Selection and characterization of single-chain recombinant antibodies against infectious haematopoietic necrosis virus from mouse phage display library.

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Selection and characterization of single-chain recombinant antibodies against infectious haematopoietic necrosis virus from mouse phage display library

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Hong Liu a , Xiaocong Zheng b , Xiujie Shi b , Li Yu b , Peng Jia b , Jinjin Wang b , Junqiang He b , Wensheng Lan b , Hong Liu b,∗ , Zhixin Wu a,∗∗ a b

College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China Shenzhen Entry-Exit Inspection and Quarantine Bureau, Shenzhen 518045, China

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Article history: Received 11 November 2013 Received in revised form 11 March 2014 Accepted 4 April 2014 Available online xxx

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Keywords: Phage display Single-chain fragment variable antibody Infectious haematopoietic necrosis virus

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1. Introduction

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Six single-chain fragment variable (scFv) antibodies against infectious haematopoietic necrosis virus (IHNV) were selected from an antibody phage display library by phage display technology. The soluble scFv antibodies showed a molecular weight 32 kDa by Western blot. Dot blot analysis revealed that the six scFv antibodies could recognize IHNV. For enzyme linked immunosorbent assay (ELISA), four scFv antibodies (P1A4, P1A12, P1D5 and P3E2) showed cross-reactivity with spring viraemia of carp virus (SVCV). However, none of the six scFv antibodies had cross-reaction with Pike fry rhabdovirus (PFRV), Soft-shelled turtle iridovirus (STIV), viral haemorrhagic septicemia virus (VHSV), or viral nervous necrosis virus (VNNV). Indirect immunofluorescence results showed that all of these scFv antibodies reacted positively with virus in the IHNV-infected cells. These scFv antibodies will be useful in diagnostic test development and pathogenesis studies for IHNV. © 2014 Published by Elsevier B.V.

Infectious haematopoietic necrosis virus (IHNV), a significant pathogen of salmon and trout (Wolf, 1988), is enzootic in river systems throughout western North America and has spread to Asia and Europe by movement of infected fish and eggs (Winton, 1991). It is an economically important fish pathogen, causing severe acute infections in young salmonid fish and resulting in high mortalities which can reach 80%–100% during outbreaks (Alonso et al., 2005). Outbreaks of the diseases have caused great economic losses and became factors restricting aquaculture and the development of fisheries. The virus is a member of the genus Novirhabdovirus in the family Rhabdoviridae, and consists of a linear single-strand, negative-sense RNA genome of approximately 11k nucleotides. The IHNV genome contains 6 genes in the order 3 -N-P-M-G-NV-L-5 , encoding the

∗ Corresponding author at: Shenzhen Entry-Exit Inspection and Quarantine Bureau, China. Tel.: +86 755 25588410. ∗ ∗ Corresponding author at: College of Fisheries, Huazhong Agricultural University, Wuhan, Hubei 430070, China. E-mail addresses: [email protected] (H. Liu), [email protected] (Z. Wu).

nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), nonvirion protein (NV) and polymerase (L), respectively (Kurath et al., 1985; Morzunov et al., 1995; Tordo et al., 2005). Phage display technology is based on recombinant DNA methods allowing co-selection of recombinant antibodies and their corresponding genes (Wittrup, 1999). If DNA fragments encoding polypeptides are fused to certain bacteriophage coat protein genes, they can be encapsulated within phage particles that also display the encoded polypeptides on their surfaces (Sidhu, 2000; Wong et al., 2001). Recombinant antibody technology allows the display of functional antibody binding sites on the surface of bacteriophage, without the immortalization of B-cell lines used in hybridoma technology (Garet et al., 2010). Generally, phage display libraries consist of a heavy and light chain variable region domains (VH and VL) linked by a flexible Glysine-Serine linker, fused to the phage minor coat protein gene III and displayed as a single-chain fragment variable (scFv) (McCafferty et al., 1990). This technique for the production of recombinant antibodies has several advantages: rapid culture of phage clones; easy handling and detection of secreted antibodies; genetic stability; lower costs compared with monoclonal antibodies (MAbs) (Johns et al., 2000; Foy et al., 2002). The major advantage of this technological advance is that antibodies could be expressed both on the surface of the

http://dx.doi.org/10.1016/j.jviromet.2014.04.008 0166-0934/© 2014 Published by Elsevier B.V.

