authors compared several methods and found FFFMALS to be a valuable technique for measuring the concentration and the aggregation state of the influenza virus [7]. In this study we used three model viruses, the bacteriophages MS2, PP7, and X174. Bacteriophages are viruses that infect bacteria. For example, MS2 is hosted by Escherichia coli bacteria and PP7 infects Pseudomonas aeruginosa. Both MS2 and PP7 are members of the family Leviviridae and are single-stranded RNA viruses [10], while X174 is a member of the family Microviridae [11] and has a circular single-stranded DNA of 5386 nucleotides [12]. Model viruses like these are often used to simulate the transport, capture, clearance (removal), and inactivation of more dangerous viruses. For instance, MS2 serves as a model virus for biodefense applications [3,13], environmental fate in ground and waste water [4,14], and disinfection studies in water [15], for hands [16] and on food [17]. PP7, X174, and other model viruses have been used to test the effectiveness of gloves [18] and virus removal filters [19–25]. Here we introduce two additional techniques to determine the total particle concentration. First, electrospray differential mobility analysis (ES-DMA) is a technique, historically used to size environmental aerosols such as soot, that has shown recent promise for the characterization of nanoparticles [26], proteins [27,28] and large macromolecular assemblies [29]. ES-DMA has recently been used to characterize small viruses including MS2 and larger ones including adenovirus (used for gene therapy) [30–34]. In ES-DMA, the samples are electrosprayed into a gas stream, where they undergo evaporation, charge neutralization, size separation in the differential mobility analyzer, and finally detection using a condensation particle counter (CPC). This detector counts the individual particles emerging from the differential mobility analyzer. To develop a reliable and rapid method for measuring viral concentration, we report the use of internal standards added to each sample to determine sample recovery. The second approach is to use amino acid analysis (AAA) to measure the mass of the viral proteins and from this calculate the equivalent concentration of virus particles. AAA is a mature technique that has proved invaluable in the characterization of proteins and peptides [35]. Modern improvements in the instrumentation, use of standards, and refinements to hydrolysis methods have significantly improved the technique for determination of protein concentration [36–38]. AAA can be used to measure the concentration of any purified protein or macromolecular assembly with the provisions that the amino acid sequence of the proteins and the protein composition of the macromolecular assemblies must be known. Our objective in this study is to develop ES-DMA and AAA as reliable measurement methods to more fully characterize virus samples that can be used as reference materials.
2. Experimental 2.1. Chemicals and reagents Chemicals and amino acid standards were obtained from Sigma Chemical Company (St. Louis, MO, USA). Gold particles (15 nm) were obtained from Ted Pella (Redding, CA USA). The initial concentration was 1.4 × 1012 particles/mL (based on supplier’s absorbance
measurements). One milliliter of gold suspension was concentrated 20-fold by centrifugation (20 min at 16,000 × g) and removal of 0.95 mL of visually clear supernatant. To prevent aggregation of the gold particles, the ammonium acetate buffer concentration remained below 4 mmol/L [39]. MS2 RNA (#10165948001) was obtained from Roche Diagnostics Corporation (Indianapolis, IN, USA). 2.2. MS2, PP7 and X174 virus samples and biological assays MS2 (#15597-B1 from American Type Culture Collection, ATCC, Manassas, VA, USA) and its E. coli host (ATCC #15597) were propagated and purified by the methods described for the bacteriophage lambda [40]. Large-scale cultures (1 L) were used to purify the virus followed by clarification, precipitation by polyethylene glycol (8000 molecular weight), and two cycles of CsCl gradient purification [40]. The virus band was collected and dialyzed for several days at 4 ◦ C against 10 mmol/L Tris, 100 mmol/L NaCl, and 1 mmol/L MgCl2 at pH 8 (TSM buffer). MS2 samples in TSM were stored at 4 ◦ C or frozen at −80 ◦ C. Phage PP7 and its host P. aeruginosa were obtained from the ATCC (Manassas, VA, USA; accession numbers 15692-B4 and 15692). Coliphage X174 and host E. coli strain C were obtained from the Félix d’Hérelle Reference Center for Bacterial Viruses (Université Laval, Québec, Canada). Stocks were prepared by CsCl gradient ultracentrifugation method as described elsewhere [41,42]. Plaque assays were done using the respective hosts in top agar. Dilutions of the virus stocks were prepared and added to the plates containing the host in the top agar. Plaques were counted to determine the biological activity of the virus stocks. 