1Umi S. Intansari
Clinical Pathology Department
Faculty of Medicine Universitas Gadjah Mada
IMMUNODIAGNOSTIC APPLICATION & INTERPRETATION
2Presentation contents
Humoral immunity:
Detection, measurement &
characterization of Ab
Cell-mediated immunity:
Isolation of lymphocytes
Characterization of lymphocytes
specificity, frequency & function
Detection immunity in vivo
3Immunoassay
An immunoassay is a test that uses antibody and antigen complexes as a means of generating a measurable result.
the specific binding of an antibody to an antigen allows the detection of analytes by a variety of immunoassay methods.
4Keith chaitoff, 2004 abbot diagnostic division
Antigen
+Antibody
+Indicator
system /
detector
Immuno-serology technique
Fundamental components
Antibody
Specific to corresponding antigen
Differentiate similar molecules
Label Generate detectable
signal
Radioisotop, enzim, Fluorochrom, chemiluminescence
Immunoassay
Precipitation Aglutination
Unlabelled
immunoassay
RIA, ELISA, CLIA, FIA Lateral flow Chromatography Flow cytometry
Labeled
immunoassay
Laboratory diagnostic method
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Ease of use
Confidence
DIRECT METHODS INDIRECT METHODS
Virus
Isolation
Genome
detection
Antigen
detection
Serology
IgM
Serology
IgG
TDR Cit. J. Cardosa
The choice of diagnostic method depends
on:
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clinical diagnosis epidemiological survey, Research, vaccine development
Purpose of the tests
laboratory facilities
technical expertise available
costs
the time of sample collection
9Immunoassays Must Be Accurate
and Precise
Keith chaitoff, 2004 abbot diagnostic division
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Detection, Measurement &
Characterization of Ab/ Ag
Precipitin reaction
Agglutination
Anti immunoglobulin Ab (Coombs test)
RIA, ELISA
Immunohistochemistry
Immunoblotting
Structure of an antibody molecule
Immunoglobulin is a general term for antibodiess
* *
The variable regions of Abs are encoded by multiple gene fragments
Variable region determine Ag binding specificity
B cells produce antibodies which can recognize antigen
An immunoglobulin molecule has two identical H-chains and two identical L-chains
Antibodies
Ag binding site
Immunoglobulin isotypes
are selectively distributed
in the body
IgG and IgM predominate in plasma,
dIgA predominates in mucosal tissues and
IgE is found in epithelia where it is associated with mast cells.
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Maturation of B cell producing antibody
IgM is the first to produced before isotype switching
After maturation B cell will express different set of isotypes depend on effector site:
low affinity but compensate by pentameric form
so far FAB determine specific
function of antibody
But, Fc also plays role
Antibody function
Fc role
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Figure A-10
Different Ab bind to distinct epitopes on Ag
Affinity?
Avidity?
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Precipitin reaction
First quantitative assay for Ab
Various amounts of soluble antigen are added to fix amount of serum containing Antibody precipitate
The amount of precipitate depends on the amount of Ag and Ab
Valence of Ab: number binding site that antibody has for Ag Valence antigen: maximum number of antibodies that can
be bound by an antigen molecule
Precipitin reaction is affected by the valence of Aband valence of Ag
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Figure A-9Precipitin curve
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Agglutination
Antibody can agglutinate to Ag on the surface of a large particle (bacteria, latex)
Hemaglutination if Ag on the surface RBC
- Widal test, C-Reactive Protein, ASTO, TPHA, RF - ABO blood grouping
Clinical application:
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Hemagglutination
B
B
BB
Anti A
B
Hemaglutination
B
B
Anti-B
+
Anti-B
Anti-B
Anti-B
B
B
Interpretation
Qualitative
Semi quantitative: titration
Dose-response effect
Visual subjective control
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Anti immunoglobulin antibodies
Immunized goat with mouse IgG
Goat anti mouse IgG
Purify using affinity chromatography
Labelled and used as a probe for bound IgG antibody
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Anti immunoglobulin antibodies
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Coombs tests and the detection of Rh
incompatibility
Use anti-immunogobulin antibodies (Coombs
reagent) to detect antibodies that cause disease
Direct : directly detect Ab bound to the surface of
fetal red blood cells
Indirect: detect nonagglutinating anti-Rh Ab in
maternal serum
Coombs tests and the detection of
Rh incompatibility
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Direct
directly detect Ab bound to the surface of fetal red blood cells
Indirect
detect nonagglutinating anti-Rh Ab in maternal serum
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Figure A-13 Rh- mothers make anti Rh Ab when they
exposed to Rh+ fetal RBC
Maternal IgG antibodies are transported across the placenta to the fetus
IgG anti Rh coated the fetal RBC
destroyed by phagocytic cells
Hemolytic anemia
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Labeled Immunoassay
Solid phase 1 reactant
Separation bound & free reagent
Color development enzyme
Basic parameters:
Enzyme: Enzyme-linked immunosorbent assay Chemiluminesce molc Radioactive molc Fluorescense molc
Label:
