Immunological diagnosis

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
Avidity
• The overall strength of binding between an Ag
with many determinants and multivalent Abs
Keq =
104
Affinity
106
Avidity
1010
Avidity
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)
6. ELISA
to detect Ab (HIV, HCV)
to detect Ag
to detect Ag
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
G
T
R
A
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
3H-TdR or 125I-UdR incorporation
MTT chromatometry
B. DTH detection: ‘OT’ test or PPD test
 Lymphoblast
( morphological features):
 Lymphoblasts are 12-20 µm 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.
3H-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.
51Cr 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