1. بسم الله الرحمن الرحيم

2. Faculty of Allied Medical Sciences
Clinical Immunology & Serology Practice
(MLIS 201)

3. Antigen Antibody Reaction
Prof. Dr. Ezzat M Hassan
Prof. of Immunology
Med Res Inst, Alex Univ

4. 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

5. 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

6. Nature of antigen-antibody interaction
i.e epitope-paratope binding forces

7. Binding of the epitope with the paratope (antigen binding site)
POOR FIT
GOOD FIT
Antigen binding site
Antigen binding site
epitope
epitope
Study Guide
What constitutes a good fit in an antigen binding site?
How does antibody affinity relate to fit?
What are the forces that are attractive and repulsive?
What is actually "fitting" into the combining site?
antigen determinant
high attraction
low repulsion
high repulsion
low attraction

8. Factors Affecting binding of Ag/Ab
Affinity
Avidity
Law of Mass Action
Physical form of Ag
Ag:Ab ratio

9. 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.
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.

10. 2-Avidity (cont.)
The overall strength of binding between a multivalent Antigen and multivalent Antibodies Y
Keq =
104
Affinity Y
106
Avidity Y
1010
Avidity

11. Specificity Cross Reactivity
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.
Cross Reactivity
The ability of an individual Ab binding site to react with more than one antigenic determinant.
The ability of a population of Ab molecules to react with more than one Ag

12. Similar epitope Shared epitope
Anti-A Ab
Ag A
Anti-A Ab
Ag C
Similar epitope
Cross reactions
Anti-A Ab
Ag B
Shared epitope
Specific Reaction

13. Antigen antibody tests
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.

14. Methods for detection of Ag-Ab Reactions
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

15. Methods for detection of Ag-Ab Reactions
1- Precipitation reactions

16. Precipitation Serological Tests

17. 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.

18. Precipitation reactions
Polyclonal Ab can form lattices, or large aggregates with polyvalent Ag.
Monoclonal antibody can link only two molecules (epitopes) of antigen and no precipitate is formed.
( Lattices or
large aggregates )
( no precipitate is formed)

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

20. Precipitation curve

21. Precipitation techniques
1. Tube precipitation test
2. Gel diffusion
Single radial
Double
3. Immuno-electrophoresis, immuno-fixation

22. Precipitation techniques
1- Tube precipitation test
Ag & Ab molecules diffuse until they reach equivalence zone
Visible Precipitation
(In gel)
PPT in Fluid
PPT in Gel

23. 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

24. 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
is proportional to the Ag concentration.

25. Ouchterlony Double Diffusion
Both Ab and Ag diffuse independently from wells into a gel medium

26. Electrophoresis Techniques
Electrophoresis separates molecules according to differences in their electrical charge.
Rocket Immunoelectrophoresis
Countercurrent Immunoelectrophoresis
Immunoelectrophoresis
Immunofixation Electrophoresis

27. 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 Ags precipitin arcs Ag - + Ag Ab Ag Ab
Interpretation- Precipitin arc represent individual antigens

29. 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

30. Methods for detection of Ag-Ab Reactions
1- Precipitation Reactions 2- Agglutination Reactions

31. Agglutination Serological Tests

32. Agglutination Principle evaluation: Qualitative (y/n),
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)

34. Agglutination Tests
Types of Agglutination Reactions
Direct agglutination Tests
Indirect agglutination Tests
Agglutination Inhibition Tests

35. (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.

36. a) Blood Grouping Y +  Y
Anti-RBC
RBC
Agglutinate

38. b) Direct Coombs (Antiglobulin)Test
detects the presence of antibodies on red blood cell
Simply add a second antibody ( Antiglobulin antibody, Coomb’ Reagent) directed against the immunoglobulin coating the red cells.
This anti-immunoglobulin antibodies cross link the red blood cells and result in agglutination.

39. C) Latex Test
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
positive
negative

40. (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.

41. Hemagglutination-Inhibition
Some viruses (eg. Measles virus) agglutinate RBCs in vitro.
Anti-virus Ab will bind the virus and inhibit hemagglutination.

42. Applications of agglutination tests in Serology
Determination of blood group antigens.
Diagnosis of cases with Rh incombatability (erythoblastosis fetalis)
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)

43. Agglutination vs. Precipitation
Soluble Ag & Ab
Ag must have at least two determinants
Ag xs results in postzone reaction
Ab xs results in prozone reactions
Reaction time: hours to days
Test results: qualitative, semi-quantitative or quantitative
Agglutination
Insoluble or particulate Ag or Ab
Ag must have at least two determinants
Ag xs results in postzone reaction
Ab xs results in prozone reactions
Reaction time: minutes to hours
Test results: qualitative or semi-quantitative

44. Methods for detection of Ag-Ab Reactions
1- Precipitation Reactions 2- Agglutination Reactions
3- Labelling methods:
a- Immuno-fluorescence
b- ELISA.
c- Flow cytometry

45. Immunofluorescence: Methods
Direct
Indirect
Fix specimen on slide
Add Flourescein labeled antibody specific for the desired antigen
Look for fluorescence
Fix specimen on slide
Add antibody specific for the desired antigen
Add second Flourescein labeled antibody
Look for fluorescence

49. Immunofluorescence: Interpretation
Fluorescence = patient has the antigen

50. Immunologic Lab Tests Outline
Agglutination reactions
DAT
IAT
Immunofluorescence
ELISA

51. 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

52. 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

54. ELISA: Interpretation
Color change = patient has the antibody

55. ELISA: Variations detects antigen (not antibody)
Sandwich immunoassay
detects antigen (not antibody)
coat well with antibody
rest is like ELISA

57. Radioimmunoassay detects antibody or antigen
Label (detector) is a radioactive substance
otherwise like ELISA or sandwich immunoassay

58. Immunologic Lab Tests Outline
Agglutination reactions
DAT
IAT
Immunofluorescence
ELISA
Western blot

59. Western Blot: Purpose Detection of antibodies in patient specimen
Most common example: HIV test

60. 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

63. Western Blot: Interpretation
Bands on strip = patient has antibodies to
corresponding proteins

64. Enough bands = patient is “positive”

65. Immunologic Lab Tests Outline
Agglutination reactions
DAT
IAT
Immunofluorescence
ELISA
Western blot
Flow cytometry

66. Flow Cytometry: Purpose
Examples:
diagnosis of leukemia and lymphoma
determination of CD4/CD8 counts in patients with HIV

71. Immunologic Lab Tests Outline
Agglutination reactions
DAT
IAT
Immunofluorescence
ELISA
Western blot
Flow cytometry

72. Study Questions: Complete:
The Factors Affecting binding of Ag/Ab are ……………………….
Compare between: Agglutination & Precipitation.

73. Assignment Write on the following subjects:
1. Factors affecting Ag-Ab reaction
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

74. Thanks