Please cite this article in press as: Liu, H., et al., Selection and characterization of single-chain recombinant antibodies against infectious haematopoietic necrosis virus from mouse phage display library. J. Virol. Methods (2014), http://dx.doi.org/10.1016/j.jviromet.2014.04.008

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phage (antibodies linked to the phage) and in the medium (soluble antibodies) (Gourdine et al., 2005). Therefore phage display is a powerful technology for selecting and engineering polypeptides with novel functions and has been used widely to produce antibodies. Due to the extensive losses caused by IHNV in salmon and trout aquaculture, rapid detection and identification of infectious viruses are important for disease control. Many methods have been developed for the diagnosis of fish viruses. These include isolating virus from candidate fish by infection of established cell lines then confirming the identity by serum neutralization assay (OIE, 2000), immunofluorescent antibody techniques (IFAT), enzymelinked immunosorbent assay (ELISA), immunohistochemical and immunogold labelling (Drolet et al., 1995), reverse transcriptasePCR (RT-PCR) (Barlic-Maganja et al., 2002; Jakob et al., 2011), and real-time RT-PCR (Purcell et al., 2006, 2013). PCR technology has high sensitivity, but it suffers from false positive rate. MAbs as useful tools, have been used widely for development of effective diagnosis of fish viral pathogens (Monini and Ruggeri, 2002; Chen et al., 2008), but the cost and time for production are main limitations. In this study, selection and characterization of single-chain fragment variable (scFv) antibody against IHNV from phage antibody library were performed. This work will serve as the basis for further development of a rapider and simpler detection kit for IHNV.

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2. Materials and methods

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2.1. Virus and phage display library

The viral pathogen infectious haematopoietic necrosis virus (IHNV) and viral haemorrhagic septicemia virus (VHSV) strain J167 88 were gifted by Centre for Environment, Fisheries and Aquacul89 ture Science (CEFAS) of UK. Spring viraemia of carp virus (SVCV) 90 strain A-1 was originally isolated from virus-infected common 91 92Q2 carp in China and preserved in our laboratory (Teng et al., 2007). Pike fry rhabdovirus (PFRV), Soft-shelled turtle iridovirus (STIV) 93 and viral nervous necrosis virus (VNNV) strain SSS0607 were 94 isolated and preserved in our laboratory. The cell lines of epithe95 lioma papulosum cyprini (EPC) were preserved in our laboratory. 96 Immunotube were purchased from Becton, Dickinson and Com97 pany (USA). The phage-displayed native mouse antibody library 98 was provided by Professor Dai Heping of Institute of Hydrobiology, 99 Chinese Academy of Sciences. 100 87

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for 1 h and then plated onto a SOBAG (SOB containing 100 ␮g/ml ampicillin and 2% glucose) plate and incubated at 30 ◦ C overnight. Then the first round of panning was completed. The titres of input and output phage were calculated for each panning round according to the numbers of colonies on petri dishes of series dilution. The other two rounds of panning were performed according to the above. 2.3. Selection of antibodies After three rounds of panning, single colonies were picked up and cultured in 2YT-AG medium (2YT medium containing 2% glucose and 100 ␮g/ml ampicillin) overnight at 30 ◦ C and 250 rpm. After that, the bacteria were cultured in 2YTAK medium (2YT medium containing 100 ␮g/ml ampicillin and 50 ␮g/ml kanamycin) supplemented with 1 × 1010 PFU/ml M13KO7 overnight at 30 ◦ C and 250 rpm. The supernatants of the cultures were collected after centrifugation and checked for ability of binding by ELISA utilizing HRP-anti M13IgG as second antibody. Absorbance values twice higher than the background level were considered to have affinity. 2.4. DNA sequencing The nucleotide sequences of scFv antibody with binding activity to IHNV were determined by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd. Shanghai, China. 2.5. Production of soluble antibody fragments After DNA sequencing, six positive clones with the highest ELISA signal and different sequences were cultured to produce soluble scFv antibody for further characterization, as described by Dai et al. (2003). Single colony was transferred from a SOBAG plate to 5 ml of freshly prepared 2YT-AG medium and incubated overnight at 30 ◦ C and 250 rpm. A 500 ml volume of 2YT-AG was then inoculated with 5 ml of overnight preculture, and grown at 30 ◦ C with shaking until OD600 nm reached 0.5–0.8. After centrifugation at 4000 × g for 20 min, the bacteria were collected and the supernatant was removed. The bacteria were then resuspended in 2YT-AI (2YT medium containing 100 ␮g/ml ampicillin and 1 mM IPTG) and incubated for 20 h at 30 ◦ C and 250 rpm. Production of soluble scFv antibody from extracellular or periplasmic extract was based on the manual for the Pharmacia Expression Module (Amersham Biosciences). The scFv antibodies were stored at −20 ◦ C until use.

