Tehnici Utilizate in Imunologie

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PETRU CIANGA

TEHNICI UTILIZATE IN IMUNOLOGIENOTIUNI INTRODUCTIVE

PETRU CIANGA

TEHNICI UTILIZATE IN IMUNOLOGIENOTIUNI INTRODUCTIVE

Editura Pim 2008

EDITURA PIM Soseaua Stefan cel Mare nr. 11 Iasi -700498 Tel. / fax: 0232-212740 e-mail:editurapim@pimcopy.ro www.pimcopy.roEDITUR ACREDITAT CNCSIS BUCURETI 66/01.05.2006

Descrierea CIP a Bibliotecii Naionale a Romniei CIANGA, PETRU Tehnici utilizate n imunologie : noiuni introductive / Petru Cianga. - Iai : PIM, 2008 Bibliogr. ISBN 978-606-520-214-6 612.017

CuprinsReacia Antigen-Anticorp................................................................1 1. Legturi implicate n formarea complexului antigen-anticorp.......2 2. Factori care influeneaz interaciunea dintre antigen i anticorp..8 2.1 Caracteristici structurale...............................................................9 2.1.1 Specificitatea situsurilor de legare a antigenului.......................9 2.1.2 Reactivitatea ncruciat............................................................9 2.1.3 Accesibilitatea antigenic........................................................10 2.1.4 Structura imunoglobulinelor....................................................10 2.1.5 Valena antigenelor..................................................................10 2.1.6 Concentraia de antigene i anticorpi.......................................11 2.2 Factori fizico-chimici care afecteaz legarea dintre antigen i anticorp..............................................................................................12 2.2.1 Factori de mediu.......................................................................12 2.2.2 Concentraia n sruri (fora ionic).........................................12 2.2.3 Potenialul zeta.........................................................................13 Anticorpi monoclonali....................................................................14 Reacia de precipitare.....................................................................24 1. Precipitarea n soluie....................................................................24 2. Precipitarea n gel..........................................................................27 2.1 Dubla imunodifuzie (Oucthterlony)............................................27 2.2 Imunodifuzia radial simpl (Mancini).......................................30 3. Imunelectroforeza.........................................................................32 3.1 Imunelectroforeza.......................................................................35 3.2 Electroforeza de imunofixare (immunofixation electrophoresis IFE)...................................................................................................36 3.3 Electroforeza contracurent (countercurrent electrophoresis)......37 3.4 Electroforeza n rachet (rocket electrophoresis)........................37 3.5 Focusarea isoelectric (isolectric focusing)................................38 Reacia de aglutinare......................................................................40 1. Aglutinarea direct........................................................................44 2. Aglutinarea indirect.....................................................................46 3. Aglutinarea pasiv.........................................................................47 4. Testele de inhibiie a aglutinrii....................................................48

5. Testul anti-imunoglobulinic (testul Coombs)...............................50 Teste de faz solid - Solid phase assay.........................................52 1. RIA - Radio Immuno Assay..........................................................52 2. RIST Radioimmunosorbent test.................................................53 3. RAST Radioallergosorbent test.................................................54 4. ELISA - Enzyme Linked Immunosorbent Assay.........................56 4.1 ELISA indirect..........................................................................57 4.2 ELISA sandwich.........................................................................59 4.3 ELISA competitiv.....................................................................60 4.4 ELISPOT.....................................................................................61 5. Chemiluminiscena........................................................................62 Imunohistochimia (Corina Cianga, Petru Cianga)..........................65 1. Noiuni de enzimologie.................................................................67 2. Metode de colorare........................................................................70 2.1 Metoda direct.............................................................................70 2.2 Metoda indirect n doi pai........................................................71 2.3 Metoda indirect n trei pai........................................................72 3. Controale.......................................................................................73 4. Colorarea de fond (nespecific sau background)..........................74 5. Contracolorarea.............................................................................77 6. Imunocitochimia...........................................................................77 7. Dubla/tripla coloraie....................................................................78 Imunofluorescena..........................................................................80 1. Imunofluorescena direct............................................................82 2. Imunofluorescena indirect.........................................................84 Microscopia confocal....................................................................85 Tissue FAXS....................................................................................86 Citometrie n flux (Flow cytometry)..............................................87 1. Principiu........................................................................................87 2. Sortarea particulelor......................................................................94 Aplicaii ale citometriei n flux......................................................95 1. Imunofenotiparea.........................................................................95 2. Analiza coninutului ADN (Ploidia ADN)...................................97

