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

10

Imunoelectroforeza bidimentionala

Masurarea inaltimii picului determinare

cantitativa

Radioimunodetectii (RIA)

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

Dezavantaje

Avantaje

Limita

de detectie < 0.001 g

11

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

12

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

13

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

14

Enzyme Immunoassay
serum (sample)

serum (sample)

conjugate
conjugate

E
E

substrate

substrate

negative reaction

positive reaction

negative reaction

positive reaction

Enzyme Immunoassay

15

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

16

Microscopie electronica

Markeri:

feritina, aur coloidal

cu microscopie electronica 
mici puncte negre

Vizualizare

Anticorpi marcati magnetic

17

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

18

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

19

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

20

Southern blotting probe DNA

Northern blotting probe RNA

21

Imunosenzori

BIOSENZOR

= elemente biologice sau cu activitate

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

ANALIT
S
BIORECEPTOR
P
TRADUCTOR

AMPLIFICATOR DE SEMNAL

22

Imunosenzor

23

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

24

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)

25