بسم الله الرحمن الرحيم
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بسم الله الرحمن الرحيم. Faculty of Allied Medical Sciences Clinical Immunology & Serology Practice (MLIS 201). Prof. Dr. Ezzat M Hassan Prof. of Immunology Med Res Inst, Alex Univ E-mail: [email protected] Antigen Antibody Reaction. TEACHING OBJECTIVES :

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بسم الله الرحمن الرحيم

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بسم الله الرحمن الرحيم


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Faculty of Allied Medical Sciences

Clinical Immunology & Serology Practice

(MLIS 201)


Antigen antibody reaction

Prof. Dr. Ezzat M Hassan

Prof. of Immunology

Med Res Inst, Alex Univ

E-mail: [email protected]

Antigen Antibody Reaction


Clinical immunology serology practice code mlis 201

TEACHING OBJECTIVES:

1. To discuss the factors affecting Ag-Ab reactions

2. To define Affinity & Avidity

3. To know different methods of Ag-Ab reactions

4. To describe the basics and types of precipitation reactions

6. To describe the basics and types of aggulination reactions

7. To describe the basics and types of immuno-flourescent techniques

8- To describe the basics and types of ELISA

Clinical Immunology & Serology Practice (Code: MLIS 201)


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Serology

The science that deals with the properties and reactions of sera, especially blood serum.

Usually refers to the diagnostic identification of Ag or Ab in the serum.

Serological assays depends on Antigen-antibody interactions


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Nature of antigen-antibody interaction

i.e epitope-paratope binding forces


Binding of the epitope with the paratope antigen binding site

POOR FIT

high repulsion

high attraction

low attraction

low repulsion

Binding of the epitopewith the paratope (antigen binding site)

GOOD FIT

Antigen binding site

Antigen binding site

epitope

epitope

antigen determinant


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Factors Affecting binding of Ag/Ab

Affinity

Avidity

Law of Mass Action

Physical form of Ag

Ag:Ab ratio


1 affinity

  • Antibody affinity is the strength of the binding between a single antigenic determinant and a single combining site on the antibody.

  • The higher the affinity of the antibody for the antigen, the more stable will be the interaction.

1-Affinity

  • 2-Avidity

  • Avidity is a measure of the overall strength of binding of multivalent Ag (i.e. antigen with many antigenic determinants) and multivalent antibodies.

  • Reactions between multivalent antigens and multivalent antibodies are more stable and thus easier to detect.


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Y

Y

Y

Y

Y

Y

Y

106

104

1010

Keq =

Avidity

Avidity

Affinity

2-Avidity (cont.)

The overall strength of binding between a multivalent Antigen and multivalent Antibodies


Specificity

The ability of an individual Ab binding site to react with only one antigenic determinant.

The ability of a population of antibody molecules to react with only one antigen.

Specificity

Cross Reactivity

  • The ability of an individual Abbinding site to react with more than one antigenic determinant.

  • The ability of a population of Abmolecules to react with more than one Ag


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Anti-A Ab

Anti-A Ab

Anti-A Ab

Ag B

Ag C

Shared epitope

Similar epitope

Ag A

Cross reactions

Specific Reaction


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All tests based on Ag/Ab reactions can be used to detect either Ag or Ab

Cannot be visualized, thus Detection of antigen/antibody reactions is difficult

Various laboratory methods have been developed to make this reaction visible eg. Precipitation, agglutination, labeled reagents (ELISA, RIA, IF), …etc.

Antigen antibody tests


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  • Precipitation reactions (Ag is soluble) precipitin.

  • Agglutination reactions (Ag is particles) clumping.

  • Complement fixation reactions.

  • Labelling methods:

    a- Immuno-fluorescence reactions.

    b- ELISA.

    c- Flow cytometry

Methods for detection of Ag-Ab Reactions


Methods for detection of ag ab reactions

1- Precipitation reactions

Methods for detection of Ag-Ab Reactions


Precipitation serological tests

Precipitation Serological Tests


Precipitation techniques

  • Precipitation reactions Involve soluble antigens

    with antibodies cross-linked and form lattice that

    Eventually develops into a visible precipitate.

  • In immunodiffusion, antigens and antibodies diffuse through a gel until they reach the zone of equivalence.

  • In immunoelectrophoresis, diffusion is combined with electrophoresis.

Precipitation techniques


Precipitation reactions

  • Polyclonal Abcan form lattices, or large aggregates with polyvalent Ag.

  • Monoclonal antibodycan link only two molecules (epitopes) of antigen andno precipitateis formed.

Precipitation reactions

( Lattices or

large aggregates )

( no precipitate is formed)


Precipitation curve

The plot of the amount of antibody precipitated versus increasing antigen concentrations (at constant total Ab) reveals 3 zones:

Precipitation curve

  • Zone of antibody excess:

  • Precipitation is inhibited.

