O ligante de merozoítos de Plasmodium vivax em DARC nos eritrócitos é PvDBPII. Aqui, nós testamos se anticorpos naturalmente adquiridos contra PvDBPII conferem proteção para malária vivax clínica em indivíduos da Amazônia. Para tanto, foram realizados ensaios multiplex com 466 indivíduos revelando uma alta proporção de respondedores. Porém, nenhuma associação entre níveis de anticorpos anti-PvDBPII e atraso para o próximo episódio de malária vivax foi encontrad a. Dessas amostras, 27,8% mostraram alta atividade inibitória e a presença desses anticorpos foi associada com atraso no próximo episódio de malária vivax. Para produzir um painel de anticorpos monoclonais (mAbs) anti-PvDBPII de indivíduos do Amazonas foi realizado um sorting de células B especificas de indivíduos com respostas inibitórias. Um desses mAbs competiu pelo mesmo sitio de ligação ou similar com anticorpos inibitórios naturalmente adquiridos. Em culturas P. vivax esse mAb foi capaz de reduzir a invasão de merozoítos em eritrócitos. / Merozoites of Plasmodium vivax has a critical parasite ligand (PvDBPII) that binds to DARC on erythrocytes. Here, we tested whether naturally acquired antibodies to PvDBPII conferred protection against clinical vivax malaria among rural Amazonians. We measured IgG antibodies from 466 individuals using a multiplex assay. A high proportion of samples tested had IgG antibodies to PvDBPII. However, found no association between high-level antibody response to PvDBPII and time to the next malaria episode. Of these samples, 27.8% displayed high blocking activity against PvDBP and these subjects had a statistically significant longer time to the next clinical P. vivax episode. To produce a panel of monoclonal antibodies (MoAbs) against PvDBPII from Amazon individuals, we sorted single PvDBPII-specific B-cell from subjects with BIAb responses. One of these MoAb generated recognizes PvDBPII and compete with naturally acquired antibodies from plasma samples to the same epitope recognized by MoAb. In short-term culture of Plasmodium vivax this Moab can reduced invasion.

by Sze Shun Fai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 132-139). / Abstract --- p.1 / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- The pineal gland --- p.5 / Chapter 1.2 --- Discovery of melatonin --- p.5 / Chapter 1.3 --- Synthesis of melatonin --- p.5 / Chapter 1.4 --- Physiology of melatonin and its derivatives --- p.6 / Chapter 1.5 --- In vitro tumor biology of melatonin and its derivatives --- p.7 / Chapter 1.6 --- In vivo tumor biology of melatonin --- p.10 / Chapter 1.7 --- Macrophages --- p.11 / Chapter 1.8 --- Lymphocytes --- p.14 / Chapter Chapter 2 --- Toxicity of pineal indoles on tumor cell lines / Chapter 2.1 --- General introduction --- p.17 / Chapter 2.2 --- Material and methods --- p.18 / Chapter 2.3 --- Results --- p.22 / Chapter 2.4 --- Discussion --- p.23 / Chapter Chapter 3 --- Activation of murine peritoneal macrophages by melatonin and methoxytryptamine / Chapter 3.1 --- General introduction --- p.37 / Chapter 3.2 --- Material and methods --- p.38 / Chapter 3.3 --- Results --- p.55 / Chapter 3.4 --- Discussion --- p.61 / Chapter Chapter 4 --- Activation of murine splenocytes by melatonin and methoxytryptamine / Chapter 4.1 --- General introduction --- p.81 / Chapter 4.2 --- Material and methods --- p.82 / Chapter 4.3 --- Results --- p.91 / Chapter 4.4 --- Discussion --- p.128 / Chapter Chapter 5 --- General discussion --- p.132 / References

