IMMUNODIAGNOSTIC APPLICATION & INTERPRETATION
Umii S. Int Um Intan ansa sari ri Clinical Pathology Department Faculty Facul ty of Medicin Medicine e Universitas Universitas Gadjah Gadjah Mada
[email protected]
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Presentation contents
Humoral immunity:
Detection, measurement & characterization of Ab
Cell-mediated immunity:
Isolation of lymphocytes Characterization of lymphocytes specificity, frequency & function
Detection immunity in vivo 2
Presentation contents
Humoral immunity:
Detection, measurement & characterization of Ab
Cell-mediated immunity:
Isolation of lymphocytes Characterization of lymphocytes specificity, frequency & function
Detection immunity in vivo 2
Immunoassay 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 analyt ana lytes es by a varie variety ty of immunoassay methods. 3
Immuno-serology technique
Keith chaitoff, 2004 abbot diagnostic division
+ Antigen
+
Indicator system / detector
Antibody 4
Fundamental components
Antibody •
•
Specific to corresponding antigen Differentiate similar molecules
Label • •
Generate detectable signal Radioisotop, enzim, Fluorochrom, chemiluminescence
Immunoassay Unlabelled immunoassay
Labeled immunoassay
Precipitation • Aglutination •
• • •
RIA, ELISA, CLIA, FIA Lateral flow Chromatography Flow cytometry
Laboratory diagnostic method Ease of use
DIRECT METHODS
Virus Isolation
Genome detection
INDIRECT METHODS
Antigen detection
Serology IgM
Serology IgG
Confidence 7
The choice of diagnostic method depends on: Purpose of the tests • • •
clinical diagnosis epidemiological survey, Research, vaccine development
laboratory facilities technical expertise available costs the time of sample collection 8
Immunoassays Must Be Accurate and Precise
9
Detection, Measurement & Characterization of Ab/ Ag Precipitin reaction Agglutination Anti immunoglobulin Ab (Coomb’s test) RIA, ELISA Immunohistochemistry Immunoblotting 10
Antibodies Structure of an antibody molecule
B cells produce antibodies which can recognize antigen
*
Ag binding site
*
An immunoglobulin molecule has two identical H-chains and two identical L-chains
The variable regions of Abs are encoded by multiple gene fragments Variable region determine Ag binding specificity
Immunoglobulin is a general term for antibodiess
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. 1
Maturation of B cell producing antibody
IgM is the first to produced before isotype switching
low affinity but compensate by pentameric form
After maturation B cell will express different set of isotypes depend on effector site:
Antibody function
Fc role
so far F AB determine specific function of antibody But, Fc also plays role
Figure A-10
Different Ab bind to distinct epitopes on Ag Affinity? Avidity? 15
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 Precipitin reaction is affected by the valence of Ab and valence of Ag •
•
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 16
Precipitin curve
Figure A-9
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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
Clinical application: • •
- Widal test, C-Reactive Protein, ASTO, TPHA, RF - ABO blood grouping 18
Hemagglutination
Anti-B
B
B
B
B
B
B
B
Anti A
B
Anti-B Anti-B
+
B
Anti-B Hemaglutination 19
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 21
Anti immunoglobulin immunoglobulin antibodies
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Coombs tests and the detection of Rh incompatibility
Use antianti-immun immunogobu ogobulin lin antib antibodies odies (Coomb (Coombs s reagent) to detect antibodies that cause disease Direct : directly Direct directly detect detect Ab bound to to the surface surface of fetal red blood cells Indirec Indi rect: t: de dete tect ct no nona nagg gglu luti tina natin ting g an anti ti-R -Rh h Ab in maternal serum
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Coombs tests and the detection of Rh in inco comp mpat atib ibililit ity y
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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•
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
Figure A-13
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Labeled Immunoassay Basic parameters: • • •
Solid phase 1 reactant Separation bound & free reagent Color development enzyme
Label: • • • •
Enzyme: Enzyme-linked immunosorbent assay Chemiluminesce molc Radioactive molc Fluorescense molc 26
Immuno-serology technique
+ Antigen
+
Indicator system / detector
Antibody
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Systems :
Direct
Indirect
Sandwich
Competitive
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Direct EIA
substrat
E
Antibody + enzyme
Ag
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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 E
Anti-human Ig+enzyme
substrat
Antibodi
Ag
BLK SUB
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Antigen sandwich EIA
Antigen + enzyme
E
substrat
Antibody
Ag
BLK SUB
32
Competitive EIA
substrat
Antibody + enzyme
E Antibody Ag
Ag
Ag
BLK SUB
33
Capture EIA
Antigen + enzyme
E
substrate
Antibody
Anti-human Ig G / M
34
Clinical Applications Ag measurement:
•
HBsAg, HBeAg
•
IgM/ IgG toxo (TORCH), HIV, anti HCV, anti HBs
Hormone :
•
TSH, Thyroxin, Estrogen, insulin
Tumor markers
•
PSA, AFP, Ca-19
Other molecules
•
Peptide, cytokine, etc
Ab measurement:
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Interpretation Results Optical density Index value : ODs/ODst Cutoff! Method Unit
Quantitative
Course & history of disease 36
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 37
Direct Fluorescent Assay
Figure A-17 part 1 of 2
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IFA? ANA
Figure A-16
39
immunohistochemistry Analogous to ELISA Detecting a protein in tissue section Specific Ab is coupled to an enzyme
convert substrate into colored product
insoluble and precipitate in situ 40
Figure A-20
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Interpretation of Ag Detection Recent infection
Depend on the marker: • •
HBsAg : acute/ chronic infection/ carrier HBeAg : replication marker, infectious
Qualitative/ quantitative 42
Interpretation of Ab Detection Recent/ Past infection : •IgM
: recent •IgG : recent/ past/secondary •Total
paired sera?
Depend on the marker:
Anti HBs: protective • Anti HCV & anti HIV: diagnostic •
Avidity •
•
high low
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Lateral flow Immunochromatograpy (rapid Test) Detect Antigen or antibody
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Automation Matrix Cell Hopper Processing Center
System Control Center
Supply Center Sampling Center
Characterization of lymphocyte specificity, frequency and function
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Distribution of lymphocyte subpopulation in human peripheral blood
Figure A-24
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Isolation of Lymphocytes
Figure A-23
Isolation of peripheral blood lymphocytes by FicollHypaque gradient
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Stimulaton of Lymphocytes
Figure A-35
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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 51
ELISPOT ASSAYS
Figure A-29
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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Identification of Figure functionalA-30 subsets of T cells by staining for cytokines
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Flow cytometry Definition:
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
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flow cytometry technology Characterize cells:
Individually
Fast
Multiparametric
Quantitatively
Specific sub population
diagnostic & prognostic information
Clinical application 55
Cells analysis using FACS subset receptoren
activatie
cel cyclus
adhesie
apoptose
chemokine cytokine 56
Subset limfosit B cell
CD19+ T-helper
CD3+ CD4+ T-cyt.
CD3+ CD8+ 57
4
2
3
1 58
Multiparameter Assessment
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60
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
