Skip Navigation


International Immunology Advance Access originally published online on January 31, 2007
International Immunology 2007 19(3):331-336; doi:10.1093/intimm/dxl150
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
19/3/331    most recent
dxl150v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (2)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Dong, L.
Right arrow Articles by Umehara, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dong, L.
Right arrow Articles by Umehara, H.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?


© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Cloning and expression of two human recombinant monoclonal Fab fragments specific for EBV viral capsid antigen

Lingli Dong1,3, Yasufumi Masaki1, Tsutomu Takegami2, Takafumi Kawanami1, Kunihiko Itoh4, Zhe-Xiong Jin1, Cheng-Ri Huang1, Xiao-Peng Tong1, Toshihiro Fukushima1, Masao Tanaka1, Toshioki Sawaki1, Tomoyuki Sakai1, Susumu Sugai1, Toshiro Okazaki5, Yuko Hirose1 and Hisanori Umehara1

1 Department of Hematology and Immunology
2 Department of Molecular Oncology and Virology, Kanazawa Medical University, Uchinada, Ishikawa 920-0293, Japan
3 Department of Hematology and Immunology, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China
4 Department of Clinical Pharmacology and Genetics, School of Pharmaceutical Science, University of Shizuoka, Shizuoka 422-8526, Japan
5 Department of Clinical Laboratory, Medicine/Hematology, Faculty of Medicine, Tottori University, Yonago, Tottori 683-8504, Japan

Correspondence to: Y. Masaki; E-mail: yasum{at}kanazawa-med.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Serum titers of antibody to Epstein–Barr virus (EBV) viral capsid antigen (VCA) have been positively correlated with malignancies of lymphoid proliferation, such as Burkitt's lymphoma and Hodgkin's lymphoma. We have constructed a phage display combinatorial antibody Fab library from a patient with marginal zone B cell lymphoma associated with Sjögren's syndrome and carrying high serum anti-EBV-VCA IgG titer. Fab fragments were selected by panning against EBV-VCA protein coated onto ELISA plates, and selected Fab clones were characterized by ELISA, western blotting (WB), indirect immunofluorescence assay and immunohistochemistry. We have established two Fab clones, Fab-aVCA1 and Fab-aVCA21, which specifically recognize EBV-VCA by ELISA and WB. Inhibition ELISA competition showed that both clones could significantly reduce the binding of specific anti-EBV-VCA mAb to its relevant proteins. Furthermore, these two Fab clones could localize VCA protein in the EBV-positive P3HR1 and Daudi cell lines, as well as in tissue samples from patients with EBV-infected lymphoid malignancies. These results indicate that our two Fab clones are novel human mAbs specific for EBV-VCA protein and may have potential benefits for development of novel diagnostic and therapeutic approaches in EBV-related lymphoid malignancies.

Keywords: antibodies, human, molecular biology


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Epstein–Barr virus (EBV) is the first virus to be directly implicated in carcinogenesis in humans. EBV infects >90% of the world's adult population, and primary infection in a young adult can lead to infectious mononucleosis. Although most humans co-exist with the virus without serious sequels, this virus is strongly associated with several malignancies, including Burkitt's lymphoma (BL), nasopharyngeal carcinoma (NPC) and Hodgkin's lymphoma (HL) (1, 2). In cancer cells, EBV is present in a latent state, in which a subset of EBV genes is expressed, or in lytic cycles, which are characterized by the production of viral particles. Because viral capsid antigen (VCA) and early antigen, like the recently described EBV transactivator protein (ZEBRA), are expressed during EBV replication, cells expressing those EBV genes are more readily recognized by the immune system (3, 4). Therefore, high anti-VCA IgG titers are considered to be a marker of EBV reactivity (5). Remarkably, anti-VCA antibody titer is considerably higher in the sera of patients with BL, HL and NPC patients than in age-matched controls (1, 2). Although the presence of serum anti-VCA IgG has been widely used for the diagnosis of EBV infection, the molecular profiles of this human antibody remain unknown. In addition, EBV was recently found to be associated with autoimmune diseases, such as systemic lupus erythematosus (6, 7). Thus, the generation of EBV-VCA-specific human mAbs from human combinatorial Fab libraries could be of assistance in studies of molecular profiles of these antibodies and in the diagnosis of EBV infection at various stages.