Tsang Kam Sze, Kent. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 155-174). / Abstract --- p.i / Acknowledgements --- p.v / List of Abbreviations --- p.viii / Table of Contents --- p.x / List of Figures --- p.xvi / List of Tables --- p.ixx / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1. --- Idiotype Network --- p.2 / Chapter 1.2. --- Anti-idiotype Classification --- p.8 / Chapter 1.3. --- Blood Transfusion Effect --- p.11 / Chapter 1.4. --- Transfusion Protocol --- p.12 / Chapter 1.5. --- Mechanism of Beneficial Transfusion Effect --- p.15 / Chapter 1.5.1. --- Donor Selection --- p.15 / Chapter 1.5.2. --- Clonal Deletion --- p.16 / Chapter 1.5.3. --- Suppressor Cells Induction --- p.18 / Chapter 1.5.4. --- Prostaglandins Mediation --- p.19 / Chapter 1.5.5. --- Mixed Chimerism Motivation --- p.20 / Chapter 1.5.6. --- Fc-receptor Blocking Antibodies Stimulation --- p.22 / Chapter 1.5.7. --- Anti-idiotypic Antibodies Instigation --- p.23 / Chapter 1.6. --- Study Aims --- p.25 / Chapter 1.7. --- Technical Strategy --- p.26 / Chapter Chapter 2. --- Materials and Methods --- p.30 / Chapter 2.1. --- Materials --- p.31 / Chapter 2.1.1. --- Patient Population --- p.31 / Chapter 2.1.2. --- Normal Control Group --- p.31 / Chapter 2.1.3. --- Serum Samples --- p.32 / Chapter 2.1.4. --- Additional Specimens --- p.32 / Chapter 2.1.5. --- Chemicals --- p.32 / Chapter 2.1.6. --- Antisera --- p.34 / Chapter 2.1.7. --- Buffers --- p.35 / Chapter 2.1.8. --- Consumables --- p.38 / Chapter 2.1.9. --- Apparatus and Equipment --- p.39 / Chapter 2.2. --- Methods --- p.40 / Chapter 2.2.1. --- Purification of Human Polyclonal Anti-HLA Antisera --- p.40 / Chapter 2.2.1.1. --- Affinity Chromatography --- p.41 / Chapter 2.2.1.2. --- Dialysis --- p.41 / Chapter 2.2.1.3. --- Concentration --- p.42 / Chapter 2.2.1.4. --- Quantitation --- p.42 / Chapter 2.2.2. --- Generation of F(ab')2 fragments from the Purified Human Anti-HLA Antibodies --- p.42 / Chapter 2.2.2.1. --- Buffer Exchange --- p.43 / Chapter 2.2.2.2. --- Pepsin Digestion --- p.43 / Chapter 2.2.2.3. --- Purification of (ab')2、 --- p.43 / Chapter 2.2.3. --- Enzyme-Linked Immunosorbent Assay for anti-Idiotypes against anti-HLA antibodies --- p.44 / Chapter 2.2.3.1. --- Optimization --- p.44 / Chapter 2.2.3.2. --- Quality Control --- p.45 / Chapter 2.2.3.2.1. --- F(ab')2 Specificity --- p.45 / Chapter 2.2.3.2.2. --- Fc Contamination --- p.46 / Chapter 2.2.3.2.3. --- Precision Test --- p.47 / Chapter 2.2.4. --- Anti-Casein Interference --- p.47 / Chapter 2.2.5. --- Test Protocol --- p.48 / Chapter 2.3. --- Statistical Analysis --- p.48 / Chapter Chapter 3. --- Purification of Anti-HLA IgG and F(ab')2 --- p.50 / Chapter 3.1. --- Immunoglobulin Concentration --- p.51 / Chapter 3.2. --- F(ab')2 Specificity --- p.51 / Chapter 3.3. --- Fc-fragments Contamination --- p.53 / Chapter 3.4. --- Discussion --- p.56 / Chapter Chapter 4. --- ELISA Optimization --- p.57 / Chapter 4.1. --- Coating F(ab')2 Quantitation --- p.58 / Chapter 4.2. --- Blocking and Diluting Agent Concentration --- p.61 / Chapter 4.3. --- Serum Analyte Dilution --- p.61 / Chapter 4.4. --- Conjugated