TES-TES IMUNOLOGIS DI LABORATORIUM

DIKOMPILASI OLEH:

TONANG DWI ARDYANTO

Beberapa Istilah penting dlm Imunoasai

Ikatan Ab-Ag adalah spesifik seperti kunci-anak

kunci. Reaksi silang dapat terjadi dengan struktur

mol Ag lain yang mirip dengan Ag pasangannya

tergantung dari :

- profil spesifitas Ab-nya &

- kemurnian Ag-nya

Ab yang amat spesifik = Ab dengan binding

sites yang hanya dapat mengikat Ag dengan

struktur molekul yang unik/tertentu saja.

a. Spesifitas dari Ab

x

y

z

x

y

z

Antigen I Antibodi I

Antigen II

v

w

x v

w

x

X

Y

Z

X

Y

Z

V

W

X

X

Y

Z

Gambar 2. Kompleks dua antigen yang memiliki satu

epitop yang sama (X) dan berbagai macam antibodi

yang mungkin terbentuk

Antibodi II

b. Ukuran kuantitas Ab

Ada beberapa cara tentukan konsentrasi Ab dalam

serum.

- Kualitatif pos. /neg. adanya perubahan fisik dari

bahan pemeriksaan. (+/-)

- Semi kuantitatif ; ditentukan dengan pengenceran

serum secara progresif Titer (1/10, 1/100, 1/640)

- Kuantitatif ; ditentukan dengan menggunakan

beberapa sera baku kurva baku. Akurasi dicek

dengan serum kontrol. (100 pg/mL, 2 L/mL)

Hasilnya diinterpolasi ke dalam kurva baku.

Primary immune phenomena

Secondary immune phenomena

+

Ag Ab Kompleks Imun

Pembentukkan presipitat terjadi apabila konsentrasi Ag dan Ab seimbang (zona ekivalen = ZE)

Konsentrasi Ag berlebih Komplek Ag-Ab yg terbentuk larut kembali disebut postzone effect

Konsentrasi Ab berlebih Komplek Ag-Ab yg terbentuk tetap larut disebut prozone effect

ZE sempit Ag bersifat mudah larut

ZE lebar Ag bersifat tdk mudah larut, BM besar, & multikomponen Ag

Prozone,

Tak ada presipitasi

Equivalent zone,

Presipitasi

Post zone,

Tak ada presipitasi

Gambar 4. Berbagai macam rasio Ag Ab dan

implikasinya

= ANTIBODI

= ANTIGEN

PR

ES

IPIT

AS

I A

NT

IGE

N-A

NT

IBO

DI

Prozone KONSENTRASI ANTIGEN Postzone

Ekses antibodi Seimbang Ekses antigen

IMUNOASAI

KADAR BAHAN

RENDAH ( ng/ml, pg/ml ) TINGGI (mg/ml,ug/ml)

Hasil reaksi tak tampak

FAKTOR PENGUAT (LABEL)

IF RIA EIA

Homogen Heterogen

= ELISA

Hasil reaksi DAPAT

DILIHAT Presipitasi/RID

UJI AGLUTINASI

ICA

UJI PRESIPTASI

UJI AGLUTINASI

UJI FIKSASI KOMPLEMEN

UJI NETRALISASI TOKSIN

I. IMUNOASAI TAK BERLABEL

Ada 2 jenis imunoasai. I. IMUNOASAI TAK BERLABEL II. IMUNOASAI BERLABEL

JENIS IMUNOASAI

Ag yang

larut Antibodi

PRESIPITASI

Gambar 5. Prinsip dasar uji presipitasi

UJI PRESIPITASI

Presipitasi adalah bila Ag + Ab dalam

bentuk larutan menghasilkan suatu

agregasi yang terlihat dengan mata

Gambar 6. Uji presipitasi tabung

Ag.

Serum dengan Ab

Inkubasi

Presipitasi

Antisera

dalam agar

GAMBAR 10. R.I.D

1

3

4

5

6

7

8 2

Sera baku

Tes serum

Tes serum

Tes serum

Tes serum

Tes serum

APLIKASI KLINIS UJI PRESIPITASI

Uji Tabung : VDRL - Makro

Uji Slide : VDRL - Mikro

Uji Tabung Kapiler : Penentuan CRP

RID : Penentuan kelas Ig

IMUNOELEKTROFORESIS

Migrasi protein serum di dalam gel dan apabila bertemu dengan antigen

yang sesuai akan terjadi presipitasi

ELEKTROFORESIS

Gb atas : Ab mau-pun Ag bebas dlm gel, Ab bergerak ke arah kutub neg., Ag bergerak ke arah kutub pos., kedua-nya bertemu ter-bentuk presipitin.

