1Umi S. Intansari

Clinical Pathology Department

Faculty of Medicine Universitas Gadjah Mada

umintasari2003@yahoo.com

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

30

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

32

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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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