Use of superparamagnetic beads for the isolation of a peptide with specificity to cymbidium mosaic virus

Journal of Virological Methods 136 (2006) 160–165

Use of superparamagnetic beads for the isolation of a peptide with specificity to cymbidium mosaic virus Diana Jia Miin Ooi a , Adriya Dzulkurnain b , Rofina Yasmin Othman b , Saw Hoon Lim a , Jennifer Ann Harikrishna a,∗ a

Biotechnology Program, Malaysia University of Science and Technology, Block C, Kelana Square, 17 Jalan SS7/26, 47301 Petaling Jaya, Selangor, Malaysia b Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia Received 27 January 2006; received in revised form 21 April 2006; accepted 2 May 2006 Available online 16 June 2006

Abstract A modified method for the rapid isolation of specific ligands to whole virus particles is described. Biopanning against cymbidium mosaic virus was carried out with a commercial 12-mer random peptide display library. A solution phase panning method was devised using streptavidin-coated superparamagnetic beads. The solution based panning method was more efficient than conventional immobilized target panning when using whole viral particles of cymbidium mosaic virus as a target. Enzyme-linked immunosorbent assay of cymbidium mosaic virus-binding peptides isolated from the library identified seven peptides with affinity for cymbidium mosaic virus and one peptide which was specific to cymbidium mosaic virus and had no significant binding to odontoglossum ringspot virus. This method should have broad application for the screening of whole viral particles towards the rapid development of diagnostic reagents without the requirement for cloning and expression of single antigens. © 2006 Elsevier B.V. All rights reserved. Keywords: Biopanning; Cymbidium mosaic virus; Superparamagnetic beads

1. Introduction Orchids are among the world’s most popular flowers and their trade as both cut flowers and potted plants plays a significant role in the economy of many Asean countries (Hew, 1994; Laws, 1995; Eun and Wong, 1999). Over 25 viruses are known to occur in orchids (Vejaratpimol et al., 1999). Of these, Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) are among the most common (Hu et al., 1998; Wong et al., 1994). These pathogens are prevalent in cultivated orchids throughout the world, are easily spread by mechanical means and are often asymptomatic during early stages of infection

Abbreviations: CymMV, cymbidium mosaic virus; ORSV, odontoglossum ringspot virus; TBS, Tris-buffered saline; PBS, phosphate buffered saline; ELISA, enzyme-linked immunosorbent assay ∗ Corresponding author at: Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia. Tel.: +603 79675817; fax: +603 79675908. E-mail addresses: [email protected] (R.Y. Othman), [email protected] (S.H. Lim), [email protected] (J.A. Harikrishna). 0166-0934/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2006.05.001

(Jensen, 1950; Lawson and Brannigan, 1986). Crop production losses attributed to plant viruses can be enormous, especially when high-value cash crops, such as ornamentals are at stake (Webster et al., 2004). The control of plant viruses is limited largely to agronomic practices and although some viral diseases can be diagnosed quickly by visual examination of symptoms, others require molecular tests for early diagnosis (Webster et al., 2004). Among the possible molecular tests available, antibody and other ligand-based tests are often preferred as they have the advantages of being relatively inexpensive, easy for lay-men to perform and where dip-stick types tests can be developed, can be performed on site. For this reason, there is much interest in the isolation of ligands with specificity to emerging and important pathogens for the development of inexpensive diagnostic kits (Willats, 2002; Ziegler and Torrance, 2002). The development of phage display technology and its use for display of combinatorial libraries such as antibody fragments (Hoogenboom et al., 1998; Johns et al., 2000; McCafferty et al., 1990) and peptides (Cabilly, 1999; Cortese et al., 1995; Cwirla et al., 1990; Devlin et al., 1990) have made it much more rapid and straightforward to isolate ligands with specificity for target

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antigens such as viral coat proteins (Barbas et al., 2000; Gough et al., 1999; Hong and Boulanger, 1995). The practicality of this method has led to its increasing importance in plant pathology (Ziegler and Torrance, 2002) as well as for the development of human therapeutics (Udo and Jurgen, 2005). Conventionally, screening of such libraries involves the binding of the target antigen to a solid support such as the surface of a well in a microtitre plate (McCafferty and Johnson, 1996). However, this may limit the amount of antigen surface available to the ligand library during panning due to low binding affinity of the antigen for the surface and the limited surface area available. A method was devised to allow the use of whole viral particles as the antigen and to allow a much larger antigen surface area per volume ratio, by the use of superparamagnetic beads in solution, for antigen display.

