ORIGINAL PAPER Anti-Digoxin Fab Variants Generated by Phage Display Viviane Midori Murata • Mariana Costa Braga Schmidt • Jorge Kalil • Lilian Rumi Tsuruta • Ana Maria Moro Published online: 29 January 2013 Ó Springer Science+Business Media, LLC 2012 Abstract 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 mono- clonal 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 pseu- dogene 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 Ò , which allowed ranking of the clones. The development of these clones allowed the selection of variants with higher affinity than the original version. Keywords Digoxin  Phage display  CDR  Framework  Biacore  Affinity  Cardiac dysfunction Introduction Digoxin is the most widely used glycoside in the control of congestive heart failure and atrial fibrillation, the latter being present in approximately 30 % of heart failure patients in the United States [1]. Digoxin remains the oldest drug still in clinical practice in cardiovascular medicine, with more than 200 years of clinical experience and research. Digoxin is a very low-cost drug, an important aspect in developing countries where patients generally do not have access to more expensive and sophisticated medicines and device therapies. Newer therapies emerged in the 1980s, such as vasodilators, angiotensin-converting (ACE) inhibitors, and newer inotropic agents, and as a consequence of this interest in digoxin diminished. How- ever, as most of these therapies appear to reduce survival, the use of digoxin for treatment has again been considered [2]. Following the results of several randomized placebo- controlled trials demonstrating the utility of digoxin and no effect of digoxin on all-cause mortality, as well as the decrease in hospitalization related to heart failure, digoxin was approved by the FDA for the treatment of heart failure. Digoxin acts by inhibiting membrane-bound alpha subunits of sodium–potassium ATPase and causing an increase in intracellular calcium ion concentrations that is required by V. M. Murata  M. C. B. Schmidt  L. R. Tsuruta  A. M. Moro (&) Laboratorio de Biofarmacos em Celulas Animais, Instituto Butantan, Sa ˜o Paulo, SP 05503-900, Brazil e-mail: anamoro@butantan.gov.br J. Kalil Laboratorio de Imunologia, InCor, Universidade de Sao Paulo, Sa ˜o Paulo, SP 05403-000, Brazil J. Kalil  A. M. Moro Institute for Immunology Investigation–Science and Technology National Institutes, INCT, Sa ˜o Paulo, Brazil 123 Mol Biotechnol (2013) 54:269–277 DOI 10.1007/s12033-012-9564-1