2.3. Amino acid analysis AAA was performed as previously described [43]. Good results with AAA were obtained using samples containing approximately 1012 total viral particles per sample (for MS2). Dilute virus samples could be concentrated by lypholyzation, filtration or other techniques to increase sensitivity. Briefly, samples were prepared in triplicate (in 6 mm by 50 mm tubes culture tubes) in 6 N HCl containing liquefied phenol (4%, v/v). The samples were evacuated, flushed with nitrogen for three cycles, evacuated, sealed, and placed in an oven at 110 ◦ C for 22 h. The samples were reduced to dryness using a vacuum centrifuge at 45 ◦ C for 90 min, and the dried samples were suspended in 20 mmol/L HCl. The samples were then analyzed in a Hitachi L-8800 amino acid analyzer using ninhydrin detection. This concentration for each amino acid is divided by the number of respective amino acid per virus particle to determine the molar concentration of the virus. Multiplying by Avogadro’s constant gives the concentration in particles per milliliter. The number of amino acids per virus particle was determined as follows. Amino acid sequences for MS2 were obtained from Swiss Prot (http://ca.expasy.org/sprot/) primary accession numbers P3612 and P03610 for the coat and assembly proteins, respectively. Similar sequences for the PP7-08 virus major coat protein (13.9 kDa, Swiss Prot accession number P03630)) and the maturation protein (50.8 kDa, Swiss Prot accession number Q38061) were also obtained. Total amino acids were calculated with the assumption that a MS2 or PP7 viral particle is composed of 180 copies of the coat protein and 1 copy of the assembly protein. Amino acid sequences for X174 were obtained from Swiss Prot (http://ca.expasy.org/sprot/) primary accession numbers of P03641, P03643, P69592, and P03646, for the F, G, J, and H proteins, respectively. Total amino acids were calculated with the assumption that a viral particle is composed of 60 copies each of the F, G, and J proteins and 12 copies of the H protein. The assumed protein ratios were based on known structural properties of Leviviridae and Microviridae [44].
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2.4. Size-exclusion chromatography
2.7. Preparation of virus samples for ES-DMA
Size-exclusion chromatography was done at ambient temperature (21 ◦ C) using one column (Biosep SEC-S 2000 column, 7.8 mm diameter and 600 mm long) or with a second column in series (Biosep SEC-S 4000, 7.8 mm diameter and 300 mm long, Phenomenex, Torrance, CA, USA). The mobile phase comprised 100 mmol/L sodium phosphate buffer at pH 7.2 with a flow rate of 0.75 mL/min. Samples (0.02 mL) were injected and monitored at 280 nm. Samples for chromatography were diluted in the sodium phosphate buffer. In some cases MS2 virus samples containing 0.45% (mass fraction) sodium dodecyl sulfate (SDS) were heated at 75 ◦ C for 7 min before injection (0.020 mL) to dissociate the protein coat from the RNA.
To reduce the concentration of nonvolatile salts that may encrust the surface of the aerosolized virus particles, the samples were dialyzed through a 10-kDa membrane into ammonium acetate. The X174 and PP7 were dialyzed into 10 mmol/L ammonium acetate for 2 days; the MS2 was dialyzed into 2 mmol/L ammonium acetate for 3 days. The volume of the sample before and after dialysis, which varied from sample to sample, was recorded to determine the dilution and to allow reporting of pre-dialysis concentrations. This dialysis step was found to not significantly diminish the infectivity of bacteriophages (data not shown). ES-DMA needs a concentration of about 1011 viral particles/mL, but a small volume is sufficient (approximately 0.05 mL) using our conditions. Virus samples spiked with gold were prepared as follows. The dialyzed MS2-03 and MS2-07 samples (0.004 mL) were mixed with 0.020 mL of the concentrated gold, and 0.176 mL of 2.12 mmol/L ammonium acetate buffer (pH 8.5). The dialyzed MS2-06 sample (0.04 mL) was mixed with 0.020 mL of the concentrated gold, and 0.140 mL of 2.12 mmol/L ammonium acetate. The dialyzed X174 sample (0.008 mL) was mixed with 0.020 mL of the concentrated gold, 0.010 mL of 20 mmol/L ammonium acetate (pH 8.5), and 0.162 mL of water. The dialyzed PP7 sample (0.001 mL) was mixed with 0.005 mL of the concentrated gold, 0.0025–0.0050 mL of 20 mmol/L ammonium acetate (pH 8.5), and 0.039 mL of water. Samples were run in triplicate.
