23/04/2014

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 pada
antigen yang terikat.
3. Antigen dan antibodi bebas dicampurkan.
4. Menilai fungsi sel kekebalan in vitro

pada prinsipnya meniru secara in vitro reaksi 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) terikat pd sel
lab. : Ab (Y)
diikatkan pd
masa padat
II : Ab (Y) bebas
vs Ag ( ) terikat
lab. :
Ag diikatkan
pd masa padat
III : Ab (Y) bebas vs Ag ( )
bebas
lab.:
Ab ( Y) dlm larutan direaksikan 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 yang
melekat 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-kisi
lebar makroskopis gumpalan halus.
Tahap 2; pembentukan sedimen antara gumpalan halus
membentuk 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.: Coombs tes ; 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.:

Eritrosit angsa

Ag. Terlarut ?

Agglutinasi

Contoh: RPHA HBs Ag

(Reverse Passive Haemagglutination)

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Mendeteksi Ab.

Agglutinasi tak langsung

E
Ag. Terlarut

Eri kambing

Eri terlapisi Ag

Ab. ?

Agglutinasi
Contoh: PHA (TPHA)-anti HBs

INDIREK HA
Direk Coombs tes.

Inkomplit Ab.
Eritrosit

Coomb' s

Agglutinasi

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Indirek Coombs tes.

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: <125 S.Todd (dewasa).
<200 S.Todd (anak).

Indikasi Pemeriksaan:
Demam Rematik Akut.
Glomerulo nepritis Akut (GNA).
Bila peningkatan titer 2X Infeksi sedang berlangsung.
Pemeriksaan dilakukan 2X fase Akut dan konvalescen.

Agglutinasi Inhibisi
Haemagglutination Inhibisi (HI) tes DHF.
Virus + Eri Angsa Agglutinasi.
Virus + Eri Angsa + Serum pend. DHF tidak
agglutinasi.
Virus + Eri Angsa + Serum orang Normal Agglutinasi.
Preparasi serum penderita;
2-5 ml darah ambil serum simpan 0oC pada fase
akut & konvalesen.
Serum diekstraksi dengan kaolin. Perbandingan serum:
saline: kaolin= 1 : 4 : 5.
Inkubasi 20, kocok putar 2.500 ppm selama 30.
Ag viral: Den 1. Den 2. Den 3

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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 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

Labeled
Anti-Ig
Ab in
Patients
sample

Immobilized

Ag
Solid
Phase

Quantitative

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

Labeled
Ab
Ag in
Patients
sample

Ag

Immobilized

Solid
Phase

Quantitative

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Enzyme-Linked ImmunoSorbant
Assay (ELISA)

Indirect ELISA

1.

Sandwich ELISA

Indirect ELISA

The steps of the general, "indirect," ELISA for

determining serum antibody concentrations are:
1.

2.

3.

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.
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.
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.

5.
6.

7.
8.
9.

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.
The plate is washed to remove any unbound detection
antibody. After this wash, only the antibody-antigen
complexes remain attached to the well.
Secondary antibodies, which will bind to any remaining
detection antibodies, are added to the wells. These
secondary antibodies are conjugated to the substratespecific enzyme. This step may be skipped if the
detection antibody is conjugated to an enzyme.
Wash the plate, so that excess unbound enzymeantibody conjugates are removed.
Apply a substrate which is converted by the enzyme to
elicit a chromogenic or fluorogenic or electrochemical
signal.
View/quantify the result using a spectrophotometer,
spectrofluorometer, or other optical/electrochemical
device.

To detect antibody (indirect ELISA):

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2.
A

Sandwich ELISA

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.
2.
3.
4.
5.
6.
7.
8.
9.

Prepare a surface to which a known quantity of capture

antibody is bound.
Block any non specific binding sites on the surface.
Apply the antigen-containing sample to the plate.
Wash the plate, so that unbound antigen is removed.
Apply primary antibodies that bind specfically to the
antigen.
Apply enzyme-linked secondary antibodies which are
specific to the primary antibodies.
Wash the plate, so that the unbound antibody-enzyme
conjugates are removed.
Apply a chemical which is converted by the enzyme into a
color or fluorescent or electrochemical signal.
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):

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

Prior to Test
+

Labeled
Ag
Test
+

Labeled Patients
Ag
sample

Competitive ELISA for Ag

Method cont.
Determine amount
of labeled Ag
bound to Ab
NH4SO4
anti-Ig
Immobilize the Ab

Test
+

Solid Labeled Patients

Ag
sample
Phase

Solid
Phase

Concentration determined from a standard curve

using known amounts of unlabeled Ag
Quantitative

Most sensitive test

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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 yg
mengandung Ab
dibuat sederet
sumuran.
Ag dimasukkan
ke dlm sumuran.
Ag akan berdifusi ke sekelilingnya dan bereaksi dg Ab dlm gel.
Luas lingkaran
presipitin berkorelasi dg konsentrasi Ag, dibandingkan dg.
Grafik standar

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Elektroforesis

Gb bawah : Ab
terikat pada gel, Ag
bebas begerak ke
arah kutub positif
shg terjadi presipitat
berbentuk spt roket.
Gb atas : Ab maupun Ag bebas dlm
gel, Ab bergerak ke
arah kutub neg., Ag
bergerak ke arah
kutub pos., keduanya bertemu terbentuk 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 pada

media gel. Akan terjadi difusi Ag maupun Ab. Pd Gb a. terjadi satu
sedangkan pada b. dua pita precipitin pd pertemuan Ag dan Ab.

Ouchterlony :

memmperlihatkan
reaksi Ag-Ab individual antara campuran
Ag dan Ab.
Gb 1 : garis presipitin
menunjukkan bahwa
Ag di sumuran kiri
dan Ag kanan
keduanya mempunyai
epitop yg identik.
Gb 2 : Ag dlm sumuran
kiri tidak identik dg Ag
di sumuran kanan,
terlihat dari adanya
presipitin yang
bersilangan.
Gb 3 : Taji pada presipitin kanan menunjukkan bahwa Ag di sumuran kiri dan kanan
adalah identik parsial.

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Pada campuran Ag yang

telah diberi
label radioaktif
ditambahkan
Ab spesifik.
Kompleks AgAb diendapkan. Endapan
dipisahkan dari
Ag yang
bebas, lalu
dielektroforesis. Dibaca
dengan skaner
radioaktivitas

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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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 mikroorganisme
(banyak Ag)
dielektroforesis
pada gel shg
terpisah satu
sama lain.
Hasil elektroforesis dipindahkan ke lempeng
nitroselulose
(blotting),
ditambahkan Ab,
dicuci, dst dibaca
dengan pelabelan
radioaktif atau dg
pewarnaan

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Adanya lisis
membuktikan terjadi
reaksi AgAb dg keterlibatan
komplemen.
Tidak ada lisis berarti
reaksi Ag vs
Ab tidak
berpengaruh pada sel
indikator.
Gb bawah
tabel bukti
adanya macam2
kompl.

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