Tehnici imunochimice

Antigen (Ag)

Anticorpi
anticorpi monoclonali
anticorpi policlonali

Haptene

1
 Interactiile antigen-anticorp
Antigen Antibody

- CH2 - O - H O

Necovalente H2N
C - CH2 - CH2 -
hydrogen bonds

Reversibile - CH2 - CH - CH3

H3C - CH - CH2 - van der Waals interactions
CH3

Specifice H3C - CH2

O
- CH2 - CH2 - C

Exoterme O
- +
H3N - CH2 - CH2 - ionic bonds

Metode imunochimice

Imunodifuzia simpla IDS

Imunodifuzia (ID)
Imunodifuzia dubla IDD

directe Contraimunoelectroforeza siImunoelectroforeza (IEF)

Aglutinarea
TEHNICILE IMUNE Fixarea complementului
(IA)
indirecte Radioimunodetectii (RIA)
(cu markeri) Enzyme IA
Metode neizotopice Flurescence IA
si Luminescence IA

Alte IA

2
Imunodifuzia simpla
(radiala, Mancini)

Imunodifuzia dubla

monospecificitate – polispecificitate
nespecificitate

3
 Imunodifuzia dubla

monospecificitate – polispecificitate
nespecificitate

Imunodifuzie dubla - Identitate

4
Imunodifuzie dubla - Neidentitate

Imunodifuzie dubla – Identitate

partiala

5
Contraimunoelectroforeza

Electroforeza serului normal

Imunoelectoforeza
- model Grabar
- in rachete
- bidimensionala

6
 Electroforeza serului normal

albumine

α-globuline

β-globuline

γ-globuline

Imunoelectroforeza

7
 Imunoelectroforeza

Ser normal

Imunoelectroforeza

IgG

8
 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

9
 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 µg

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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 Molar λex(nm) λem(nm) Decay time
absorption (ns)
ε (L/mol)
Tetramethylrhodamine isothiocyanate 105 000 540 575 2

Rhodamine B-isothiocyanate 103 000 550 585 3

Fluorescein isothiocyanate 66 000 492 520 4.5

Lucifer yellow 13 000 430 540 14

Dansyl chloride 4 300 345 535 14

Pyrenebutyrate 40 000 340 385 100

Erythrosin B 101 000 492 517 108

Ethyl Eosin 101 000 520 545

Methyl umbelliferone 16 000 323 386

Chlorophyll 430- 648-

453 669
Eu3+-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

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
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 µM 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 

Vizualizare
mici puncte negre

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
pH variation
- unspecific antigen

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 SDS-
poliacrilamida
- transfer pe membrana
- identificare - interactia cu anticorpul primar si
anticorpul secundar marcat

19
Western blotting (Immunoblotting)

Radioizotopi

Enzime cuplate cu anticorpi secundari

Aur 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

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 Relative sensitivities of tests (approx)

Usual operating range

[Ab] or [Ag]

precipitation
immunoelectrophoresis 10 µg/ml - 1 mg/ml
double/radial diffusion

immunofluorescence 0.1 - 10 µg/ml

ELISA (colour) 0.1 - 10 ng/ml

(chemiluminescence) 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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