The preparation of combinatorial libraries on the filamentous phage surface has been demonstrated to be an efficient route for the production of high affinity human mAbs (8, 9). Their advantages over mouse mAb include the ability to alter antibody fragments by protein engineering methods. Thus, there have been many studies of recombinant Fab selected by target antigens, including viruses such as HIV types 1 and 2, human rotavirus and SARS by phage display (10–12).

We have constructed a human antibody library from a patient carrying a high titer of serum anti-EBV-VCA IgG and successfully established two Fab clones by panning the Fab library against EBV-VCA. These two Fab fragments were found to bind to EBV-VCA with high affinity and were suitable for detecting EBV-VCA in EBV-infected malignant cells.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Library construction and phage display
After obtaining informed consent, we isolated 10 million bone marrow lymphocytes from a patient with Sjögren's syndrome and marginal zone B cell lymphoma carrying a high titer of EBV-VCA IgG. Total RNA was isolated and used to generate cDNA, which was used as a template for PCR amplification of sequences in the region of Vh-Ch1 of the heavy chain and light chain {kappa}/{lambda} (13). An antibody Fab library was constructed by the pComb3 M13 surface display system (13, 14).

Panning selection of EBV-VCA antigen binding phage
Human Fab antibodies were selected by panning against commercially obtained EBV-VCA (Biogenesis Company, UK) as described (13). A total of four cycles of panning were performed on 96-well ELISA plates pre-coated with 5 µg per well of EBV-VCA. Unbound phages were removed by washing 10 times with PBS/0.05% Tween 20. Bound human Fabs were detected with alkaline phosphatase (AP)-labeled goat anti-human IgG F(ab')2 (Pierce, Rockford, IL, USA), and visualized with p-Nitrophenyl phosphate disodium salt (pNPP) substrate (Pierce) by reading the absorbance at 405 nm.

DNA sequencing
DNA was amplified using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA). The primers for the variable region of the heavy (VH) chain were T3 (5'-GAAGTAGTCCTTGACCAG-3' forward) and Gz (5'-GAAGTAGTCCTTGACCAG-3', reverse), and the primers for the variable region of the light (VL) chain were KEF (5'-GAATTCTAAACTAGCTAGTCG-3', forward) and Kb (5'-ATAGAAGTTGTTCAGCAGGCA-3', reverse). The products were sequenced on the ABI PRISM 310 Genetic Analyzer (Applied Biosystems).

Purification of Fab fragments
Monoclonal Fabs were purified by immunoaffinity chromatography, using polyclonal Goat anti-human IgG F(ab')2 (Pierce) conjugated to Gammabind G Sepharose (Amersham Bioscience, Piscataway, NJ, USA). After cross-linking with dimethyl pimelimidate (Pierce), Fab was purified (15) and its concentration was determined using the Bio-Rad protein microassay (CA, USA), and antibody purity was assessed by 12% SDS–PAGE followed by Quick-CBB staining (Wako, Osaka, Japan).

ELISA and inhibition analysis
To determine the reactivity of Fabs to EBV-VCA, 96-well microtiter plates were coated with 5 µg ml–1 EBV-VCA protein. After blocking with 3% BSA in PBS, the plates were washed with PBS/0.05% Tween 20 and incubated with two-fold serial dilutions of purified Fab (0.625–10 µg per well in PBS), followed by 1:2500 diluted AP-conjugated goat anti-human IgG F(ab')2 and pNPP. The unrelated proteins, ovalbumin peptide (OVA) and human IgG Fc fragments, were used as control antigens to test non-specific binding (data not shown), and human recombinant Fab N28 anti-rotavirus was used as negative control (14).

