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Antigen (Ag)

Anticorpi
anticorpi monoclonali
anticorpi policlonali
Tehnici imunochimice
Haptene

Structura imunoglobulinelor Structura imunoglobulinelor


Clase de imunoglobuline

Ig G
Ig D
Ig E
Ig A
Ig M

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Structura imunoglobulinelor Subclase IgG
 fragment cristalizabil (Fc)
IgG
 fragment de legare a Ag (Fab)
 70-75% din totalul Ig.

 Traverseaza prin
bariera placentara.

Legarea IgE la nivel mastocitar


IgD IgE
 Concentratie scazuta in ser  Reactii acute de  Degranularea
 Structura monomera hipersensibilitate: febra,
mastocitara
astm bronşic, şoc anafilactic.
 Nu poate traverse placenta

 Implicata in generarea
memoriei imunologice

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IgA sSerica IgA dimer IgM
 10% din totalul Ig serice
 15% dintre Ig serice.
 Molecula pentamera
Monomer –
 Faza acuta

Dimer – legare cu lant J.  Nu poate traversa


bariera plancentara
 Dimer – la nivel secretii
 Componenta secretorie (CS)
 Bariera imuna locala

Derivatizarea haptenelor si
Haptena
biosinteza anticorpilor
 Mici molecule organice, care nu au caracter de
antigen
Pot deveni antigene dupa cuplare cu molecula de
transportor mai mare (albumină din lapte,
albumina din ou, albumina serică bovină)

Exemple: pesticide, medicamente, molecule


organice

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Anticorpi monoclonali si policlonali

Recunoasterea antigenului Interactiile antigen-anticorp Metode imunochimice


Antigen Antibody

- CH2 - O - H O Imunodifuzia simpla IDS


Imunodifuzia (ID)
 Necovalente H2N
C - CH2 - CH2 -
hydrogen bonds Imunodifuzia dubla IDD

directe Contraimunoelectroforeza si Imunoelectroforeza (IEF)


 Reversibile - CH2 - CH - CH3 Aglutinarea
H3C - CH - CH2 - van der Waals interactions
TEHNICILE IMUNE Fixarea complementului
CH3
(IA)
 Specifice H3C - CH2
indirecte Radioimunodetectii (RIA)
O (cu markeri) Enzyme IA
- CH2 - CH2 - C Metode neizotopice Flurescence IA
 Exoterme O
- +
H3N - CH2 - CH2 - ionic bonds
si Luminescence IA

Alte IA

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Imunodifuzia dubla

monospecificitate – polispecificitate
nespecificitate

Imunodifuzie dubla - Identitate Imunodifuzie dubla - Neidentitate


Imunodifuzia dubla

monospecificitate – polispecificitate
nespecificitate

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Imunodifuzie dubla – Identitate Imunodifuzia simpla
partiala (radiala, Mancini)

Contraimunoelectroforeza

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Electroforeza serului normal Imunoelectroforeza
Electroforeza serului normal
Imunoelectoforeza
- model Grabar
- in rachete
- bidimensionala

 albumine
 α -globuline
 β -globuline
 γ-globuline

Imunoelectroforeza Imunoelectroforeza Imunoelectroforeza

 IgG
 Ser normal
 IgG  IgA
 IgM

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Imunoelectroforeza tip racheta Imunoelectroforeza tip racheta Imunoelectroforeza bidimensionala
Antigen incarcat negativ Gelul Se aplica pentru analize
complexe de amestecuri
contine Ac
de Ag
1. Ag se separa prin
Precipitate Ag-Ac -> forma de electroforeza.
racheta
2. a 2-a electroforeza pe o
directie perpendiculara,
Inaltimea rachetei este direct printr-un gel care contine
proportionala cu concentratia Ac
Ag din godeu

Imunoelectroforeza bidimensionala
Agglutination Agglutination
 Ag - a membrane protein on a bacterial cell or red
blood cell

 Agglutination -> visible aggregates or clumps


 Agglutination -
formation.
no drug in sample
 The agglutination is doing because the electrostatic
repulsive forces between the particles
decrease and because are realized some binding
bridges using the antibodies

 More sensitive than precipitation reaction


 No agglutination -
 Masurarea inaltimii picului – determinare
May detect [Ab] as low as 0.001 µg/ml
drug in sample
cantitativa 

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Complement fixation Complement fixation Complement fixation
 Indicator cell and a
 Very sensitive small amount of anti-
 When complement is erythrocyte antibody
 The fixed amount of added, if complexes are added to the two
antigens added to both
are present, they tubes.
tubes
fix complement and  If the indicator cells
 If the antibody is consume it will lyse -> negative test
present in the test  If the complement is
tube serum, immune consumed -> no lysis will
complexes form occur -> positive test

Radioimunodetectii (RIA)

 125I, 3H, 14C, 32P, 35S

 Dezavantaje

 Avantaje

 Limita de detectie < 0.001 µg

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Fluoroimunodetectii Markeri fluorescenti
Fluorophore Molar λex(nm) λem(nm) Decay time
absorption (ns)