2.5. Gel electrophoresis RNA gel electrophoresis proceeded as follows. The MS2 RNA and the phage samples were mixed with 10 mmol/L 3-(N-Morpholino)propanesulfonic acid (MOPS) pH 7.0 buffer, 0.25% (mass fraction) sodium dodecyl sulfate (SDS), 1.5% (mass fraction) Ficoll 400, and a trace of bromophenol blue and then heated at 75 ◦ C for 10 min to release the RNA from the virus [45,46]. Gel electrophoresis was then performed in a 1% (mass fraction) agarose gel in 10 mmol/L MOPS pH 7.0 at 50 ◦ C for 55 min at 3 V/cm. The gel was stained in ethidium bromide (1 g/mL) for 1 h and then twice rinsed for 10 min with water, before imaging using a UV transilluminator. Precast denaturing gels (1.5 mm thick 15% polyacrylamide, bis tris (Invitrogen, Carlsbad, CA, USA)) were run using Tris glycine buffer at 150 V for 90 min. Samples were denatured and reduced using the sodium dodecyl sulfate (SDS) sample buffer containing 50 mmol/L dithiothreitol by heating at 70 ◦ C for 10 min. The gels were microwaved (high setting) in 100 mL of water for 1 min and gently rocked for 1 min; this washing step was repeated twice for a total of three washes to remove SDS and buffer. Gels were then placed in 100 mL of Coomassie brilliant blue G-250 (0.075 mg/mL in 8 mM HCl) [47], microwaved for 1 min, gently rocked for 1–18 h, and then washed in water to destain. 2.6. MS2 immunoassay using a suspension array An immunoassay for MS2 was developed using the suspension array format (Luminex 100, Luminex Corp., Austin TX, USA). Polyclonal antibodies against MS2 were obtained from Tetracore (rabbit polyclonal, Rockville, MD, USA). The antibody preparations were coupled to the Luminex beads using the recommended procedures (Luminex Corp.). Another sample of the same antibody preparation was biotinylated using a commercial kit (Biotin-XX Protein Labeling Kit, Invitrogen, Molecular Probes, Carlsbad, CA, USA). The immunoassay for MS2 virus was done as follows. In subdued light, diluted samples of MS2 samples (0.02 mL) were mixed with the microspheres immobilized with the antibody (1000 spheres in 0.014 mL) in a 96-well plate. After 30 min, 0.03 mL of the biotinylated-antibody preparation (13 g/mL) was added to each sample. After an additional 30 min, 0.01 mL of a streptavidin–phycoerythrin dye complex (0.1 mg/mL, Invitrogen, Molecular Probes, Carlsbad, CA, USA) was added to each sample. After each addition, the samples were mixed by pipetting up and down a few times. After 10 min the plate was placed in the suspension assay instrument, and the samples were analyzed by interrogating 100 microspheres per well. Samples, including blanks, were run in triplicate. All reagents and sample dilutions were done using blocking buffer (0.01 mol/L phosphate, 0.138 mol/L sodium chloride, 0.0027 mol/L KCl, 10 mg/mL bovine serum albumin, pH 7.4).
