IMMUNODIAGNOSTIC

APPLICATION & INTERPRETATION

Umi S. Intansari
Clinical Pathology Department
Faculty of Medicine Universitas Gadjah Mada
umintasari2003@yahoo.com

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
analytes by a variety of
immunoassay methods.
3

Immuno-serology technique

Keith chaitoff, 2004 abbot diagnostic division

+
Antigen

Indicator
system /
detector

Antibody
4

Fundamental components

Antibody

Label

Specific to
corresponding
antigen
Differentiate similar
molecules

Generate detectable
signal
Radioisotop, enzim,
Fluorochrom,
chemiluminescence

Immunoassay
Unlabelled
Precipitation
immunoassay Aglutination

RIA, ELISA, CLIA, FIA

Labeled
Lateral flow Chromatography
immunoassay Flow cytometry

Laboratory diagnostic method

Ease of use

DIRECT METHODS

Virus
Isolation

Genome
detection

INDIRECT METHODS

Antigen
detection

Serology
IgM

Serology
IgG

Confidence
7

TDR Cit. J. Cardosa

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

Keith chaitoff, 2004 abbot diagnostic division

Detection, Measurement &

Characterization of Ab/ Ag
Precipitin reaction
Agglutination
Anti immunoglobulin Ab (Coombs 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 FAB 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

Anti A
B

Anti-B
Anti-B

Anti-B
Hemaglutination
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Interpretation
Qualitative
Semi quantitative: titration
Dose-response effect
Visual
subjective
control

20

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

22

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

23

Coombs tests and the detection of

Rh incompatibility

Direct

Indirect

directly detect
Ab bound to the
surface of fetal
red blood cells

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

Figure A-13

Maternal IgG antibodies are transported

across the placenta to the fetus

IgG anti Rh coated the fetal RBC

destroyed by phagocytic cells

Hemolytic anemia

25

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
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Immuno-serology technique

+
Antigen

Indicator
system /
detector

Antibody

27

Systems :
Direct
Indirect
Sandwich
Competitive

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Direct EIA

substrat

Antibody + enzyme

Ag

29

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

substrat

Antibody

Ag

BLK SUB

32

Competitive EIA

substrat

Antibody + enzyme

E
Antibody
Ag

Ag

Ag

BLK SUB

33

Capture EIA

Antigen + enzyme

substrate

Antibody

Anti-human Ig G / M

34

Clinical Applications
Ag measurement:

HBsAg, HBeAg

Ab measurement:

IgM/ IgG toxo (TORCH), HIV, anti HCV, anti

HBs

Hormone :
Tumor markers

Other molecules

TSH, Thyroxin, Estrogen, insulin

PSA, AFP, Ca-19
Peptide, cytokine, etc
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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
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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
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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

43

Lateral flow Immunochromatograpy

(rapid Test)
Detect Antigen or antibody

44

Automation
Matrix Cell Hopper
Processing Center

System Control
Center

Supply Center
Sampling
Center

Characterization of lymphocyte specificity,

frequency and function

47

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

49

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

52

Identification of Figure
functionalA-30
subsets of T cells
by staining for cytokines

53

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

54

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

1
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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

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