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A digoxina é um medicamento usado para tratar distúrbios cardíacos, com janela terapêutica muito estreita. Para combater seu efeito tóxico, fragmentos Fab do anticorpo policlonal anti-digoxina estão disponíveis comercialmente. Nosso objetivo foi a obtenção de variantes de fragmentos Fab do anticorpo monoclonal anti-digoxina usando a tecnologia phage display, que permite gerar fragmentos de anticorpos de alta afinidade e especificidade. Uma biblioteca combinatória de fragmentos Fab anti-digoxina foi construída no vetor pComb3X a partir do RNA total do hibridoma anti-digoxina. Seis clones foram isolados, todos com sequência idêntica na cadeia pesada. A cadeia leve apresentou 2 clones idênticos, um pseudogene e um clone com um aminoácido distinto no CDR2. Quatro clones apresentando variações na sequência do framework1 da cadeia leve foram expressos como fragmentos Fab solúveis. Todos apresentaram ligação à digoxina-BSA por ELISA e Western blotting. A ligação específica do anticorpo também foi confirmada pelo BIAcore, que permitiu ranqueamento entre os clones. / Digoxin is a pharmaceutical used in the control of cardiac dysfunction. Its therapeutic window is very narrow. To counteract the toxic effect, polyclonal anti-digoxin Fab fragments are commercially available. Our goal was to obtain variants of monoclonal anti-digoxin Fab fragments by phage display technology, which allows the generation of high affinity and specificity antibody fragments. Anti-digoxin Fab fragments combinatorial library was constructed into pComb3X vector from total RNA of anti-digoxin hybridoma. Six clones were isolated and the heavy chain presented the same sequence. For the light chain, 2 clones were identical, one was a pseudogene and other one presented a distinct amino acid in the CDR2. Four clones presenting variations in the framework 1 were induced to express soluble Fab fragments, all positive for anti-digoxin binding in ELISA assays and Western blotting. The specific binding of the antibody was further confirmed by BIAcore, which allowed ranking of the clones.

by Xiao Gang. / Thesis (M.Sc.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 91-99). / Abstract also in Chinese. / ACKNOWLEDGEMENTS --- p.i / SUMMARY --- p.1 / Chapter 1 --- LITERATURE REVIEW / Chapter 1.1 --- MAGNESIUM --- p.3 / Chapter 1.1.1 --- GENERAL FUNCTION OF MAGNESIUM --- p.3 / Chapter 1.1.2 --- ABSORPTION AND METABOLISM OF MAGNESIUM --- p.6 / Chapter 1.1.3 --- CLINICAL ASPECTS --- p.8 / Chapter 1.1.4 --- METHODS OF DETERMINATION OF MAGNESIUM --- p.11 / Chapter 1.2 --- ZINC --- p.13 / Chapter 1.2.1 --- GENERAL FUNCTION OF ZINC --- p.13 / Chapter 1.2.2 --- ABSORPTION AND METABOLISM OF ZINC --- p.14 / Chapter 1.2.3 --- CLINICAL ASPECTS --- p.16 / Chapter 1.2.4 --- ASSESSMENT OF THE BODY ZINC STATUS --- p.19 / Chapter 1.2.5 --- METHODS OF DETERMINATION OF ZINC --- p.20 / Chapter 1.3 --- COPPER --- p.21 / Chapter 1.3.1 --- GENERAL FUNCTION OF COPPER --- p.21 / Chapter 1.3.2 --- ABSORPTION AND METABOLISM OF COPPER --- p.21 / Chapter 1.3.3 --- CLINICAL ASPECT --- p.25 / Chapter 1.3.4 --- LABORATORY ASSESSMENT OF COPPER STATUS --- p.28 / Chapter 1.3.5 --- METHODS FOR THE DETERMINATION OF COPPER --- p.29 / Chapter 1.4 --- SELENIUM --- p.31 / Chapter 1.4.1 --- GENERAL FUNCTION OF SELENIUM --- p.31 / Chapter 1.4.2 --- ABSORPTION AND METABOLISM OF SELENIUM --- p.31 / Chapter 1.4.3 --- CLINICAL ASPECTS --- p.34 / Chapter 1.4.4 --- ASSESSMENT --- p.36 / Chapter 1.4.5 --- METHODS OF DETERMINATION OF SELENIUM --- p.37 / Chapter 1.5 --- INTRODUCTION TO ATOMIC ABSORPTION