IHK Labelling 2013

7/24/2019 IHK Labelling 2013

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Labelling_Conjugated


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Monoclonal & Polyclonal

Polyclonal Monoclonal

Contains many antibodies

recognizing many

detedminants on an antigen

Contains a singleantibody

recognizing only a single

determination

Various classesof

antibodies are present(IgG,IgM,and so on)

Single classof antibody

produced

Can make a specific antibody using only

a igly purified antigen

Can make a specific antibody using an

impure antigen

!eproducibility andstandardization difficult

"igly reproducible


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Antibody labelling methods:

1. Immunoenzyme method: An enzyme is used as the label.Peroxidase, alkalin phosfatase, glucose oxidaseChromogen staining chemical! must be used

". Immunofluorescence method: A fluorochrome is used as

the label.A#CA, $luorescein, $I%C, &odomine, %&I%C, %exas &ed.$luorescence microscope 'ith appropriate filter or confocallaser scanning microscope must be used to (isualize.

). Immunogold method: colloidal gold particles are used asthe label.*sually used in electron microscopy.


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Immunolabelling methods:

1. +irect method: %he antigen directly binds to its specificlabelled antibody primary antibody!

$ast to get the resultsabeling intensity is lo'

*sed for kidney or skin biopsies.


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+irect method


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". Indirect method:Primary antibody is unlabelled. Asecondary antibody 'hich is labeled! is used.

-econdary antibody must be raised against the immunoglobulin of the

species 'hich the primary antibody is made in.

etting results takes longer#ore sensiti(e#ore economic


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Indirect method


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$luorescence #ethod

%exas &ed,

&odamin,

Cy)

A#CA

$I%C,

Cy"


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). *nlabelled antibody methods:/nzym0antienzym methodPeroxidase antiperoxidase PAP!Alkaline phosphatase 0 anti0Alkaline phosphatase APAAP!

#ost sensiti(e results2idely usedApplied on paraffin, cryostat sections or on smears.


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Peroxidase

antiperoxidasePAP!

Alkaline phosphatase 0

anti0Alkalinephosphatase APAAP!


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3. A(idin04iotin method:*ses the high affinity of A(idin glycoprotein! for biotin

(itamin!.A complex of a(idin0biotin0enzym peroksidaz! is

necessary.

-treptoa(idin can be used instead of a(idin.

%he secondary antibody is labelled 'ith biotin 'hichinturn binds to a(idin in the a(idin0biotin0enzym complex.

5ery high sensiti(ity*sed in research more than routine studies. It is longer andmore expensi(e.


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In order to (isualize the enzymes labelling the antibodies'ith light microscope, enzyme substrate reactions, 'hich

con(ert colorless chromogens into (isible colored end0products, is used.:

Peroxidase0 hydrogen peroxide0 diaminobenzidine+A4!:

)0amino060ethylcarbazole A/C!:

70chloro010naphthol C8!: 9;* #A5

to detect:

Proteins, carbohidrates, nucleic acids, lipits

%ypes of the secreting cells

#embrane antigens

-tructural antigens 'ithin the cytoplasm Antigen localised in the nucleus

Immunohistochemistry is fre=uently used both inexperimental and diagnostical studies

ight, flouresans, confocal laser scanning or electronmicroscope is used in order to (isualize the labeledantibody0antigen complex.


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%he aim of the immunohistochemistry

to perform most specific immunohistochemicalstaining?

by cousing least damage on the cell or tissue,

by using least amount of antibody, in shortest time,

'ith least backgroung staining.


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Immunohistochemistry protocol 01

4efore incubation 'ith antibody: +eparaffinization.

&emo(ing the fixati(e: 'ashing in buffer solutionsphosphate buffer, %ris0@Cl buffer, @/P/- buffer, etc!

8eutralization of the endogeneus peroxidase 4locking: co(ering the non0immunological sticky

sites on tissues bo(ine serum albumine, non0immune normal serum, gelatine, milk!

4locking the surface tension: %'een ", %riton B01,

8aCl!


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%issue Preparation

$ixation: Immersion or perfusion fixation 8eutral formaline

Paraformaldehyde

Paraformaldehyde (e picric acid 4ouins solution!

-ectioning:

#icrotome: paraffin blocks

Cryostat: frozen tissue

5ibratome: fixed hard tissue

-ections on a slide PAP Pen! $loating sections


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Incubation:Primary antibody: *sed in a solution at different dilutions 'iththe blocking agent. %he incubation time changes according tothe properties of the antigen or antibody as 'ell as dependingon the temperature.

