文本教案免疫学

1、Immunological diagnosis(Institute of Immunology, ZJU) 1. Principles and influencing factors of Ag-Ab reaction1) Principles of Ag-Ab reactiona. Specificity Binding between Ab and Ag has very high specificity. Affinity: the strength of the binding between a single binding site of an Ab and an Ag Avidi

2、ty: the overall strength of interaction between an Ab and an Ag.b. reversal combinationnoncovalent forcehydrogen bondelectrostatic attractionVan der Wals forces hydrophobic bonddegree of dissociationAg-Ab affinityEnvironmental factorsC. Concentration and ratio of Ag and AbWhen the antigens and antib

3、odies are present in an appropriate ratio, they form insoluble immune complexes (e.g. aggregation or precipitation) large enough to be seen. As increasing concentrations of Ag are added to a constant amount of Ab, the amount of IC precipitated rises and then falls. The precipitin curve generated in

4、this way has three zones. Ab excess zone (prozone) equivalence zone Ag excess zone (postzone)Immune complexAntibody excess zonePrecipitin curved. Two phasesSpecific combinationVisible phase2) influencing factors of Ag-Ab reactionelectrolytes Temperature:37 degreepH:pH6-82 Methods for detection of Ag

5、 or AbA. Agglutination reactiona. Principle When the particle Ags interact with the appropriate Ab, they clump together and eventually form masses that e large enough to be seen.b. Types direct agglutination reaction indirect agglutination reaction Direct coombs testindirect coombs testB. Precipitat

6、ion reactiona. Principle When soluble Ags come in contact with specific Ab, they precipitate. Precipitation can be demonstrated via immunodiffusion in a semisolid medium (e.g. agar).b. Types immunonephelometry: the formation of IC in solution is monitored by spectrometry. single immunodiffusion doub

7、le immunodiffusion immunoelectrophoresis D. Immuno-labeling techniquesa. Principle Specific Abs (or Ags ) labelled with fluorescein, enzymes, colloidial gold or radioisotopes are used as probes for the detection of Ags (or Abs).b. Types Enzyme immunoassay (EIA)EIA is to use enzyme-labeled Abs or Ags

8、 to detect Ag and Ab interactions.The enzyme converts a colorless substrate (chromogen) to a colored product.ELISA: Ag or Ab in solutionEnzyme immunohistochemistry: Ag in tissueEnzyme linked immunosorbent assay, ELISAThe advantages of ELISA include specificity, sensitivity, rapidity, inexpensiveness

9、, and safety.Enzyme: horseradish peroxidase, HRPSubstrates: diaminobenzidine (DAB) 3,3,5,5-tetramethylbenzidine (TMB) to detect Ab (HIV, HCV)to detect Ag to detect Ag 6. ELISA ELISABAS（Biotin-avidin system）-ELISABiotin-avidin system）-ELISAImmunofluorescenceImmunofluorescence assay is to use a fluore

10、scent 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 immunofluorescenceRadioimmunoassay, RIAChemilum

11、inescence immunoassay, CLIABioluminescence immunoassay, BLIAImmuno-PCR, IM-PCR Immunologic colloidal gold signature, ICSImmunoblotting, Western blottingProtein microassay ImmunoblottingGold nanoparticle labeled anti-HCG （mouse IgG）Ag（HCG，human chorionic gonadotropin） B G T R Amouse anti-HCG (immobil

12、ized)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 separatio

13、n c. FACS d. peptide-MHC tetramer techniqueFigure A-23Magnetic cell sorting (MACS) Three basic steps Target cells are labeled with antibody-conjugated magnetic particles.2) The labeled cells are placed within a magnetic field. The labeled cells are retained in the magnetic field while the unlabeled

14、cells are washed away Figure A-26MACS: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 awayFACS separationThe ba

15、sic 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 populat

16、ions by FACS relies on the different expression of surface Ags.Biotin MHC IAg-MHC tetramer techniquePeptide:MHC complexes coupled to streptavidin to form tetramers are able to stain antigen-specific T cells 2) Lymphocyte function assaysT cell function assayA. Lymphocyte proliferation test Lymphocyte

17、 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 testPolyclonal mitogens3H-TdR incorporation method

18、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

19、 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 measu

20、red 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 es 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