pemeriksaan penunjang dalam imunologi - dr tonang dwi ardyanto

7/26/2019 Pemeriksaan Penunjang Dalam Imunologi - Dr Tonang Dwi Ardyanto

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TES-TES IMUNOLOGIS DI

LABORATORIUM

Uji lab. imunologis:1. Reagen antigen ditambahkan pada

antibodi yang terikat pada gel, lempengan(plate) atau manik-manik.

2. Reagen antibodi ditambahkan padaantigen yang terikat.

3. Antigen dan antibodi bebas dicampurkan.

4. Menilai fungsi sel kekebalan in vitro

pada prinsipnya meniru secara in vitro reak-si imunologis yang terjadi dalam tubuh.


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Reaksi Coombs & Gell

Reaksi tipe I : Ab terikat, Ag bebas

Reaksi tipe II : Ab bebas, Ag terikat

Reaksi tipe III : Ab bebas, Ag bebas

Reaksi tipe IV: hipersensitivitas

Uji fungsi limfosit

Reaksi Coombs

& Gell uji :I : Ag ( ) bebas

vs Ab (Y) ter-ikat pd sellab. : Ab (Y)diikatkan pdmasa padat

II : Ab (Y) bebasvs Ag ( ) ter-ikat lab. :

Ag diikatkanpd masa padat

III : Ab (Y) be-bas vs Ag ( )bebas lab.:Ab ( Y) dlm la-rutan direaksi-kan dg Ag ( )dlm larutan


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Reaksi tipe I : Ab terikat, Ag bebas(asma, syok anafil.)

Reaksi tipe II : Ab bebas, Ag terikat (reaksi transfusi)


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IMMUNO AGGLUTINASI

Metoda pemeriksaan interaksi Ag-Ab, Ab spesifik interaksi dg. Ag yangmelekat pada permukaan partikel/sel.

Ukuran partikel: 200-250 nm.

Reaksi:

Berlangsung cepat (menit/jam).Sensitivitas analitik > tinggi dp. Reaksi presipitasi.

Mekanisme:

Tahap 1; Aggregasi partikel/sel oleh Ab membentuk kisi-kisilebar makroskopis gumpalan halus.

Tahap 2; pembentukan sedimen antara gumpalan halusmembentuk gumpalan > besar.

Ig M mempunyai 10 binding site agglutinasi > kuat dp. Ig G.


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Syarat reaksi imunoagglutinasi:

Ag melekat pada partikel/sel.

Ag-Ab ada dalam proporsi optimal.Perlu media elektrolit, dipengaruhi; suhu, daya ionik, pH.

Jenis Agglutinasi:

I 1. Agglutinasi Langsung

2. Agglutinasi tak langsung

Ag yang larut diadsorbsikan pada carier (lateks, bentonit,

eritrosit).

Pemeriksaan carier lateks;

Rheumatoid faktor, CRP, anti TG Ab., Aso, Cryptococcal

Ag, histoplasmosis, Trichinosis

Pemeriksaan carier eritrosit:

a. Active haemagglutination.

Direk A.H. : tes golongan darah.

Indirek A.H.: Coombstes ; direk

indirek

b. Passive haemagglutination.

Direk: TPHA anti HBs, RPHA HBs Ag, Rubella PHA.

Indirek: Deteksi non agglutinating Ab ( anti serum terhadap

IgG).

3. Agglutinasi Inhibisi.

HI Virus dengue.HCG .

II. Agglutinasi bakteri:- tabung

- Slide.

III. Agglutinasi degan zarah kaku: Lateks, bentonite, colloidon.


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Agglutinasi langsung.

Reaksi Widal.

Agglutinasi

Ab. O

Ag. Ohne Hauch

Ag O/H

atau

Agglutinasi

Salmonella tifi Ab. S. tifi

Reaksi Golongan Darah.

Agglutinogen A Agglutinin Agglutinasi


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Protein A

Ag. ?