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potassium phosphate buffer (pH 7.5) and kept at −20 ◦ C. The presence of CymMV was verified using ELISA and the protein concentration of each preparation was quantified using a standard Bradford assay (Bradford, 1976) using reagents from BioRad with serially diluted bovine serum albumin standards for calibration. 2.2. Enzyme-linked immunosorbent assay (ELISA)

2. Materials and methods

The presence of CymMV in infected leaves and purified virus samples of CymMV and ORSV were verified using ELISA reagent sets from Agdia Inc., USA. Samples of infected leaves were prepared by grinding the leaves in a mortar and pestle with 0.03 M potassium phosphate buffer (pH 7.0). 100 ␮L of sample extract were used for each test well. Positive and negative controls as supplied with the kits were used for all ELISA experiments.

2.1. Preparation of viral antigen

2.3. Biotinylation of purified virus particles

Cymbidium mosaic virus (CymMV) ATTC number PV-317 and Odontoglossum ringspot virus (ORSV) ATTC number PV318 were obtained from the American Type Culture Collection in the form of lyophilized infected leaves. The CymMV viral sample was rehydrated in 0.03 M potassium phosphate buffer (pH 7.0) and ground with a mortar and pestle together with a small amount of cellite to form a paste. Cucumis sativus (cucumber) plants with six to eight fully expanded leaves were mechanically inoculated by gently applying the CymMV viral paste to the leaves using a finger covered in a clean latex glove. The plants were incubated for 5 min and then rinsed with tap water to remove debris. The plants were returned to a growth chamber and kept in the dark for 24 h followed by a regular 16 h daily photoperiod with watering daily or as required. CymMV infected plants showing characteristic chlorotic lesions were confirmed to be infected by ELISA assay. Infected leaf tissues were collected 10–14 days post-inoculation and either stored at −20 ◦ C or immediately used for virus purification using the method of AbouHaidar et al. (1998) as follows: Infected leaves were homogenized in 0.1 M potassium phosphate buffer (pH 7.5) containing 0.2% ␤-mercaptoethanol using 1–2 mL buffer per gram of leaves. Amine-free buffers were used for all subsequent steps to avoid interference with viral particle biotinylation in later steps. The homogenized tissues were squeezed through four layers of cheesecloth and n-butanol added to the filtrate to a final concentration of 6%. The mixture was kept on ice for 45 min with constant stirring then clarified by centrifugation at 15 000 g for 10 min. Viral particles were precipitated by adding PEG 8000 to the supernatant to a final concentration of 8% in the presence of 2% NaCl. The mixture was kept at 4 ◦ C for 30–60 min then centrifuged at 15 000 g for 10 min. The viral pellet was left to resuspend overnight in 0.1 M potassium phosphate buffer (pH 7.5) at 4 ◦ C. The resuspended solution was further processed through three rounds of centrifugation at 7500 g for 5 min and the supernatant laid over a 4 ml 30% sucrose cushion prior to ultracentrifugation at 86 500 g for 3 h at 4 ◦ C. The pellets were resuspended by gentle pipetting in 0.1 M

