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Tehnici imunochimice

Antigen (Ag)
Anticorpi
anticorpi monoclonali
anticorpi policlonali

Haptene

Interactiile antigen-anticorp
Antibody

Antigen
- CH2 - O - H

 Necovalente
 Reversibile

O
C - CH2 - CH2 -

hydrogen bonds

H2N

- CH2 - CH - CH3
CH3

 Specifice

H3C - CH - CH2 -

van der Waals interactions

H3C - CH2
O
- CH2 - CH2 - C

 Exoterme

H3N - CH2 - CH2 -

ionic bonds

Metode imunochimice
Imunodifuzia simpla IDS

Imunodifuzia (ID)
Imunodifuzia dubla IDD

directe

Contraimunoelectroforeza siImunoelectroforeza (IEF)


Aglutinarea

TEHNICILE IMUNE
(IA)

Fixarea complementului
indirecte
(cu markeri)

Radioimunodetectii (RIA)
Enzyme IA

Metode neizotopice

Flurescence IA
si Luminescence IA
Alte IA

Imunodifuzia simpla
(radiala, Mancini)

Imunodifuzia dubla

monospecificitate polispecificitate
nespecificitate

Imunodifuzia dubla

monospecificitate polispecificitate
nespecificitate

Imunodifuzie dubla - Identitate

Imunodifuzie dubla - Neidentitate

Imunodifuzie dubla Identitate


partiala

Contraimunoelectroforeza

Electroforeza serului normal


Imunoelectoforeza
- model Grabar
- in rachete
- bidimensionala

Electroforeza serului normal

 albumine
 -globuline
 -globuline
 -globuline

Imunoelectroforeza

Imunoelectroforeza

 Ser

normal

Imunoelectroforeza

 IgG

Imunoelectroforeza

 IgG
 IgA
 IgM

Imunoelectroforeza tip racheta


Antigen incarcat negativ Gelul
contine Ac

Precipitate Ag-Ac -> forma de


racheta

Inaltimea rachetei este direct


proportionala cu concentratia
Ag din godeu

Imunoelectroforeza tip racheta

Imunoelectroforeza bidimentionala
Se aplica pentru analize
complexe de amestecuri
de Ag
1. Ag se separa prin
electroforeza.
2. a 2-a electroforeza pe o
directie perpendiculara,
printr-un gel care contine
Ac

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Imunoelectroforeza bidimentionala

Masurarea inaltimii picului determinare


cantitativa

Radioimunodetectii (RIA)


 125I, 3H, 14C, 32P, 35S

 Dezavantaje
 Avantaje
 Limita

de detectie < 0.001 g

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Fluoroimunodetectii
 Tehnica

foarte sensibila

 imunofluorescenta

imagistica =

imunohistochimie
 Aplicatii: chimie analitica, biochimie,

biologie celulara, fiziologie, nefrologie,


cardiologie, stiintele mediului.

Markeri fluorescenti
Fluorophore

Tetramethylrhodamine isothiocyanate

Molar
absorption
(L/mol)
105 000

ex(nm) em(nm)

Decay time
(ns)

540

575

103 000
66 000

550
492

585
520

3
4.5

Lucifer yellow
Dansyl chloride

13 000
4 300

430
345

540
535

14
14

Pyrenebutyrate
Erythrosin B

40 000
101 000

340
492

385
517

100
108

Ethyl Eosin

101 000

520

545

16 000

323

386

36 000
18 800
36 000

430453
340
320
340

648669
613
572
642

Rhodamine B-isothiocyanate
Fluorescein isothiocyanate

Methyl umbelliferone
Chlorophyll
Eu3+-
-naphthoyltrifluoroacetone
Dy-TMP-DPA
Sm-2-NTA 3

500 000
16 000
50 000

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Fluoroimunodetectii

Imunohistochimie
Detectia caspazei-3 activate

(a)

(b)

Activated caspase-3 was detected using a band pass filter (EX 490nm,
EM 520nm) on a microscope (a). An UV-filter (EX 365nm, EM 460nm)
was used to visualize nuclear staining (b).