SPECTROPHOTOMETRY --- p.38 / Chapter 1.5.1 --- PRINCIPLE OF AAS --- p.38 / Chapter 1.5.2 --- INSTRUMENTATION --- p.39 / Chapter 2 --- INTRODUCTION --- p.43 / Chapter 2.1 --- TRACE ELEMENT DEFICIENCY IN HOSPITAL PATIENT --- p.43 / Chapter 2.2 --- MICRONUTRIENT DEFICIENCY AMONG PATIENTS WITH ACUTE MYOCARDIAL INFARCTION --- p.44 / Chapter 2.2.1 --- MAGNESIUM DEFICIENCY --- p.44 / Chapter 2.2.2 --- CHANGE OF ZINC AND COPPER IN AMI --- p.47 / Chapter 2.2.3 --- SELENIUM DEFICIENCY --- p.50 / Chapter 2.3 --- OBJECTIVES OF THIS PROJECT --- p.52 / Chapter 3 --- MATERIALS AND METHODS / Chapter 3.1 --- PATIENTS SELECTION --- p.53 / Chapter 3.1.1 --- CONTROL GROUP --- p.53 / Chapter 3.1.2 --- AMI GROUP --- p.53 / Chapter 3.1.3 --- DIGOXIN TREATMENT GROUP --- p.54 / Chapter 3.2 --- ANALYTIC METHODS --- p.55 / Chapter 3.2.1 --- DETERMINATION OF SERUM Magnesium --- p.55 / Chapter 3.2.2 --- DETERMINATION OF SERUM ZINC --- p.57 / Chapter 3.2.3 --- DETERMINATION OF SERUM COPPER --- p.60 / Chapter 3.2.4 --- DETERMINATION OF SERUM SELENIUM --- p.63 / Chapter 4 --- RESULTS --- p.68 / Chapter 4.1 --- RESULTS OF EVALUATION OF ANALYTICAL --- p.68 / Chapter 4.1.1 --- RESULTS OF METHODS EVALUATION OF SERUM ZINC ASSAY --- p.68 / Chapter 4.1.2 --- RESULTS OF METHODS EVALUATION OF SERUM COPPER ASSAY --- p.71 / Chapter 4.1.3 --- RESULTS OF METHODS EVALUATION OF SERUM SELENIUM ASSAY --- p.73 / Chapter 4.2 --- RESULTS OF PATIENTS STUDY --- p.76 / Chapter 4.2.1 --- CONTROL GROUP --- p.76 / Chapter 4.2.2 --- PATIENTS WITH ACUTE MYOCARDIAL INFARCTION I DEMOGRAPHIC DATA --- p.77 / Chapter 4.2.3 --- DIGOXIN GROUP --- p.83 / Chapter 5 --- DISCUSSION --- p.85 / Chapter 5.1 --- AMI PATIENTS AND TRACE ELEMENT STATUS --- p.85 / Chapter 5.2 --- MAGNESIUM AND ZINC STATUS IN PATIENTS RECEIVING DIGOXIN TREATMENT --- p.88 / REFERENCE --- p.91 / LIST OF TABLES --- p.99 / LIST OF TABLES --- p.102 / TABLES & FIGURES

Doctor of Philosophy (Medicine) / Glutamate transporters play a role in removing extracellular excitatory neurotransmitter, L-glutamate into the cells. The rate of the uptake depends on the density of the transporters at the membrane. Some studies claimed that glutamate transporters could transit between the cytoplasm and the membrane on a time-scale of minutes. The present study examined the distribution of glutamate transporter GLAST predominantly expressed in rat cortical cultured astrocytes between the membrane and the cytoplasm by using deconvolution microscopy and then analyzing the images. The regulation of the distribution of GLAST was studied in the presence of glutamate transporter substrate (D-aspartate), purinergic receptor activators (α,β-methylene ATP, adenosine), neuroleptic drugs (clozapine, haloperidol), ammonia (hyperammonia) and Na+/K+-ATPase inhibitors (ouabain, digoxin and FCCP). It was demonstrated that the translocation of GLAST towards the plasma membrane was induced by D-aspartate, α,β-methylene ATP, adenosine, clozapine and ammonia (at 100 μM and very high concentrations of 10 mM). However, the inhibition of Na+/K+-ATPase activity had an opposite effect, resulting in redistribution of GLAST away from the membrane. It has previously been claimed that the membrane-cytoplasm trafficking of GLAST was regulated by phosphorylation catalysed by protein kinase C delta (PKC-delta). Involvement of this mechanism has, however, been put to doubt when rottlerin, a PKC-delta inhibitor, used to test the hypothesis showed to inhibit Na+/K+-ATPase-mediated uptake of Rb+, suggesting that rottlerin influenced the activity of Na+/K+-ATPase. As Na+/K+-ATPase converts ATP to energy