Biotinylation of purified CymMV was carried out using a MSDTM Biotin-LC-Sulfo-NHS-Ester Kit (Meso Scale Discovery, USA) according to the manufacturer’s instructions. Unbound biotin was removed from the virus solution using a microconcentrator (Amicon Centricon-10, Millipore, USA). Biotin labeling efficiency was determined spectrophotometrically using HABA/Avidin reagent (H 2153, Sigma) according to the manufacturer’s instructions by measuring change in absorbance at 500 nm (A500) due to dye displacement (Green, 1965). Following this, the ability of the viral particles to participate in binding reactions was confirmed by ELISA. 2.4. Immobilized target panning Wells of a 96-well microtitre plate were coated with various dilutions of purified CymMV in 0.03 M potassium phosphate (pH 7.0) overnight, followed by incubation with blocking buffer (0.1 M NaHCO3 (pH 8.6), 5 mg/mL BSA, 0.02% NaN3 ) for 1 h at 4 ◦ C. Drained plates were washed six times with 1×TBS, 0.1% Tween 20. Prepared wells were then incubated with a pre-titred Ph.D.-12 Random Peptide Phage Display Library according to the manufacturer’s instructions (“Rapid screening of peptide ligands with a phage display peptide library” Ph.D.-12 Phage Display Peptide Library Kit Manual, 2003. New England BioLabs. USA) 2.5. Solution-phase panning: capture with streptavidin-coated magnetic beads Solution-phase panning was carried out using a modification of the method above where biotinylated virus particles were first mixed together with the phage display library then recovered via mixing with strepatvidin-coated magnetic beads: For the first round of panning, a sample containing 1011 –1013 phage particles from the titred, unamplified Ph.D.-12 Random Peptide Phage Display Library (Ph.D.-12 Random Peptide Phage Dis-

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play Library Kit, New England Biolabs, USA) was incubated with 1 mL of blocking buffer (0.1 M NaHCO3 (pH 8.6), 5 mg/mL BSA, 0.02% NaN3 ) at room temperature for 30 min. Biotinylated CymMV particles were added at a range of dilutions and the solution was incubated for a further 1–2 h at room temperature. Meanwhile, Dynabeads M-280, streptavidin-coated magnetic beads (Invitrogen, USA) were washed to remove preservatives and the volume of the beads used was determined following the manufacturer’s protocols. Ten volumes of TBS containing 2% BSA and 0.2% NaN3 were added. The supernatant was aspired and the washing procedure repeated twice. The washed beads were then resuspended in TBS containing 2% BSA and 0.2% NaN3 to a final streptavidin concentration of 10 mg/mL. The beads were then mixed with blocking solution (0.1 M NaHCO3 (pH 8.6), 5 mg/mL BSA, 0.02% NaN3 ) for 30 min at room temperature. The blocked streptavidin-coated magnetic beads were then added to an equal volume of the mixture of random peptide phage display library and biotinylated virus particles, mixed by inversion and incubated for 15 min. Magnetic beads with bound CymMV particle:phage complex were recovered by placing the microcentrifuge tubes in a magnetic particle concentrator (Invitrogen, USA) and removing the supernatant. The beads were washed in 1 mL of blocking solution containing 0.1% Tween 20. The washed beads were then transferred to a new tube, washed three times with PBS (10 mM phosphate, 150 mM NaCl, pH 7.4) then resuspended in 100 ␮L of TBS. Bound phage particles were eluted by addition of 1 mL 0.2 M glycine–HCL (pH 2.2), 1 mg/mL BSA for 10 min followed by removal of the supernatant for neutralisation in a new tube containing 150 ␮L of 1 M Tris–HCl (pH 9.1). 2.6. Secondary rounds of panning For both immobilized and solution-phase panning methods, panning was repeated for two further rounds using the amplified eluate from the previous round in each case. The amplified phage titer at each stage was determined by counting the number of blue plaques.

