title: Production and characterization of recombinant scFv against digoxin by phage display technology.
abstract: The cardiac glycoside digoxin is widely used for the treatment of congestive heart failure and cardiac arrhythmias. Digoxin is a highly toxic drug and consequently is routinely measured in sera of treated patients. In such cases, antibodies are required against digoxin for detection as well as detoxification purposes. To obtain recombinant single chain antibody against digoxin, RNA was extracted from spleen of BALB/c mice immunized with digoxin-BSA and converted to cDNA. The gene fragment corresponding to the variable regions of the repertoire of antibody genes were amplified by PCR. ScFv construct was generated by randomly joining individual heavy- and light-chain variable domains through gene splicing by overlapping extension PCR. Recombinant phage library expressing scFv polypeptides were produced. Phages with higher affinity toward digoxin were selected in the biopanning process. Sensitivity of produced recombinant MAb (AR85) was determined to be about 100 pg/well, while intact MAb (BBA) produced by hybridoma technology (data not shown) was reported to be around 100 pg/well too. The saturation value for recombinant scFv MAb was found to be 1000 ng/well while that for hybridoma MAb was reported to be 10 ng/well. The affinity constant of recombinant MAb (AR85) towards digoxin was also found to be around ka=3.8×10(7) M(-1) while that for hybridoma MAb (BBA) was reported to be ka=2.6×10(8) M(-1).
authors.affiliation: Department of Clinical Biochemistry, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Behruz
authors.initials: B
authors.lastname: Alirezapour
authors.forename: Masoumeh
authors.initials: M
authors.lastname: Rajabibazl
authors.forename: Mohhamad Javad
authors.initials: MJ
authors.lastname: Rasaee
authors.forename: Kobra
authors.initials: K
authors.lastname: Omidfar
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology*; D000915:Antibody Affinity / Q000235:genetics; D001435:Bacteriophages / Q000235:genetics; D061505:Cell Surface Display Techniques; D018076:DNA, Complementary / Q000235:genetics; D004077:Digoxin / Q000097:blood / Q000276:immunology*; D005784:Gene Amplification; D051379:Mice; D008807:Mice, Inbred BALB C; D019151:Peptide Library; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D019151:Peptide Library; D012333:RNA, Messenger; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D004077:Digoxin