To determine the ability of our Fab clones to competitively inhibit the binding of mouse mAb to EBV-VCA, we performed inhibition ELISA. After determining the dilution of mouse mAb (Argene, clone F3.23, France) that bound 90% of EBV-VCA protein in mouse mAb capture ELISA (1:100; data not shown), microtiter plates were coated with 5 µg per well of EBV-VCA, and 50 µl per well of serially diluted Fabs in PBS and 50 µl per well of diluted mouse mAb (1:50) were added. The plates were incubated for 1.5 h at room temperature (RT), washed, and incubated with HRP-conjugated goat anti-mouse IgG (ICN Pharmaceutical Inc., CA, USA) (1:2000) and 1-StepTMABTS [2-2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt] (Pierce) substrates, then the absorbance at 405 nm was determined.

Western blotting analysis
One-half microgram aliquots of EBV-VCA protein loaded onto each lane were separated by 10% SDS–PAGE, followed by electrophoresis and transfer to a nitrocellulose membrane (Bio-Rad) by the semi-dry system (Bio-Rad). After blocking with 3% skimmed milk, the membrane was incubated overnight at 4°C with purified Fab fragment (1ug ml–1). The membrane was subsequently incubated with HRP-labeled anti-human IgG F(ab')2 (Pierce) for 1 h at RT and the reaction was developed and visualized with ECL-plus (Amersham Life Science, UK) (16).

Indirect immunofluorescence assay
Recombinant Fabs (10 µg ml–1) were detected by indirect immunofluorescence assay (IFA) with FITC-conjugated goat anti-human IgG F(ab')2 antibody (Jackson Immunoresearch, PA, USA). EBV-infected P3HR1 and Daudi cells [American Type Culture Collection (ATCC), VA, USA] were fixed with acetone after cytospin on glass slides, with EBV-negative Jurkat cells (ATCC) used as control. 4'-6-Diamidino-2-phenylindole (DAPI) (Wako) was used to counterstain the nucleus. Mouse mAb was used as positive reference control and detected using PE-conjugated rabbit anti-mouse IgG antibody (Jackson Immunoresearch) (17).

Immunohistochemistry and in situ hybridization
Paraffin-embedded tumor tissue samples from five patients with EBV-positive lymphoid malignancies were used for immunohistochemical identification of EBV-VCA by our Fab clones and mouse mAb. They were one BL patient, two HL (mixed cellularity) patients and two diffuse large B cell lymphoma patients. As control, we used spleen samples from a patient with EBV-negative BL. Deparaffinized slides were treated for 10 min with 3% H2O2 to block endogenous peroxidase and 30 min with 3% skimmed milk. After antigen retrieval by autoclave heating, the slides were incubated for 1 h at RT with 10 µg ml–1 Fabs or 1:50 diluted mouse mAb. As control, slides were incubated with an anti-rotavirus Fab. After extensive washing with PBS, the slides were incubated for 1 h at RT with HRP anti-human IgG F(ab)2 for Fabs and second antibody (Envision + Dual link system peroxidase, DAKO, Denmark) for mouse mAb. The slides were stained with DAB substrate (Envision DAB kit, DAKO) and counterstained with hematoxylin (DAKO). In situ hybridization (ISH) for EBV-encoded small RNA1 was performed simultaneously as described (18, 19).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Selection of Fab fragments
After four rounds of panning, two Fab clones (Fab-aVCA1 and Fab-aVCA21) were considered positive for EBV-VCA. The reactivity of these Fab clones against the relevant antigen was higher than 1.0 optical density (OD), whereas their reactivity against human IgG Fc fragment and OVA were <0.3 OD by ELISA. Both purified Fab fragments appeared as clear bands of molecular weight 28 kDa on SDS–PAGE (Fig. 1).


Figure 1
View larger version (76K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1. SDS–PAGE analysis of purified Fab fragments. Fab fragments (2 µg) were subjected to 12% SDS–PAGE, and the gel was visualized with CBB staining. Both purified Fab fragments, Fab-aVCA1 and Fab-aVCA21, have molecular weights of ~28 kDa (M, molecular marker; lane 1, Fab-aVCA1; lane 2, Fab-aVCA21).