2.7. Sequence analysis Phage samples taken from plaques from the third round of panning were purified using the Wizard® Plus SV Miniprep DNA Purification kit (Promega, USA) and the respective peptide sequences determined by DNA sequencing using an automated sequencing service with the -96gIII primer (New England Biolabs, USA). Sequence alignments for phage display peptide clones were carried out with ClustalW (http://www. ebi.ac.uk/clustalw/). 2.8. Analysis of binding specificity A standard sandwich ELISA method was used to determine the binding efficiency of purified phage display clones with CymMV and ORSV. Wells of a 96 well microtitre plate were coated with either purified CymMV or purified ORSV, or were left empty for controls. The microtiter plate was sealed and incubated overnight at 4 ◦ C. Excess solution was removed by slapping the microtiter plate face-down onto a paper towel. Each well was filled completely with blocking buffer (0.1 M NaHCO3 (pH 8.6), 5 mg/mL BSA, 0.02% NaN3 ) and plates were incubated at 4 ◦ C for 1–2 h. The blocking buffer was then shaken out and plates were washed 4 times with 1× TBS, 01% Tween 20. Excess wash solution was removed by slapping the plate facedown onto a clean section of paper towel. Freshly purified phage from selected phage display clones was then transferred to the microtiter plate coated with the target and incubated at room temperature for 1–2 h. The microtiter plate was then washed 4 times with 1× TBS/Tween as above. HRP-conjugated antiM13 antibody (Pharmacia # 27-9411-01) was diluted 1:5000 in blocking buffer. 200 ␮L of diluted conjugate were added to each well and incubated at room temperature for 1 h with agitation. The wells were washed 4 times with 1X TBS/Tween. HRP substrate solution was prepared by dissolving 22 mg ABTS (Sigma #A1888) in 100 mL of 50 mM sodium citrate at pH 4.0, filter sterilized and stored at 4 ◦ C. Immediately prior to the detection step, 30 ␮L of 30% H2 O2 was added to 21 mL of ABTS stock

Table 1 Cymbidium mosaic virus (CymMV) levels in infected cucumber leaf samples and after binding directly to microtitre plate for immobilized target panning Sample

Dilution

Absorbance unitsa

% Relative bindingb

CymMV crude leaf extract CymMV from ATTC (undiluted) CymMV bound after washing CymMV bound after washing CymMV bound after washing CymMV bound after washing CymMV bound after washing CymMV bound after washing CymMV bound after washing Positive control Positive control after washing Negative control Negative control after washing

– – 1:1 1:10 1:100 1:1 000 1:10000 1:100 000 1:1000000 – – – –

2.426 2.219 0.789 0.661 0.607 0.562 0.521 0.457 0.391 1.530 0.834 0.039 0.047

– – 100.0 83.8 76.9 71.2 66.0 57.9 49.6 – – – –

a b

ELISA absorbance readings averaged from two samples. Relative binding, determined as the percentage of the absorbance value seen when comparing the diluted to the undiluted CymMV samples bound after washing.

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Table 2 Numbers of plaque forming units (PFU) obtained after panning of peptide phage library against CymMV and amplification of selected phage clones Round of panning

Immobilized target panning Post-panning

1 2 3

8.51 × 103 920 230

Solution-phase panning Post-amplification

Post-panning

Post-amplification

1.02 × 109

9.55 × 106

4.72 × 1013 4.49 × 1014 7.71 × 1015

6.21 × 106 5.98 × 105

solution per plate to be analyzed. 200 ␮L of substrate solution was added to each well and incubated at room temperature for 10–60 min. The microtiter plates were then read at 405 nm. Controls included wells not coated with either CymMV or ORSV but incubated with phage, and wells coated with CymMV or ORSV with no phage added.

7.13 × 107 2.99 × 108

similar ELISA absorbance readings with ORSV (clones C1, B1, B2, B3, B5 and B6, Table 3) showing closely comparable affinity for this virus. The remaining clone, phage display peptide C2 (GenBank accession number AM182757), showed much lower absorbance values in ELISA with ORSV, only slightly higher than that of the negative control, indicating that this clone has low affinity for ORSV and is specific for CymMV.