2.8. ES-DMA operation The electrospray was operated with gas flow rates of 1.0 L/min of house air and 0.2 L/min of CO2 . A vial containing the Au colloids (>100 L) was placed under pressure, which drove the sample solution to the nozzle through a capillary (25 m in diameter, 24 cm in length) at approximately 66 nL/min. Voltages between 1.0 kV and 3.5 kV were applied as needed until the meniscus at the exit of the electrospray capillary formed into a Taylor cone and the measured current remained stable. Electrospray currents varied substantially based on the conductivity of the solution and the protein concentration, ranging from 29 nA to 182 nA. Achieving a stable Taylor cone as described in the Supporting information of a previous study [26] is critical because other meniscus regimes at higher or lower voltages failed to produce the narrow distribution of small droplet sizes necessary to achieve high quality, reproducible results. Highly charged droplets formed at the tip of the Taylor cone passed into a bipolar charger where the charge distribution on the droplets, and consequently the virus particles (or components thereof) remaining as the droplet evaporates, achieves a steady-state distribution dominated by +1, 0, −1 as described by Wiedensohler [48]. The aerosolized, charged, and essentially dry particles passed into the differential mobility analyzer. The DMA was run with a sheath-tosample flow rate ratio of 30:1.2 and voltages ranging from 0.0 kV to −11 kV were applied to the central electrode to classify the particles. The flow rate exiting the DMA was 1.0 L/min which was supplemented by 0.5 L/min of HEPA filtered air as it entered the condensation particle counter (CPC). Data points were collected at a rate of three per minute with a step interval of 0.2 nm. 2.9. Data reduction for ES-DMA samples Because a negative bias is applied to the DMA electrode, only positively charged particles are counted by the CPC. The raw CPC count is divided by the fraction of positively charged particles described by Wiedensohler [48] to obtain the count for all particles regardless of their charge state when entering the DMA. The DMA voltage is converted into a spherically equivalent size as described elsewhere [26,49] to obtain a size distribution. Because the DMA
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Table 1 Compiled measurements of the values biological assays, amino acid analysis, and ES-DMA for the virus samples. Values are means and one standard deviation (in parenthesis) of at least three experiments. Values without standard deviations are the means of two measurements. Sample
Biological activity (plaques/mL)
Predicted by amino acid (particles/mL)
ES-DMA (particles/mL)
MS2-03 (stored at 4 ◦ C) MS2-06 MS2-07 PP7-08 X174-07
4.5 (±0.3) × 1011 a 1.2 (±0.7) × 1012 4.3 (±2.4) × 1012 8 × 1012 3 × 1012
6.22 (±0.37) × 1014 6.99 (±0.13) × 1013 3.40 (±0.67) × 1014 1.69 (±0.02) × 1014 7.7 (±1.3) × 1013
5.9 (±1.4) × 1014 2.7 (±0.4) × 1013 3.9 (±0.2) × 1014 1.93 (±0.76) × 1014 4.1 (±0.2) × 1013 b 8.8 (±0.3) × 1013 c
a b c
Initial biological activity (measured in 2003) was 1.3 (±0.3) × 1013 . Based on monomer peak alone. Based on monomer, dimer, trimer, and tetramer peaks.
may bin particles of the same size at two neighboring voltages, Pease et al. [50] recently developed a correction to account for this overlap, which is essential to determining the true concentration. This correction is given for the operating conditions here as foverlap = 10.94/d − 29.94/d2 . The raw count divided by the fraction positively charged and multiplied by foverlap is reported for each size versus a spherically equivalent diameter, d, termed the mobility diameter. The portion of the spectrum attributable to each type of peak (e.g. gold, virus, etc.) is determined and the sum of all corrected counts within that range of diameters is determined. The concentration reported is the product of the known concentration of the gold particle and the summed count due for the virus all divided by the summed count for the gold particles. This concentration is multiplied by the dilution factor as necessary to determine the concentration prior to dilution. Where significant aggregates of the virus were observed, concentrations for both the individual virus particles and all virus particles including small aggregates of the virus were reported. The summed count was multiplied by two for the dimer peak, three for the trimer peak, etc. Because free protein, capsomer or capsomer assembly have a distinctly smaller size, they do not overlap with either the gold or intact virus peaks, and thus their presence does not bias the concentration reported by ES-DMA.
measurement. This sample was limited in amount and not further characterized. SDS PAGE was used to examine the protein composition of the virus samples. The samples gave the expected bands. The purities of the virus samples were 95% or greater, based on the stained intensity of the bands (results not shown). SEC was also used to assess the heterogeneity of the samples under native conditions (in phosphate buffer at pH 7.2). The MS2 and PP7 samples eluted as single peaks in Fig. 1A, while the X174 sample gave two peaks: one with retention times similar to MS2 and PP7, and the other peak eluting earlier at the exclusion volume of the columns. This latter peak indicates aggregation of X174 virus sample.