 
Western blotting analysis of recombinant Fabs targeting EBV-VCA protein
Western blotting (WB) against EBV-VCA proteins was performed to define the targets of the recombinant monoclonal antibodies. Fab-aVCA1 recognized bands, of molecular weight 160, 85, 58 and 30 kDa (Fig. 2, lane 3), whereas Fab-aVCA21 detected bands of 85, 58 and 30 kDa (Fig. 2, lane 4). To determine why the Fab fragments recognized multiple bands, the EBV-VCA proteins were blotted with mouse mAb, which recognized EBV-VCA proteins of molecular weight 160, 85, 58, and 30 kDa (Fig. 2, lane 2), suggesting that the VCA protein may be proteolytically degraded (20).


Figure 2
View larger version (99K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2. Western blot analysis of EBV-VCA proteins with Fab-aVCA1 and Fab-aVCA21. EBV-VCA protein (0.5 µg) was separated by 10% SDS-PAGE, and transferred to a nitrocellulose membrane. After blocking, the membrane was incubated overnight at 4°C with purified Fab fragment (1 µg ml–1), followed by incubation for 1 h at RT with HRP-labeled anti-human IgG F(ab')2 and visualization with ECL system (lane 1, protein profiles of EBV-VCA; lane 2, mouse anti-VCA mAb; lane 3, Fab-aVCA1; lane 4, Fab-aVCA21).

 
Amino acid sequences of Fab H and L chains
The amino acid sequences of the VH and VL chains of both Fab clones were deduced from DNA sequencing and compared with the closest known germ line proteins (Fig. 3). The VH chain sequence of Fab-aVCA1 was derived from the VH4 family whereas the VH chain sequence of Fab-aVCA21 was from the VH3 family. The two Fab clones were found to share the same {kappa} light chain. Homologies to the closest germ line gene were 94% for both VL chains and 95 and 93% for the VH chains of Fab-aVCA1 and Fab-aVCA21, respectively. The nucleotide sequence data of our Fabs reported in this paper have been submitted to the DDBJ database and were assigned the following accession number: AB266511 (Fab-aVCA1 light chain), AB266512 (Fab-aVCA1 heavy chain) and AB266513 (Fab-aVCA21 heavy chain).


Figure 3
View larger version (9K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3. Comparison of the deduced amino acid sequences of both VH and VL domains to the closest germ line sequence. (A) VH sequences; (B) VL sequences. Amino acid identity within a group is indicated by dots.

 
ELISA and inhibition ELISA
Binding of Fab-aVCA1 and Fab-aVCA21 to EBV-VCA was examined using ELISA. Both Fab fragments showed high affinity, concentration-dependent binding to EBV-VCA, whereas the negative control, Fab anti-rotavirus, did not bind to EBV-VCA (Fig. 4A). To further characterize the affinity of Fab-aVCA1 and Fab-aVCA21 to EBV-VCA, we compared their binding ability with that of mouse mAb (clone F3.23), which is specific for EBV-VCA, using inhibition ELISA. We found that Fab-aVCA21 significantly reduced the binding of mouse mAb to EBV-VCA, whereas Fab-aVCA1 showed a similar, but relatively weak effect, on binding (Fig. 4B).


Figure 4
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4. Specificity of Fab fragments by ELISA. (A) Binding of Fab-aVCA1 and Fab-aVCA21 to EBV-VCA. Microtiter plates were coated with 5 µg µl–1 EBV-VCA protein, and two-fold serially diluted Fabs were added to the wells. Bound Fabs were detected by anti-human IgG F(ab')2–HRP and measured by optical density at 405 nm. (B) Inhibition ELISA: Microtiter plates were coated with 5 µg per well EBV-VCA and blocked with 3% BSA/PBS, and to each well was added 50 µl serially diluted Fabs in PBS and 50 µ1 1:50 diluted mouse mAb. After incubation for 1.5 hour at RT, HRP-conjugated goat anti-mouse IgG was added and visualized by absorbance at 405 nm. Each experiment was done in triplicate. Error bars represent SEM.