  • Ab not bound to Ag can be detected in the supernatant;

  • 2. Zone equivalence:

  • Maximal precipitation in which antibody and antigen form large insoluble complexes.

  • No Ab or Ag can be detected in the supernatant

  • 3. Zone of antigen excess:

  • Precipitation is inhibited.

  • Ag not bound to Ab can be detected in the supernatant


Precipitation curve1

Precipitation curve


Precipitation techniques1

1. Tube precipitation test

2. Gel diffusion

  • Single radial

  • Double

    3. Immuno-electrophoresis, immuno-fixation

Precipitation techniques


Precipitation techniques2

Precipitation techniques

  • 1- Tube precipitation test

  • Ag & Ab molecules diffuse until they reach equivalence zone

    Visible Precipitation

(In gel)

PPT in Gel

PPT in Fluid


Precipitation techniques3

Precipitation techniques:

2. Gel Immunodiffusion Techniques

  • Radial Immunodiffusion

    • A single diffusion technique where Ab is put into a gel and Ag is measured by the size of a precipitin ring formed when it diffused out in all directions from a well cut into the gel

  • Ouchterlony Double Diffusion

    • Both Ab and Ag diffuse from wells into a gel medium


Radial immunodiffusion

Radial Immunodiffusion

  • A quantitative technique based upon the reaction between an Ag placed in a well diffuses into an agar containing the Ab.

  • During diffusion period the Ag & Ab

  • continue to react until the zone of

  • equivalence is reached with

  • formation of a well-defined ring of

  • precipitation around the Ag well which

  • isproportional to the Ag concentration.


Ouchterlony double diffusion

Ouchterlony Double Diffusion

  • Both Ab and Ag diffuse independently from wells into a gel medium


Electrophoresis techniques

Electrophoresis Techniques

  • Electrophoresis separates molecules according to differences in their electrical charge.

    • Rocket Immunoelectrophoresis

    • Countercurrent Immunoelectrophoresis

    • Immunoelectrophoresis

    • Immunofixation Electrophoresis


3 immuno electrophoresis

3. Immuno-electrophoresis

Ag molecultes are separated according to differences in their electrical charge by electrophoresis

Ab is placed in a trough cut in the agar

Ab diffuse towards Agsprecipitin arcs

-

+

Ag

Ag

Ab

Ag

Ab

  • Interpretation- Precipitin arc represent individual antigens


Measurement of precipitation

Measurement of Precipitation

  • Turbidimetry

    • Passing light through a cloudy solution of Ag-Ab complexes.

    • Net decrease in light intensity correlates to the concentration of the Ag

  • Nephelometry

    • Measuring light scattered at a particular angle after being passed through a solution of Ag-Ab complexes.

    • Amount of light scattered correlates to the concentration of the Ag


Methods for detection of ag ab reactions1

1- Precipitation Reactions

2- Agglutination Reactions

Methods for detection of Ag-Ab Reactions


Agglutination serological tests

Agglutination Serological Tests


Agglutination

Agglutination

  • Principle

    • Particulate antigen + antibody –> clumping

    • The clumps will be called agglutinates

    • Lattice formation (antigen binds with Fab sites of 2 antibodies forming bridges between antigens)

    • Agglutination Curve follow

      the same roles as in precipitation curve

  • evaluation:

  • Qualitative (y/n),

  • Semi-quantitative (serum titration)


Agglutination tests

Agglutination Tests

Types of Agglutination Reactions

  • Direct agglutination Tests

  • Indirect agglutination Tests

  • Agglutination Inhibition Tests


I direct agglutination tests

(I) Direct Agglutination Tests

  • Used to determine antibody titer against particulate antigens (Bacteria, Fungi or RBCs)

  • Particulate antigen reacts directly with antibodies.

  • Applications

    • Blood group typing

    • Direct Coomb’s Test

    • The diagnosis of certain diseases (Bacterial infection

    • Eg. Syphilis)

    • The identification of some viruses.


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+

Y

Y

Y

a) Blood Grouping

Y

Anti-RBC

RBC

Agglutinate


B direct coombs antiglobulin test

b) Direct Coombs (Antiglobulin)Test

  • detects the presence of antibodies on red blood cell

  • Simply add a second antibody ( Antiglobulinantibody, Coomb’ Reagent)directed against the immunoglobulin coating the red cells.

  • This anti-immunoglobulin antibodies cross link the red blood cells and result in agglutination.


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Ag or Ab are not part of particulate matter but are attached to it (ANTIBODIES OR ANTIGENS ARE ATTACHED TO PLASTIC BEADS)

EASY TO DO

Eg. Latex test for RF & CRP

C) Latex Test

positive

negative


Ii indirect agglutination coomb s test

(II) indirect agglutination (Coomb’s)test

To detect anti- RBC’s antibodies in a serum sample.

  • This test is done by incubating RBC’s with the serum sample

  • washing out any unbound antibodies

  • then

  • adding a second anti-immunoglobulin reagent (Coomb’s Reagent) to cross link the cells resulting in agglutination.