2.2. Panning

2.6. Characterization analysis of antibody fragments

The antibody library was screened by three rounds of panning against IHNV, as described by Zhao et al. (2010). The transformed Escherichia coli TG1 cells were infected with M13KO7 helper phage to rescue the phagemid with its scFv insert. The phage library was precipitated by addition of PEG/NaCl (PEG8000 in 2.5 M NaCl (20%, w/v)) for more than 1 h on ice bath and collected by centrifugation. Then the phages were resuspended in 5 ml 2YT medium. Panning of the phage scFv library was performed against IHNV which was coated on immunotube at a concentration of 50 ␮g/5 ml PBS overnight at 4 ◦ C. After 3 rinses with PBS, the unoccupied sites in the immunotube were blocked with 5% bovine serum albumin (BSA) in PBS at 37 ◦ C for 1 h. Then the prepared phages were added and incubated at 37 ◦ C for 2 h. Unbound phage was removed by 20 rinses with PBST (PBS containing 0.1% Tween-20) followed by 20 rinses with PBS. Bound phages were eluted by adding 1 ml 100 mM triethylamine to the tube and rotating for 10 min. Eluted phages were neutralized with 0.5 ml of 1 M Tris–HCl (pH 7.5) and then added to 5 ml E. coli TG1 mid-log cells. The mixture was incubated at 37 ◦ C

2.6.1. Dot blot and Western blot analysis For dot blot assays, the indicated quantities of IHNV, cell debris and PBS were spotted on nitrocellulose membrane. The membrane was then blocked with 3% BSA in PBS at 37 ◦ C for 1 h. Diluted scFv antibodies were incubated with coated IHNV on the nitrocellulose membrane at room temperature for 1 h. After washing, the binding of the first antibody was assayed with rabbit polyclonal to E-Tag (HRP), and then its substrate, 3,3 -diaminobenzidine (DAB) was used for colour development. For Western blot assays, the soluble scFv antibodies were subjected to SDS-PAGE (12%) and then transferred to a polyvinylidene fluoride (PVDF) membrane as described by Bhaskaran et al. (2005). The membrane was then blocked in 3% (w/v) BSA in PBS for 1 h at room temperature on a rocker. The membrane was washed for three times, 5 min for each with PBS and PBST. Then the membrane was incubated with rabbit polyclonal to E-Tag (HRP) diluted 1:5000 in PBS containing BSA for 1 h at room temperature. Finally, DAB was used for colour development.


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Table 1 Selective enrichment of scFv antibodies from the libraries during panning. scFv antibody library

1st round

2nd round

3rd round

Phage input (CFU) Phage output (CFU) Output rate (%)

4 × 1011 7.53 × 104 1.88 × 10−5

4 × 1011 1.79 × 105 4.48 × 10−5

4 × 1011 1.10 × 106 2.75 × 10−4

Output rate (%) = (no. of phage output × 100)/(no. of phage input).

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After that, IHNV was subjected to SDS-PAGE (12%) and then transferred to a PVDF membrane. Expressed soluble scFv antibodies as first antibody were detected by Western blot. 2.6.2. Binding specificity of scFv antibodies The binding specificity of soluble scFv antibodies was determined by ELISA. 96-well microplates were coated with the indicated concentration (0.5 ␮g/well) of IHNV, SVCV, PFRV, STIV, VNNV, and VHSV. Cell debris as negative control was prepared in the same way. After overnight incubation at 4 ◦ C, the plates were washed with PBS and then blocked with 3% BSA in PBS at 37 ◦ C for 1 h. The soluble scFv antibodies were added to the wells and incubated further for 1 h at 37 ◦ C. After washing, bound scFv were detected with rabbit polyclonal to E-Tag (HRP) (1:5000). Finally, 3,3 ,5,5 -tetramethylbenzidine (TMB) was used for the colour reaction with peroxidase and the absorbance was determined at 450 nm. Absorbance values twice higher than the background level reactivity of cell debris were considered to be positive. 2.6.3. Determination of scFv affinity constants The affinity constant (Kaff ) of scFv antibodies for IHNV coated onto the 96-well microplate was determined using the protocol described by Beatty et al. (1987). The method is based upon the law of Mass Action and using solid-phase noncompetitive enzymelinked immunoadsorbent assay (EIA) curves for quantification of antibody content: Kaff = (n − 1)/2(n[Ab2] − [Ab1]) where [Ab1] and [Ab2] represent the respective scFv concentrations required to