Detector Antibody Titration --- p.64 / Chapter 4.5. --- Discussion --- p.66 / Chapter Chapter 5. --- Quality Control --- p.70 / Chapter 5.1. --- Avoidance of Prozone Phenomenon --- p.71 / Chapter 5.2. --- Inter-assay and Intra-assay Precision --- p.71 / Chapter 5.3. --- Discussion --- p.74 / Chapter Chapter 6. --- Adjustment of Anti-casein Interference --- p.77 / Chapter 6.1. --- Casein Allergy --- p.78 / Chapter 6.2. --- Prevalence of Anti-casein --- p.80 / Chapter 6.3. --- Discussion --- p.81 / Chapter Chapter 7. --- Prevalence of Anti-idiotypic Antibodies --- p.86 / Chapter 7.1. --- Formation Kinetics --- p.87 / Chapter 7.2. --- Occurrence in Transplant Patients --- p.87 / Chapter 7.3. --- Transfusion Effect --- p.101 / Chapter 7.3.1. --- Comparison between Transfused Transplant Patients and Normal Controls --- p.103 / Chapter 7.3.2. --- Comparison between Transfused Transplant Patients and Non-transfused Transplant Patients --- p.116 / Chapter 7.3.3. --- Association with Graft Survival --- p.117 / Chapter 7.4. --- Discussion --- p.128 / Chapter Chapter 8. --- Correlation of Transfusion with the Outcome of Transplant --- p.137 / Chapter 8.1. --- Rejection Episode --- p.138 / Chapter 8.2. --- Graft Survival --- p.139 / Chapter 8.3. --- Discussion --- p.142 / Chapter Chapter 9. --- General Conclusions --- p.149 / References --- p.153

Chun Sing Chu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 139-149). / Acknowledgements --- p.i / Abstract --- p.ii / Abbreviations --- p.vi / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter Chapter 2 --- "Development of Polyclonal Antibodies against 5α-Estrane-3β,17α-diol" / Chapter 2.1 --- Introduction --- p.39 / Chapter 2.2 --- Materials and Methods --- p.45 / Chapter 2.3 --- Results --- p.55 / Chapter 2.4 --- Discussion --- p.64 / Chapter Chapter 3 --- "Development of Monoclonal Antibodies against 5α-Estrane-3β,17α-diol" / Chapter 3.1 --- Introduction --- p.69 / Chapter 3.2 --- Materials and Methods --- p.86 / Chapter 3.3 --- Results --- p.107 / Chapter 3.4 --- Discussion --- p.126 / Chapter Chapter 4 --- General Conclusion --- p.134 / References --- p.139

Wong Yuk-Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 132-152). / abstract --- p.1 / ACKNOWLEDGEMENT --- p.4 / content --- p.6 / Chapter I. --- INTRODUCTION --- p.8 / Chapter II. --- literature reviews --- p.11 / Chapter 1. --- Menstrual cycle --- p.11 / Chapter 2. --- oestrogen receptor and progesterone receptor --- p.18 / Chapter 3. --- Monoclonal antibody assays for the study of oestrogen and progesterone receptors --- p.30 / Chapter III. --- materials and methods --- p.35 / Chapter 1. --- study population --- p.35 / Chapter 2. --- sample collection and analysis JO --- p.36 / Chapter 3. --- Histological dating of endometrial biopsies --- p.37 / Chapter 4. --- Determination of oestrogen and progesterone receptors using immunocytochemical assay --- p.38 / Chapter 5. --- Determination of oestrogen and progesterone receptors using enzyme immunoassay --- p.52 / Chapter 6. --- Determination of serum oestradiol and progesterone --- p.66 / Chapter 7. --- Data handling and statistical analysis --- p.76 / Chapter IV. --- results --- p.77 / Chapter 1. --- Study population --- p.77 / Chapter 2. --- Histological dating of endometrial biopsies --- p.77 / Chapter 3. --- oestrogen and progesterone receptors in frozen section of endometrium in menstrual cycle --- p.80 / Chapter 4. --- oestrogen and progesterone receptors in paraffin section of endometrium in menstrual cycle --- p.95 / Chapter 5. --- Oestrogen and progesterone receptors in endometrium in menstrual cycle determined by enzyme immunoassay --- p.113 / Chapter 6. --- Serum oestradiol and progesterone --- p.116 / Chapter V. --- DISCUSSIONS --- p.120 / Chapter 1. --- oestrogen and progesterone receptors in frozen section of endometrium in menstrual cycle --- p.121 / Chapter 2. --- Oestrogen and progesterone receptors in paraffin section of endometrium in menstrual cycle --- p.124 / Chapter 3. --- oestrogen and progesterone receptors in endometrium in menstrual cycle determined by enzyme immunoassay --- p.127 / Chapter 4. --- Potential application of oestrogen and progesterone receptors in endometrium in menstrual cycle --- p.129 / REFERENCE --- p.132

Poon Kwok Man. / Thesis submitted in: December 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 118-123). / Abstracts in English and Chinese. / ABSTRACT --- p.ii / 摘要 --- p.iv / DECLARATION --- p.vi / ACKNOWLEDGEMENTS --- p.vii / TABLE OF CONTENTS --- p.viii / LIST OF FIGURES --- p.xii / LIST OF TABLES --- p.xv / ABBREVIATIONS --- p.xvi / Chapter CHAPTER 1: --- INTRODUCTION / Chapter 1.1. --- Antibody structure and diversity --- p.1 / Chapter 1.2. --- Antibody genes --- p.5 / Chapter 1.3. --- The antibody response to phosphorylcholine --- p.10 / Chapter 1.3.1. --- Group I antibodies --- p.11 / Chapter 1.3.2. --- Group II antibodies --- p.14 / Chapter 1.3.3. --- Fine specificity of group I antibodies --- p.14 / Chapter 1.4. --- Anti-phosphorylcholine antibody structure --- p.15 / Chapter 1.5. --- Recombinant antibody --- p.22 / Chapter 1.5.1. --- Phage biology --- p.24 / Chapter 1.5.2. --- Phage-displayed antibodies --- p.29 / Chapter 1.5.3. --- Helper phage --- p.32 / Chapter 1.6. --- Objectives and scope of study --- p.34 / Chapter CHAPTER 2 --- METHODOLGY / Chapter 2.1. --- Antibody --- p.41 / Chapter 2.1.1. --- Hybridoma culture --- p.41 / Chapter 2.1.2. --- Production of antibody by induction of ascitic fluid --- p.41 / Chapter 2.1.3. --- Antibody purification --- p.41 / Chapter 2.1.3.1. --- Ammonium sulfate precipitation --- p.42 / Chapter 2.1.3.2. --- Affinity purification by Protein A-sepharose --- p.42 / Chapter 2.1.4. --- Production of Fab fragment by papain digestion --- p.43 / Chapter 2.2. --- Antigens --- p.43 / Chapter 2.2.1. --- Preparation of TsAg form infected ICR mouse --- p.44 / Chapter 2.2.2. --- Purification of Trichinella spairalis PC antigen --- p.44 / Chapter 2.2.2.1. --- Preparation of Mab2 affinity column --- p.44 / Chapter 2.2.2.2. --- Purification of TsAg --- p.45 / Chapter 2.2.3. --- Preparation of PC-HSA --- p.45 / Chapter 2.2.3.1. --- Preparation of p-diazonium phenylphosphorylcholine (DPPC) --- p.45 / Chapter 2.2.3.2. --- Conjugation of PC to HSA --- p.45 / Chapter 2.2.4. --- Commercial available antigens --- p.46 / Chapter 2.2.4.1. --- Pneumovax® 23 --- p.46 / Chapter 2.2.4.2. --- Lipopolysaccharide --- p.46 / Chapter 2.2.5. --- Standardization of PC-antigens --- p.46 / Chapter 2.3. --- Cloning of Mab2-scFv into phage display form --- p.47 / Chapter 2.3.1. --- Total RNA extraction --- p.50 / Chapter 2.3.2. --- cDNA synthesis --- p.50 / Chapter 2.3.3. --- Heavy chain variable region gene amplification --- p.51 / Chapter 2.3.4. --- Light chain variable region gene amplification --- p.51 / Chapter 2.3.5. --- Joining of heavy and light chain gene with linker --- p.52 / Chapter 2.3.6. --- Ligation of scFv gene with pCANTAB-5E vector --- p.52 / Chapter 2.3.7. --- Transformation --- p.53 / Chapter 2.3.7.1. --- E.coli strains --- p.53 / Chapter 2.3.7.2. --- E.coli cell preparation for electroporation --- p.54 / Chapter 2.3.7.3. --- Electroporation --- p.54 / Chapter 2.3.7.4. --- Competent E.coli preparation by CaCl2 --- p.55 / Chapter 2.3.7.5. --- Heat shock --- p.55 / Chapter 2.4. --- Expression of phage display scFv --- p.55 / Chapter 2.5. --- Enrichment and screening of Mab2-scFv phage --- p.56 / Chapter 2.5.1. --- Biopanning --- p.56 / Chapter 2.5.2. --- Restricition fragment analysis --- p.58 / Chapter 2.5.3. --- PCR screening --- p.58 / Chapter 2.5.4. --- DNA sequencing --- p.58 / Chapter 2.5.4.1. --- Manual sequencing --- p.58 / Chapter 2.5.4.2. --- Auto sequencing --- p.59 / Chapter 2.6. --- Mutagenesis --- p.59 / Chapter 2.6.1. --- Preparation of Uracil containing ssDNA --- p.60 / Chapter 2.6.2. --- Phosphorylation of mutagenic oligonucleotide --- p.60 / Chapter 2.6.3. --- Hybridization and secondary strand synthesis...…… --- p.60 / Chapter 2.6.4. --- Transfection and screening of mutants --- p.61 / Chapter 2.7. --- Expression of soluble scFv-E-tag --- p.61 / Chapter 2.7.1. --- SDS-PAGE analysis --- p.62 / Chapter 2.7.2. --- Anti-E-tag ELISA --- p.62 / Chapter 2.8. --- ELISA binding assay --- p.63 / Chapter 2.8.1. --- Specificity of Mab2 antibody Fab --- p.63 / Chapter 2.8.1.1. --- Carrier specifcity assay --- p.63 / Chapter 2.8.1.2. --- Free hapten inhibition assay --- p.64 / Chapter 2.8.2. --- Specificity of the scFv --- p.64 / Chapter 2.8.2.1. --- Antigen binding assay --- p.65 / Chapter 2.8.2.2. --- Free hapten inhibition assay --- p.65 / Chapter 2.8.2.3. --- Inhibition on Ts2 and Mab2 antibody assay --- p.65 / Chapter 2.9. --- Affinity assay --- p.66 / Chapter 2.10. --- Mutants analysis --- p.66 / Chapter CHAPTER 3 --- RESULTS / Chapter 3.1. --- Cloning VH and VL gene of Mab2 into scFv --- p.67 / Chapter 3.1.1. --- Amplification of variable region of H and L chain --- p.67 / Chapter 3.1.2. --- Biopanning --- p.70 / Chapter 3.1.3. --- Genetic composition of isolated clones --- p.70 / Chapter 3.2. --- Mutagenesis --- p.84 / Chapter 3.3. --- Expression and characterisation of wild-type scFv --- p.88 / Chapter 3.3.1. --- ScFv soluble protein --- p.88 / Chapter 3.3.2. --- Phage displayed scFv --- p.91 / Chapter 3.3.3. --- Standardization of PC antigens --- p.91 / Chapter 3.3.4. --- Binding acticity of scFv --- p.94 / Chapter 3.3.4.1. --- Influence of the avidity on carrier specificity binding --- p.96 / Chapter 3.4. --- Antigen specificity --- p.99 / Chapter 3.4.1. --- Free hapten inhibiton --- p.99 / Chapter 3.4.2. --- Inhibition on the binding of Ts2 --- p.102 / Chapter 3.4.3. --- Binding affinity --- p.104 / Chapter 3.5. --- Binding activities of mutants --- p.106 / Chapter CHAPTER 4 --- GENERAL DISCUSSION --- p.109 / REFERENCE --- p.118

