DIAGNOSTIK IMUNOLOGI

DOSEN IMUNOLOGI
FAKULTAS FARMASI
UNIVERSITAS PANCASILA

Penerapan uji imunologi

Diagnosis Penyakit

infectious diseases
Immunodeficiency diseases
Autoimmune disease
hypersensitivity
Tumour
Imunosurveylance
Virus hepatitis B (HBV)
Virus imunodeficiency (HIV)

PRINSIP UJI IMUNOLOGI

REAKSI ANTARA
Antigen dengan Antibodi

AG >< AB

Prinsip
A. Spesifisiti
Ikatan

antara Ab dengan Ag mempunyai

spesifisitas yg tinggi (high specificity)
Afinitas: daya afinitas/gabung antara satu Ab
dengan satu Ag sangat kuat
Aviditas: kekuatan ikatan antara Ag dngan
banyak determinan dan multivalen Ab secara
keseluruhan sangat kuat

B. Rasio konsentrasi Ag dan Ab:

Bilamana Ag dan Ab berada dalam
konsentrasi yg sesuai, maka mereka
akan membentuk reaksi imun komplek
yg insolubel (agregat atau presipitat)
cukup besar dan jelas untuk dilihat

Immune complex

Precipitin curve

Antibody
excess zone

2C. Faktor yg mempengaruhi

reaksi

1. electrolytes
2. Temperature:37 degree

3. pH:pH6-8

METODE UJI
1. Reaksi Aglutinasi
2. Reaksi Presipitasi
3. Complement Fixatio test (CFT)
4. Tehnik imunolabel

A. Enzyme imunoassay (EIA)

B. Enzym link immunosorbent asasay (ELISA)
Indirect ELISA: mengukur Ab
Sandwich ELISA: Deteksi Ag
Competitive ELISA: deteksi Ag atau Ab

C, Immunofluorescent
D. Radio immunou assay (RIA)

UJI IMUNOLOGI LAINNYA

1 Deteksi fungsi sel imun

A. Isolasi sel imun

Isolasi PBMC

B. Isolasi limfosit dan subsetnya

a. Immunosorbent assay
b Immunomagnetic separation
c.FACS
d.tehnik MHC-tetramer-peptida

2. limfosit sel essay

Limfosit proliferation tes

Lectin stimulasi sel T
Penghitungan bentuk sel morfologi

3. Deteksi limfosit activation essay

deteksi Ig
deteksi Ab forming sel
sitolytik tes
fagositik diysfungsi
produksi sitokin

REAKSI AGLUTINASI
a. Prinsip
b. Bilamana partikel Ag berinteraksi dengan Ab

yg sesuai, mereka memebntuk ikatan

(clamp) dan cukup terlihat dg jelas
b. Tipe aglutinasi
direct agglutination reaction
indirect agglutination reaction

Direct
Ag

Ab

Indirectd

REAKSI PRESIPITASI
Prinsip
When soluble Ags come in contact
with specific Ab, they precipitate.
Precipitation can be demonstrated via
immunodiffusion in a semisolid medium
(e.g. agar).
bTipe Presipitasi
immunonephelometry: the formation
of IC in solution is monitored by
spectrometry. single immunodiffusion
double immunodiffusion
immunoelectrophoresis

REAKSI PENGIKATAN KOMPLEMEN

(CFT)
Ag and Ab reactions lead to the formation of

IC that activates complement system by

classical pathway.
This may be exploited to detect the amount
of unknown Ag or Ab.

TEHNIK IMUNOLABEL
Prinsip

Specific Abs (or Ags ) labelled with

fluorescein, enzymes, colloidial gold or
radioisotopes are used as probes for the
detection of Ags (or Abs).
Tipe
EIA
ELISA
Indirect
Sandwich

Competitiv

Enzyme linked immunosorbent

assay, ELISA
The advantages of ELISA include

specificity, sensitivity, rapidity,

inexpensiveness, and safety.
Enzyme: horseradish peroxidase,

HRP
Substrates:

diaminobenzidine (DAB)
3,3,5,5-tetramethylbenzidine
(TMB)

todetectAb(HIV,HCV)

todetectAg

todetectAg

6.ELISA

IMMUNOFLUORESCENCE
- Immunofluorescence assay is to use
a fluorescent compound (usually
fluorescein) to detect the binding
of Ag and Ab.
- The Ab is labeled with the
fluorescent compound and its
presence is revealed using a
fluorescence microscope.
- Direct, indirect immunofluorescence
and indirect complement amplified
immunofluorescence