and pumps Na+, K+ ions, thus helping to maintain normal electrochemical and ionic gradients across the cell membrane. Its inhibition also reduced D-aspartate transport and could impact on the cytoplasm-to-membrane traffic of GLAST molecules. Furthermore, rottlerin decreased the activity of Na+/K+-ATPase by acting as a mitochondrial inhibitor. The present study has focused on the inhibition of Na+/K+-ATPase activity by rottlerin, ouabain and digoxin in homogenates prepared from rat kidney and cultured astrocytes. The activity of Na+/K+-ATPase was measured by the absorption of inorganic phosphate product generated from the hydrolysis of ATP and the fluorescent transition of the dye RH421 induced by the movement of Na+/K+-ATPase. This approach has a potential to test whether the rottlerin effect on Na+/K+-ATPase is a direct inhibition of the enzyme activity. Rottlerin has been found to block the activity of Na+/K+-ATPase in a dose-dependent manner in both rat kidney and astrocyte homogenates. Therefore, rottlerin inhibited the activity of Na+/K+-ATPase directly in a cell-free preparation, thus strongly indicating that the effect was direct on the enzyme. In parallel experiments, ouabain and digoxin produced similar inhibitions of Na+/K+-ATPase activity in rat kidney while digoxin blocked the activity of Na+/K+-ATPase to a greater extent than ouabain in rat cortical cultured astrocytes. In a separate set of experiments, Na+/K+-ATPase in the astrocytic membrane was found to be unsaturated in E1(Na+)3 conformation in the presence of Na+ ions and this could explain the differences between the effects of digoxin and ouabain on the activity of Na+/K+-ATPase in rat astrocytes. In addition, it was found that at low concentrations of rottlerin, the activity of Na+/K+-ATPase was increased rather than inhibited. This effect was further investigated by studying rottlerin interactions with membrane lipids. The activity of Na+/K+-ATPase has been reported to be regulated by membrane lipids. The enzyme activity can be enhanced by increasing fluidity of the lipid membrane. I have, therefore, proposed that rottlerin binds to the membrane lipids and the effects of rottlerin on Na+/K+-ATPase are mediated by changes in the properties (fluidity) of the membrane. The hypothesis was tested by comparing rottlerin and a detergent, DOC (sodium deoxycholate), for their binding to the lipids by using a DMPC (1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine) monolayer technique. DOC has been shown to both increase and inhibit activity of Na+/K+-ATPase in a manner similar to that displayed by rottlerin. The effects of rottlerin and DOC on the DMPC monolayers were studied by measuring the surface pressure of DMPC monolayers and surface area per DMPC molecule. I established that both rottlerin and DOC decreased the surface pressure of DMPC monolayers and increased the surface area per DMPC molecule. This indicates that both rottlerin and DOC penetrated into the DMPC monolayers. If rottlerin can interact with the lipids, changes in fluidity of the lipid membrane cannot be ruled out and should be considered as a possible factor contributing to the effects of rottlerin on the activity of Na+/K+-ATPase. Overall, the study demonstrates that rottlerin is not only a PKC-delta inhibitor but can have additional effects, both on the enzyme activities (Na+/K+-ATPase) and/or on lipid-containing biological structures such as membranes. The findings have implication not only for studies where rottlerin was used as a supposedly specific PKC-delta inhibitor but also for mechanisms of its toxicity.