The aim of this thesis was to establish the therapeutic value of the IFITM1 monoclonal antibodies and to design and develop therapeutically valuable recombinant monoclonal antibodies so as to study the implication of these novel antibodies in cancer therapy. Cancer metastasis is one of the main interests that has given rise to the design and development of innovative strategies for cancer therapeutics. The Interferon Induced Transmembrane Protein 1(IFITM1), a notable member of the IFITM family of proteins has been identified as one of the most up-regulated trans-membrane proteins in metastatic breast cancer and cervical adenocarcinoma. This interferon-regulated protein is also involved in cell migration, invasion in glioma and squamous cancers. This PhD aimed to study IFITM1 as a pro-invasive cancer target by the use of IFITM1 monoclonal antibodies that were raised against the extracellular domain of the human IFITM1 gene. The epitope mapping of IFITM1 revealed the binding activity of the IFITM1 monoclonal antibody. This gave the opportunity to design and generate to new IFITM1-specific molecular tools, in the form of recombinant IFITM1 targeted murine scFv antibody, IFITM1-CPG2 yeast fusion protein antibody for potential application in ADEPT as well as a Mouse-Human Chimeric IFITM1 antibody secreting mammalian cell line. The immunohistochemical staining of IFITM1 in tissue micro array from breast, colon and oeosphegal cancer has revealed that the majority of these cancers produce this protein. However, IFITM1 is over produced in cervical cancer indicating it’s selective over expression in cervical cells. This PhD endeavored to investigate the expression of IFITM1 at a translational and transcriptional level and to study the clinical significance of IFITM1 in cervical cancer. The antibody dependent cell mediated cytotoxic activity of the chimeric IFITM1 antibody was found to be cytotoxic to SiHa cells in vitro. In the future these molecular tools could be used to regulate and further characterize the activity of this transmembrane protein antibody. In an effort to better understand the mechanisms that regulate the activity and the over production of the IFITM1 gene and its interacting proteins, a proteomic screen of cervical cancer cells was carried out using data-independent SWATH-MS on an AB SCIEX TripleTOF™ mass spectrometer. This Mass Spec analysis provided us with a host of IFITM1 biomarkers and revealed that the IFITM1 gene and its binding proteins also cross link with the IRF1 pathway. The data presented in this thesis, demonstrates that the IFITM1 gene can be targeted to either stimulate or inhibit IFITIM1 signaling to engage IFITM1 as a potential pro-invasive extracellular receptor as a target in antibody cancer therapy. In summary, this thesis aimed to confirm the activity and the binding specificity of the IFITM1 antibody. Additionally, this thesis demonstrated a promising application of the recombinant antibody in the ADEPT technology. Characterization of IFITM1 mAb effector functions indicated that the antibody was cytotoxic to cervical cancer cells. This highlights an important element in the immune suppressive tumour microenvironment. And finally, this thesis also provides the basis for the production of recombinant mouse human chimeric antibodies that are a part of a new group of immunotherapeutic molecules paving the way for cancer therapeutics.