achieve 50% of the maximum absorbance obtained at two different concentrations of coated antigen ([Ag]1 = n[Ag]2), and n is the dilution factor between the two concentrations of antigen used. Values for the affinity constant (Kaff ) can be calculated by this equation. The method utilized the OD50 of the sigmoid curve and obviated the need for affinity purification of the specific antibody used as the standard. Although the exact values of the antigen adsorbed onto the plate are unknown, the relationship of the antigen adsorptions to one another is assumed. In the experiment, microplates were coated with four concentrations (5, 2.5, 1.25 and 0.625 ␮g/ml) of IHNV in PBS at 4 ◦ C overnight. Serial twofold dilutions of scFv antibodies in 3% BSA in PBS were then performed in duplicate for each concentration of coated IHNV. The subsequent steps were identical to those described above for ELISA.

2.6.4. Immunofluorescence test Immunofluorescence was performed according to the protocol described by Takekoshi et al. (1998). EPC cells were cultured on a 6-well plate and infected with IHNV at a multiplicity of infection (MOI) of 0.01 and incubated at 15 ◦ C. After 24 h post-infection, monolayers were rinsed with PBS and fixed in icy methanol for 15 min. Then the fixed cells were incubated with scFv antibodies at 37 ◦ C for 60 min. Then FITC-conjugated rabbit anti-mouse IgG probe was added at 1:200 and incubated for 60 min. After washed in PBS, the cells were mounted and examined under a fluorescence microscope.

Fig. 1. Amino acid sequence of scFv antibodies.


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Fig. 2. The binding ability of 6 soluble scFv antibodies was determined by dot blot analysis. 5 ␮l of PBS (A, blank control), cell debris (B, negative control) and IHNV (C) per spot; 1–6 represent P1A4, P1A12, P1D5, P3B3, P3E2, P4H6.

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The phage antibody library was obtained through three rounds of panning against IHNV. After incubation of the library with the virus and washing away unbound and weakly bound phage, phage particles were eluted and used to infect E. coli culture. In this manner, specifically binding clones were selected and amplified. As shown in Table 1 a significant enrichment of specific antibody was observed after the third panning round. Along the three rounds of panning, the output rate showed a trend of gradual increase, which indicate that scFv antibodies carrying gene against IHNV were condensed. After third rounds of panning, 384 clones (four 96-well plates) were randomly picked and assessed for binding affinity to IHNV by ELISA. Among these 384 clones, 85 displayed IHNV binding activity. Positive clones, which had relatively higher binding affinity to IHNV, were chosen for further characterization.

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Thirty positive clones with the highest ELISA signal were picked out and sequenced. The complete nucleotide sequence and predicted amino acid sequence of the heavy- and light-chain variable region of the anti-IHNV scFv antibodies from the positive colonies were determined. 253 amino acids were encoded based on gene sequences of the clones. Among the 30 clones, 6 clusters were determined according to the homogeneity of the amino acid sequences of heavy-chain CDR3 regions (Fig. 1). The clone with highest binding affinity in each cluster, namely totally 6 representative clones were selected for production of soluble antibody.

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The binding ability to IHNV of these soluble scFv antibodies was determined by dot blot analysis. Dot blot signal were shown in Fig. 2. The results indicated that the six soluble scFv antibodies could recognize IHNV.

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Fig. 3. Western blot of soluble scFv antibodies. Soluble antibody was detected using HRP/Anti-E Tag. M: low MW protein Marker. Lanes 1–6: P1A4, P1A12, P1D5, P3B3, P3E2, P4H6, respectively.

Fig. 4. Western blot showing binding of scFv antibodies to denatured IHNV. Lanes 1–6: IHNV were run on a 12% SDS-PAGE gel and transferred to PVDF membrane and detected with six scFv antibodies (lane 1–6 detected with scFv P1A4, P1A12, P1D5, P3B3, P3E2 and P4H6, respectively), followed by HRP/Anti-E Tag and visualization with DAB. M: low MW protein Marker.

