Development of a Recombinant Antibody with Specificity for Chelated Uranyl Ions

Conference · Mon Apr 18 00:00:00 EDT 2005

OSTI ID:895003

Li, X; Kriegel, A M; Bishop, T C; Blake, R C; Figueiredo, E; Yu, H; Blake, D A

The goal of our project is to continue the development of new techniques for rapid, automated identification of radionuclides, metals, and chelators that may contaminant sur face and groundwater at DOE sites. One of the four specific aims of the present project is to develop new technologies in antibody engineering that will enhance our immunosensor program. Recombinant antibodies have potential advantages over monoclonal antibodies produced by standard hybridoma technology. The cloned genes represent a stable, recoverable source for antibody production. In addition, the recombinant format offers opportunities for protein engineering that enhances antibody performance and for studies that relate antibody sequence to binding activity. In this study, a hybridoma that synthesized an antibody (12F6) that recognized a 1:1 complex between 2,9-dicarboxyl-1,10- phenanthroline (DCP) and UO{sub 2}{sup 2+} was used as a source of RNA for the development of a recombinant (Fab){sub 2} fragment. RNA was isolated from the 12F6 hybridoma and the cDNA encoding the entire {kappa} light chain and the linked VH and C1 portions of the heavy chain were amplified from total RNA. cDNA sequences were verified by comparison with the N-terminal amino acid sequences of the light and heavy chains of the native 12F6 monoclonal antibody. A leader sequence and appropriate restriction sites were added to each chain, and the fragments were ligated into a commercial dicistronic vector (pBudCE4.1, Invitrogen, Inc.). COS-1 cells were transfected with this vector and the culture supernatant was assayed for activity and the (Fab){sub 2} protein. Cells transfected with vector containing 12F6 cDNA synthesized and secreted recombinant (Fab){sub 2} fragments that bound to the UO{sub 2}{sup 2+}-DCP complex with an affinity indistinguishable from that of a (Fab){sub 2} fragment prepared from the native antibody. Molecular models of the heavy and light chain variable domains were constructed according to the canonical structures method detailed by Morea et al. (J. Mol. Biol. 275:269), and the participation of specific residues in antigen recognition was assessed using site-directed mutagenesis. Three amino acids in the light chain variable region, H39, Y54 and F103, were particularly important in antigen recognition. In a separate series of experiments, a recombinant phage-displayed antibody library has been prepared using RNA isolated from the spleens of sheep and rabbits immunized with specific metal-chelate complexes. Phage-display libraries produced from an immunized source are inclined to include variable genes specific for the immunized antigen(s), many of which are already affinity matured. An antibody fragment specific for the UO{sub 2}{sup 2+}-DCP complex was isolated from this combined phage display library. While the binding affinity of this antibody fragment for UO{sub 2}{sup 2+}-DCP was not as high as that of the 12F6 monoclonal antibody, the beauty of antibody phage display technology is that it allows for the potential manipulation and saturation of the antibody's binding affinity, which may drastically improve and ultimately surpass that of monoclonal antibodies.

• Research Organization: Tulane University, New Orleans, LA; Xavier University of Louisiana, New Orleans, LA
• Sponsoring Organization: USDOE Office of Science (SC)
• OSTI ID: 895003
• Report Number(s): CONF-NABIR2005-6; TRN: US0700366
• Resource Relation: Conference: Annual NABIR PI Meeting, April 18, 2005, Warrenton, VA
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
60 APPLIED LIFE SCIENCES
38 RADIATION CHEMISTRY, RADIOCHEMISTRY, AND NUCLEAR CHEMISTRY
54 ENVIRONMENTAL SCIENCES
AFFINITY
AMINO ACID SEQUENCE
AMINO ACIDS
ANTIBODIES
ANTIGENS
GENES
HYBRIDOMAS
MOLECULAR MODELS
MONOCLONAL ANTIBODIES
MUTAGENESIS
PHENANTHROLINES
PROTEIN ENGINEERING
RABBITS
RADIOISOTOPES
RESIDUES
RNA
SATURATION
SHEEP
SPECIFICITY
VECTORS