 
Specificity of Fab fragments by IFA, immunohistochemistry and ISH
To further investigate the interaction of the purified Fab fragments and EBV-VCA, we assayed the binding of purified Fab fragments to the EBV-positive P3HR1 and Daudi cell lines and the EBV-negative Jurkat cell line by IFA. Confocal microscopy showed that both Fab-aVCA1 and Fab-aVCA21 stained P3HR1 and Daudi (Fig. 5), but not Jurkat cells (data not shown). In contrast, no staining was observed using Fab anti-rotavirus (Fig. 5). Immunohistochemically, we found that Fab-aVCA1 and Fab-aVCA21 specifically recognized EBV-VCA protein in EBV-infected lymphoma cells with good concordance with mouse mAb in five patients with EBV-positive lymphoid malignancies. Figure 6 showed staining of paraffin-embedded lymph node tissues from a patient with EBV-positive diffuse large B cell lymphoma by Fab-aVCA1 and Fab-aVCA21 (Fig. 6 B and C). Staining with Fab anti-rotavirus (Fig. 6 D) and staining of control tissues (spleen from a patient with EBV-negative BL) were negative (Fig. 6 G–I). These findings were confirmed by ISH (Fig. 6 E and J).


Figure 5
View larger version (38K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5. Indirect immunofluorescence of EBV-infected P3HR1 (A) cells and Daudi cells (B) using recombinant Fab fragments. Recombinant Fabs (10 µg ml–1) were detected by indirect immunofluorescence assay (IFA) with FITC-conjugated goat anti-human IgG F(ab')2 antibody. EB virus-infected P3HR1 and Daudi cells were fixed with acetone after cytospin on glass slides. DAPI was used to counterstain the nucleus. (A) P3HR1 cells. (B) Daudi cells. Nuclei counterstained with DAPI (left panel), Fab-aVCA1, Fab-aVCA21 and Fab anti-rotavirus (right panel).

 

Figure 6
View larger version (41K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6. EBV-VCA detection with Fab-aVCA1 and Fab-aVCA21 by IHC and EBAR detection by ISH study. Deparaffinized slides were treated for 10 min with 3% H2O2. After antigen retrieval, the slides were incubated with 10 µg ml–1 Fabs for 1 h at RT. After extensive washing with PBS, the slides were incubated for 60 min at RT with HRP anti-human IgG F(ab’)2 for Fabs. The slides were stained with DAB substrate and counterstained with hematoxylin. Immunohistochemistry of EBV-positive LN tissues (A–E) and EBV-negative spleen tissues (F–J) with Fab-aVCA1 (B and G), Fab-aVCA21 (C and H), Fab anti-rotavirus (D and I), as well as ISH studies of EBV-positive (E) and negative (J) lymphoma tissues. (A) and (F) are stained with H&E.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
VCA is a complex of incompletely defined EBV late gene products associated with virion particles (21, 22) and expressed in the late period of EBV replication (17, 20). The presence of EBV-VCA in EB-related malignant cells may be associated with EBV reactivation, which usually entails virus replication and excretion. It is likely that the exposure of EBV-infected cancer cells to the immune system leads to an increase in titer of anti-VCA antibodies. In this regard, anti-VCA IgG titers have been reported to be associated with EB viral load in patients with HL, patients infected with HIV and children receiving solid-organ transplants (23–25). In addition, the presence of EBV-positive tumor cells has been associated with poorer survival in older patients with HL (26, 27) and advanced NPC (28). Therefore, new effective implications for diagnosis of EBV infection and potentially clinical marker to monitor the presence of EBV-related lymphoid malignancies are required.

Phage display is a rapid and convenient way to isolate recombinant mAbs specific for various antigens (8), including the isolation of a human monoclonal Fab fragment specific for EBV envelope glycoprotein, the gp350/220 antigen (29). We have successfully identified two EBV-specific Fab fragments by panning a Fab library against EBV-VCA, and subsequently characterized these fragments by DNA sequencing and serial approaches.