<p>Neuroblastoma is one of the most common solid cancers of early childhood. In Sweden, approximately 10-15 cases occur annually. The overall five-year neuroblastoma survival in Europe is approximately 45%. Since cancer treatment involves drugs with risks of side effects in the growing child, there is a need for more effective and less toxic drugs. One new approach in cancer treatment is inhibition of tumor angiogenesis, i.e., of new blood vessel growth into the tumor. An angiogenesis inhibitor may be combined with cytostatic drugs to enhance the efficacy. The aim of this study was to investigate how drugs could be used to inhibit angiogenesis and tumor growth in a xenograft model of human neuroblastoma in nude mice. </p><p>The tumors express the angiogenesis stimulator vascular endothelial growth factor (VEGF) on both protein and mRNA levels. The angiogenesis inhibitors SU5416 (an inhibitor of VEGF signalling) and TNP-470 (an inhibitor of endothelial cell proliferation) inhibited angiogenesis in our model. TNP-470, however, inhibited angiogenesis without significant reduction of the tumor growth, in contrast to SU5416. </p><p>We also discovered that the cytostatic drug CHS 828 could cause regression of neuroblastoma tumors in the model when given orally at a low daily dose, alone or in combination with the angiogenesis inhibitor SU5416 or TNP-470. </p><p>Furthermore, a new use of the cardiac glycoside digoxin was found. Digoxin inhibited FGF-2 -stimulated bovine capillary endothelial cell growth in vitro, and inhibited angiogenesis in vivo in the chick chorioallantoic membrane assay (CAM). It also inhibited neuroblastoma growth by approximately 50% in our neuroblastoma model. </p><p>In conclusion, CHS 828 and digoxin represent two classes of drugs with potent antitumor effects that may be valuable in treatment of neuroblastoma, either alone or in combination with angiogenesis inhibitors.</p>

Neuroblastoma is one of the most common solid cancers of early childhood. In Sweden, approximately 10-15 cases occur annually. The overall five-year neuroblastoma survival in Europe is approximately 45%. Since cancer treatment involves drugs with risks of side effects in the growing child, there is a need for more effective and less toxic drugs. One new approach in cancer treatment is inhibition of tumor angiogenesis, i.e., of new blood vessel growth into the tumor. An angiogenesis inhibitor may be combined with cytostatic drugs to enhance the efficacy. The aim of this study was to investigate how drugs could be used to inhibit angiogenesis and tumor growth in a xenograft model of human neuroblastoma in nude mice. The tumors express the angiogenesis stimulator vascular endothelial growth factor (VEGF) on both protein and mRNA levels. The angiogenesis inhibitors SU5416 (an inhibitor of VEGF signalling) and TNP-470 (an inhibitor of endothelial cell proliferation) inhibited angiogenesis in our model. TNP-470, however, inhibited angiogenesis without significant reduction of the tumor growth, in contrast to SU5416. We also discovered that the cytostatic drug CHS 828 could cause regression of neuroblastoma tumors in the model when given orally at a low daily dose, alone or in combination with the angiogenesis inhibitor SU5416 or TNP-470. Furthermore, a new use of the cardiac glycoside digoxin was found. Digoxin inhibited FGF-2 -stimulated bovine capillary endothelial cell growth in vitro, and inhibited angiogenesis in vivo in the chick chorioallantoic membrane assay (CAM). It also inhibited neuroblastoma growth by approximately 50% in our neuroblastoma model. In conclusion, CHS 828 and digoxin represent two classes of drugs with potent antitumor effects that may be valuable in treatment of neuroblastoma, either alone or in combination with angiogenesis inhibitors.

