Immunological Immunological diagnosisdiagnosis

(Institute of Immunology, ZJU)

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

1) 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

Avidity: the overall strength of interaction between an Ab and an Ag.

Avidity• The overall strength of binding between an Ag

with many determinants and multivalent Abs

Keq = 104

Affinity

106

Avidity

1010

Avidity

b. reversalb. reversal combinationnoncovalent forcenoncovalent forcehydrogen bondhydrogen bondelectrostatic electrostatic attractionattractionVan der Wals forcesVan der Wals forces hydrophobic bondhydrophobic bonddegree of dissociationdegree of dissociationAg-Ab affinityAg-Ab affinityEnvironmental factorsEnvironmental factors

C. Concentration and ratio C. Concentration and ratio of Ag and Abof Ag and Ab

When the antigens and When the antigens and antibodies are present in an antibodies are present in an appropriate ratio, they form appropriate ratio, they form insoluble immune complexes insoluble immune complexes (e.g. aggregation or (e.g. aggregation or precipitation) large enough to precipitation) large enough to be seen.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 this way has three zones.

Ab excess zone (prozone) equivalence zone Ag excess zone (postzone)

Imm

un

e co

mp

lex

Antibody excess zone

Precipitin curve

d. Two d. Two phasephasessSpecific combinationSpecific combinationVisible phaseVisible phase

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)

Methods Methods

• Indirect ELISA: Ab measurement

• Sandwich ELISA: Ag detection

• Competitive ELISA: Ag or Ab detection

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

Magnetic cell sorting (MACS)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-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.

Fluoresceinated avidin

Biotin

MHC I

Antigenic peptide

MHC I

Ag-MHC tetramer technique

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.

D. phagocytic dysfunctionE. Cytokine production biological activity immunoassay:ELISA, intracellular

CKs, ELISPOT PCR

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