We have identified the genes encoding the Fab-aVCA1 and Fab-aVCA21 antibodies. Both Fab fragments share the same VL chain, with homology to the closest germ line gene of 94%. The homologies of VH chain of Fab-aVCA1 and Fab-aVCA21 were 95 and 93%, respectively. In addition, we found that both Fab-aVCA21 and Fab-aVCA1 specifically bind to EBV-VCA in a concentration-dependent manner, as well as significantly reducing the binding of mouse mAb to EBV-VCA, suggesting that Fab-aVCA1 and Fab-aVCA21 bind to VCA epitopes with high affinity, similar to that of mouse mAb. By WB, we detected minor differences in the proteins bound by both Fab fragments and mouse mAb, suggesting that reactive epitopes may differ in EBV-infected humans and EBV-immunized mice (11). Using IFA, we found that both Fab fragments could detect VCA protein in the EBV-positive P3HR1 cell line, which has been used for EBV serodiagnosis (30), as well as in Daudi cell line. These findings strongly suggest that our two human Fabs can recognize EBV-VCA with high affinity. Finally, we confirmed the specificity of these two Fab fragments in tissues from five patients with EBV-positive lymphoid malignancies by immunohistochemistry and ISH. These results indicate that these two Fab fragments are novel human EBV-VCA specific mAbs. Altogether, these results help us to further understand the molecular profiles of human anti-EBV-VCA antibody and may provide the insight for future study leading to intracellular expression of antibodies (31).

Hybridoma techniques to produce murine mAbs are quite expensive and require animal handling. In addition, the inherent immunogenicity of murine sequences in humans has presented obstacles to the clinical application of mouse mAbs. Human Fab should have reduced immunogenicity in humans, and may therefore have enhanced efficacy, safety and ease of use. In addition to phage display, transgenic mice can generate a full repertoire of human therapeutic mAbs and may facilitate the optimization of mAb production and open new therapeutic avenues (32, 33). Although further studies are necessary to determine whether our two Fab fragments specific to EBV-VCA have protective activity in vitro and in vivo, these results may provide building blocks for the development of novel diagnostic and therapeutic approaches.


   Acknowledgements
 
We gratefully thank Dennis R. Burton (The Scripps Research Institute, La Jolla, CA, USA) for the generous gift of the pCombs vector. This work was supported by grants from the Japanese Ministry of Education, Culture, Sports, Science and Technology (13557160, 15024236, 15390313, 13877075, 15024236, 15390313 to H.U. and 17591060 to Y.M.), the Uehara Memorial Foundation (to H.U.), the Hokkoku Cancer Fund (to Y.M.) and Kanazawa Medical University Research Foundation (C2006-1 to H.U. and S2004-16 to Y.M.).


   Abbreviations
 
AP, alkaline phosphatase
BL, Burkitt's lymphoma
DAPI, 4'-6-Diamidino-2-phenylindole
EBV, Epstein–Barr virus
HL, Hodgkin's lymphoma
IFA, indirect immunofluorescence assay
ISH, in situ hybridization
NPC, nasopharyngeal carcinoma
OD, optical density
OVA, ovalbumin peptidep
pNPP, p-Nitrophenyl phosphate disodium salt
RT, room temperature
VCA, viral capsid antigen
WB, western blotting