Gb bawah : Ab terikat pada gel, Ag bebas begerak ke arah kutub positif shg terjadi presipitat berbentuk spt roket

Ag. pada permukaan sel

Ab.

Aglutinasi

Gambar 11. Prinsip dasar reaksi aglutinasi

UJI AGLUTINASI

Tak larut

+ - Gambar 12. Uji Aglutinasi Slide

Gambar 13. Uji Aglutinasi tabung

Serum ( Ab )

Susp.

Ag

Inkubasi

Aglutinasi

AGLUTINASI TAK LANGSUNG

A. AGLUTINASI PASIF

B. Ab TAK LENGKAP

a. Ab Monovalen

b. Lokasi Tersembunyi / Ukuran Terlalu Kecil ( Ig. G )

+ +

Ag Larut Partikel

Partikel disalut Ag

Ab dalam serum

Aglutinasi Gambar 14. Uji aglutinasi pasif

Partikel:

Sel darah merah

Lateks

Carbo adsorben

(Ko-aglutinasi)

APLIKASI KLINIS UJI AGLUTINASI

Uji Slide (lempeng): uji Widal slide

Uji Tabung : uji Widal tabung

Aglutinasi Tak Langsung: uji Rose-Waaler

III. UJI HEMAGLUTINASI : KULIAH Bank Drh

IV. UJI LISIS IMUN & FIKSASI KOMPLEMEN

Hampir sama dengan uji aglut. tak langsung,

Hanya Anti Ig diganti C Lisis Imun

UJI LISIS IMUN & FIKSASI KOMPLEMEN

Komplemen di dalam plasma sebanyak 3 mg/ml dalam bentuk inaktif

Jika bertemu dengan kompleks Ag-Ab komplemen menjadi aktif (melalui jalur klasik), dan menghasilkan

berbagai kaskade aktivasi, misalnya lisis dari sel target

Komplemen

Sensitized cell

Ab

Ag pada

permukaan sel

= Komplemen

Gambar 15 . Prinsip dasar uji lisis imun

Uji Lisis Imun

Gambar 16 . Uji Fiksasi Komplemen

A.

C C Tak ada

Lisis

Komplemen Komplemen

Terikat

Sensitized SDM

B.

C C Lisis Komplemen Komplemen

Bebas

Serum

dgn. Ab

Serum

tanpa Ab

Uji Positif

Uji Negatif

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

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

Gb 3. Komplemen bebas di tabung kiri melisis sel indikator

AN EXAMPLE OF THE COMPLEMENT FIXATION TEST.

Fig. 17.14 Complement fixation test.

II. IMUNOASAI BERLABEL

1. CAT FLUORESENS: IF

2. RADIOISOTOP: RIA

3. ENZIM: IMUNOASAI ENZIM ( EIA )

A. EIA HOMOGEN

B. EIA HETEROGEN (ELISA)

C. UJI IMUNO-PEROKSIDASE

4. EMAS KOLOIDAL: ASAI IMUNOKROMATOGRAFIK (ICA)

CUCI

Mikroskop

Fluoresens

Ab diket berlabel cat

fluoresens Ag tak diket.

Fiksasi pada

slide

Kompleks Ag-Ab

Berfluoresensi

Gambar 18. Prinsip dasar uji imunofluoresens

langsung (direct).

1. IMUNOASAI FLUORESENS (IF)

Cuci Ag diket.

Ab tak

diket

Cuci

Mikroskop

Fluoresens

Kompleks Ag Ab

tak tampak

AHG dilabel

Fluorescein

Kompleks Ag Ab AHG

berfluoresensi

Gambar 19. Prinsip dasar uji imunofluoresens

tak langsung (indirect).

Imunofloresen assay

Fig. 17.15 Immunofluorescence testing

Fig. 17.15 Immunofluorescence testing

KELEMAHAN UJI IF

Peralatan canggih dan mahal

Perlu tenaga terlatih

Per hari maks 25 slide / analis

Sukar dibuat otomatis

Pelaksanaan agak kompleks & membosankan

Gambar 20. Prinsip dasar Uji RIA

R = label radioisotop

R

R R R

R R

R

R

R

Radiation

Counter

2. Uji RIA

Radioisotop : 3H Thymidin, 131 I

Gambar 21. Prinsip dasar Uji RIA kompetitif

R

R

R

R

R

R R

RADIATION

COUNTER

Cuci

KELEMAHAN UJI RIA

Butuh alat mahal & tenaga terlatih

Waktu paruh reagens amat pendek ( 1,5 2 bln )

Perlu perlindungan khusus pd petugas lab.

