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 Presentation


Immunological diagnosis

Dosen Imunologi Fakultas farmasi Universitas PancasilaDIAGNOSTIK IMUNOLOGIPenerapan uji imunologiDiagnosis Penyakit infectious diseases Immunodeficiency diseases Autoimmune disease hypersensitivity Tumour ImunosurveylanceVirus hepatitis B (HBV)Virus imunodeficiency (HIV)PRINSIP UJI IMUNOLOGIREAKSI ANTARA

Antigen dengan Antibodi

AG >< AbPrinsipA. SpesifisitiIkatan antara Ab dengan Ag mempunyai spesifisitas yg tinggi (high specificity)Afinitas: daya afinitas/gabung antara satu Ab dengan satu Ag sangat kuatAviditas: 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 komplekyg insolubel (agregat atau presipitat)cukup besar dan jelas untuk dilihatImmune complexAntibody excess zonePrecipitin curve

2C. Faktor yg mempengaruhi reaksi1. electrolytes 2. Temperature:37 degree3. pH:pH6-8METODE UJI1. Reaksi Aglutinasi2. Reaksi Presipitasi3. Complement Fixatio test (CFT)4. Tehnik imunolabelA. Enzyme imunoassay (EIA)B. Enzym link immunosorbent asasay (ELISA)Indirect ELISA: mengukur AbSandwich ELISA: Deteksi AgCompetitive ELISA: deteksi Ag atau AbC, ImmunofluorescentD. Radio immunou assay (RIA)UJI IMUNOLOGI LAINNYA1 Deteksi fungsi sel imunA. Isolasi sel imunIsolasi PBMCB. Isolasi limfosit dan subsetnyaa. Immunosorbent assayb Immunomagnetic separationc.FACSd.tehnik MHC-tetramer-peptida

2. limfosit sel essayLimfosit proliferation tesLectin stimulasi sel TPenghitungan bentuk sel morfologi3. Deteksi limfosit activation essaydeteksi Igdeteksi Ab forming selsitolytik tesfagositik diysfungsiproduksi sitokin

REAKSI AGLUTINASIPrinsip 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

AgAbDirectIndirectdREAKSI PRESIPITASIPrinsip 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 IMUNOLABELPrinsipSpecific Abs (or Ags ) labelled with fluorescein, enzymes, colloidial gold or radioisotopes are used as probes for the detection of Ags (or Abs).TipeEIAELISAIndirectSandwichCompetitivEnzyme linked immunosorbent assay, ELISAThe advantages of ELISA include specificity, sensitivity, rapidity, inexpensiveness, and safety.Enzyme: horseradish peroxidase, HRPSubstrates: 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 immunofluorescenceIMMUNOFLUORESCENCEImmunofluorescenceImmunofluorescence 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


Gold nanoparticle labeled anti-HCG mouse IgGAgHCGhuman chorionic gonadotropin B G T R Amouse anti-HCG (immobilized)Anti-mouse IgG (immobilized)Absorbent material

positive negative33Is there anything wrong ? (first,positive;second,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 techniqueFigure 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-26

MACS:magnetic cell sorting1,The target cell are labeled with Ab-conjugated magnetic paticles2,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 separationThe 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 assaysT 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 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.

41Small resting lymphocyte are 6-8 m in diameter

3H-TDR incorporation method 2) Lymphocyte function assaysB cell function assayA. Detection of IgB. Ab-forming cell detection

2) Lymphocyte activation assaysC. 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,



The overall strength of binding between an Ag with many determinants and multivalent Abs