3 Antibodies Polyclonal Monoclonal recognize multiple antigenic sites Individual B lymphocyte hybridomais cloned and cultured.Secreted antibodies are collectedfrom culture mediaAntibodies that are collectedfrom sera of exposed animalrecognizemultiple antigenic sitesof injected biochemical.recognizeONE antigenic siteof injected biochemical
4 Monoclonal Antibodies Populações homogêneas de anticorpos derivados de uma única célula produtora de anticorpo, na qual todos os anticorpos possuem a mesma especificidade para um determinante antigênico.
7 Uses of Monoclonal Antibodies: Protein purificationIdentification and isolation of cell sub-populations using fluorescence cell sorting.Tumor detection and imagingTumor killing (magic bullets)Diagnostic reagents
8 How does we make a monoclonal antibody? Immunize miceTest the serumPerform a fusionScreen the fusion for the right cellsGrow the hybridomasHarvest the antibodyConcentrate and purify the product
13 Test the serum - Decision time When the serum titer of the mice has reached a plateau, an additional ELISA test is performed to determine the predominant isotype present. The two isotypes that are most common in mouse serum are IgG and IgM.A fusion is done when the IgG level is high and the IgM level is low.Sometimes additional testing is done (Western blots, immunoflurorescence) to determine whether the serum response is specific for the selected antigen.The mouse with the strongest, most specific response is chosen for the fusion.
14 Anticorpos Monoclonais - Produção HybridomaAnticorpos Monoclonais - Produção
19 Detecção dos clones de interesse Imunofluorescência Indireta ELISA
20 Perform a fusion - Growing the cells (hybridomas) Cells are grown in a 37o C incubator.Cells are kept in an atmosphere of about 6% CO2.The cells are fed after 7 days of incubation.The cells are checked for growth after 10 days of incubation.
21 Harvest the antibody1) The antibody is produced by the cells and released into the cell supernatant.2) Media is added to the flasks until the desired volume of antibody-containing supernatant is obtained.3) When the desired volume is reached, the supernatant is refrigerated and tested by ELISA.4) If the testing is successful, the supernatant can be used by the investigator, or concentrated and purified.
22 Expand in mice or expand in vitro in vitro material is less concentratedand containsbovine serumascites fluidcontain high [mAb]and minor contaminationwith mouse Ig
23 Concentrate and Purify the Product The antibody can be concentrated by precipitation with a solution of saturated ammonium sulfate.Concentrated antibody can then be purified by passing it through a column.
28 Dynabeads: Subpopulations of cells can by physically separated using antibodiescoupled to magnetic beadsDynabeads:Which human cell typedo you want to isolate?T CellsB CellsNK CellsStem CellsMonocytesDendritic CellsGranulocytesEndothelial CellsLeucocytesTumour Cells
32 Immunotoxin: Antibody-Toxin Conjugate is tumor-specificricin from the castor bean(Ricinus communis).waynesword.palomar.edu/ images/antibod2.gif
33 Only the targeted tumor cells are killed by the toxin Common side effect –Vascular leak syndrome.Could be relievedby usage of mutated toxinBunch of other toxinscould be used
34 Saponin (from soapwort) potentsingle chain ribosome inactivating proteinGoal:treatment ofleukemiasand lymphomasFor T-cell leukemia could betargeted via CD7: DFlavell/djf2.htm
35 How Radioimmunotherapy Works Cancer CellRadioactive Antibodies
36 Radioactive Antibodies How Radioimmunotherapy WorksCancer CellRadioactive Antibodies
37 Monoclonal antibodies, which are specific for a certain antigen, i. e Monoclonal antibodies, which are specific for a certain antigen, i.e. prostate carcinoma cells, can be tagged with a radioactive tracer, which when injected into the body will localize in areas of these recurrent carcinoma cellsOctreoScan in a patientwith metastatic small cell carcinoma of the prostate
38 FDA Approved RIT 2 current FDA-approved RIT drugs: Zevalin™ (2002) Bexxar™ (2003)
39 Human MAb production Immune response to mouse antibodies (HAMA response: clearance + disease)HAMA: human anti-mouse antibodies
40 Anticorpos monoclonais humanos DIFICULDADES (mielomas): Human MAb productionAnticorpos monoclonais humanosDIFICULDADES (mielomas):Falta de linhagens de células de mieloma humano bem estabelecidaMieloma secretores de IgCrescimento deficiente
41 Anticorpos monoclonais humanos DIFICULDADES (linfócitos B): Human MAb productionAnticorpos monoclonais humanosDIFICULDADES (linfócitos B):Obtenção das células B antígenos-específicas ativadassangue x baço/linfonodosAlternativa: imunizar indivíduos antes de retirar os linf. BEstimular células B in vitro com Agprodução de IgM de baixa afinidadeTransferência de linfócitos humanos para SCDI miceTransformação de linfócitos B com Epstein-Barr
42 Produção de Anticorpos Monoclonais AlternativasAnticorpos monoclonais quiméricosAnticorpos monoclonais humanizadosPhage displayCamundongos transgênicos
49 Humanized antibodiesClone those genesand express themas a recombinant proteinin the protein expression systemSales of Rituxan/MabThera by YearMouse antigen-interacting sequencesintragrad/micro/micro297/4Anti-CD20 MAbs (Rituxan) for treatment of non-Hodgkin’s lymphoma
55 Selection of antibodies from phage display libraries: the enrichment ofan antigen-specificphage-antibody (circle) on a background ofnon specificphage-antibodies (square).protocols/schema.jpgAntigen comesfrom serum of cancer patients
57 Xenomouse (mouse with human immunoglobulin loci) AbgenixXenoMouse genetically engineeredTo replace murine IgH and Igk lociwith their human Ig counterparts on YACHuman Ig transgenescarry the majorityof the human variable repertoireand can undergoclass switchingfrom IgM to IgG isotypes.intragrad/micro/micro297/4
58 Unconjugated MAbs as a molecular drugs Target Antigen Clinical Use 1. Drugs Overdose 2. TNF- Septic shock 3. Viruses Infection 4. CD3 Transplants 5. IL-2R Leukaemia/ lymphoma 6. Anti-venoms Snake and Bee BitesOrgan Transplants:Donor T cells cause graft vs host (GvH) reaction.Donor T cells eliminated using anti-CD3 Mabs (OKT3) Muromonab; .Treat patients with anti-CD3 after solid organ transplants.
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