11 Tehnici Imunochimice (1)
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Transcript of 11 Tehnici Imunochimice (1)
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Tehnici imunochimice
Antigen (Ag)
Anticorpi
anticorpi monoclonalianticorpi policlonali
Haptene
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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
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Imunodifuzia simpla
(radiala, Mancini)
Imunodifuzia dubla
monospecificitate polispecificitatenespecificitate
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Imunodifuzia dubla
monospecificitate polispecificitatenespecificitate
Imunodifuzie dubla - Identitate
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Imunodifuzie dubla - Neidentitate
Imunodifuzie dubla Identitatepartiala
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Contraimunoelectroforeza
Electroforeza serului normal
Imunoelectoforeza- model Grabar- in rachete
- bidimensionala
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Electroforeza serului normal
albumine-globuline
-globuline
-globuline
Imunoelectroforeza
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Imunoelectroforeza
Ser normal
IgG
Imunoelectroforeza
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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
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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
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Masurarea inaltimii picului determinarecantitativa
Imunoelectroforeza bidimentionala
Radioimunodetectii (RIA)
125I, 3H, 14C, 32P, 35S
Dezavantaje
Avantaje
Limita de detectie < 0.001g
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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
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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)
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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
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Enzyme Immunoassay
serum (sample)
conjugate
E E
substrate
negative reaction positive reaction
serum (sample)
conjugate
EE
substrate
negative reaction positive reaction
Enzyme Immunoassay
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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
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Microscopie electronica
Markeri: feritina, aur coloidal
Vizualizare cu microscopie electronica
mici puncte negre
Anticorpi marcati magnetic
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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
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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 SDS-
poliacrilamida
- transfer pe membrana
- identificare - interactia cu anticorpul primar si
anticorpul secundar marcat
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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
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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
S
P
ANALIT
BIORECEPTOR
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 -1997Fluorescenta - 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]
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)