11 tehnici imunochimice (1)

8/10/2019 11 Tehnici Imunochimice (1)

1/25

1

Tehnici imunochimice

Antigen (Ag)

Anticorpi

anticorpi monoclonalianticorpi policlonali

Haptene


2/25

2

Interactiile antigen-anticorp

Necovalente

Reversibile

Specifice

Exoterme

C - CH2- CH2-

O

H2N

- CH2- O - H

- CH2- CH - CH3

CH3H3C - CH - CH2-

H3C - CH2

- CH2- CH2- CO

O-

H3N+- CH2- CH2-

Antigen Antibody

van der Waals interacti

hydrogen bonds

ionic bonds

Metode imunochimice

Contraimunoelectroforeza si

TEHNICILE IMUNE (IA)

directe

indirecte(cu markeri)

Imunodifuzia (ID)IDD

IDS

Imunodifuzia dubla

Imunodifuzia simpla

Imunoelectroforeza (IEF

Aglutinarea

Fixarea complementului

Radioimunodetectii (RIA)

Metode neizotopice

Enzyme IA

Flurescence IAsi LuminescenceIA

Alte IA


3/25

3

Imunodifuzia simpla

(radiala, Mancini)

Imunodifuzia dubla

monospecificitate polispecificitatenespecificitate


4/25

4

Imunodifuzia dubla

monospecificitate polispecificitatenespecificitate

Imunodifuzie dubla - Identitate


5/25

5

Imunodifuzie dubla - Neidentitate

Imunodifuzie dubla Identitatepartiala


6/25

6

Contraimunoelectroforeza

Electroforeza serului normal

Imunoelectoforeza- model Grabar- in rachete

- bidimensionala


7/25

7

Electroforeza serului normal

albumine-globuline

-globuline

-globuline

Imunoelectroforeza


8/25

8

Imunoelectroforeza

Ser normal

IgG

Imunoelectroforeza


9/25

9

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


10/25

10

Imunoelectroforeza tip racheta

Imunoelectroforeza bidimentionala

Se aplica pentru analize

complexe de amestecuri

de Ag

1. Ag se separa prinelectroforeza.

2. a 2-a electroforeza pe o

directie perpendiculara,

printr-un gel care contine

Ac


11/25

11

Masurarea inaltimii picului determinarecantitativa

Imunoelectroforeza bidimentionala

Radioimunodetectii (RIA)

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

Dezavantaje

Avantaje

Limita de detectie < 0.001g


12/25

12

Fluoroimunodetectii

Tehnica foarte sensibila

imunofluorescenta imagistica =

imunohistochimie

Aplicatii: chimie analitica, biochimie,

biologie celulara, fiziologie, nefrologie,cardiologie, stiintele mediului.

ar er uorescentFluorophore Molar

absorption

(L/mol)

ex(nm) em(nm) Decay time

(ns)

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-

453

648-

669

Eu3+--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


13/25

13

Fluoroimunodetectii

ImunohistochimieDetectia caspazei-3 activate

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

(a) (b)


14/25

14

EIA - Enzyme ImmunoassayELISATehnici imunoenzimatice

Enzime: Peroxidaza (horseradish

peroxidase), PAL (fosfataza alacalina),

glucozoxidaza

Interactie Ag- Ac (o specie marcata

enzimatic) si interactie E-S

Enzyme Immunoassay


15/25

15

Enzyme Immunoassay

serum (sample)

conjugate

E E

substrate

negative reaction positive reaction

serum (sample)

conjugate

EE

substrate

negative reaction positive reaction

Enzyme Immunoassay


16/25

16

ImunohistochimieApoptosis test - TUNEL, pentru celule C2C12 crescutein mediu cu deficit de colina (5 M cholina)

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

Ac marcati cu peroxidaza detectie su sistemABC apa oxigenta - diaminozenzidina

Tehnici biologie moleculara

Biosenzori


17/25

17

Microscopie electronica

Markeri: feritina, aur coloidal

Vizualizare cu microscopie electronica

mici puncte negre

Anticorpi marcati magnetic


18/25

18

Anticorpi marcati magnetic

Imunocromatografie- Cromatografie de afinitate

antigens + mobile phase

stationary phase

with immobilized antibody

column

pH variation

signal

pH variation

- antibody

- specific antigen

- unspecific antigen


19/25

19

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


20/25

20

Western blotting (Immunoblotting)Radioizotopi

Enzime cuplate cu anticorpi secundari

Aur cuplat cu anticorpi secundari

Sistem avidina-biotina

Chemiluminescenta

Western blotting (Immunoblotting)

Identificare HIV

1 - refeinta2 - blank

A, B, C - probe reale


21/25

21

Southern blotting probe DNA

Northern blotting probe RNA


22/25

22

Imunosenzori

BIOSENZOR

= elemente biologice sau cu activitate

biologica (enzime, anticorpi, DNA,

celule, tesuturi) + traductori

S

P

ANALIT

BIORECEPTOR

TRADUCTOR

AMPLIFICATOR DE SEMNAL


23/25

23

Imunosenzor


24/25

24

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 -1997Fluorescenta - 0.3 mg/L -1997

Electrochimici - 0.1 mg/L - 1998

Electrochimici - 0.01 mg/L - 1997

Amperometrici - 0.02 mg/L - 1997


25/25

Relative sensitivities of tests (approx)

Usual operating range [Ab] or [Ag]

precipitationimmunoelectrophoresisdouble/radial diffusion

10 g/ml - 1 mg/ml

immunofluorescence 0.1 - 10 g/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)

11 tehnici imunochimice (1)

Documents