3. Results and discussion 3.1. Biological activity, gel electrophoresis, and SEC to characterize sample purity Table 1 shows the five lots of virus samples that were characterized by ES-DMA and quantitative AAA. These samples were evaluated as prototype virus reference materials in this study. The samples are named by the year the lot was produced. The MS2 samples included three lots prepared in three different years. The lots are identified by their years of production, for instance MS2-03, MS2-06, and MS2-07 were produced in years 2003, 2006, and 2007, respectively. We first ascertained the biological activity, purity, and aggregation state of the samples using plaque assays, gel electrophoresis, and size-exclusion chromatography (SEC). The biological activities of the samples were measured by plaque assays using their respective host bacteria (Table 1). MS2-03 (prepared in 2003) used for the ES-DMA and amino acid analysis was stored at 4 ◦ C maintaining its initial activity for approximately two years (results not shown), but when the activity was again measured after over four years of storage, the activity decreased by approximately 30-fold (Table 1). This sample thus represented an old sample that had degraded biological activity and was used as comparison to results using the new lots. A small amount of the MS2-03 lot was stored at −80 ◦ C for the entire period, in contrast, retained the initial biological activity when thawed but once. This sample is referred to as MS2-03 (−80 ◦ C) was only used to confirm the effect of storage conditions on the biological activity
Fig. 1. Size-exclusion chromatography. (A) Virus samples analyzed on a SEC 2000 (6 mm × 600 mm) column and a SEC 4000 (6 mm × 300 mm) column. (B) MS2 virus and MS2 RNA analyzed on a SEC 2000 (6 mm × 600 mm) column. In some cases, MS2 virus samples were heated at 75 ◦ C for 7 min in the presence of SDS (0.4%) to strip the coat proteins from the RNA. The insert has the absorbance (y-axis) multiplied by 10 to show details. Flow rate was 0.75 mL/min using 0.1 M sodium phosphate pH 7.2.
K.D. Cole et al. / J. Chromatogr. A 1216 (2009) 5715–5722 Table 2 Amino acid compositions of MS2, PP7, and X174. Amino acid
MS2 a
Aspartic acid Threonine Serine Glutamic acida Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Tryptophan Arginine Proline Total
2,550 1,647 2,376 2,015 1,648 2,555 363 2,550 367 1,456 1,296 736 736 1,094 5 372 749 1,097 23,612
PP7 2,016 2,369 2,195 2,374 1,114 1,645 367 3,094 5 735 2,746 555 202 1,280 191 189 1,662 569 23,308
PhiX174 4,620 3,288 3,060 3,396 3,192 2,952 480 2,736 1,104 1,992 3,288 1,800 2,196 1,956 1,056 516 2,196 2,508 42,336
a The aspartic acid numbers include asparagines and the glutamic acid numbers include glutamines because the amides are converted to their respective amino acids during the hydrolysis conditions. The source of the viral protein amino acid sequences is described in Section 2.
SEC was also used to examine the integrity of the virus particles in the samples at gross level (large structural changes in the virus). If the virus particles had degraded by dissociation of the coat proteins, then there would be a shift of the peaks to lower molecular weights, indicated by longer retention times on SEC column. Heating the MS2 virus in SDS detergent was a way to cause the complete disaggregation of the virus structure and this served as a control for detection of virus disaggregation. SEC was thus used to detect the presence of significant amounts free protein not associated with the intact virus particle. Fig. 1B shows that MS2-03 (stored at 4 ◦ C)
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and MS2-07 are predominantly intact virus particles and do not contain significant amounts of dissociated proteins. In contrast, the chromatogram of the MS2-07 sample, which had been heated in SDS to dissociate the capsid shell into smaller free proteins, did display a peak corresponding to the dissociated proteins (seen in the insert of Fig. 1B with expanded scale). The MS2 samples that were dissociated with SDS detergent contain the RNA peak that elutes near the excluded volume of the column and strongly absorbs at the monitored wavelength (280 nm) due to the broad absorbance spectrum of RNA. This is in addition to the proteins that do not absorb as strongly (Fig. 1B). This can be seen from the MS2 RNA sample (Fig. 1B). Thus, the SEC data indicated that the virus samples did not contain detectable amounts of virus protein that was not associated into larger macromolecular complexes. This is an important result because the presence of any free protein could have lead to an overestimate of the concentration of virus particles when measured by AAA.
3.2. Electrophoresis of MS2 RNA The quality of the RNA in the MS2 virus samples was also analyzed using agarose gel electrophoresis. The MS2 virus and MS2 RNA (as an internal standard) samples were heated in the presence of SDS detergent before loading onto the gel (to strip the viral coat proteins and to help denature any RNA secondary structure). The agarose gel was also heated to 50 ◦ C to reduce interference from the secondary structure of the MS2 RNA [45]. The MS2-03 that had been stored at 4 ◦ C for almost four years, showed considerable degradation as indicated by the smearing of low molecular weight components (results not shown). This degradation explains in part the decrease in MS2-03’s infectivity (see Table 1). However, samples of the MS2-07 appeared very similar to the MS2 RNA commercial preparation when analyzed in this manner.