Stapilokokus Ab. Spesifik

Agglutinasi

Agglutinasi tak langsung

Mendeteksi Ag.:

E E

Eritrosit angsa Ag. Terlarut ? Agglutinasi

Contoh: RPHA HBs Ag

(Reverse Passive Haemagglutination)


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Mendeteksi Ab. Agglutinasi tak langsung

E E

Ag. Terlarut Eri kambing Eri terlapisi Ag Ab. ?

Agglutinasi

Contoh: PHA (TPHA)-anti HBs

INDIREK HA

Direk Coombstes.

E

Inkomplit Ab. Coomb' s Agglutinasi

Eritrosit


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Indirek Coombstes.

E

Coombs

Inkomplit Ab

serum

Agglutinasi

CRP (C Reactive Protein)

L

Ab pd Latek Ag protein

CRP serum pend.

Agglutinasi

Kelebihan CRP dibandingkan LED:

_ Infeksi akut: CRP +, LED , Infeksi sembuh: CRP , LED masih .


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ASTO (ASO)

Ag :Streptococcus Hemolyticus

Kadar Normal:


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Reaksi tipe II, komplemen terlibat

Reaksi tipe III : Ab bebas, Ag bebas), terbentuk

kompleks Ag-Ab (GNA, PJR)


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Reaksi tipe IV : tipe selular (peny. granuloma,

uji tuberkulin)

Pembacaan hasil

langsung (mata telanjang):

presipitasi

aglutinasi

dengan alat :

nefelometri

scanning

dengan bahan radioaktif

dengan pewarnaan

dengan fluoresensi


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Meningkatkan reaksi Ag-Ab

menggunakan media yang tepat :Larutan

gel

massa padat : plate, butir manik-manik

mengatur suhu optimum

menggunakan medan listrik

mengatur suhu optimum

menambahkan bahan tertentu

ELISA

Uses an enzyme system to show thespecific combination of antigen antibody

An enzyme labeled or linked to a specificantigen

A substrate

A color reader

Double antibody technique to detect andassay antigen

Indirect technique to Assay and antibody


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Indirect ELISA

Ab detection

Immobilize Ag

Incubate with sample

Add labeled anti-Ig

Amount of labeled Abbound is proportionalto amount of Ab inthe sample

Quantitative

SolidPhase

AgImmobilized

Ab inPatientssample

LabeledAnti-Ig

Double Antibody ELISA

Ag detection

Immobilize Ab

Incubate with sample

Add labeled antibody

Amount of labeled Ab

bound is proportionalto the amount of Agin the sample

Quantitative

SolidPhase

Ag

Immobilized

Ag inPatientssample

LabeledAb


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Enzyme-Linked ImmunoSorbant

Assay (ELISA)

Indirect ELISA Sandwich ELISA

1. Indirect ELISA

The steps of the general, "indirect," ELISA fordetermining serum antibody concentrations are:

1. Apply a sample of known antigen of known concentration toa surface, often the well of a microtiter plate. The antigen isfixed to the surface to render it immobile. Simple adsorptionof the protein to the plastic surface is usually sufficient.These samples of known antigen concentrations willconstitute a standard curveused to calculate antigenconcentrations of unknown samples. Note that the antigenitself may be an antibody.

2. The plate wells or other surface are then coated with serumsamples of unknown antigen concentration, diluted into thesame buffer used for the antigen standards. Since antigenimmobilization in this step is due to non-specific adsorption,it is important for the total protein concentration to besimilar to that of the antigen standards.

