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  1. DIAGNOSTIK IMUNOLOGI Dosen Imunologi Fakultas farmasi Universitas Pancasila

  2. Penerapan uji imunologi • Diagnosis Penyakit infectious diseases Immunodeficiency diseases Autoimmune disease hypersensitivity Tumour Imunosurveylance Virus hepatitis B (HBV) Virus imunodeficiency (HIV)

  3. PRINSIP UJI IMUNOLOGI • REAKSI ANTARA • Antigen dengan Antibodi • AG >< Ab

  4. 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

  5. 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

  6. Precipitin curve Immune complex Antibody excess zone

  7. 2C. Faktor yg mempengaruhi reaksi 1. electrolytes 2. Temperature:37 degree 3. pH:pH6-8

  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)

  9. 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

  10. 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

  11. REAKSI AGLUTINASI Prinsip 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

  12. Direct Ag Ab Indirectd

  13. 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

  14. 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.

  15. 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

  16. 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)

  17. 6. ELISA to detect Ab (HIV, HCV) to detect Ag to detect Ag

  18. 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

  19. 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

  20. Radioimmunoassay, RIAChemiluminescence immunoassay, CLIAImmunoblotting, Western blottingImmuno-PCR, IM-PCR Immunologic colloidal gold signature, ICE

  21. Immunoblotting

  22. B G T R A Absorbent material Gold nanoparticle labeled anti-HCG (mouse IgG) Ag(HCG,human chorionic gonadotropin) mouse anti-HCG (immobilized) Anti-mouse IgG (immobilized)

  23. positive negative

  24. 2. Detection the Function of Immune cells 1) Isolation of immune cells A Isolation of PBMC: FicollUrografindensity-gradient separation B: Isolation of lymphocytes and subsets. a,immunoabsorbing assay b. immunomagnetic separation c. FACS d. peptide-MHC tetramer technique

  25. Figure A-23

  26. 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

  27. 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

  28. 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.

  29. 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

  30. 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 irregularin 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.

  31. 3H-TDR incorporation method

  32. 2)Lymphocyte function assays B cell function assay A. Detection of Ig B. Ab-forming cell detection

  33. 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 byradioisotopes. 51Cr releasing LDH cell staining method Apoptosis cell detection

  34. 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.

  35. Fragmented DNA can be labeled by terminal deoxynucleotidyltransferase (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.

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