1. Antigen – Antibody Reactions
DIL – 7
Nov. 5th 2013
Mohammed El-Khateeb

2. General characteristic of Ab/Ag Reactions
Non-covalent interaction (similar to “lock and key” fit of enzyme-substrate)
Does not lead to irreversible alteration of Ag or Ab
This exact and specific interaction has led to many immunological assays used to:
detect Ag or Ab
diagnose disease
measure magnitude of humoral IR
identify molecules of bio and med interest

3. Ag-Ab Interactions Bonds: Hydrogen Ionic Hydrophobic interactions
Van der Waals forces
Each bond is weak; many are strong
To “hold” they must be close  requiring high amts of complementarity!

4. Three Distinct Phases of Ag-Ab Reactions
Primary phenomenon: Sensitization – binding of antibody to antigen – not visible
Secondary Phenomenon :
Lattice Formation
The Fab portion of the Ig molecule attaches to antigens on 2 adjacent cells-visible results in agglutination
If both antigen and antibody are SOLUBLE reaction will become visible over time, ie, precipitation
Tertiary Phenomenon: Reaction not visible, detected by affect of reaction on tissues or cells.

5. Factors Affecting Measurement of Ag/Ab Reactions
Affinity
Avidity
Ag:Ab ratio
Physical form of Ag

6. Affinity =  attractive and repulsive forces
Strength of the reaction between a single antigenic determinant and a single Ab combining site Ab Ag
High Affinity Ab Ag
Low Affinity
Affinity =  attractive and repulsive forces

7. Avidity Y Y Y Keq = Affinity Avidity Avidity 104 106 1010
Incorporates affinity of multiple binding sites
True strength of the Ab-Ag interaction within biological systems
The interaction at one site will increase the possibility of reaction at a second site
High avidity can compensate for low affinity ( secreted pentameric IgM has a higher avidity than IgG ) Y
Keq =
104
Affinity Y
106
Avidity Y
1010
Avidity

8. Specificity
The ability of an individual antibody combining site to react with only one antigenic determinant.
The ability of a population of antibody molecules to react with only one antigen.

9. Antigen Binding
Antigen 2
Antigen 1
Antigen 3
Variable
Light
Heavy

10. Ccroos Reactionsross Reactivity
The ability of an individual Ab combining site to react with more than one antigenic determinant.
The ability of a population of Ab molecules to react with more than one Ag
Anti-A Ab
Ag C
Similar epitope
Cross reactions
Anti-A Ab
Ag A
Anti-A Ab
Ag B
Shared epitope

11. Secondary Manifestations of Ag-Ab Reactions
1. Precipitation 2. Agglutination 3. Complement dependent reactions a. cytolysis b. chemotaxis c. opsonization 4. Neutralization LATUCE FORMATION

12. Primary Manifestation of Ag-Ab reactions
1. Immunofluorescence (IF) 2. Radioimmunoassay (RIA) 3. Enzyme-linked immunosorbent assay (ELISA)

13. Factors affecting solubility
Size
Charge
Temperature
Solvent ionic strength

14. Antibody-antigen Interactions
Antibodies and antigens can both be multivalent.
The flexibility of the hinge region improves the efficiency of antigen binding and cross-linking.

15. Polyclonal Antibodies
Produced by immunizing an animal with the appropriate antigen.
The immunized animal’s serum is collected.
Antibodies can then be purified from the serum.
Since one antigen induces the production of many antibodies the result is a ‘polyclonal’ mixture of antibodies.
Polyclonal antibodies are much less expensive than monoclonal antibodies.
Problems related to specificity. You can enrich for antibodies produced from a particular antigen, but they won’t be alone unless the serum is subsequently affinity purified.

16. Monoclonal Antibodies
Much more complicated to produce than polyclonal antibodies.
Process begins by immunizing an animal (most commonly a mouse) with an antigen.
The animal’s spleen is removed.
B-cells are fused with myeloma cells resulting in hybridomas.
Hybridomas are screened to find those producing antibodies to the antigen with which they were immunized.
Each hybridoma cell is derived from one B-cell so the antibodies that a clonal population of hybridoma cells produce are monoclonal antibodies.
Monoclonal antibodies recognize one epitope only.
Spleen is the site of B-cell production.
Hybridomas are ‘immortal’ cell lines.

17. PRECIPITATION TESTS
Lattice Formation

18. Precipitin Reactions
Reaction of soluble antigens with IgG and IgM antibodies
Form visible molecular aggregates called LATTICES
Precipitation only occurs where the ratio of antigen to antibody is optimal

19. TYPES OF PRECIPITATION REACTIONS
Double Diffusion (Ouchterlony)
Counter electrophoresis
Radial Immunodiffusion (Mancini)
Roquete Technique
Immunoelectrophoresis
Immunofixation
Nephlometry / Turbidimetry

20. Precipitation reactions in fluids

21. Precipitin ring test Principles of Immunological Tests
Precipitation – formation of an insoluble complex when a specific antibody is reacted with a soluble antigen (usually in a gelatin-like substance)

22. Ouchterlony Gel Diffusion
Both antigen and antibody can diffuse independently.
Holes punched in agar.
Known antibody or antigen added to center well.
Known sample added to outer well.
Unknown sample added to outer well next to unknown sample.
Wait for bands to form.