Orientador: Elenice Deffune / Coorientador: Márjorie de Assis Golim / Banca: Rosimeire Aparecida Roela / Banca: Paulo Inácio da Costa / Banca: Rosimeire Aparecida Roela / Resumo: O sistema imunológico é dividido em imunidade natural e adquirido (humoral e celular). Os linfócitos B são os principais efetores da resposta humoral. Junto aos linfócitos T, mediam diversas reações imunológicas. Todos os leucócitos possuem antígenos de superfície (clusters of differentiation - CD) determinados que possuem as mais diferentes funções. As expressões destes CDs podem variar na maturação e na presença de patologias, sendo as de maior prevalência e gravidade as leucemias e linfomas, tornando-se marcadores importantes que podem ser avaliados por citometria de fluxo ou imunoquímica através do uso de anticorpos monoclonais murinos (AcMm). Após produção dos AcMm é necessário caracterizar e validá-los. Utilizou-se 11 clones que apresentaram especificidade somente contra linfócitos B. Pela técnica de Western Blotting, 5 anticorpos (3 do tipo IgM e 2 do tipo IgG) foram escolhidos de acordo com sua possível especificidade e importância clínica. A validação dos anticorpos tipo IgM foi realizada por citometria de fluxo utilizando anticorpo comercial para comparação de quantidade de células marcadas, sendo testados em 20 amostras de indivíduos normais e 20 de indivíduos portadores de neoplasias hematológicas diversas. O LINB B, que foi comparado com o anti-CD171 e anti-CD45RA, apresentando diferença estatística somente em relação ao anti-CD45RA, e identidade com o anti-CD171. O LINB C, que foi comparado com o anti-CD20 e anti-CD19, não apresentou diferença estatística significante quando frente a ambos anticorpos comerciais. No teste de regressão linear, houve maior correlação dos resultados com o anti-CD19. O LINB E foi comparado somente contra o anti-CD107b, havendo grande identidade entre os dois. Dos resultados apresentados, conclui-se que o LINBs B e E apresentam grandes chances de serem específicos contra... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The immunological system is divided into: natural immunity and acquired immunity (celular and humoral responses). The B lymphocytes are the main effectors of the humoral response. Together with the T lymphocytes, they make several immunological reactions. All leucocytes have antigens on the surface (clusters of differentiation - CD) that possess lot of functions. The expressions of these CDs may be altered during maturation and pathologies, like leukemia and lymphomas, becoming important markers that can be evaluated by flow cytometry or immunochemistry thought murine monoclonals antibodies (Mab). After production of Mabs it's necessary characterize and validated them. We used 11 clones that presented Mab against B lymphocytes only. By Western Blotting method 5 Mab (3 IgM and 2 IgG) were chosen according you possible especifity and clinical importance. The validation of IgM Mabs were made by flow cytometry using commercial antibody to compare the quantity of marked cells, being used 20 samples from normal people and 20 samples from person with hematological cancer. The LINB B, compared to anti-CD171 and anti-CD45RA, presented statistical difference from anti-CD45RA and identity to anti-CD171. The LINB C, compared to anti-CD19 and anti-CD20, didn't presented any statistical difference from both commercial antibodies, although it correlates better with anti-CD19. The LINB E was compared to anti-CD107b, where it appears great identity between then. By the present results, we conclude that LINB B and E need multicentre studies to expand validation, and LINB C, needs to increase the samples to have a statistical validation. The two IgGs LINB, possible anti-CD138, weren't test yet. / Mestre

Monoclonal antibodies (mAbs) represent one of the fastest growing classes of therapeutic proteins. This success is due to a number of attractive properties such as high binding affinity, specificity, low immunogenicity and high aqueous solubility. Despite this, mAbs can suffer from undesirable physical instabilities, especially reversible self-association (RSA), which can lead to aggregation and phase separation. One aspect of formulation is therefore to find solution conditions which minimise mAb aggregation propensity during storage at high concentrations. Hence, the buffer, excipient and pH must be carefully considered to obtain the optimal formulation. Currently, if a platform formulation process is non-ideal for a particular candidate mAb, then an alternative strategy is to utilise high-throughput screening to measure various physical parameters indicative of physical stability. Arginine (in the form of hydrochloride salt Arg·HCl) is often used in formulations exhibiting high RSA and a propensity for aggregation. The interaction of Arg with the protein surface is complex and dependent on both the salt form and concentration. Here the focus was on the glutamate salt of arginine (Arg·Glu), to quantify its effect on mAb conformational and colloidal stability under different pH conditions. Arg·Glu was able to decrease the propensity of the mAbs to aggregate, particularly at pH values closer to their pI.The work also included the use of in vitro cell culture models to examine cell viability in the presence of the various arginine salts over a range of osmolalities. Whilst Arg·Glu is composed of two naturally occurring amino acids and both of which are considered non-toxic individually, the effect of the increased concentrations of their combination, on cells has not been explored previously. In vitro cell lines were chosen to represent the subcutaneous tissue, the effect of Arg·Glu on cell viability was compared against NaCl, Arg·HCl and sodium glutamate (NaGlu). The work concluded there was no additional toxicity associated with the presence of Arg·Glu in the cell culture models studied, therefore Arg·Glu has the potential as an excipient as it reduces aggregation and is nontoxic. Another aspect of the work was to assess the use of solution NMR spectroscopy as an orthogonal technique in mAb formulation characterisation. 1H NMR spectroscopy was used to measure a number of experimental parameters for high concentration mAb solution. The work proposed that 1H NMR spectroscopy can serve as a valuable orthogonal method for mAb characterization and formulation.

