Tes Laboratorium

Kompetensi Imun Tubuh

Uleng Bahrun

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 Pokok Bahasan

A.Tes Lab utk deteksi Ag & Ab

B.Tes untuk Deteksi Imunitas Seluler

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 Basic Immunochemistry

1. Antigen-antibodi reaksi in vitro

a. interaksi Ag-Ab primer
- butuh indikator (label)
- bermanfaat bila kadar rendah
- RIA, ELISA, imunofluoresensi
b. interaksi Ag-Ab sekunder
- presipitasi
- aglutinasi
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 Basic Immunochemistry

2. Spesifisitas dan sensitivitas

- antigenik deteminan

Ag1 Ag2 Ag3 Ag3

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 A. Lab. Methods for Detection Ag & Ab

1. IMUNOPRESIPITASI
- Ag + Ab
kompleks yg tdk larut (presipitat)
- media cair atau semisolid (gel)

TesLabKompImun/UlengBahrun
 A. Lab. Methods for Detection Ag & Ab
- Teknik Imunopresipitasi:
a. Double Imunodifusion (kualitatif,
semi kuantitatif)
b. Radial Imunodifusion (kuantitatif)

a b

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 A. Lab. Methods for Detection Ag & Ab

c. Imunoelektroforesis; (gabungan imunodif.

ganda & elektroforesis)
d. Elektroimunodifusi / imunodifusi aktif
(kualitatif)
e. Rocket imunoelektroforesis
(elektroimunodifusi, kuantitatif)
f. Imunonefelometri: mengukur cahaya yg
dipencarkan oleh partikel Ag-Ab kompleks

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 A. Lab. Methods for Detection Ag & Ab

2. AGLUTINASI
(


2 tahap)
a. Direct Agglutination
- Ag harus berupa partikel atau sel
- Contoh: Tes Widal (Salmonella typhi), Tes
Golongan Darah)
b. Indirect Agglutination / Passive agglutination
- mendeteksi Ag yg larut setelah dilekatkan pada
carrier (eritrosit, lateks, karbol, kolodion)
- Contoh: TPHA, Uji Faktor Reumatoid, Tes anti-HBs
TesLabKompImun/UlengBahrun
 A. Lab. Methods for Detection Ag & Ab

c. Reverse passive hemagglutination assay = RPHA

- mendeteksi Ag yg larut
- Ab dilekatkan pada eritrosit, kmd direaksikan dg Ag
- Kualitatif
Kuantitatif
- Contoh: Tes HBsAg
d. Agglutination Inhibition Test
- untuk mendeteksi Ag yg larut
- prinsip: cairan sampel yg diuji + Ab spesifik + Ag-
carrier
ada aglutinasi = Ag sampel NEG
tdk ada aglutinasi = Ag spl POS
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 A.UJI
3. Lab. Methods
FIKSASI for Detection Ag & Ab
KOMPLEMEN
e. Complement Fixation Test
- Prinsip: ikatan Ag-Ab
aktivasi komplemen
(kompleks eritrosit-hemolisin
aktivasi
komplemen
hemolisis)
- 2 tahap: 1. ikatan komplemen oleh Ag-Ab kompleks
2. penghancuran eritrosit yg tlh dilapisi
hemolisin oleh komplemen yg tersisa
↓
parameter penilaian: banyaknya
komplemen tersisa pd thp 1 / hemolisis pd thp 2
- contoh Uji fiksasi komplemen untuk infeksi virus,
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parasit
 A. Lab. Methods for Detection Ag & Ab

3. RADIOIMMUNOASSAY (RIA)
- 2 METODE:
1. Reaksi Ag-Ab dalam larutan (liquid phase)
2. Reaksi Ag-Ab pd benda padat / partikel (solid
phase)
- Prinsip: - kompetitif Ag berlabel & non label
(met.1)
- non kompetitif (sandwich) (metode 2)
- sensitif
- kelemahan: bahaya radioaktif, half life
radioisotop pendek, mahal
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 A. Lab. Methods for Detection Ag & Ab

4. ENZYME IMMUNOASSAY (EIA) / ENZYME

LINKED IMMUNOSORBENT ASSAY (ELISA)
- prinsip: Ag(or Ab) is fixed to a surface → test
sample is applied → bound material
detected by secondary-enzimatically
labeled Ab
- Kelebihan: * rapid, sensitif,
* half life reagen ckp panjang,
* dpt menggunakan spektrofotometer
biasa
* mudah automatisasi
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* tdk ada bahaya radioaktif