3.5. Specificity and affinity constants of soluble scFv antibodies For ELISA, all scFv antibodies reacted positively with IHNV. Four scFv antibodies (P1A4, P1A12, P1D5 and P3E2) showed positive reactions with SVCV, whereas no positive reaction between all six scFv antibodies and PFRV, STIV, VHSV and VNNV was detected (Table 2). The results indicated that scFv P1A4, P1A12, P1D5 and P3E2 had cross-reactivity between IHNV and SVCV. The relative affinity constant for the scFv antibodies against IHNV was calculated based on the formation of antibody-antigen complex in a solid-phase EIA, described in Section 2. Based on the four concentrations of IHNV used for coating on the immuno-plate (5, 2.5, 1.25 and 0.625 ␮g/ml), six affinity constants (three for n = 2, two for n = 4 and one for n = 8) were calculated for the scFv antibodies. The mean Kaff value for scFv antibodies were 3.95 ± 0.57 × 105 M−1 (P1A4), 1.62 ± 1.07 × 105 M−1 (P1A12), 1.49 ± 0.66 × 105 M−1 (P1D5), 6.61 ± 2.36 × 105 M−1 (P3B3), 4.31 ± 1.92 × 105 M−1 (P3E2), and 2.30 ± 0.56 × 105 M−1 (P4H6). 3.6. Immunofluorescence

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For the Western blot assay, the expected molecular weight of approximately 32 kDa specific protein bands were confirmed by Western blot (Fig. 3). No specific band was observed in Fig. 4. The results show that all scFv antibodies can only bind to native IHNV (Fig. 2), instead of denatured IHNV (Fig. 4).

Reactivity of the six scFv antibodies against IHNV was determined by immunofluorescence microscopy. The specific fluorescence signals dispersed in the infected zone of cells cytoplasm were observed. No fluorescence staining was observed in negative


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Table 2 Binding specificity the scFv antibodies by ELISA. Virus isolates

scFv antibodies P1A4

P1A12

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IHNV SVCV PFRV STIV VHSV VNNV

+ + − − − −

+ + − − − −

+ + − − − −

+ − − − − −

+ + − − − −

+ − − − − −

+: positive reaction; −: negative reaction. 289 290

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control cells (Fig. 5). It is indicated that all of the six scFv antibodies can react with IHNV infected EPC cells. 4. Discussion Phage display antibody technology has become increasingly popular for producing recombinant antibodies. There are several examples of successful isolation of antibodies against various antigens from different phage displayed antibody libraries (Hoogenboom and Winter, 1992; Takekoshi et al., 1998; Kirsch

et al., 2008), and also applied to the antibody isolation against aquatic virus (Dai et al., 2003). Unfortunately, the libraries are not available commercially. Researchers interested in using this technique are required to construct a library by themselves, or obtain existing libraries with restrictions. There are two types of antibody libraries (immunized and non-immunized libraries), and the nonimmunized one is of more general use, because it can be applied to generate antibodies against any desired antigen (Sheets et al., 1998; Pansri et al., 2009). In this study the phage-displayed native mouse antibody library was non-immunized.

Fig. 5. Immunofluorescence staining of IHNV-infected EPC cells with different soluble scFv antibodies. Photographs were taken at 24 h post-infection (400×). (1A–6A): IHNV infected EPC cells stained by scFv antibodies P1A4, P1A12, P1D5, P3B3, P3E2 and P4H6, respectively. (1B–6B): normal EPC cells stained by scFv antibodies P1A4, P1A12, P1D5, P3B3, P3E2 and P4H6, respectively.