3. Investigarea unor molecule solubile............................................99 4. Medicina reproducerii.................................................................100 5. Cross-match................................................................................100 6. Biologia celular.........................................................................101 7. Analiza proliferrii celulare........................................................101 8. Analiza morii celulare prin apoptoz.........................................104 9. Analiza citotoxicitii celulare....................................................104 10. Microbiologie............................................................................105 11. Botanic....................................................................................106 12. Farmacologie.............................................................................106 Histocompatibilitatea n transplant............................................107 Tiparea tisular (tiparea HLA I i II)........................................110 1. Citometria n flux.......................................................................110 2. Microlimfocitotoxicitatea sau citotoxicitatea dependent de complement (CDC).................................................110 3. Metode de biologie molecular...................................................112 3.1 Metoda SSP - sequence-specific priming.................................113 3.2 Metoda SSOP - sequence-specific oligonucleotide probing.....114 3.3 SBT sequence-based typing...................................................116 4. Cultura limfocitar mixt............................................................116 Cross-match (X-match)................................................................117 Evaluarea funcionalitii componentelor sistemului imun......120 1. Limfocitele B..............................................................................120 1.1 Teste in vivo..............................................................................120 1.2 Teste in vitro.............................................................................122 2. Limfocitele T..............................................................................122 2.1 Teste in vivo..............................................................................122 2.2 Teste in vitro.............................................................................124 3. Celulele NK (natural killer)........................................................126 4. Fagocite.......................................................................................127 5. Bazofile.......................................................................................130 6. Sistemul complement (SC).........................................................131 Modele experimentale animale....................................................134 1. Animale inbred (singenice).........................................................135 2. Animale congenice......................................................................136 3. Animale chimerice (chimeras)....................................................138

4. oareci SCID (severe combined immunodeficiency imunodeficiena sever combinat)...............................................140 5. oareci nuzi.................................................................................141 6. oareci transgenici......................................................................143 7. oareci knock out (KO)..............................................................145 8. oareci knock-in.........................................................................146 Tehnici de biologie molecular (manipulare a acizilor nucleici i producie de proteine) Metode de manipulare a genelor.................................................149 1. Extragerea ADN din esuturi i culturi de celule........................149 2. Extragerea ARN-ului din esuturi sau culturi de celule..............151 3. Electroforeza ADN n gel...........................................................152 3.1 Electroforeza n gel de agaroz.................................................153 3.2 Electroforeza n gel de poliacrilamid......................................156 3.3 Electroforeza n cmp electric pulsatil .....................................157 4. Southern blot...............................................................................157 5. Northern blot...............................................................................161 Metode de evideniere a secvenelor nucleotidice n celule i esuturi...........................................................................................161 1. Hibridizarea in situ......................................................................161 2. FISH Fluorescence in situ hybridization..................................168 3. CISH Chromogenic in situ hybridization.................................169 Manipularea enzimatic a ADN-ului..........................................170 1. Digestia cu enzime de restricie..................................................170 2. Utilizarea ligazelor n ingineria genetic....................................171 3. Amplificarea prin PCR................................................................172 4. RT-PCR (reverse transcription-PCR).........................................177 5. Real time PCR PCR n timp real..............................................178 6. Secvenierea ADN.......................................................................179 Metode de introducere a ADN-ului recombinant n bacterii i celule eucariote 1. Vectori.........................................................................................182 1.1 Plasmide....................................................................................182 1.2 Virusuri.....................................................................................186

1.3 Bacteriofagi...............................................................................187 1.4 Cosmide....................................................................................188 1.5 Cromosomi bacterieni artificiali...............................................188 1.6 Cromosomii artificiali din drojdii (YACs)................................188 2. Transformarea bacterian i purificarea ADN-ului din plasmide....................................................................................189 Producerea proteinelor n gazde bacteriene 1. Inducerea sintezei proteice n gazde bacteriene..........................192 2. Purificarea proteinelor.................................................................193 2.1 Filtrarea n gel (cromatografie de excludere)............................194 2.2 Purificarea proteinelor cu ajutorul rinilor schimbtoare de ioni.......................................................................195 2.3 Tehnologia Ni-NTA (nichel-acid nitriloacetic)........................195 3. Inhibitori de proteaze..................................................................199 4. Metode de caracterizare a produsului genelor manipulate..........201 4.1 Electroforeza proteinelor...........................................................206 4.2 Western blot..............................................................................203 4.3 Caracterizarea funcional prin analiza asocierii cu alte proteine.....................................................................................205 4.4 Alte metode de analiz funcional...........................................206 4.4.1 Analiza Farwestern................................................................206 4.4.2 Imunoprecipitarea..................................................................206 4.5 Surface plasmon resonance (SPR)............................................208 Bibliografie....................................................................................213

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