TES-TES IMUNOLOGIS DI LABORATORIUM

DIKOMPILASI OLEH:
TONANG DWI ARDYANTO
Beberapa Istilah penting dlm Imunoasai
a. Spesifitas dari Ab
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.
 Antigen I Antibodi I
X x X x
Y y Y y
Z z Z z

Z
Antigen II Antibodi II
Y
V v X x
W w w
X x v

Gambar 2. Kompleks dua antigen yang memiliki satu

epitop yang sama (X) dan berbagai macam antibodi
yang mungkin terbentuk
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 µL/mL)
Hasilnya diinterpolasi ke dalam kurva baku.
Primary immune phenomena

Ag Ab Kompleks Imun

Secondary immune phenomena

—  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, Equivalent zone, Post zone,
Tak ada presipitasi Presipitasi Tak ada presipitasi

Gambar 4. Berbagai macam rasio Ag – Ab dan

implikasinya
= ANTIBODI
= ANTIGEN
 Ekses antibodi Seimbang Ekses antigen
PRESIPITASI ANTIGEN-ANTIBODI

Prozone KONSENTRASI ANTIGEN Postzone

IMUNOASAI

KADAR BAHAN

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

Hasil reaksi tak tampak Hasil reaksi DAPAT

DILIHAT
♦ Presipitasi/RID
FAKTOR PENGUAT (LABEL)
♦ UJI AGLUTINASI

IF RIA EIA ICA

Homogen Heterogen
= ELISA
 JENIS IMUNOASAI
Ada 2 jenis imunoasai.
I. IMUNOASAI TAK BERLABEL
II. IMUNOASAI BERLABEL

I. IMUNOASAI TAK BERLABEL

♦ UJI PRESIPTASI
♦ UJI AGLUTINASI
♦ UJI FIKSASI KOMPLEMEN
♦ UJI NETRALISASI TOKSIN
UJI PRESIPITASI

Ag yang
larut Antibodi

Presipitasi adalah bila Ag + Ab dalam

bentuk larutan menghasilkan suatu
agregasi yang terlihat dengan mata PRESIPITASI

Gambar 5. Prinsip dasar uji presipitasi

 Ag.

Inkubasi

Serum dengan Ab Presipitasi

Gambar 6. Uji presipitasi tabung

 Sera baku
1
Tes serum

8 2

3
Antisera
7
dalam agar
Tes serum
4
6
Tes serum
Tes serum 5
Tes serum
GAMBAR 10. R.I.D
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
UJI AGLUTINASI
Ag. pada permukaan sel Aglutinasi
Ab.

Tak larut

Gambar 11. Prinsip dasar reaksi aglutinasi

 + -
Gambar 12. Uji Aglutinasi Slide
 Susp.
Ag

Inkubasi

Aglutinasi

Serum ( Ab )

Gambar 13. Uji Aglutinasi tabung

 AGLUTINASI TAK LANGSUNG

A. AGLUTINASI PASIF

B. Ab TAK LENGKAP

a. Ab Monovalen

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

 + +
Partikel Ab dalam serum
Ag Larut Partikel disalut Ag

Partikel:
Sel darah merah
Lateks
Carbo adsorben

(Ko-aglutinasi)
Aglutinasi
Gambar 14. Uji aglutinasi pasif
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
 Uji Lisis Imun
Komplemen

Ab
Ag pada Sensitized cell
permukaan sel

= Komplemen

Gambar 15 . Prinsip dasar uji lisis imun

A.

Komplemen Komplemen
Tak ada
C C Lisis
Serum Terikat
dgn. Ab
Sensitized SDM Uji Positif
B.
Komplemen Komplemen

Serum
C C Lisis
tanpa Ab Bebas
Uji Negatif
Gambar 16 . Uji Fiksasi Komplemen
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)
1. IMUNOASAI FLUORESENS (IF)
Mikroskop
Fluoresens
CUCI
Ab diket berlabel cat
fluoresens
Ag tak diket.
Fiksasi pada
slide

Kompleks Ag-Ab
Berfluoresensi
Gambar 18. Prinsip dasar uji imunofluoresens
langsung (direct).
 Cuci AHG dilabel
Fluorescein

Ab tak
Ag diket. diket Kompleks Ag – Ab Cuci
tak tampak

Mikroskop
Fluoresens
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

2. Uji RIA
R
R R
R
R
R RR
R

Radioisotop : 3H Thymidin, 131 I

Radiation
Gambar 20. Prinsip dasar Uji RIA
Counter
R = label radioisotop
 R R
R R
R
R
R Cuci

RADIATION
COUNTER

Gambar 21. Prinsip dasar Uji RIA kompetitif

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
Labeled
Anti-Ig
•  Immobilize Ag
Ab in
•  Incubate with sample Patient’s
sample
•  Add labeled anti-Ig
•  Amount of labeled Ab bound Immobilized
Ag
is proportional to amount of
Ab in the sample Solid
Phase

•  Quantitative
DOUBLE ANTIBODY ELISA
•  Ag detection
•  Immobilize Ab Labeled
•  Incubate with sample Ab
•  Add labeled antibody Ag in
•  Amount of labeled Ab bound is proportional to Patient’s
the amount of Ag in the sample sample Ag
Immobilized

Solid
Phase

•  Quantitative
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 Prior to Test
needed to bind to a known
amount of labeled Ag + !

Labeled
–  Use predetermined
Ag
amounts of labeled Ag
and Ab and add a Test
sample containing
unlabeled Ag as a + + ! +
competitor
Labeled Patient’s
Ag sample
 COMPETITIVE ELISA FOR AG
•  Method cont.
•  Determine amount of
labeled Ag bound to Ab Test
•  NH4SO4 + + ! +

•  anti-Ig
Solid Labeled Patient’s Solid
•  Immobilize the Ab Phase Ag sample
Phase

–  Concentration determined from a standard curve

using known amounts of unlabeled Ag

•  Quantitative
–  Most sensitive test
 IMUNOKROMATOGRAFI

Imunokromatografi
§  Lateral flow test
§  Membacanya cukup dgn mata saja
§  Tidak membutuhkan substrat
§  Penggunaan colloidal gold waktu inkubasi pendek (<15
menit)

Kerugian :
§  Nitrocelulose membrane tdk stabil pada suhu ↑

Keuntungan :
§  Prosedur cepat (<15 menit) dan praktis
§  Nilai diagnostik baik
§  Stabil untuk jangka panjang
§  Relatif tidak mahal
PRINSIP DASAR ICT

A. Melacak Analit (Ag)

a.  Reaksi langsung (Double Antibody Sandwich)/Asai
Imunometrik à untuk melacak analit yang besar dan
memiliki > 1 epitop (LH,hCG dan HIV)
b.  Reaksi kompetitif/Hambatan kompetitif (competitive
Inhibition) à untuk melacak molekul kecil dengan
epitop tunggal

B. Melacak Ab à Indirect Assay