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Immuno-serology technique
Antigen
+Antibody
+Indicator
system /
detector
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Systems :
Direct
Indirect
Sandwich
Competitive
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Direct EIA
Ag
EAntibody + enzyme
substrat
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Direct binding assay for
Ab or Ag
Specific binding is
detected by enzyme
(ELISA) label Ab/Ag
The unlabeled
component is attached to
solid support
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Indirect EIA
Ag
Antibodi
EAnti-human Ig+enzyme
substrat
BLK SUB
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Antigen sandwich EIA
Ag
Antibody
substratEAntigen + enzyme
BLK SUB
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Competitive EIA
Ag AgAg
Antibody
EAntibody + enzyme
substrat
BLK SUB
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Capture EIA
EAntigen + enzyme
Antibody
substrate
Anti-human Ig G / M
Clinical Applications
HBsAg, HBeAgAg measurement:
IgM/ IgG toxo (TORCH), HIV, anti HCV, anti HBsAb measurement:
TSH, Thyroxin, Estrogen, insulinHormone :
PSA, AFP, Ca-19Tumor markers
Peptide, cytokine, etcOther molecules
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Interpretation
Results
Optical density
Index value : ODs/ODst
Cutoff!
Method
Unit Quantitative
Course & history of disease36
Immunofluorescence microscopy
use Ab for identifying a particular molecule in cells, tissue or biolgical fluids
Antibody or anti-Ig antibody is labeled with fluorescent dye
The dyes are excited by light and emit light of a different wavelength in visible spectrum
By selective filter, only the light coming from the dye is detected in microscope
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Figure A-17 part 1 of 2Direct Fluorescent Assay
IFA? ANA
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Figure A-16
immunohistochemistry
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Specific Ab is coupled to an enzyme
convert substrate into colored product
insoluble and precipitate in situ
Detecting a protein in tissue section
Analogous to ELISA
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Figure A-20
Interpretation of Ag Detection
Recent infection
HBsAg : acute/ chronic infection/ carrier HBeAg : replication marker, infectious
Depend on the marker:
Qualitative/ quantitative
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Interpretation of Ab Detection
IgM : recent
IgG : recent/ past/secondary paired sera?
Total
Recent/ Past infection :
Anti HBs: protective Anti HCV & anti HIV: diagnostic
Depend on the marker:
high low
Avidity
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Lateral flow Immunochromatograpy
(rapid Test)
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Detect Antigen or antibody
Processing Center
Supply Center
Matrix Cell Hopper
System Control
Center
Sampling
Center
Automation
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Characterization of lymphocyte specificity,
frequency and function
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Figure A-24Distribution of lymphocyte subpopulation in human peripheral blood
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Figure A-23Isolation of Lymphocytes
Isolation of peripheral blood lymphocytes by Ficoll-
Hypaque gradient
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Figure A-35Stimulaton of Lymphocytes
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Assay for CD4 T cells
CD4 T-cell function is usually studied by
measuring the type and amount of these
released proteins different amount &
types of cytokines
Cytokine can be detected by:
- sandwich ELISA
- ELISPOT
- Flow cytrometric measurement
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Figure A-29ELISPOT ASSAYS
A modification of ELISA antigen-capture assay
Measure frequency of T cell response (T cells secreting particular cytokines)
Each T cell that secreted cytokine give rise to single spot of color
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Figure A-30Identification of functional subsets of T cells by staining for cytokines
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Flow cytometry
a technique which cells/ events passed individually through a beam of laser light, amplified and converted to digital signal and can be plotted to form a scatter gram.
Cells or other particles can be analyzed using fluorescence technique resulted after previously fluorochrome labeled
Definition:
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flow cytometry technology
Characterize cells:
Individually
Fast
Multiparametric
Quantitatively
diagnostic & prognostic
informationSpecific
sub population
Clinical application
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Cells analysis using FACS
activatie
subset
cytokine
apoptose
cel cyclus
receptoren
adhesie
chemokine
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Subset limfosit
T-helper
T-cyt.
B cell
CD19+
CD3+ CD4+
CD3+ CD8+
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1
4
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Multiparameter Assessment
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Methods for describing the histogram distribution of signal intensities from a population of cells.
The plots show the number of cells on the vertical axis against channel numbers (related to scatter or FLorescence intensity) on the horizontal
markers are placed to delineate a region of positive intensity
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