Anti-digoxin Fab variants generated by phage display.
Digoxin is a pharmaceutical used in the control of cardiac dysfunction. Its therapeutic window is narrow, with effect dosage very close to the toxic dosage. To counteract the toxic effect, polyclonal Fab fragments are commercially available. Our study is based on a monoclonal anti-digoxin antibody, which would provide a product with a specific potency and more precise dosage for the detoxification of patients under digoxin treatment. Phage display technology was used to select variants with high affinity. From an anti-digoxin hybridoma, RNA was extracted for subsequent cDNA synthesis. Specific primers were used for the LC and Fd amplifications, then cloned sequentially in a phagemid vector (pComb3X) for the combinatorial Fab library construction. Clones were selected for their ability to bind to digoxin-BSA. The presence of light and heavy chains was checked, randomly selected clones then sequenced and induced to produce soluble Fabs, and subsequently analyzed for anti-digoxin expression. Out of ten clones randomly chosen, six resulted positive expression of the product. The sequencing of these revealed two identical clones and one presenting a pseudogene in the LC. Four clones presenting variations in the framework1 showed binding to digoxin-BSA by ELISA and western blotting. The specific binding was further confirmed by Biacore(®), which allowed ranking of the clones. The development of these clones allowed the selection of variants with higher affinity than the original version.
Laboratorio de Biofarmacos em Celulas Animais, Instituto Butantan, São Paulo, Brazil.
Viviane Midori
VM
Murata
Mariana Costa Braga
MC
Schmidt
Jorge
J
Kalil
Lilian Rumi
LR
Tsuruta
Ana Maria
AM
Moro
D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology; D061505:Cell Surface Display Techniques / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics / Q000276:immunology; D004077:Digoxin / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology; D014644:Genetic Variation; D005822:Genetic Vectors / Q000235:genetics / Q000276:immunology; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology; D019151:Peptide Library
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D019151:Peptide Library; D004077:Digoxin