   Notes
 
Transmitting editor: K. Okumura

Received 4 September 2006, accepted 22 December 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Thompson MP and Kurzrock R. (2004) Epstein-Barr virus and cancer. Clin. Cancer Res. 10:803.[Abstract/Free Full Text]
  2. Williams H and Crawford DH. (2006) Epstein-Barr virus: the impact of scientific advances on clinical practice. Blood 107:862.[Abstract/Free Full Text]
  3. Sugimoto M, Tahara H, Ide T, Furuichi Y. (2004) Steps involved in immortalization and tumorigenesis in human B-lymphoblastoid cell lines transformed by Epstein-Barr virus. Cancer Res. 64:3361.[Abstract/Free Full Text]
  4. Drouet E, Brousset P, Fares F, et al. (1999) High Epstein-Barr virus serum load and elevated titers of anti-ZEBRA antibodies in patients with EBV-harboring tumor cells of Hodgkin's disease. J. Med. Virol. 57:383.[CrossRef][Web of Science][Medline]
  5. Hess RD. (2004) Routine Epstein-Barr virus diagnostics from the laboratory perspective: still challenging after 35 years. J. Clin. Microbiol. 42:3381.[Free Full Text]
  6. Gross AJ, Hochberg D, Rand WM, Thorley-Lawson DA. (2005) EBV and systemic lupus erythematosus: a new perspective. J. Immunol. 174:6599.[Abstract/Free Full Text]
  7. Chen CJ, Lin KH, Lin SC, et al. (2005) High prevalence of immunoglobulin A antibody against Epstein-Barr virus capsid antigen in adult patients with lupus with disease flare: case control studies. J. Rheumatol. 32:44.[Abstract/Free Full Text]
  8. Paschke M. (2006) Phage display systems and their applications. Appl. Microbiol. Biotechnol. 70:2.[CrossRef][Web of Science][Medline]
  9. Barbas CF III. (1993) Recent advances in phage display. Curr. Opin. Biotechnol. 4:526.[CrossRef][Medline]
  10. Zhang MY, Xiao X, Sidorov IA, et al. (2004) Identification and characterization of a new cross-reactive human immunodeficiency virus type 1-neutralizing human monoclonal antibody. J. Virol. 78:9233.[Abstract/Free Full Text]
  11. Higo-Moriguchi K, Akahori Y, Iba Y, Kurosawa Y, Taniguchi K. (2004) Isolation of human monoclonal antibodies that neutralize human rotavirus. J. Virol. 78:3325.[Abstract/Free Full Text]
  12. Flego M, Di Bonito P, Ascione A, et al. (2005) Generation of human antibody fragments recognizing distinct epitopes of the nucleocapsid (N) SARS-CoV protein using a phage display approach. BMC Infect. Dis. 5:73.[CrossRef][Medline]
  13. Ditzel HJ, Masaki Y, Nielsen H, Farnaes L, Burton DR. (2000) Cloning and expression of a novel human antibody-antigen pair associated with Felty's syndrome. Proc. Natl Acad. Sci. USA 97:9234.[Abstract/Free Full Text]
  14. Itoh K, Nakagomi O, Suzuki K, Inoue K, Tada H, Suzuki T. (1999) Recombinant human monoclonal Fab fragments against rotavirus from phage display combinatorial libraries. J. Biochem. (Tokyo) 125:123.[Abstract/Free Full Text]
  15. Ditzel HJ, Binley JM, Moore JP, et al. (1995) Neutralizing recombinant human antibodies to a conformational V2- and CD4-binding site-sensitive epitope of HIV-1 gp120 isolated by using an epitope-masking procedure. J. Immunol. 154:893.[Abstract]
  16. Miyaji M, Jin ZX, Yamaoka S, et al. (2005) Role of membrane sphingomyelin and ceramide in platform formation for Fas-mediated apoptosis. J. Exp. Med. 202:249.[Abstract/Free Full Text]
  17. Liu MY, Huang YT, Sheen TS, Chen JY, Tsai CH. (2004) Immune responses to Epstein-Barr virus lytic proteins in patients with nasopharyngeal carcinoma. J. Med. Virol. 73:574.[CrossRef][Web of Science][Medline]
  18. Hirose Y, Masaki Y, Yoshioka R, Takiguchi T. (1997) Aggressive natural killer cell lymphoproliferative disorder associated with Epstein-Barr viral RNA. Am. J. Hematol. 54:314.[CrossRef][Web of Science][Medline]