 Tehnica foarte sensibila Tetramethylrhodamine isothiocyanate


ε (L/mol)
105 000 540 575 2
Fluoroimunodetectii
Rhodamine B-isothiocyanate 103 000 550 585 3
 imunofluorescenta imagistica = Fluorescein isothiocyanate 66 000 492 520 4.5

imunohistochimie Lucifer yellow


Dansyl chloride
13 000
4 300
430
345
540
535
14
14

Pyrenebutyrate 40 000 340 385 100


Erythrosin B 101 000 492 517 108

 Aplicatii: chimie analitica, biochimie, Ethyl Eosin 101 000 520 545

biologie celulara, fiziologie, nefrologie, Methyl umbelliferone 16 000 323 386

Chlorophyll 430- 648-


cardiologie, stiintele mediului. 453 669
Eu 3+-β
β-naphthoyltrifluoroacetone 36 000 340 613 500 000
Dy-TMP-DPA 18 800 320 572 16 000
Sm-2-NTA 3 36 000 340 642 50 000
Tb –TMP -DPA 18 800 320 545 1 050 000

Imunohistochimie
Detectia caspazei-3 activate (celule musculare C2C12 Imunohistochimie
dezvoltate in mediul Dulbecco’s modified Eagle’s medium
(DMEM; Gibco Invitrogen, Grand Island, NY) Detectia caspazei-3 activate

(a) (b)
(a) (b)  Activated caspase-3 was detected using a band pass filter (EX 490nm,
 Celule tratate cu FAM-DEVD-FMK (CaspaTag™ kit; Intergen, Purchase, EM 520nm) on a microscope (a). An UV-filter (EX 365nm, EM 460nm)
NY)  se leaga de caspase-3 acticate. was used to visualize nuclear staining (b).
 Nuclei colorati cu DAPI.
 Pentru cuantificarea caspazei -3 program computerizat - NIH based
Scion Image program

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EIA - Enzyme Immunoassay Enzyme Immunoassay
ELISA
Tehnici imunoenzimatice
 Enzime: Peroxidaza (horseradish
peroxidase), PAL (fosfataza alcalina),
glucozoxidaza

 Interactie Ag- Ac (o specie marcata


enzimatic) si interactie E-S

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Enzyme Immunoassay
Enzyme Immunoassay
serum (sample) serum (sample)

conjugate
conjugate

E E
E E
substrate
substrate

negative reaction positive reaction negative reaction positive reaction

Imunohistochimie
Apoptosis test - TUNEL, pentru celule C2C12 crescute
in mediu cu deficit de colina (5 µM cholina)

 TUNEL (Apop-Tag; Intergen, Purchase, NY; 25)


 Ac marcati cu peroxidaza – detectie su sistem
ABC apa oxigenta - diaminozenzidina

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Microscopie electronica

Tehnici biologie moleculara


 Markeri: feritina, aur coloidal
 Vizualizare cu microscopie electronica 
Biosenzori
mici puncte negre

Anticorpi marcati magnetic Anticorpi marcati magnetic Imunocromatografie


- Cromatografie de afinitate
antigens + mobile phase

column

pH variation

stationary phase
with immobilized antibody
- antibody
signal
- specific antigen
pH variation
- unspecific antigen

time

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Imuno blotting Western blotting (Immunoblotting) Western blotting (Immunoblotting)
 Proteine  Radioizotopi
 Blotting – transferul moleculelor mari (proteine,
- solubilizare cu detergenti si agenti reducatori  Enzime cuplate cu anticorpi secundari
acizi nucleici) pe suprafata unei membrane, dupa - separare electroforetica pe gel SDS-  Aur cuplat cu anticorpi secundari
poliacrilamida  Sistem avidina-biotina
o separare electroforetica
- transfer pe membrana
- identificare - interactia cu anticorpul primar si  Chemiluminescenta
 Se identifica in final specia imuna
anticorpul secundar marcat

Southern blotting – probe DNA


Western blotting (Immunoblotting)
Northern blotting – probe RNA
Identificare HIV

1 - refeinta
2 - blank
A, B, C - probe reale

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Imunosenzori
ANALIT
S
BIOSENZOR BIORECEPTOR
P
= elemente biologice sau cu activitate
TRADUCTOR
biologica (enzime, anticorpi, DNA,
celule, tesuturi) + traductori AMPLIFICATOR DE SEMNAL

Imunosenzor Imunosenzori - Aplicatii Imunosenzori - limita detectie


 Pesticide: 2,4-D, Imidazolinone, Paration,  Piezoelectrici - 1 mg/L-1994
Triazine  Piezoelectrici - 0.3 mg/L -1997
 Cu fibre optice - 0.2 mg/L -1997
 Compusi organici : Benzo[a]pyrene,
 Fluorescenta - 0.3 mg/L -1997
Polychlorinated Biphenyls, TNT
 Electrochimici - 0.1 mg/L - 1998
 Parametrii biologici: identificare bacterii
 Electrochimici - 0.01 mg/L - 1997
 Amperometrici - 0.02 mg/L - 1997

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Relative sensitivities of tests (approx)
Bibliografie
Usual operating range
[Ab] or [Ag] o Kuby, J., Immunology, W.H. Freeman and Company, New York, 1997, 159-60.

o Prescott, L.M., Harley, J.P., Klein, D.A., Microbiology, Wm.C. Brown Publishers
precipitation London, 1996, 590-595.
immunoelectrophoresis 10 µg/ml - 1 mg/ml
o Roitt, I., Essential Immunology, Bleckwell Science Ltd., Oxford, 1997, 112-
double/radial diffusion
114.
immunofluorescence 0.1 - 10 µg/ml o Analytical Biotechnology. Principles and Applications, Billiet, H., Draghici, C.,
(Eds.), Editura Universitatii Transilvania din Brasov, (2004)
ELISA (colour) 0.1 - 10 ng/ml
(chemiluminescence) 0.01 - 10 ng/ml o Learning Guide Immunoassay, Abott

o Curs Proteomica de la fundamente la clinica – note de curs 2012


radioimmunoassay 0.01 - 10 ng/ml
o

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