Fig. 2. ES-DMA of (A) MS2, (B) PP7, (C) X174, and (D) size distribution showing both gold particles and MS2 shortly after mixing (circle symbols) and stored for 10 days at ambient temperature later (× symbols).
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3.3. Amino acid analysis of the virus samples The total virus concentration can be determined from AAA. The total number of each amino acid present in a virus particle was calculated from available amino acid sequences of the viral proteins and the number of each protein present in a mature virus particle. Table 2 represents the expected amino acid composition for a theoretical virus particle. To decompose the viruses into their individual proteins, the samples were hydrolyzed. An internal standard (norvaline, which is not naturally found in the native species) was added to the samples prior to hydrolysis to quantify the recovery of the hydrolyzed samples. The recovery of nor-valine exceeded 95%, indicating that hydrolysis, sample preparation, and amino acid analysis sustained minimal sample loss. The amino acid concentrations were compared to the expected virus composition to calculate the molar concentration of virus particles in the samples. Of the amino acids shown in Table 2, only those with the highest recoveries were used to determine the concentration [37]. These include aspartic acid, glutamic acid, glycine, alanine, leucine, and arginine. Aspartic acid includes asparagine, and glutamic acid includes glutamine residues, because the amides are converted to their respective acids during hydrolysis. The use of six analytes improves the reliability of the measurement because the concentrations determined from all six amino acids were aggregated to obtain an average and standard deviation for each sample. If the amino acid compositions deviate significantly from the predicted values, this would have indicated a sample purity problem. 3.4. ES-DMA of the virus samples The total virus concentration was also determined from ESDMA. Prior to analysis by ES-DMA, the samples were dialyzed into a low ionic strength, volatile buffer (ammonium acetate). This was done to avoid the formation of a salt layer on the dry particles and provide an acceptable conductivity for the electrospray. Samples were analyzed at several dilutions, until consistent results were obtained to ensure that any aggregation was present in the dialyzed sample and not an artifact of the measurement technique [27]. MS2 and PP7 viruses gave predominantly single peaks when analyzed by ES-DMA (Fig. 2). For our system the detection limit is somewhat less than 10 kDa as particles as small as 3 nm are also detectible [28]. The smaller peaks seen in the MS2 sample (Fig. 2A) are probably due to amounts of either impurities or small amounts of protein or salt remaining in the sample. A small amount of larger sized material was observed in the PP7 sample (Fig. 2 B) that may be due to a small amount of aggregation. In contrast, the X174 sample was more heterogeneous with additional peaks in the spectrum (Fig. 2C). The presence of additional peaks is not unexpected, as X174 is generally more difficult to purify than PP7, leading to the choice of the latter for virus filter standardization [19]. Peaks smaller than the primary peaks (i.e. <25 nm) represent capsomer, free protein, or other contaminants. Indeed, Fig. 2 shows the presence of several peaks suggesting that there is a broad range of partially disintegrated viral structures present in the sample. These structures are similar to the degradation products observed by Pease et al., for the larger virus PR772 [50]. The figure also shows several peaks larger than the primary peak. These peaks represent aggregates of X174 and the figure shows peaks attributable to dimers, trimers and tetramers. While the free protein may contribute to the protein content modestly (less than approximately 10%), the aggregate peaks represent at least as many virus particles as the primary peak, and thus can substantially influence the concentration reported. It should also be noted that phage aggregates, while possibly infectious could lead to titer underestimates because several phage in one aggre-
Fig. 3. Calibration curves for an immunoassay of MS2 virus using a suspension array. The calculated concentrations of the indicated samples of MS2 were determined by amino acid analysis (A) and by biological assay (B). The samples were run in triplicate and the results averaged (the bars show the standard deviation of the mean).
