Immunological diagnosis (Institute of Immunology, ZJU)

1.Detection of Antigen and antibodies 2.Detection of Cellular Immunity Immunodiagnosis

Agglutination(aggregation) Assays: Immunodiffusion Complement Fixation EIA (IHC/ELISA/ELISPOT) Immunofluorescence (IFA FACS) CLIA （ Chemiluminescence immumoassay ） Traditional Immunoassays Anitgen-Ab reaction Modern Immunoassays

1. Principles and 1. Principles and influencing factors of Ag-Ab reaction 1) Principles of Ag-Ab reaction a.Specificity b.reversal b.reversal combination c.Concentration and ratio of Ag and Ab

Immune complex Antibody excess zone Precipitin curve

2) influencing factors of Ag-Ab reaction A.electrolytes B.Temperature:37 degree C.pH:pH6-8

2 Methods for detection of Ag or Ab A. Agglutination reaction a. Principle When the particle Ags interact with the appropriate Ab, they clump together and eventually form masses that become large enough to be seen. b. Types direct agglutination reaction indirect agglutination reaction

B. Precipitation reaction a. 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 double immunodiffusion immunoelectrophoresis

C. Complement fixation test 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.

D. Immuno-labeling techniques a. 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 to detect Ag and Ab interactions. The enzyme converts a colorless substrate (chromogen) to a colored product. ELISA: Ag or Ab in solution Enzyme immunohistochemistry: Ag in tissue

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)

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

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

Radioimmunoassay, RIA Chemiluminescence immunoassay, CLIA Immunoblotting, Western blotting Immuno-PCR, IM-PCR Immunologic colloidal gold signature, ICE 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

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

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.

Identification of cell subsets by FACS Immune Cell types and subtypes defined by surface markers (CDs) B cell T cell CD4+ T cell CD8+ Tcells Tregs (CD4+CD25+) Conventional CD4 Type of cellCD markers stem cellsCD34+,CD31- all leukocyte groupsCD45+ GranulocyteCD45+,CD15+ MonocytesCD45+,CD14+ T lymphocyteCD45+,CD3+ T helper cellCD45+,CD3+,CD4+ Cytotoxic T cellCD45+,CD3+,CD8+ B lymphocyte CD45+,CD19+ or CD45+,CD20+ ThrombocytesCD45+,CD61+ Natural killer cellCD16+,CD56+,CD3-

2) Lymphocyte function assays T cell function assay --Proliferation --DTH --Apoptosis --Phagocytosis --Cytokine

T cell proliferation - MTT Mitochondria enzyme catalyze the reduction of MTT – Turn blue

T cell proliferation FACS-CFSE staining

CTL Assay Supernatant

Tetramers can bind to three TCRs at once, allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I- peptide-TCR interaction. Tetramer

Immunization Challenge (Ear/Foogpad) Measurement (Calipers) DTH

Detection of Cytokine production 1.Real-time PCR (mRNA Level) 2.ELISA/ELISPOT 3.Intracellular staining (FACS)

Intracellular Staining Identification of different T helper cells characterized by different cytokine production

Application of Immunoassay Diagnosis of Diseases infectious diseases Immunodeficiency diseases Autoimmune disease hypersensitivity Tumour

Application of Immunoassay Immune surveilence HBV HIV