Doctor of Philosophy (Medicine) / Glutamate transporters play a role in removing extracellular excitatory neurotransmitter, L-glutamate into the cells. The rate of the uptake depends on the density of the transporters at the membrane. Some studies claimed that glutamate transporters could transit between the cytoplasm and the membrane on a time-scale of minutes. The present study examined the distribution of glutamate transporter GLAST predominantly expressed in rat cortical cultured astrocytes between the membrane and the cytoplasm by using deconvolution microscopy and then analyzing the images. The regulation of the distribution of GLAST was studied in the presence of glutamate transporter substrate (D-aspartate), purinergic receptor activators (α,β-methylene ATP, adenosine), neuroleptic drugs (clozapine, haloperidol), ammonia (hyperammonia) and Na+/K+-ATPase inhibitors (ouabain, digoxin and FCCP). It was demonstrated that the translocation of GLAST towards the plasma membrane was induced by D-aspartate, α,β-methylene ATP, adenosine, clozapine and ammonia (at 100 μM and very high concentrations of 10 mM). However, the inhibition of Na+/K+-ATPase activity had an opposite effect, resulting in redistribution of GLAST away from the membrane. It has previously been claimed that the membrane-cytoplasm trafficking of GLAST was regulated by phosphorylation catalysed by protein kinase C delta (PKC-delta). Involvement of this mechanism has, however, been put to doubt when rottlerin, a PKC-delta inhibitor, used to test the hypothesis showed to inhibit Na+/K+-ATPase-mediated uptake of Rb+, suggesting that rottlerin influenced the activity of Na+/K+-ATPase. As Na+/K+-ATPase converts ATP to energy and pumps Na+, K+ ions, thus helping to maintain normal electrochemical and ionic gradients across the cell membrane. Its inhibition also reduced D-aspartate transport and could impact on the cytoplasm-to-membrane traffic of GLAST molecules. Furthermore, rottlerin decreased the activity of Na+/K+-ATPase by acting as a mitochondrial inhibitor. The present study has focused on the inhibition of Na+/K+-ATPase activity by rottlerin, ouabain and digoxin in homogenates prepared from rat kidney and cultured astrocytes. The activity of Na+/K+-ATPase was measured by the absorption of inorganic phosphate product generated from the hydrolysis of ATP and the fluorescent transition of the dye RH421 induced by the movement of Na+/K+-ATPase. This approach has a potential to test whether the rottlerin effect on Na+/K+-ATPase is a direct inhibition of the enzyme activity. Rottlerin has been found to block the activity of Na+/K+-ATPase in a dose-dependent manner in both rat kidney and astrocyte homogenates. Therefore, rottlerin inhibited the activity of Na+/K+-ATPase directly in a cell-free preparation, thus strongly indicating that the effect was direct on the enzyme. In parallel experiments, ouabain and digoxin produced similar inhibitions of Na+/K+-ATPase activity in rat kidney while digoxin blocked the activity of Na+/K+-ATPase to a greater extent than ouabain in rat cortical cultured astrocytes. In a separate set of experiments, Na+/K+-ATPase in the astrocytic membrane was found to be unsaturated in E1(Na+)3 conformation in the presence of Na+ ions and this could explain the differences between the effects of digoxin and ouabain on the activity of Na+/K+-ATPase in rat astrocytes. In addition, it was found that at low concentrations of rottlerin, the activity of Na+/K+-ATPase was increased rather than inhibited. This effect was further investigated by studying rottlerin interactions with membrane lipids. The activity of Na+/K+-ATPase has been reported to be regulated by membrane lipids. The enzyme activity can be enhanced by increasing fluidity of the lipid membrane. I have, therefore, proposed