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Most of the phage libraries that have been constructed can display the antibody fragment on the surface of the phage minor coat proteins (pIII). scFv can be expressed on the surface of M13 without apparent loss of specificity and affinity. To compare with traditional hybridoma technology, phage display antibody technology has the possibility of storing bacterial cells indefinitely and they can be thawed rapidly and fermented on an industrial scale (Johns et al., 2000; Foy et al., 2002). MAbs have been used in protocols developed for the rapid and sensitive detection and diagnosis for IHNV (Kim et al., 2008). However, MAbs production is expensive and timeconsuming. To overcome these difficulties, isolate of antibodies against IHNV by antibody phage display technology was conducted. The present work describes the screening and isolation of antiIHNV antibody fragments from a native mouse antibody gene library by phage display. After three rounds of panning, six scFv antibodies with the highest ELISA signal and different sequences were selected for characterization further. The amino acid sequences of the six scFv have high similarity, especially the light-chain variable region of the scFv antibodies, but that of heavy-chain variable region is lower. Although the six scFv antibodies shared high homology, their properties were different. scFv antibodies P1A4, P1A12, P1D5 and P3E2 had cross reactions with SVCV, while the other two (P3B3 and P4H6) had no cross reactions with all these viruses (SVCV, PFRV, STIV, VNNV, and VHSV). And the relative affinity constant of the scFv also have some differences. One of the reasons may be their different CDR regions. Another reason may be that IHNV is a member of the family Rhabdoviridae, cross reactions with other fish rhabdoviruses may exist and affect the reliability (Hill et al., 1975). The scFv antibodies with high affinity could not bind to the denatured IHNV caused by SDS polyacrylamide gel electrophoresis. It indicated that the scFv antibodies and IHNV binding sites was not linear epitope. The sites were determined by the conformation of amino acid sequence. The soluble scFv antibodies showed specific bindings to IHNV as confirmed by dot blot and immunofluorescence. For IFA, immunofluorescent staining dispersing in the cytoplasm of IHNV-infected EPC cells provided visualized evidence regarding epitopes localization in infected cells. The model for rhabdoviral replication displays that virus replication occurs in some sites or microdomains of the cytoplasm (Ni et al., 1996; Jayakar et al., 2004) which is consistent with the finding in the present study. Cell culture isolation and molecular methods both require complicated equipment and laboratory conditions. These methods take several days to complete the detection. Furthermore, most fish culture fields are far away from a laboratory. Dot blot can be performed in short time for detection and does not require any equipment. Farmers can use this method routinely, and once they get a positive dot blot signal, they will have enough time to make a meaningful decision on treatments. This method is more convenient and economical, and they may be useful in developing specific and sensitive diagnostic assays. In summary, the isolation of specific antibodies against IHNV from a phage display antibody library was demonstrated in the present study. Moreover, single-chain antibodies can be produced very efficiently using bacterial protein synthesis systems and this is beneficial for reducing time and cost. Easy and economic scFvphage production could be a powerful tool for the detection of IHNV, and a realistic alternative to current methods.

Acknowledgements This work was supported by General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China (AQSIQ) (No. 2012IK032 and 201210055). The authors are

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Selection of recombinant anti-SH3 domain antibodies by high-throughput phage display.

Screening and selection of peptides specific for esophageal cancer cells from a phage display peptide library.

Selection and characterization of single-chain recombinant antibodies against phosphoprotein of Newcastle disease virus.

Utilization of Multi-Immunization and Multiple Selection Strategies for Isolation of Hapten-Specific Antibodies from Recombinant Antibody Phage Display Libraries.

Polyreactive monoclonal autoantibodies in multiple sclerosis: functional selection from phage display library and characterization by deep sequencing analysis.

Generation of Potent Anti-Vascular Endothelial Growth Factor Neutralizing Antibodies from Mouse Phage Display Library for Cancer Therapy.

Discovery and Characterization of Phage Display-Derived Human Monoclonal Antibodies against RSV F Glycoprotein.

Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library.

High-content analysis of antibody phage-display library selection outputs identifies tumor selective macropinocytosis-dependent rapidly internalizing antibodies.

Generation of Recombinant Antibodies against the beta-(1,6)-Branched beta-(1,3)-D-Glucan Schizophyllan from Immunized Mice via Phage Display.

Epitope mapping with random phage display library.

Specific ligands for classical swine fever virus screened from landscape phage display library.

Selection and characterization of human PCSK9 antibody from phage displayed antibody library.

Phage display library selection of a hypoxia-binding scFv antibody for liver cancer metabolic marker discovery.

An affinity improved single-chain antibody from phage display of a library derived from monoclonal antibodies detects fumonisins by immunoassay.

Chemical Synthesis and In Vitro Evaluation of a Phage Display-Derived Peptide Active against Infectious Salmon Anemia Virus.

A protocol for phage display and affinity selection using recombinant protein baits.

Construction of a filamentous phage display peptide library.

Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone.

Selection of cell-type specific antibodies on tissue-sections using phage display.

Bivalent DNA vaccine induces significant immune responses against infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus in rainbow trout.

Diversity of the antibody response to tetanus toxoid: comparison of hybridoma library to phage display library.

Preliminary study of an oral vaccine against infectious hematopoietic necrosis virus using improved yeast surface display technology.

Infectious bronchitis virus S2 expressed from recombinant virus confers broad protection against challenge.