TEHNICI UTILIZATE IN IMUNOLOGIE

9 786065 202146

PETRU CIANGA

ISBN 606-520-214-2

PETRU CIANGA

TEHNICI UTILIZATE IN IMUNOLOGIE

NOTIUNI INTRODUCTIVE

PETRU CIANGA

TEHNICI UTILIZATE IN IMUNOLOGIE

NOTIUNI 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.ro
EDITUR 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

Cuprins
Reacia 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