 A. Lab. Methods for Detection Ag & Ab

Membran
selulosa

Ag AbE Ag-AbE
(sample)

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 Prinsip tes Anti TPO

TPO dilekatkan masukkan masukkan

ke microwell serum pasien HRP anti-IgG

Ket: = TPO = Anti TPO = HRP anti-IgG

TesLabKompImun/UlengBahrun
 A. Lab. Methods for Detection Ag & Ab
5. IMMUNOMICROSCOPY
- Teknik histokimia / sitokimia untuk mendeteksi
kompleks Ag-Ab pada jaringan atau sel
- Indikator:
* zat warna fluorokrom (Fluorescein
isothyocyanate/FITC), Tetramethyl rhodamin
isothyocyanate)
- Teknik: * Direk
* Indirek
* Sandwich

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 A. Lab. Methods for Detection Ag & Ab
6. ELEKTROFORESIS
→ Pemisahan protein pada medan listrik
2 basic procedure:
1. zone electrophoresis (berdasarkan muatan listrik
permukaan)
2. denaturing electrophoresis (berdasarkan berat
molekul)
↓
Serum sample are placed on support media → subjected to
electric field → protein migration

TesLabKompImun/UlengBahrun
 A. Lab. Methods for Detection Ag & Ab
Serum Protein Electrophoresis (SPEP)
→ patient serum is placed in the centre of a shallow well
within a strip of cellulose acetate → electric field → the
film stained to localized the protein bands

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 A. Lab. Methods for Detection Ag & Ab
7. SDS-PAGE & WESTERN BLOT (Immunoblotting)
→ dilakukan untuk menentukan adanya protein
tertentu
SDS-PAGE (PolyAcrylamide Gel Electrophoresis) digunakan
untuk memisahkan molekul berdasarkan ukuran, bentuk atau
muatan listrik.

Protein denatured by incubation with ionic detergent (SDS) at 100C

↓
Elecrophoresis
↓
Western Blot
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 A. Lab. Methods for Detection Ag & Ab

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 B. Lab. Methods for Detection Cellular Immunity

- determination of number & types of the cell

surface (marker) → for assessing the cells of
immune system
- Development of mAbs
- 1983, 1st International Workshop on Human
Leucocyte
Differentiation Antigens → Cluster Differentiation
(CD)

TesLabKompImun/UlengBahrun
 B. Lab. Methods for Detection Cellular Immunity

Flow Cytometry → FACS

->> detecting binding of mAb to leucocytes surface
- is made up of three main systems: fluidics, optics, and electronics.

The fluidics system transports particles in

a stream to the laser beam for
interrogation.

• The optics system consists of lasers to

illuminate the particles in the sample stream
and optical filters to direct the resulting
light signals to the appropriate detectors.

• The electronics system converts the

detected light signals into electronic signals
that can be processed by the computer.
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 B. Lab. Methods for Detection Cellular Immunity

Flow Cytometry

Light scattering properties of a cell

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 Contoh Tes Imun

1. Tes Widal
- Prinsip: agglutinin (Ab) + agglutinogen (Ag) →aglutinasi
- Dasar: identifikasi pengenceran tertinggi dari serum
pasien yang masih menyebabkan aglutinasi dari
suspensi bakteri
- Metode:
1. Metode Slide
2. Metode tabung
(antigen pada Salmonella!)
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ahrun
2. HBs Ag (Rapid Immunochromatography test)
- HBsAg: - salah satu Ag pd permuk. virus hepatitis B
- terdeteksi 3-5 mgg sebelum timbul ikterus
- merup. petunjuk infeksi HVB akut/kronik
- Prinsip:
- HBsAg serum/plasma + partikel anti-HBs
(membran) →kompleks Ag-Ab, bmigrasi
→kompleks Ab-Ag-Ab → grs merah
- Interpretasi: positif, negatif, invalid
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Imun/UlengB
ahrun
3. Dengue Test (Rapid test)
- deteksi IgG & IgM → dpt membedakan inf. primer &
sekunder
- Prinsip tes:
- Interpretasi: positif: inf.primer, post inf, inf.sekunder
negatif
invalid

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The end
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