1. Immunological diagnosis
(Institute of Immunology, ZJU)

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

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

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

5. Precipitin curve
Immune complex
Antibody excess zone

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

7. 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

9. 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

11. 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.

14. 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

15. 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

17. 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)

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

22. 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

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

25. Immunoblotting

27. 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)

28. Is there anything wrong ? (first,positive;second,negative)
positive negative

29. 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

30. Figure A-23

31. 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

32. 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.

34. Identification of cell subsets by FACS
Type of cell
CD markers
stem cells
CD34+,CD31-
all leukocyte groups
CD45+
Granulocyte
CD45+,CD15+
Monocytes
CD45+,CD14+
T lymphocyte
CD45+,CD3+
T helper cell
CD45+,CD3+,CD4+
Cytotoxic T cell
CD45+,CD3+,CD8+
B lymphocyte
CD45+,CD19+ or CD45+,CD20+
Thrombocytes
CD45+,CD61+
Natural killer cell
CD16+,CD56+,CD3-
B cell
T cell
CD4+ T cell
CD8+ Tcells
Tregs
(CD4+CD25+)
Conventional
CD4
Immune Cell types and subtypes defined by surface markers (CDs)

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

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

37. T cell proliferation
FACS-CFSE staining

38. CTL Assay
Supernatant

39. Tetramer
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.

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

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

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

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

44. Application of Immunoassay
Immune surveilence
HBV
HIV