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