gate are likely to form one plaque due to spatial and infectivity constraints. To establish ES-DMA as a tool to measure the total concentration of virus particles, a solution of nominally 15 nm gold nanoparticles was added to each virus sample before analysis to serve as an internal standard with which to quantify the overall recovery. Table 1 shows the concentrations determined for the three viruses. The concentrations measured by ES-DMA are in good agreement with those measured by amino acid analysis, mutually confirming the analyses. For X174, the presence of aggregates (i.e. dimers, trimers, and tetramers) affects the concentration reported. Table 1 reports two concentrations for X174: one derived from the count of individual particles and a second that includes the individual particles and the aggregates. The difference of a factor of two shows the importance of accounting for the aggregation state of the virus when using ES-DMA to determine the viral load. It is important to confirm that the gold nanoparticles did not form a complex with the virus samples. Fig. 2D shows that the gold nanoparticles did not react with the MS2 virus even under prolonged storage (i.e. 10 days), while the PP7 samples had to be analyzed shortly after the gold particle were added to prevent the formation of complexes. We note that ES-DMA is also a rapid means of measuring the size of particles. We find the measured sizes for MS2, PP7, and X174 to be 23.4 nm, 24.0 nm, and 27.0 m, respectively, consistent with previous measurements [19].
K.D. Cole et al. / J. Chromatogr. A 1216 (2009) 5715–5722 Table 3 Ratio of biological activity to total viral particle concentration as measured by amino acid analysis and ES-DMA. Sample
Percentage infectivity to concentration (amino acid analysis)
Percentage infectivity to concentration (ES-DMA)
MS2-03 MS2-06 MS2-07 PP7-08 X14-07
2.0a (0.072b ) 1.7 1.3 4.7 3.9
2.2a (0.076b ) 4.4 1.1 4.1 3.4c
a b c
Calculated using initial biological activity shown in Table 1. Calculated using biological activity at time of analysis (Table 1). Calculated values for X174 included aggregated forms.
3.5. Immunoassay application of MS2 Common methods used to detect viruses and to measure titers are immunoassays. Immunoassays need to be calibrated with ether well-characterized virus reference materials or purified antigens of known concentrations. We included data on the immunoassay of MS2 to further emphasize the importance and value of measuring the total particle concentration. The three MS2 samples (03, 06, and 07 lots) were used in an immunoassay implemented in a suspension array format. To calibrate the assay, the virus concentration is correlated with the fluorescence intensity, and the slope of these curves varies substantially depending on the concentration metric selected. For example, fluorescence intensity in Fig. 3A of 10,000 corresponds to a narrow range of concentration around 1.6 × 1010 virus/mL, whereas the same intensity in panel B corresponds to a broad range of concentration ranging from just 0.2 × 108 virus/mL to 2.2 × 108 virus/mL. This is due in part to the 30-fold decrease in biological activity of the MS2-03 sample stored for four years at 4 ◦ C. Yet when the AAA derived concentration of the three samples was used to calibrate the assay (values shown for the x-axis in Fig. 3A) the samples gave very similar slopes, in contrast to the results obtained if the biological activity was used to calibrate the assays (Fig. 3B). 4. Conclusions ES-DMA and AAA gave similar results in most cases, and the mean concentration values were within one or two standard deviations of their means. The difference between the concentration of biologically active particles and the total particle concentration is very large. Table 3 shows that only 1–5% of the total particle concentration leads to a plaque in the infectivity assay. Studies on a related RNA virus (phage R17) revealed heterogeneity in the functional characteristics of the virus samples [51]. They observed that approximately 80% of the total particles did not efficiently bind to the F pilli of their host bacteria and consequently did not transfer the RNA to the host cell. Another class of R17 virus lacked the A protein, which is essential to viral assembly, and these virus particles were not infective [51]. It was also shown that the RNA in defective particles lacking the assembly protein was susceptible to degradation by RNase [51]. An important observation is that even though the MS2-03 sample stored at 4 ◦ C lost the majority of its biological activity, the gross structural integrity of the virus remained intact as evidenced by the SEC and ES-DMA data showed that the virus proteins did not dissociate significantly. Minor changes to the structure of the virus particle may not be detected by either ES-DMA or SEC. This indicates that either sizing technique did not resolve live from dead virus particles. Minor structural changes could result in compromising the biological activity. ES-DMA and AAA techniques both require sample preparation. Samples for ES-DMA must be relatively free of salt and this can