Hemagglutination inhibition

Hemagglutination-Inhibition

  • Some viruses (eg. Measles virus) agglutinate RBCs in vitro.

  • Anti-virus Ab will bind the virus and inhibit hemagglutination.


Applications of agglutination tests in serology

Applications of agglutination tests in Serology

  • Determination of blood group antigens.

  • Diagnosis of cases with Rhincombatability (erythoblastosisfetalis)

  • Diagnostics of autoimmune hemolytic diseases

  • Latex test for detection of Ags or Abs (eg. RF, CRP)

  • To assess bacterial infections (e.g. Typhoid Fever, Syphilis)


Agglutination vs precipitation

Agglutination

Insoluble or particulate Ag or Ab

Ag must have at least two determinants

Agxs results in postzone reaction

Abxs results inprozonereactions

Reaction time: minutes to hours

Test results: qualitative or semi-quantitative

Precipitation

Soluble Ag & Ab

Ag must have at least two determinants

Agxs results in postzone reaction

Abxs results inprozonereactions

Reaction time: hours to days

Test results: qualitative, semi-quantitative or quantitative

Agglutination vs. Precipitation


Methods for detection of ag ab reactions2

1- Precipitation Reactions

2- Agglutination Reactions

Methods for detection of Ag-Ab Reactions

3- Labelling methods:

a- Immuno-fluorescence

b- ELISA.

c- Flow cytometry


Immunofluorescence methods

Immunofluorescence: Methods

  • Fix specimen on slide

  • Add Flourescein labeled antibody specific for the desired antigen

  • Look for fluorescence

Direct

Indirect

  • Fix specimen on slide

  • Add antibody specific for the desired antigen

  • Add second Flourescein labeled antibody

  • Look for fluorescence


Immunofluorescence interpretation

Immunofluorescence:Interpretation

Fluorescence = patient has the antigen


Immunologic lab tests outline

Immunologic Lab Tests Outline

  • Agglutination reactions

  • DAT

  • IAT

  • Immunofluorescence

  • ELISA


Enzyme linked immunosorbent assay purpose

Enzyme-Linked Immunosorbent Assay: Purpose

  • Detection of antibodies in patient specimen

  • Examples:

    • home pregnancy tests

    • HIV tests

    • tests for some coagulation factors, cytokines, and autoantibodies


Elisa method

ELISA:Method

  • Fix the target (AG or Ab) to wells of microtiter plate

  • Add patient specimen to well coated with ligand

  • Add AHG with enzyme attached

  • Add substrate

  • Measure color change


Elisa interpretation

ELISA:Interpretation

Color change = patient has the antibody


Elisa variations

ELISA: Variations

  • Sandwich immunoassay

    • detects antigen (not antibody)

    • coat well with antibody

    • rest is like ELISA


Radioimmunoassay

  • detects antibody or antigen

  • Label (detector) is a radioactive substance

  • otherwise like ELISA or sandwich immunoassay

Radioimmunoassay


Immunologic lab tests outline1

Immunologic Lab Tests Outline

  • Agglutination reactions

  • DAT

  • IAT

  • Immunofluorescence

  • ELISA

  • Western blot


Western blot purpose

Western Blot: Purpose

  • Detection of antibodies in patient specimen

  • Most common example: HIV test


Western blot method

Western Blot: Method

  • Make a protein suspension of the target you’re looking for (e.g., HIV)

  • Electrophorese the suspension onto a little gel strip

  • Apply the patient’s specimen (containing antibodies) to the strip

  • Add AHG that has an enzyme attached

  • Add substrate and look for bands


Western blot interpretation

Western Blot: Interpretation

Bands on strip = patient has antibodies to

corresponding proteins


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Enough bands = patient is “positive”


Immunologic lab tests outline2

Immunologic Lab Tests Outline

  • Agglutination reactions

  • DAT

  • IAT

  • Immunofluorescence

  • ELISA

  • Western blot

  • Flow cytometry


Flow cytometry purpose

Flow Cytometry: Purpose

  • Examples:

    • diagnosis of leukemia and lymphoma

    • determination of CD4/CD8 counts in patients with HIV


Immunologic lab tests outline3

Immunologic Lab Tests Outline

  • Agglutination reactions

  • DAT

  • IAT

  • Immunofluorescence

  • ELISA

  • Western blot

  • Flow cytometry


Study questions

Complete:

The Factors Affecting binding of Ag/Ab are ……………………….

Compare between: Agglutination & Precipitation.

Study Questions:


Assignment

Write on the following subjects:

1. Factors affecting Ag-Abreaction

أميرة رفعت – اميرة صلاح – ايمان محمود – ايمن شكرى – داليا ناصر

2. Precipitation reactions

دعاء عبد الله – رغدة رشدى – رنا ابراهيم – سمر عبد الحميد – سمية جمال

Assignment


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Thanks


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