Pentru cuantificarea caspazei -3 program computerizat - NIH based


Scion Image program

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EIA - Enzyme Immunoassay


ELISA
Tehnici imunoenzimatice
 Enzime:

Peroxidaza (horseradish

peroxidase), PAL (fosfataza alacalina),


glucozoxidaza
 Interactie

Ag- Ac (o specie marcata

enzimatic) si interactie E-S

Enzyme Immunoassay

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Enzyme Immunoassay
serum (sample)

serum (sample)

conjugate
conjugate

E
E

substrate

substrate

negative reaction

positive reaction

negative reaction

positive reaction

Enzyme Immunoassay

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

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

Ac marcati cu peroxidaza detectie su sistem


ABC apa oxigenta - diaminozenzidina

Tehnici biologie moleculara


Biosenzori

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Microscopie electronica
 Markeri:

feritina, aur coloidal

cu microscopie electronica 
mici puncte negre

 Vizualizare

Anticorpi marcati magnetic

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Anticorpi marcati magnetic

Imunocromatografie
- Cromatografie de afinitate
antigens + mobile phase
column
pH variation

stationary phase
with immobilized antibody
- antibody

signal

- specific antigen
- unspecific antigen

pH variation
time

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Imuno blotting
 Blotting transferul moleculelor mari (proteine,

acizi nucleici) pe suprafata unei membrane, dupa


o separare electroforetica

Se identifica in final specia imuna

Western blotting (Immunoblotting)


 Proteine
-

solubilizare cu detergenti si agenti reducatori

separare electroforetica pe gel SDSpoliacrilamida

transfer pe membrana

- identificare - interactia cu anticorpul primar si


anticorpul secundar marcat

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Western blotting (Immunoblotting)


 Radioizotopi
 Enzime
 Aur

cuplate cu anticorpi secundari

cuplat cu anticorpi secundari

 Sistem

avidina-biotina

 Chemiluminescenta

Western blotting (Immunoblotting)


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

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Southern blotting probe DNA

Northern blotting probe RNA

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Imunosenzori
BIOSENZOR

= elemente biologice sau cu activitate


biologica (enzime, anticorpi, DNA,
celule, tesuturi) + traductori

ANALIT
S
BIORECEPTOR
P
TRADUCTOR

AMPLIFICATOR DE SEMNAL

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Imunosenzor

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Imunosenzori - Aplicatii
 Pesticide: 2,4-D, Imidazolinone, Paration,

Triazine
 Compusi

organici : Benzo[a]pyrene,

Polychlorinated Biphenyls, TNT


 Parametrii

biologici: identificare bacterii

Imunosenzori - limita detectie


 Piezoelectrici

- 1 mg/L-1994

 Piezoelectrici

- 0.3 mg/L -1997

 Cu

fibre optice - 0.2 mg/L -1997

 Fluorescenta

- 0.3 mg/L -1997

 Electrochimici

- 0.1 mg/L - 1998

 Electrochimici

- 0.01 mg/L - 1997

 Amperometrici

- 0.02 mg/L - 1997

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Relative sensitivities of tests (approx)


Usual operating range
[Ab] or [Ag]
precipitation
immunoelectrophoresis
double/radial diffusion
immunofluorescence

10 g/ml - 1 mg/ml

0.1 - 10 g/ml

ELISA (colour)
(chemiluminescence)

0.1 - 10 ng/ml
0.01 - 10 ng/ml

radioimmunoassay

0.01 - 10 ng/ml

Bibliografie
1.

Kuby, J., Immunology, W.H. Freeman and Company, New


York, 1997, 159-60.

2.

Prescott, L.M., Harley, J.P., Klein, D.A., Microbiology,


Wm.C. Brown Publishers London, 1996, 590-595.

3.

Roitt, I., Essential Immunology, Bleckwell Science Ltd.,


Oxford, 1997, 112-114.

4.

Analytical Biotechnology. Principles and Applications,


Billiet, H., Draghici, C., (Eds.), Editura Universitatii
Transilvania din Brasov, (2004)

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