Perlu tempat pembuangan reagens yang khusus

ELISA

Uses an enzyme system to show the specific combination of antigen antibody

An enzyme labeled or linked to a specific antigen

A substrate

A color reader

Double antibody technique to detect and assay antigen

Indirect technique to Assay and antibody

INDIRECT ELISA

Ab detection

Immobilize Ag

Incubate with sample

Add labeled anti-Ig

Amount of labeled Ab bound is proportional to amount of

Ab in the sample

Quantitative

Solid Phase

Ag Immobilized

Ab in Patients sample

Labeled Anti-Ig

DOUBLE ANTIBODY ELISA

Ag detection

Immobilize Ab

Incubate with sample

Add labeled antibody

Amount of labeled Ab bound is proportional to the amount of Ag in the sample

Quantitative

Solid Phase

Ag

Immobilized

Ag in Patients sample

Labeled Ab

ENZYME-LINKED IMMUNOSORBANT

ASSAY (ELISA)

Indirect ELISA Sandwich ELISA

1. INDIRECT ELISA

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

1. Apply a sample of known antigen of known concentration to a surface, often the well of a microtiter plate. The antigen is fixed to the surface to render it immobile. Simple adsorption of the protein to the plastic surface is usually sufficient. These samples of known antigen concentrations will constitute a standard curve used to calculate antigen concentrations of unknown samples. Note that the antigen itself may be an antibody.

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

3. A concentrated solution of non-interacting protein, such as Bovine Serum Albumin (BSA) or casein, is added to all plate wells. This step is known as blocking, because the serum proteins block non-specific adsorption of other proteins to the plate.

4. The plate is washed, and a detection antibody specific to 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 the blocking proteins.

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

6. Secondary antibodies, which will bind to any remaining detection antibodies, are added to the wells. These secondary antibodies are conjugated to the substrate-specific enzyme. This step may be skipped if the detection 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 electrochemical signal.

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

TO DETECT ANTIBODY (INDIRECT ELISA):

2. SANDWICH ELISA

A sandwich ELISA:

Plate is coated with a capture antibody

sample is added, and any antigen present binds to capture antibody

detecting antibody is added, and binds to antigen

enzyme-linked secondary antibody is added, and binds to detecting antibody

substrate is added, and is converted by enzyme 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 capture antibody 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 to determine the presence and quantity of antigen.

TO DETECT ANTIGEN (SANDWICH ELISA):

3. COMPETITIVE ELISA

A third use of ELISA is through competitive binding. The steps for this ELISA are somewhat different than the first two examples:

1. Unlabeled antibody is incubated in the presence of its antigen.

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

3. The plate is washed, so that unbound antibody is removed. (The more antigen in the sample, the less antibody will be able to bind to the antigen in the well, hence "competition.")

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

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

For competitive ELISA, the higher the original antigen concentration, the weaker the eventual signal.

COMPETITIVE ELISA FOR ANTIGEN

Method

Determine amount of Ab needed to bind to a known

amount of labeled Ag +

Prior to Test

Labeled Ag

+

Test

+

Patients sample

Labeled Ag

+

Use predetermined amounts of labeled Ag and Ab and add a sample containing unlabeled Ag as a competitor

COMPETITIVE ELISA FOR AG

Method cont.

Determine amount of labeled Ag bound to Ab

NH4SO4

anti-Ig

Immobilize the Ab

Quantitative

Most sensitive test

+

Test

+

Patients sample

Labeled Ag

+

Concentration determined from a standard curve using known amounts of unlabeled Ag

Solid Phase

Solid Phase

IMUNOKROMATOGRAFI

Imunokromatografi Lateral flow test Membacanya cukup dgn mata saja Tidak membutuhkan substrat Penggunaan colloidal gold waktu inkubasi pendek (

PRINSIP DASAR ICT

A. Melacak Analit (Ag)

a. Reaksi langsung (Double Antibody Sandwich)/Asai Imunometrik untuk melacak analit yang besar dan memiliki > 1 epitop (LH,hCG dan HIV)

b. Reaksi kompetitif/Hambatan kompetitif (competitive Inhibition) untuk melacak molekul kecil dengan epitop tunggal

B. Melacak Ab Indirect Assay