3. Results 3.1. Preparation and binding of cymbidium mosaic virus (CymMV) to microtitre plates CymMV infection of cucumber plants for antigen preparation was confirmed by ELISA of crude leaf extracts as shown in Table 1. After purification, a ten-fold dilution series of the purified virus was used to coat wells of a microtitre plate in preparation for immobilized target panning. The amount of purified non-diluted CymMV remaining bound to the plate after washing and prior to panning, as measured by ELISA, was found to be relatively low (0.789), with a value approximately half of that for normal ELISA (unwashed) positive control (1.530) as shown in Table 1. A similarly lower level was also seen for the positive control provided in the ELISA reagent kit when treated in the same manner (0.834). The levels of diluted CymMV remaining attached to the microtitre plate wells after washing in preparation for library panning, decreased with each level of dilution, as expected, however, the decrease was of much lesser magnitude than the dilution factor with the highest dilution (1:1 000 000) giving approximately half of the value seen for non-diluted sample (49.6%, Table 1), suggesting that the higher concentrations of CymMV were close to saturation of the available binding surface.

Fig. 1. Multiple sequence alignment of phage display peptide clones. B1–B6: Clones picked from the third round of solution phased panning. C1–C6: clones picked from the third round of immobilized target panning. Bold, underlined AM numbers indicate GenBank accession numbers. Boxes and shading indicate subsets of clones with identical sequences: Peptides C1 and C3 (light grey), peptides C2, C4, C5 and C6 (white text on black background) and peptides B2 and B4 (outlined with box) are duplicate clones, most probably resulting from amplification of selected fractions of the peptide library. The isolation of more than one clone with the same sequence provides further support to the data indicating that these peptides have good affinity for CymMV. Table 3 Binding of phage display peptide clones to CymMV and ORSV Plate coated with

Peptide clone

Absorbance unitsa

CymMV

C1 C2 B1 B2 B3 B5 B6 –

0.186 0.181 0.184 0.180 0.187 0.183 0.183 0.035

C1 C2 B1 B2 B3 B5 B6 – –

0.181 0.047 0.185 0.183 0.187 0.183 0.184 0.036 0.508

3.2. Selection and analysis of peptide display library clones The phage titre (PFU) after each round of biopanning was found to decrease markedly for the immobilized target panning method whilst the titre increased for each round with solutionphase panning using the magnetic beads, as shown in Table 2. Random sequencing of six clones from the third round of panning from each panning method resulted in seven different peptide encoding sequences, two from the immobilized target panning and five from the solution-phase panning (Fig. 1). All of the selected clones exhibited very similar levels of binding to CymMV in terms of ELISA absorbance readings, with values ranging from 0.180 to 0.187, compared to 0.035 for the negative control (Table 3). This indicates that these clones have comparable affinities to CymMV. Six of the seven clones also gave very

ORSV

Positive control a

ELISA absorbance readings averaged from two samples.

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4. Discussion Phage display peptide libraries offer a powerful resource for the isolation of novel ligands and thus can be of great value to diagnostic and therapeutic research (Udo and Jurgen, 2005; Ziegler and Torrance, 2002). A major advantage of this technology is that no prior knowledge of the antigen structure or sequence is required and by use of the whole virus particle, ligands which bind to the native structure may be isolated for subsequent diagnostic use. A typical peptide display library, such as the one used in this research, contains approximately 2.7 × 109 different 12-mer peptides (Hoogenboom et al., 1998). With this large diversity of peptides, there will be a significant probability of finding sequences with affinity and specificity for any target, in this case the CymMV virion. The peptides selected by biopanning will be a mixture of those exhibiting preferential affinity for CymMV, those exhibiting favorable growth properties, and those randomly binding non-specifically to the substrate (Rodi et al., 2001) thus isolation of multiple candidates allows further screening for those with specificity as well as affinity to the target antigen. In this study, multiple clones with identical sequences were isolated among the randomly selected clones with affinity for CymMV. This most likely occurred due to the amplification of the selected clones between rounds of panning, resulting in redundancy in the selected fraction of the peptide display library. The isolation of identical sequences provides further confirmation of the affinity of these peptides for CymMV as it would be improbable to find the same sequences among unselected random clones. The fact that the majority of the peptides isolated also showed affinity to ORSV may be due to the known similarity between the coat proteins of CymMV and ORSV (Eun et al., 2000; Seoh et al., 1998). As these peptides were isolated based on their affinity to intact virions, in which the coat protein is in native conformation, this result adds more evidence to suggest surface structural similarities between the two viruses. Therefore, these peptides might provide useful research tools for study and comparison of the native coat protein structures of CymMV and ORSV. As these two viruses are frequently found to co-infect orchids and to show synergism (Ajjikuttira et al., 2005; Hu et al., 1998) ligands that are capable of detecting both viruses as well as those differentiating them can be of value to phytopathological research as well as have diagnostic application. Strategies for selection are equally important as library quality when using phage display (Griffiths and Duncan, 1998). Thus a number of panning strategies have been developed and optimized for various applications (Hoogenboom et al., 1998; Rodi et al., 2001; Watters et al., 1997). Among these, solution-phase panning methods have the advantage of allowing larger surface areas of antigen to be accessible during panning and have been used for the enrichment of phage display libraries prior to solid phase methods such as use of microtitre plate wells or nitrocellulose membranes (Moulard et al., 2004; Nakamura et al., 2001; Zhou et al., 2002). In particular, the use of bead antigen display systems has an advantage over solid phase systems as the higher surface area and the ability of beads to freely rotate in solution