3. A concentrated solutionof non-interacting protein, such asBovine Serum Albumin(BSA) or casein, is added to all platewells. This step is known as blocking, because the serumproteins block non-specific adsorption of other proteins tothe plate.
http://en.wikipedia.org/wiki/Microtiter_platehttp://en.wikipedia.org/wiki/Microtiter_platehttp://en.wikipedia.org/wiki/Standard_curvehttp://en.wikipedia.org/wiki/Standard_curvehttp://en.wikipedia.org/wiki/Blood_plasmahttp://en.wikipedia.org/wiki/Bovine_Serum_Albuminhttp://en.wikipedia.org/wiki/Caseinhttp://en.wikipedia.org/wiki/Bovine_Serum_Albuminhttp://en.wikipedia.org/wiki/Blockinghttp://en.wikipedia.org/wiki/Blockinghttp://en.wikipedia.org/wiki/Caseinhttp://en.wikipedia.org/wiki/Blockinghttp://en.wikipedia.org/wiki/Blockinghttp://en.wikipedia.org/wiki/Caseinhttp://en.wikipedia.org/wiki/Bovine_Serum_Albuminhttp://en.wikipedia.org/wiki/Blood_plasmahttp://en.wikipedia.org/wiki/Standard_curvehttp://en.wikipedia.org/wiki/Microtiter_plate


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4. The plate is washed, and a detection antibody specificto the antigen of interest is applied to all plate wells.This antibody will only bind to immobilized antigen on

the well surface, not to other serum proteins or theblocking proteins.

5. The plate is washed to remove any unbound detectionantibody. After this wash, only the antibody-antigencomplexes remain attached to the well.

6. Secondary antibodies, which will bind to any remainingdetection antibodies, are added to the wells. Thesesecondary antibodies are conjugated to the substrate-specific enzyme. This step may be skipped if thedetection antibody is conjugated to an enzyme.

7. Wash the plate, so that excess unbound enzyme-antibody conjugates are removed.

8. Apply a substrate which is converted by the enzyme to

elicit a chromogenic or fluorogenic or electrochemicalsignal.

9. View/quantify the result using a spectrophotometer,spectrofluorometer, or other optical/electrochemicaldevice.

To detect antibody (indirect ELISA):


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2. Sandwich ELISA

A sandwich ELISA: Plate is coated with a capture antibody

sample is added, and any antigenpresent binds to capture antibody

detecting antibody is added, and bindsto antigen

enzyme-linked secondary antibody is

added, and binds to detecting antibody substrate is added, and is converted byenzyme to detectable form.

A less-common variant of this technique, called "sandwich" ELISA, is

used to detect sample antigen. The steps are as follows:

1. Prepare a surface to which a known quantity of captureantibody is bound.

2. Block any non specific binding sites on the surface.3. Apply the antigen-containing sample to the plate.4. Wash the plate, so that unbound antigen is removed.5. Apply primary antibodies that bind specfically to the

antigen.6. Apply enzyme-linked secondary antibodies which are

specific to the primary antibodies.7. Wash the plate, so that the unbound antibody-enzyme

conjugates are removed.8. Apply a chemical which is converted by the enzyme into a

color or fluorescent or electrochemical signal.9. Measure the absorbance or fluorescence or

electrochemical signal (e.g., current) of the plate wells todetermine the presence and quantity of antigen.


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To detect antigen (sandwich ELISA):

3. Competitive ELISA

A third use of ELISA is through competitivebinding. The steps for this ELISA are somewhatdifferent than the first two examples:

1. Unlabeled antibody is incubated in the presence of itsantigen.

2. These bound antibody/antigen complexes are thenadded to an antigen coated well.

3. The plate is washed, so that unbound antibody is

removed. (The more antigen in the sample, the lessantibody will be able to bind to the antigen in the well,hence "competition.")

4. The secondary antibody, specific to the primaryantibody is added. This second antibody is coupled tothe enzyme.

5. A substrate is added, and remaining enzymes elicit achromogenic or fluorescent signal.

For competitive ELISA, the higher the original antigenconcentration, the weaker the eventual signal.
http://en.wikipedia.org/wiki/Secondary_antibodyhttp://en.wikipedia.org/wiki/Secondary_antibody


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Competitive ELISA for antigen

Method

Determine amount ofAb needed to bind toa known amount oflabeled Ag

+

Prior to Test

LabeledAg

+

Test

+

Patients

sample

Labeled

Ag

+

Use predeterminedamounts of labeled Agand Ab and add asample containingunlabeled Ag as acompetitor

Competitive ELISA for Ag Method cont.