Immunofluorescence
Immunofluorescence assay is to use a

fluorescent compound (usually

fluorescein) to detect the binding of Ag
and Ab.
The Ab is labeled with the fluorescent

compound and its presence is revealed

using a fluorescence microscope.
Direct, indirect immunofluorescence and

indirect complement amplified

immunofluorescence

Radioimmunoassay, RIA
Chemiluminescence immunoassay,
CLIA
Immunoblotting, Western blotting
Immuno-PCR, IM-PCR
Immunologic colloidal gold signature,
ICE

Immunoblotting

B
Absorbent
material

Gold nanoparticle labeled anti-HCG

mouse IgG
Ag HCG human chorionic
gonadotropin
mouse anti-HCG (immobilized)

Anti-mouse IgG (immobilized)

positive
negative

2. Detection the Function

of Immune cells
1) Isolation of immune
cells
A Isolation of PBMC:
Ficoll Urografin density-gradient separation
B: Isolation of lymphocytes and subsets.
a,immunoabsorbing assay
b. immunomagnetic separation
c. FACS
d. peptide-MHC tetramer technique

Figure A-23

Magnetic cell sorting (MACS)

Three basic steps
1) Target cells are labeled with antibodyconjugated magnetic particles.
2) The labeled cells are placed within a
magnetic
field.
3) The labeled cells are retained in the
magnetic
field while the unlabeled cells are
washed
away

Figure A-26

MACS:magnetic cell sorting

1,The target cell are labeled
with Ab-conjugated magnetic
paticles
2,The labeled cells are
placed within a magnetic
fields.
3, The labeled cells are
retained in the magnetic
fields while the unlabeled
cells are washed away

FACS separation
The basic principle of FACS is

immunofluorescence and therefore flow

cytometers can be considered to be
specialized fluorescence microscopes.
The modern flow cytometer consists of a
light source, collection optics, electronics
and a computer to translate signals to
data
Isolation of different cell populations by
FACS relies on the different expression of
surface Ags.

2) Lymphocyte function
assays
T cell function assay
A. Lymphocyte proliferation test
Lymphecyte proliferation is usually
determined using polyclonal
activators of lymphocytes or
lymphocyte mitogens.
T cell stimuli are lectins (PHA, Con
A).
Morphologic counting
3
H-TdR or 125I-UdR incorporation
MTT chromatometry
B. DTH detection: OT test or PPD
test

 Lymphoblast

( morphological features):
Lymphoblasts are 12-20 m in
diameter with a round to oval
nucleus. The periphery of both
the nucleus and the cell may be
irregular in outline.
The fine, highly dispersed
nuclear chromatin stains a light
reddish-purple, and one or two
pale blue or colorless large
nucleoli are visible. The
cytoplasm is usually basophilic,
with marginal (peripheral)
intensity a common
characteristic.

H-TDR incorporation method

2) Lymphocyte function
assays

B cell function assay

A. Detection of Ig
B. Ab-forming cell detection

2) Lymphocyte activation
assays
C. Cytolytic test
Assays for CTL in patients can be
performed as a variant of a mixed
cell culture using the target cells
that labelled by radioisotopes.
51
Cr releasing
LDH
cell staining method
Apoptosis cell detection

Cytotoxic T-cell activity is often assessed by chromium

release from labeled target cells. Target cells are labeled with
radioactive chromium as Na251CrO4, washed to remove excess
radioactivity and exposed to cytotoxic T cells. Cell destruction is
measured by the release of radioactive chromium into the medium,
detectable within 4 hours of mixing target cells with T cells.

Fragmented DNA can be labeled by terminal deoxynucleotidyl

transferase (TdT) to reveal apoptotic cells. When cells undergo
programmed cell death, or apoptosis, their DNA becomes fragmented (left
panel). The enzyme TdT is able to add nucleotides to the ends of DNA
fragments; most commonly in this assay, biotin-labeled nucleotides (usually
dUTP) are added (second panel). The biotinylated DNA can be detected by
using streptavidin, which binds to biotin, coupled to enzymes that convert a
colorless substrate into a colored insoluble product (third panel). Cells
stained in this way can be detected by light microscopy, as shown in the
photograph of apoptotic cells (stained red) in the thymic cortex. Photograph
courtesy of R. Budd and J. Russell.

phagocytic dysfunction
Cytokine production
biological activity
immunoassay:ELISA,
intracellular CKs,
ELISPOT
PCR