results in a greater concentration of target antigen in the reaction and reduces the average distance between the target antigen and phage particles, thus improving reaction kinetics (Kay and Hoess, 1996; Lu and Sloan, 1999). A useful development in solution-phase panning has been the use of magnetic beads to support and present the antigen, allowing selection and washing to take place in solution followed by separation of selected particles in a magnetic field. This method has been applied to peptide antigens such as type I plasminogen activator inhibitor (Lang et al., 1996) and mutant EGF receptor (Lorimer et al., 1996) however, there have been no reports of its use with whole virions as antigens. In this study, it was observed that the levels of CymMV remaining bound to the microtitre wells after washing and prior to panning, did not reflect the concentrations in solution, in that the lower dilutions did not give proportionately higher ELISA values (Table 1). This was also reflected in the numbers of plaque forming units recovered after each stage of panning and amplification of the peptide phage display library (Table 2) where each subsequent round of immobilized target panning resulted in a decrease in the phage titre. These results indicate that there was limited binding of the CymMV to the microtitre well surface. One possible explanation for this is the limited surface area available in the microtitre well: the surface area of a typical 96 well microtiter plate well is approximately 0.9 cm2 whereas the surface area of 10 ␮L of the superparamagnetic beads used in this research is 14–17 cm2 . Thus, if the available surface area does affect the amount of target present, it can be assumed that far higher numbers of target were present in the solution-based panning procedure originating from an equivalent initial number of target virions, compared to the numbers of target available in the microtitre plate well. This is consistent with the much higher numbers of selected clones at each round of panning seen with the solution-phase method (Table 2). The apparently low binding affinity of the CymMV virion to the microtitre plate well might also be a consequence of using intact virus particles, rather than a heterologously expressed epitope, such as coat protein. Whilst the use of single cloned epitopes for selection and isolation of specific ligands is advantageous for certain applications, the use of whole intact virus particles may be more appropriate in the development of viral diagnostics: use of intact virions offers the combination of speed, as no cloning steps are required, as well as the certainty of the antigen used for selection being in the same conformation as that to be tested diagnostically. In this study, use of solution-phase panning was able to address these limitations, thus this method offers a useful addition for the specific application of viral diagnostic development. Despite the limited binding of target antigen, panning of the peptide phage display library using the immobilized technique was successful in the isolation of peptides with affinity for CymMV. However, far higher numbers of peptide display clones were isolated from the solution-phase panning (Table 3). The solution-phase panning also had a lower amount of redundancy among the selected clones (Fig. 1). Whilst this might be a consequence of the small numbers of clones examined in this study, it nevertheless resulted in a greater diversity among the peptide sequences selected, which would be an advantage when

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