Immunological Immunological diagnosisdiagnosis

(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 Immunoassa

ys

Traditional Immunoassa

ys

Anitgen-Ab reaction

Modern Immunoassays

Modern Immunoassays

1. 1. PrinciplesPrinciples and and influencing factors of Ag-Ab reaction

1) Principles of Ag-Ab reactiona.Specificity

b.b.reversalreversal combination

c.c.Concentration and ratio of Ag Concentration and ratio of Ag and Aband Ab

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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 2 Methods for detection of Ag or AbAb

A. Agglutination reactiona. 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 reactionB. Precipitation 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

double immunodiffusion immunoelectrophoresis

C. Complement fixation testC. 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 D. Immuno-labeling techniquestechniques

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)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 Enzyme linked immunosorbent assay, ELISAassay, 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

to detect Ag

6. ELISA 6. ELISA

ImmunofluorescenceImmunofluorescence

• 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, RIARadioimmunoassay, RIA

Chemiluminescence immunoassay, Chemiluminescence immunoassay,

CLIACLIA

Immunoblotting, Western blotting Immunoblotting, Western blotting

Immuno-PCR, IM-PCR Immuno-PCR, IM-PCR

Immunologic colloidal gold signature, Immunologic colloidal gold signature,

ICEICE

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-23Figure A-23

Figure A-26Figure 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 separationFACS 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+)

ConventionalCD4

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-

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 proliferationFACS-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 ImmunoassayApplication of Immunoassay

• Diagnosis of Diseases

infectious diseases

Immunodeficiency diseases

Autoimmune disease

hypersensitivity

Tumour

Application of ImmunoassayApplication of Immunoassay

• Immune surveilence

HBV

HIV