that rottlerin binds to the membrane lipids and the effects of rottlerin on Na+/K+-ATPase are mediated by changes in the properties (fluidity) of the membrane. The hypothesis was tested by comparing rottlerin and a detergent, DOC (sodium deoxycholate), for their binding to the lipids by using a DMPC (1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine) monolayer technique. DOC has been shown to both increase and inhibit activity of Na+/K+-ATPase in a manner similar to that displayed by rottlerin. The effects of rottlerin and DOC on the DMPC monolayers were studied by measuring the surface pressure of DMPC monolayers and surface area per DMPC molecule. I established that both rottlerin and DOC decreased the surface pressure of DMPC monolayers and increased the surface area per DMPC molecule. This indicates that both rottlerin and DOC penetrated into the DMPC monolayers. If rottlerin can interact with the lipids, changes in fluidity of the lipid membrane cannot be ruled out and should be considered as a possible factor contributing to the effects of rottlerin on the activity of Na+/K+-ATPase. Overall, the study demonstrates that rottlerin is not only a PKC-delta inhibitor but can have additional effects, both on the enzyme activities (Na+/K+-ATPase) and/or on lipid-containing biological structures such as membranes. The findings have implication not only for studies where rottlerin was used as a supposedly specific PKC-delta inhibitor but also for mechanisms of its toxicity.

Thesis (MSc.(Anatomy)--Faculty of Health Sciences)-University of Pretoria, 2007. / Includes bibliographical references.

Made available in DSpace on 2014-07-29T15:29:05Z (GMT). No. of bitstreams: 1 dissertacao isabel cristina.pdf: 549593 bytes, checksum: ff591d0e143ca66263716f60188e9801 (MD5) Previous issue date: 2008-10-27 / It was investigated the clinical, lab and plasma digoxin concentration profiles in 15 cardiac heart failure (CHF) outpatients of the Cardiac Heart Service of the Goias Federal University Clinical Hospital. It was aimed to know plasma digoxin concentration profile in 15 cardiac heart failure (CHF) outpatients by two analytical methods, taking account clinical, laboratorial, habits, anthropometric data and drug usage. Digoxin dosage was developed by LC/MS/MS and immunoassay methods; questionnaire and consults handbooks were performed. Results and Conclusions: 87% of the patients who over 46 years of age (33% above 61), the masculine majority; the IC of Chagas disease origin presented greater occurrence, followed of the hipertensive and idiopatic (59%); IC functional classroom II (53.33%); hypertension and diabetes had been distinguished as co-morbidities (26.67% and 20%); tobaccoism, overweight and obesity degree I had presented low occurrence. No patients presented relevant clinical data suggestive of digitalis intoxication. No observed changes in biochemical and hematological exams. The ejection fractions were good by means 41.7 ± 9%. Some drugs with interaction potential had been associated to the treatment, apparently without alterations. Statistical significative difference between both methods was observed (P < 0.05, ANOVA, Tukey Test). In the immunoassay method, all the patients were inside of the therapeutical range (0, 5-2, 0 ng/mL), whereas for the LC-MS/MS method, 8 patients they would be in subtherapeutical concentrations. However, no patient presented signals or symptoms of poisoning or inefficacy of the digoxin, demonstrating biological variability. The two methods are useful, since that it has a correlation with the clinical and laboratorial state of the patients. / Investigou-se o perfil clínico, laboratorial e as concentrações de digoxina plasmática em 15 pacientes com insuficiência cardíaca (IC) atendidos no Ambulatório de