Cloning and expression of two human recombinant monoclonal Fab fragments specific for EBV viral capsid antigen.
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.
Dong
Lingli
L
Department of Hematology and Immunology, Kanazawa Medical University, Uchinada, Ishikawa 920-0293, Japan.
Masaki
Yasufumi
Y
Takegami
Tsutomu
T
Kawanami
Takafumi
T
Itoh
Kunihiko
K
Jin
Zhe-Xiong
ZX
Huang
Cheng-Ri
CR
Tong
Xiao-Peng
XP
Fukushima
Toshihiro
T
Tanaka
Masao
M
Sawaki
Toshioki
T
Sakai
Tomoyuki
T
Sugai
Susumu
S
Okazaki
Toshiro
T
Hirose
Yuko
Y
Umehara
Hisanori
H
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D000914:Antibodies, Viral / Q000097:blood*; D000956:Antigens, Viral / Q000276:immunology*; D015153:Blotting, Western; D036022:Capsid Proteins / Q000276:immunology*; D002460:Cell Line; D003001:Cloning, Molecular; D020650:Combinatorial Chemistry Techniques; D004797:Enzyme-Linked Immunosorbent Assay; D020031:Epstein-Barr Virus Infections / Q000150:complications / Q000276:immunology*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D016393:Lymphoma, B-Cell / Q000209:etiology / Q000276:immunology* / Q000821:virology; D008969:Molecular Sequence Data; D019151:Peptide Library; D020539:Sequence Analysis, Protein; D012859:Sjogren's Syndrome / Q000150:complications* / Q000276:immunology
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral; D036022:Capsid Proteins; C041788:Epstein-Barr viral capsid antigen; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library