Orientador: Mário de Beni Arrigoni / Banca: Paulo Roberto Leme / Banca: Rafael da Costa Cervieri / Resumo: O presente estudo teve por objetivo avaliar os possíveis impactos causados pela suplementação de anticorpos policlonais Y contra Streptococcus bovis, Fusobacterium necrophorum e algumas cepas de bacterias proteolíticas, ou monensina sódica na carcaça e qualidade de carne de bovinos jovens. Foram usados 72 animais, sendo 24 nelores puros, NE, 24 canchins (3/8 Nelore e 5/8 Charolês, CC) e 24 Tri-cross (½ sangue Brangus, ¼ Nelore e ¼ Angus, TC), desmamados com sete meses de idade. Os animais foram alimentados com dieta de alta proporção de concentrado duas vezes ao dia, no período da manhã e tarde e monitorados a cada 28 dias com ultra-som em tempo real. Não houve interação (P>0,05) aditivo x grupo genético. NE apresentaram menor (P<0,05) peso inicial, final, área de olho de lombo e quantidade de ácidos graxos saturados e maior CLA (P<0,01) quando comparados aos outros grupos genéticos. CC apresentou maior (P<0,05) área de olho de lombo e menor (P<0,05) espessura de gordura subcutânea, comparando TC e NE. TC obteve menor (P<0,05) rendimento de carcaça e força de cisalhamento. O grupo suplementado com anticorpos obteve menor rendimento de carcaça, enquanto não houve efeito de aditivo para os demais parâmetros avaliados. O uso de anticorpos não afetou negativamente os parâmetros estudados neste trabalho, salvo o rendimento de carcaça. / Abstract: The objective of this study was to evaluate effects of feed additive (300 mg monensin/hd, MO, vs 10 mL/hd of a polyclonal antibody preparation against lactateproducing bacteria, PAP ) or biotype (Nellore, NE, Canchim cross, 5/8 Charolais, 3/8 Nellore, CC, or a 3-way cross, ½ Brangus, ¼ Nellore and ¼ Angus, TC) on ultrasound (US)-assessed measures of fat and ribeye area, carcass characteristics, and longissimus dorsi tenderness (shear force, SF, and myofibrillar fragmentation index, MFI) of bullocks fed high-concentrate diets. 72 bullocks were allocated in a 2 X 3 factorial arrangement replicated thrice (4 bullocks/pen) of feed additive (FA) and biotype (GG), and monitored monthly for a 107-d (CC and TC) or 147-d (NE) feeding period. Analyses of variance included the initial measurement covariate when appropriate (P < 0.05). Final (BW) and hot carcass weight (HCW) were unaffected (P < 0.05) by FA, but were lower (P < 0.05) for NE than CC and TC. Dressing percentage (DP) was lower (P < 0.05) for TC than NE and CC bullocks. Monensin had greater (P < 0.05) DP than PAP. There was no effect (P > 0.05) of FA on monthly measurements of fat depth (BFT), rump fat (P8), visceral fat (VF), ribeye area (REA), or SF and MFI. Bullocks of CC biotype were leaner (P < 0.05; less BFT and P8) than those of TC and NE biotypes. Bullocks of NE biotype had smaller (P < 0.05) REA than those of CC and TC biotypes. Steaks of TC biotype had lower (P < 0.05) SF values than those of the other biotypes. There were no differences (P > 0.05) in MFI or VF due to biotypes. NE presented greater (P<0,01) concentrations of unsaturated fatty acids and CLA. Other than effects of PAP on DP, PAP did not affect carcass fat, REA or tenderness. / Mestre