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be accomplished by dilution (if samples are concentrated) or by exchange to a low ionic strength using dialysis, membranes concentrators or size-exclusion chromatography. A concentration of about 1011 viral particles/mL was used for ES-DMA using our conditions. ES-DMA can count millions of particles per hour, providing a lot of data for analysis in a relatively short time. The samples for AAA need to be hydrolyzed to their amino acids. Sample preparation for AAA requires overnight hydrolysis, removal of the acid, and suspension of the amino acids in a buffer. This is best accomplished by batching a number of samples together for processing. The amino acid samples are stable and can be analyzed later. The chromatographic separation of the amino acids is done in less than an hour. Advances in the AAA instruments have increased the reliability and sensitivity of the analysis, and automation of sample injection increases measurement productivity. We have demonstrated that protein content determined by AAA and ES-DMA are two methods to measure the particle concentration of viral samples that are independent of each other, and provide complimentary information. ES-DMA is a counting method that relies upon a particle standard (in this case gold nanoparticles), whose concentration has been determined independently by the manufacturer. ES-DMA also provides information on the aggregation state of the sample that AAA does not provide. AAA is a technique that is used to measure the total mass of the proteins of the samples. The standard used to calibrate the AAA instrument is a standard whose mass concentrations (of the individual amino acids) were determined by the manufacturer. Our results indicate that the two methods provide complementary information on virus reference samples that are pure and not aggregated, such as the MS2 and PP7 samples. Acknowledgements We wish to thank Dr. Bert Coursey (U.S. Department of Homeland Security) for support of this project. Reference to commercial equipment, supplies, or software neither implies its endorsement by the National Institute of Standards and Technology (NIST) nor that it is necessarily best suited for this purpose. Views expressed in this article reflect those of the authors and do not constitute official positions of the Food and Drug Administration or the U.S. Government. Inclusion or exclusion of any product in this study does not constitute an endorsement by the Food and Drug Administration or the U.S. Government. References [1] J.D. Callahan, S.J. Wu, A. Dion-Schultz, B.E. Mangold, L.F. Peruski, D.M. Watts, K.R. Porter, G.R. Murphy, W. Suharyono, C.C. King, C.G. Hayes, J.J. Temenak, J. Clin. Microbiol. 39 (2001) 4119. [2] M.A. Espy, J.R. Uhl, L.M. Sloan, S.P. Buckwalter, M.F. Jones, E.A. Vetter, F.R. Cockerill III, T.F. Smith, Clin. Microbiol. Rev. 19 (2006) 165. [3] K.P. O’Connell, J.R. Bucher, P.E. Anderson, C.J. Cao, A.S. Khan, M.V. Gostomski, J.J. Valdes, Appl. Environ. Microbiol. 72 (2006) 478. [4] M.D. Sobsey, D.A. Battigelli, G.-A. Shin, S. Newland, Water Sci. Technol. 38 (1998) 91. [5] PDA Virus Preparation Standardization Task Force, Technical Report: Preparation of Virus Spikes used for Virus Clearance Studies, PDA (Bethesda, MD) in preparation. [6] E.K. Wagner, M.J. Hewlet, Basic Virology, Blackwell Publisher, 2004. [7] Z. Wei, M. Mcevoy, V. Razinkiv, A. Polozova, E. Li, J. Casas-Finet, G.I. Tous, P. Balu, A.A. Pan, H. Mehta, M.A. Schenerman, J. Virol. Methods 144 (2007) 122. [8] C.P.D. Brussaard, Appl. Environ. Microbiol. 70 (2004) 1506. [9] K. Wen, A.C. Ortmann, C.A. Suttle, Appl. Environ. Microbiol. 70 (2004) 3862. [10] J. van Duin, N. Tsareva, in: R. Calendar (Ed.), The Bacteriophages, second ed., Oxford University Press, Oxford, 2006, p. 175. [11] B.A. Fane, K.L. Brentlinger, A.D. Burch, M. Chen, S. Hafenstein, E. Moore, C.R. Novak, A. Uchiyama, in: R. Calendar (Ed.), The Bacteriophages, second ed., Oxford University Press, Oxford, 2006, p. 129. [12] F. Sanger, A.R. Coulson, T. Friedmann, G.M. Air, B.G. Barrell, N.L. Brown, J.C. Fiddes, C.A.I. Hutchison, P.M. Slocombe, M. Smith, J. Mol. Biol. 125 (1978) 225. [13] M.T. McBride, S. Gammon, M. Pitesky, T.W. O’Brien, T. Smith, J. Aldrich, R.G. Langlois, B. Colston, K.S. Venkateswaran, Anal. Chem. 75 (2003) 1924.
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