Determine amountof labeled Agbound to AbNH4SO4

anti-Ig

Immobilize the Ab

Quantitative

Most sensitive test

+

Test

+

Patientssample

LabeledAg

+

Concentration determined from a standard curveusing known amounts of unlabeled Ag

SolidPhase

SolidPhase


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Antibodi diikatkan pada lempengan

(meniru reaksi tipe I)

Gb 1 dan 2 : Ab dilekatkan pada massa solid,

Gb 1 : Ag berlabel ditambahkan, kemudian Ag uji ditambahkan,

Gb 2 : Ag uji ditambahkan, kemudian Ab berlabel ditambahkan

Pengukuran : dilakukan pd Ag berlabel yg terdesak oleh Ag uji(Gb1) atau pada Ab berlabel yang terikat (Gb 2).


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Antibodi diikatkan pada gel

(meniru reaksi tipe II)

Pada gel ygmengandung Abdibuat sederetsumuran.

Ag dimasukkanke dlm sumur-an.

Ag akan berdifu-si ke sekeliling-nya dan bereak-si dg Ab dlm gel.

Luas lingkaranpresipitin berko-relasi dg kon-sentrasi Ag, di-bandingkan dg.Grafik standar


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Elektroforesis

Gb bawah: Abterikat pada gel, Agbebas begerak kearah kutub positifshg terjadi presipitatberbentuk spt roket.

Gb atas: Ab mau-pun Ag bebas dlmgel, Ab bergerak kearah kutub neg., Ag

bergerak ke arahkutub pos., kedua-nya bertemu ter-bentuk presipitin.

Antigen diikatkan pada massa

padat(meniru reaksi tipe II)


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Antigen bebas, antibodi bebas(meniru reaksi tipe III)


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Ag maupun Ab dimasukkan ke dalam sumuran yang dibuat padamedia gel. Akan terjadi difusi Ag maupun Ab. Pd Gb a. terjadi satusedangkan pada b. dua pita precipitin pd pertemuan Ag dan Ab.

Ouchterlony :memmperlihatkanreaksi Ag-Ab indivi-dual antara campuranAg dan Ab.

Gb 1 : garis presipitinmenunjukkan bahwaAg di sumuran kiridan Ag kanankeduanya mempunyaiepitop yg identik.

Gb 2 : Ag dlm sumurankiri tidak identik dg Ag

di sumuran kanan,terlihat dari adanyapresipitin yangbersilangan.

Gb 3 : Taji pada presipi-tin kanan menunjuk-kan bahwa Ag di su-muran kiri dan kananadalah identik parsial.


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Pada campur-an Ag yangtelah diberi

label radioaktifditambahkanAb spesifik.Kompleks Ag-Ab diendap-kan. Endapandipisahkan dariAg yangbebas, laludielektrofore-

sis. Dibacadengan skanerradioaktivitas

Gb 1. Pd tabung kanan maupun kiri terdapat Ag. Tbg kanan :terjadi reaksi Ag dg serum uji yang sesuai komplekAg-Ab

Gb 2. Setelah penambahan komplemen : pd tabung kirikomplemen yg ditambahkan tetap bebas.

Gb 3. Komplemen bebas di tabung kiri melisis sel indikator


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Gb 1. Komplemen diikatkan pada fase padat.

Gb 2. Kompleks Ag-Ab terikat pada komplemen

Gb 3. Pembacaan dg anti-IgG yg telah dilabel radioaktif

Antigen mikro-organisme(banyak Ag)dielektroforesispada gel shgterpisah satusama lain.

Hasil elektrofo-resis dipindah-kan ke lempengnitroselulose(blotting),ditambahkan Ab,dicuci, dst dibacadengan pelabelanradioaktif atau dgpewarnaan


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Adanya lisismembukti-kan terjadireaksi Ag-Ab dg ke-terlibatankomplemen.

Tidak ada li-sis berartireaksi Ag vsAb tidak

berpenga-ruh pada selindikator.

Gb bawahtabel buktiadanya ma-cam2kompl.

pemeriksaan penunjang dalam imunologi - dr tonang dwi ardyanto

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