Insuficiência Cardíaca do HC-UFG. Objetivou-se estudar o perfil de concentrações plasmáticas de digoxina em pacientes com IC, utilizando dois métodos analíticos, e descrever a digoxinemia considerando os dados clínicos, laboratoriais, hábitos, IMC e consumo de medicamentos. A metodologia utilizada consistiu de cromatografia líquida de alta eficiência acoplada a espectrometria de massas (LC/MS/MS) e Imunoensaio, aplicação de questionário e consulta a prontuários. Como resultados, observou-se: 87% dos pacientes maiores que 46 anos de idade (33% acima de 61), a maioria masculina, a IC de origem chagásica apresentou maior ocorrência, seguida da hipertensiva e idiopática (59%); IC classe funcional II (53,33%); hipertensão e diabetes destacaram-se como co-morbidades (26,67% e 20%); tabagismo, sobrepeso e obesidade grau I apresentaram baixa ocorrência. Nenhum dos pacientes apresentou dados clínicos relevantes sugestivos de intoxicação digitálica. Não houve alterações em exames bioquímicos e hematológicos. A fração de ejeção média foi 41,7 ± 9%, portanto nenhum paciente apresentou FE como preditor de mau prognóstico. Vários fármacos com potencial de interação estiveram associados ao tratamento, aparentemente sem alterações. Houve diferença significativa (P < 0,05, ANOVA, Teste de Tukey) entre os métodos analíticos. No método de imunoensaio, todos os pacientes estavam dentro da faixa terapêutica (0,5-2,0 ng/mL), enquanto que pelo método LC-MS/MS, 8 pacientes estariam em sub-dosagens. Entretanto, nenhum paciente apresentou sinais ou sintomas de intoxicação ou de ineficácia da digoxina, demonstrando variabilidade biológica. Os dois métodos são úteis, desde que haja uma correlação com o estado clínico e laboratorial dos pacientes.

A digoxina é um medicamento usado para tratar distúrbios cardíacos, com janela terapêutica muito estreita. Para combater seu efeito tóxico, fragmentos Fab do anticorpo policlonal anti-digoxina estão disponíveis comercialmente. Nosso objetivo foi a obtenção de variantes de fragmentos Fab do anticorpo monoclonal anti-digoxina usando a tecnologia phage display, que permite gerar fragmentos de anticorpos de alta afinidade e especificidade. Uma biblioteca combinatória de fragmentos Fab anti-digoxina foi construída no vetor pComb3X a partir do RNA total do hibridoma anti-digoxina. Seis clones foram isolados, todos com sequência idêntica na cadeia pesada. A cadeia leve apresentou 2 clones idênticos, um pseudogene e um clone com um aminoácido distinto no CDR2. Quatro clones apresentando variações na sequência do framework1 da cadeia leve foram expressos como fragmentos Fab solúveis. Todos apresentaram ligação à digoxina-BSA por ELISA e Western blotting. A ligação específica do anticorpo também foi confirmada pelo BIAcore, que permitiu ranqueamento entre os clones. / Digoxin is a pharmaceutical used in the control of cardiac dysfunction. Its therapeutic window is very narrow. To counteract the toxic effect, polyclonal anti-digoxin Fab fragments are commercially available. Our goal was to obtain variants of monoclonal anti-digoxin Fab fragments by phage display technology, which allows the generation of high affinity and specificity antibody fragments. Anti-digoxin Fab fragments combinatorial library was constructed into pComb3X vector from total RNA of anti-digoxin hybridoma. Six clones were isolated and the heavy chain presented the same sequence. For the light chain, 2 clones were identical, one was a pseudogene and other one presented a distinct amino acid in the CDR2. Four clones presenting variations in the framework 1 were induced to express soluble Fab fragments, all positive for anti-digoxin binding in ELISA assays and Western blotting. The specific binding of the antibody was further confirmed by BIAcore, which allowed ranking of the clones.