Isolation and characterization of the human monoclonal antibodies C10 in single-chain fragment variable (scFv) format to glucose oxidase from Aspergillus niger.
Despite biotechnological and clinical applications very few monoclonal antibodies (MAbs) directed to the enzyme glucose oxidase, have been produced so far because of the heavy side effects of the immunization schedule for conventional MAb preparation. In contrast, the phage display method allows for the selection of monoclonal human antibody fragments against any antigens, including toxic proteins. Furthermore, cDNA sequences encoding selected antibody fragments are readily identified, facilitating various molecular targeting approaches. In order to obtain such human fragments recognizing glucose oxidase, we used the large synthetic ETH-2 library based on the principle of protein design. Phage displaying glucose oxidase reactive scFvs were obtained after three rounds of selection on glucose oxidase-coated immunotubes and subsequent amplification in TG1 E. coli cells. Eventually, one high reactive scFv clone was selected and further examined. The anti-glucose oxidase scFv C10 was found suitable for Western blot; Biacore analysis showed that the binding affinity of the glucose oxidase-reactive scFv is almost equal that of MAbs prepared with conventional hybridoma technology. Finally, the cDNA sequence of this human scFv may be exploited to generate bispecific antibodies to target in the tumor environment-specific toxic enzymatic reaction.
Ascione
Alessandro
A
Section of Pharmacogenetics, Drug Resistance and Experimental Therapeutics, Department of Drug Research and Evaluation, Istituto Superiore di Sanità, Rome, Italy.
Flego
Michela
M
Zamboni
Silvia
S
De Cinti
Emanuela
E
Dupuis
Maria Luisa
ML
Cianfriglia
Maurizio
M
D000911:Antibodies, Monoclonal / Q000276:immunology*; D001234:Aspergillus niger / Q000276:immunology*; D005949:Glucose Oxidase / Q000276:immunology*; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000276:immunology*; D017422:Sequence Analysis, DNA
D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D005949:Glucose Oxidase