Download Immunological diagnosis

Immunological
diagnosis
(Institute of Immunology, ZJU)
Immunodiagnosis
1.Detection of Antigen and
antibodies
2.Detection of Cellular
Immunity
Anitgen-Ab reaction
Agglutination(aggregation) Assays:
Traditional
Immunoassays
Immunodiffusion
Complement Fixation
EIA (IHC/ELISA/ELISPOT)
Modern
Immunoassays
Immunofluorescence (IFA FACS)
CLIA （Chemiluminescence immumoassay）
1. Principles and influencing
factors of Ag-Ab reaction
1) Principles of Ag-Ab reaction
a.Specificity
b.reversal combination
c.Concentration and ratio of Ag and
Ab
Immune complex
Precipitin curve
Antibody
excess zone
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)
6. ELISA
to detect Ab (HIV, HCV)
to detect Ag
to detect Ag
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
Immunoblotting
B
Absorbent
material
G
T
R
A
Gold nanoparticle labeled anti-HCG
（mouse IgG）
Ag（HCG，human chorionic gonadotropin）
mouse anti-HCG (immobilized)
Anti-mouse IgG (immobilized)
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-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 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
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)
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
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 Ipeptide-TCR
interaction.
DTH
Immunization
Challenge (Ear/Foogpad)
Measurement (Calipers)
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