  19. Hirose Y, Masaki Y, Sawaki T, et al. (2006) Association of Epstein-Barr virus with human immunodeficiency virus-negative peripheral T-cell lymphomas in Japan. Eur. J. Haematol. 76:109.[CrossRef][Web of Science][Medline]
  20. Johannsen E, Luftig M, Chase MR, et al. (2004) Proteins of purified Epstein-Barr virus. Proc. Natl Acad. Sci. USA 101:16286.[Abstract/Free Full Text]
  21. van Grunsven WM, van Heerde EC, de Haard HJ, Spaan WJ, Middeldorp JM. (1993) Gene mapping and expression of two immunodominant Epstein-Barr virus capsid proteins. J. Virol. 67:3908.[Abstract/Free Full Text]
  22. Serio TR, Angeloni A, Kolman JL, et al. (1996) Two 21-kilodalton components of the Epstein-Barr virus capsid antigen complex and their relationship to ZEBRA-associated protein p21 (ZAP21). J. Virol. 70:8047.[Abstract]
  23. Besson C, Amiel C, Le-Pendeven C, et al. (2006) Positive correlation between Epstein-Barr virus viral load and anti-viral capsid immunoglobulin G titers determined for Hodgkin's lymphoma patients and their relatives. J. Clin. Microbiol. 44:47.[Abstract/Free Full Text]
  24. Stevens SJ, Blank BS, Smits PH, Meenhorst PL, Middeldorp JM. (2002) High Epstein-Barr virus (EBV) DNA loads in HIV-infected patients: correlation with antiretroviral therapy and quantitative EBV serology. AIDS 16:993.[CrossRef][Web of Science][Medline]
  25. Rogers BB, Conlin C, Timmons CF, Dawson DB, Krisher K, Andrews WS. (1997) Epstein-Barr virus PCR correlated with viral histology and serology in pediatric liver transplant patients. Pediatr. Pathol. Lab. Med. 17:391.[CrossRef][Web of Science][Medline]
  26. Jarrett RF, Stark GL, White J, et al. (2005) Impact of tumor Epstein-Barr virus status on presenting features and outcome in age-defined subgroups of patients with classic Hodgkin lymphoma: a population-based study. Blood 106:2444.[Abstract/Free Full Text]
  27. Keegan TH, Glaser SL, Clarke CA, et al. (2005) Epstein-Barr virus as a marker of survival after Hodgkin's lymphoma: a population-based study. J. Clin. Oncol. 23:7604.[Abstract/Free Full Text]
  28. Yang X, Goldstein AM, Chen CJ, et al. (2006) Distribution of Epstein-Barr viral load in serum of individuals from nasopharyngeal carcinoma high-risk families in Taiwan. Int. J. Cancer 118:780.[CrossRef][Web of Science][Medline]
  29. Bugli F, Bastidas R, Burton DR, Williamson RA, Clementi M, Burioni R. (2001) Molecular profile of a human monoclonal antibody Fab fragment specific for Epstein-Barr virus gp350/220 antigen. Hum. Immunol. 62:362.[CrossRef][Web of Science][Medline]
  30. Liao G, Huang J, Fixman ED, Hayward SD. (2005) The Epstein-Barr virus replication protein BBLF2/3 provides an origin-tethering function through interaction with the zinc finger DNA binding protein ZBRK1 and the KAP-1 corepressor. J. Virol. 79:245.[Abstract/Free Full Text]
  31. Cattaneo A and Biocca S. (1999) The selection of intracellular antibodies. Trends Biotechnol. 17:115.[CrossRef][Web of Science][Medline]
  32. Weiner LM. (2006) Fully human therapeutic monoclonal antibodies. J. Immunother. 29:1.[Medline]
  33. Sharon J, Liebman MA, Williams BR. (2005) Recombinant polyclonal antibodies for cancer therapy. J. Cell. Biochem. 96:305.[CrossRef][Web of Science][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
19/3/331    most recent
dxl150v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (2)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Dong, L.
Right arrow Articles by Umehara, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dong, L.
Right arrow Articles by Umehara, H.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?