uji imunologi - fk uns 2015 - tonang
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TES-TES IMUNOLOGIS DI LABORATORIUM
DIKOMPILASI OLEH:
TONANG DWI ARDYANTO
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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
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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.
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Primary immune phenomena
Secondary immune phenomena
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Ag Ab Kompleks Imun
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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
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Prozone,
Tak ada presipitasi
Equivalent zone,
Presipitasi
Post zone,
Tak ada presipitasi
Gambar 4. Berbagai macam rasio Ag Ab dan
implikasinya
= ANTIBODI
= ANTIGEN
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PR
ES
IPIT
AS
I A
NT
IGE
N-A
NT
IBO
DI
Prozone KONSENTRASI ANTIGEN Postzone
Ekses antibodi Seimbang Ekses antigen
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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
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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
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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
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Gambar 6. Uji presipitasi tabung
Ag.
Serum dengan Ab
Inkubasi
Presipitasi
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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
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APLIKASI KLINIS UJI PRESIPITASI
Uji Tabung : VDRL - Makro
Uji Slide : VDRL - Mikro
Uji Tabung Kapiler : Penentuan CRP
RID : Penentuan kelas Ig
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IMUNOELEKTROFORESIS
Migrasi protein serum di dalam gel dan apabila bertemu dengan antigen
yang sesuai akan terjadi presipitasi
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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
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Ag. pada permukaan sel
Ab.
Aglutinasi
Gambar 11. Prinsip dasar reaksi aglutinasi
UJI AGLUTINASI
Tak larut
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+ - Gambar 12. Uji Aglutinasi Slide
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Gambar 13. Uji Aglutinasi tabung
Serum ( Ab )
Susp.
Ag
Inkubasi
Aglutinasi
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AGLUTINASI TAK LANGSUNG
A. AGLUTINASI PASIF
B. Ab TAK LENGKAP
a. Ab Monovalen
b. Lokasi Tersembunyi / Ukuran Terlalu Kecil ( Ig. G )
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+ +
Ag Larut Partikel
Partikel disalut Ag
Ab dalam serum
Aglutinasi Gambar 14. Uji aglutinasi pasif
Partikel:
Sel darah merah
Lateks
Carbo adsorben
(Ko-aglutinasi)
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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
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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
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Komplemen
Sensitized cell
Ab
Ag pada
permukaan sel
= Komplemen
Gambar 15 . Prinsip dasar uji lisis imun
Uji Lisis Imun
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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
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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
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AN EXAMPLE OF THE COMPLEMENT FIXATION TEST.
Fig. 17.14 Complement fixation test.
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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)
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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)
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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).
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Imunofloresen assay
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Fig. 17.15 Immunofluorescence testing
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Fig. 17.15 Immunofluorescence testing
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KELEMAHAN UJI IF
Peralatan canggih dan mahal
Perlu tenaga terlatih
Per hari maks 25 slide / analis
Sukar dibuat otomatis
Pelaksanaan agak kompleks & membosankan
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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
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Gambar 21. Prinsip dasar Uji RIA kompetitif
R
R
R
R
R
R R
RADIATION
COUNTER
Cuci
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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
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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
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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
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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
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ENZYME-LINKED IMMUNOSORBANT
ASSAY (ELISA)
Indirect ELISA Sandwich ELISA
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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.
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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.
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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 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.
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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.
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TO DETECT ANTIGEN (SANDWICH ELISA):
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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.
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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
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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
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IMUNOKROMATOGRAFI
Imunokromatografi Lateral flow test Membacanya cukup dgn mata saja Tidak membutuhkan substrat Penggunaan colloidal gold waktu inkubasi pendek (
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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