-econdary antibody: #ust be raised in the species otherthan the species of 'hich the cells or tissues are taken orthe primary antibody is raised. It should specificallyrecognize the immunoglobuline of the species in 'hich theprimary antibody is raised.

abelling: Immunoenzyme, immunoflourescence, immunogold

#icroscopical analyses

Immunohistochemistry protocol 0"


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+etermining the -ecretory

Contents of the8euroendocrine 8eurones


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%he use of the Immunohistochemistry:

Intercellular antigens, for instance: immunoglobulines ofthe kidney glomerular basal membrane

Cell surface antigens, tissue antigen for diagnosingautoimmune diseases

Protein hormones in histopathological diagnosis -oluble antigens of the cell +iagnosis of the endocrine tumors -mall amounts of peptides in endocrine or neuroendocrine

cells Immunodeposits %umoral markers %umor typing


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The basic p"inciple of

i!!noflo"escence To se a flo"escent co!pond -sally

flo"escein. to detect the binding of antigen and

antibody

The Ab is labelled 'ith the flo"escent co!pond

/nde" a flo"escence !ic"oscope0 flo"escein

appea"s b"ight g"een 'he"eve" the binding occ"s


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/sing the flo"escence !ic"oscope

Select the co""ect filte" bloc) fo" the

flo"escent co!pond

Flo"escence fades 1ic)ly nde" /# light2t"y to li!it the ti!e of expos"e to /# as

!ch as possible

/se high speed fil!s fo" photog"aphy


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Di"ect *!!noflo"escence

The ai! is to identify the p"esence and

location of an antigen by the se of a

flo"escent labelled specific antibody


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Medical applications of di"ect *F

Renal diseases fo" evidence of i!!ne

deposition

S)in diseases fo" evidence of i!!nedeposition

Detection of specific antigens0 especially

those of infective o"ganis!s


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Application in "enal diseases

*g+


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A section of )idney is placed on a slide2 a

flo"escein3labeled antigloblin -specific fo" *g+0

in this case. is added0 then "insed a'ay

The p"esence of flo"escence in the glo!e"li

indicates that *g+ 'as deposited p"io" to the biopsy

*g+ is deposited in g"anla" cl!ps along the

capilla"y 'alls0 enabling a diagnosis of!e!b"anos glo!e"loneph"itis in this case


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,he!il!inescent labels

Advantages

Size

Sensitivity

Disadvantages

%a"d'a"e


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Doble labelling


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*ndi"ect *!!noflo"escence


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The ai! is to identify the p"esence of

antigen specific antibodies in se"!4 The

!ethod is also be sed to co!pa"econcent"ation of the antibodies in se"a4


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A )no'n antigen is placed on a slide2 the

patient5s se"! is added0 then "insed a'ay4

A flo"escein3labeled antigloblin is added0

then "insed a'ay4

The p"esence of flo"escence ove" the

antigen indicates the p"esence of antibodiesto this antigen in the patient4


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Diagnosis of (acte"ial Diseases

,lost"idial diseases -di"ect.

Brucella canis-indi"ect.

Afipia catei0 cat sc"atch disease -indi"ect.

Borrelia burgdorferi-indi"ect.

Coxiella burnetii0 6 Feve" -indi"ect.

Rickettsia rickettsiae0 Roc)y Montain

Spotted Feve" -indi"ect.


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Diagnosis of #i"al Diseases

"abies vi"s -di"ect.

bovine i!!nodeficiency3li)e vi"s -indi"ect.

canine co"onavi"s -indi"ect.

canine diste!pe" -indi"ect.

feline infectios pe"itonitis -co"ona3. vi"s -di"ect.

po"cine "espi"ato"y and "ep"odctive synd"o!e-indi"ect.


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Diagnosis of P"otozoal Diseases

(abesia species -indi"ect.

h"lichia species -indi"ect.

Toxoplas!a gondii -indi"ect.

T"ypanoso!a c"zi -indi"ect.

,"yptospo"idia7+ia"dia -di"ect.

ncephalitozoon cnicli -indi"ect.

8eospo"! canin! -di"ect0 indi"ect.


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So!e exa!ples



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Indirect #luorescent $ntibody %est for

$ntibodies to Toxoplasma gondii

Toxoplasmao"ganis!s a"e )illed and placed on the slide2

the patient9s se"! is added0 then 'ashed a'ay4

A flo"escein3labeled antigloblin is added0 then 'ashed

a'ay4

The p"esence of the g"een flo"escence otlining the T.

gondii o"ganis!s indicates the p"esence of antibodies in

the patient5s se"!4


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*!!ne3Mediated Diso"de"s

antinclea" antibody -A8A. test -fo"

diagnosis of syste!ic lps e"ythe!atoss.

Di"ect flo"escent antibody test fo"deposition of Abs in tisses0 e4g4 )idney0

s)in


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Indirect #luorescent $ntibody %est

for $ntinuclear $ntibodies ,ells f"o! a clt"ed cell line a"e placed on a

slide2 the patient5s se"! is added0 then "insed

a'ay4 A flo"escein3labeled antigloblin is added0 then

"insed a'ay4

The p"esence of flo"escence in the ncles ofthese cells indicates the p"esence of antibodies to

nclea" antigens in the patient4


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T%A8:S


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$*SA

The $*SA techni1e is sed 'idely to

detect and 1antitate o"ganis!s and7o" thei"

p"odcts in foods0 and synopses of so!e ofthese applications a"e p"esented belo'4 Fo"

!o"e details0 the cited "efe"ences shold be

conslted4


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nzy!e3lin)ed i!!noso"bent assay

Mic"otite" 'ell

E E E E E

Speci!en ;nd antibody

E

Sbst"ate

S P


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$*SA -va"iation


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$*SA -va"iation ;.

Mic"otite" 'ell

Speci!en $abeled antibody

E

E E E E

EEE

Documents

IHK Labelling 2013