diagnostik imunologi
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DIAGNOSTIK IMUNOLOGI. Dosen Imunologi Fakultas farmasi Universitas Pancasila. P enerapan uji imunologi. Diagnosis Penyakit infectious diseases Immunodeficiency diseases Autoimmune disease hypersensitivity Tumour Imunosurveylance Virus hepatitis B (HBV) - PowerPoint PPT PresentationTRANSCRIPT
DOSEN IMUNOLOGI FAKULTAS FARMASI UNIVERSITAS PANCASILA
DIAGNOSTIK IMUNOLOGI
Penerapan uji imunologiDiagnosis 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
PrinsipA. 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
Affinity106
Avidity1010
Avidity
B. Rasio konsentrasi Ag dan Ab:Bilamana Ag dan Ab berada dalam konsentrasi yg sesuai, maka mereka akan membentuk reaksi imun komplekyg insolubel (agregat atau presipitat)cukup besar dan jelas untuk dilihat
Imm
une
com
plex
Antibody excess zone
Precipitin curve
2C. Faktor yg mempengaruhi reaksi
1. electrolytes 2. Temperature:37 degree3. pH:pH6-8
METODE UJI1. Reaksi Aglutinasi2. Reaksi Presipitasi3. 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 essaydeteksi Igdeteksi Ab forming selsitolytik tesfagositik diysfungsiproduksi 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
Ag Ab
Direct
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).
TipeEIAELISA
IndirectSandwichCompetitiv
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 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
ImmunofluorescenceImmunofluorescence 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
Gold nanoparticle labeled anti-HCG (mouse IgG)Ag( HCG, human chorionic gonadotropin)
B G T R A
mouse anti-HCG (immobilized)
Anti-mouse IgG (immobilized)
Absorbent material
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 antibody- conjugated 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-26MACS: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 assayA. 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 chromatometryB. 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 assayA. Detection of IgB. 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 dysfunctionCytokine production
biological activity
immunoassay:ELISA, intracellular CKs,
ELISPOT
PCR