Generation and selection of immunized Fab phage display library against human B cell lymphoma.
The approval of using monoclonal antibodies as a targeted therapy in the management of patients with B cell lymphoma has led to new treatment options for this group of patients. Production of monoclonal antibodies by the traditional hybridoma technology is costly, and the resulting murine antibodies often have the disadvantage of triggering human anti-mouse antibody (HAMA) response. Therefore recombinant Fab antibodies generated by the phage display technology can be a suitable alternative in managing B cell lymphoma. In this study, we extracted total RNA from spleen cells of BALB/c mice immunized with human B lymphoma cells, and used RT-PCR to amplify cDNAs coding for the kappa light chains and Fd fragments of heavy chains. After appropriate restriction digests, these cDNA fragments were successively inserted into the phagemid vector pComb3H-SS to construct an immunized Fab phage display library. The diversity of the constructed library was approximately 1.94x10(7). Following five rounds of biopanning, soluble Fab antibodies were produced from positive clones identified by ELISA. From eight positive clones, FabC06, FabC21, FabC43 and FabC59 were selected for sequence analysis. At the level of amino acid sequences, the variable heavy domains (V(H)) and variable light domains (V(L)) were found to share 88-92% and 89-94% homology with sequences coded by the corresponding murine germline genes respectively. Furthermore, reactivity with membrane proteins of the B cell lymphoma was demonstrated by immunohistochemistry and western blotting. These immunized Fab antibodies may provide a valuable tool for further study of B cell lymphoma and could also contribute to the improvement of disease therapy.
Shen
Yongmei
Y
Department of Radioimmunoassay, Second Affiliated Hospital of Soochow University, Suzhou, China. yongmei_shen@yahoo.com
Yang
Xiaochun
X
Dong
Ningzheng
N
Xie
Xiaofang
X
Bai
Xia
X
Shi
Yizhen
Y
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007106:Immune Sera / Q000032:analysis / Q000276:immunology; D007114:Immunization; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000276:immunology; D007145:Immunoglobulin kappa-Chains / Q000235:genetics / Q000276:immunology; D016393:Lymphoma, B-Cell / Q000276:immunology / Q000473:pathology; D051379:Mice; D008807:Mice, Inbred BALB C; D008957:Models, Genetic; D008969:Molecular Sequence Data; D019151:Peptide Library; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
D007106:Immune Sera; D007140:Immunoglobulin Fab Fragments; D007145:Immunoglobulin kappa-Chains; D019151:Peptide Library