24. Ouchterlony Analysis: 2-Dimensional Precipitation in Agarose

25. Ouchterlony Immunodiffusion
Identity
Partial Identity
Non-Identity
The precipitation appears as a continuous line in the form of an angle between those two wells and the C well. There are no spurs at the angle and this type of reaction is termed a band of identity
If Ag A (patient) and Ag A1 (control) share a common element but are not exactly the same (Abs to A), a single spur is formed. This is the line of partial identity.
If the material in wells 1 and 2 do not possess common antigens and the antiserum in well 3 possesses specificities for both materials, the reaction will appear as two crossed lines

26. Countercurrent electrophoresis
Method
Ag and Ab migrate toward each other by electrophoresis
Used only when Ag and Ab have opposite charges
Qualitative
Rapid Ag Ab - + Ag Ab - + PS

27. 1 2 Single radial immunodiffusion Method Ab in gel Ag in a well
Quantitative
Interpretation
1. Diameter of ring
is proportional
to the concentration
2. Highet of the
Rocket 1 2
Electroimmunodiffusion

28. Serum Protein Electrophoresis (SPE)
Elevated total protein suggests hypergammaglobulinemia
Total protein = Albumin + Globulins
Quantitative immunoglobulins (Measures amount of IgM, IgG and IgA )
In myeloma, typically see “reciprocal depression” of uninvolved Igs

29. The Monoiclonal Gammopathies
IgG C
IgM
IgA
IgM
IgG
IgA

30. Serum protein electrophoresis
Albumin 1 2   + -

31. Protein Electrophoresis

32. Monoclonal Gammopathy
Normal Pattern
Albumin decreased and sharp peak in gamma region
Albumin 1 2  

33. Immunoelectrophoresis
Combines serum protein electrophoresis with immunometric detection
Electrophoresis provides separation
Immunoprecipitation provides detection
Two related applications:
Immunoelectrophoresis
Immunofixation electrophoresis

34. IMMUNOELECTROPHORESIS

35. Immunofixation Electrophoresis
Immunofixation Electrophoresis (IFE) combines zone electrophoresis with immunoprecipitation.
This technique may be used to identify and characterise serum proteins.
In IFE, proteins of sample are first separated by electrophoresis on a support (agarose) according to their charge and after that the medium is overlaid with monospesific antiserá reactive with specific protein - antigen.
If the antigen is present a characteristic immunoprecipitin band will be formed.

36. Immunofixation Electrophoresis

37. IMMUNOFIXATION IgG Lambda IgM Kappa IgG Kappa 1. Normal Plasma
2. Polyclonal
Hyperglobulinemia
3. Monoclonal Spike
4. Bence Jones proteins in urine

38. Western blotting
Separates the components according to their molecular weight.
The proteins in the gel are transferred to the sheet of nitrocellulose or nylon by the passage of an electric current.
Probed with Ab & then radiolabeled or
enzyme-linked 2nd Ab.
A position is visualized by means of an ELISA reaction.

39. The Western blot procedure
[INSERT FIGURE 17.17]

40. Tests Based on Ag/Ab Reactions
Ab’s and Ag’s in aqueous solution form a lattice Precipitin
Lattice formation requires:
polyvalent Ab’s
Ag must be bivalent, polyvalent
All tests based on Ag/Ab reactions will have to depend on lattice formation or they will have to utilize ways to detect small immune complexes
All tests based on Ag/Ab reactions can be used to detect either Ag or Ab

41. Agglutination Tests
Lattice Formation

42. Agglutination
Visible clumping together of particulate matter by antigen combining with its specific antibody.
The clumps will be called agglutinates
Performed
Slide
Tube
Tile
Micrtitration plates

43. TYPES OF AGGLUTINATION REACTIONS
Direct aggultination
Hemagglutination
Indirect (Passive) Agglutination
Agglutination inhibition

44. Direct Agglutination
Bacterial Antigen
- ve
+ ve

46. Direct agglutination
Figure Overview

47. Agglutination Reactions
Particulate antigens, such as cells, and antibodies
Form visible clumps or aggregates
Very sensitive and easy to read
Direct agglutination tests
Detect antibodies against large cellular antigens – RBC’s, bacteria, fungi
Antibody titer
Indirect agglutination tests
Antigens absorbed onto latex spheres

48. Agglutination / Hemagglutination
Definition - tests that have as their endpoint the agglutination of a particulate antigen both Ag and Ab can be determined
Qualitative
1/2
1/4
1/8
1/16
1/32
1/64
1/128
1/256
1/512
1/1024
Pos.
Neg.
Titer 64 8
512
<2 32
128 4
Patient 1 2 3 5 6 7
Applications
Blood typing
Bacterial infections
Fourfold rise in titer
Practical considerations
Easy
Semi-quantitative
Quantitative test

49. Passive Agglutination/Hemagglutination
Definition - agglutination test done with a soluble antigen coated onto a particle Y + 
Applications
Measurement of
antibodies to soluble
antigens

50. Agglutination/Hemagglutination Inhibition
Definition - test based on the inhibition of agglutination due to competition with a soluble Ag Y + 
Prior to Test Y + 
Test
Patient’s sample

51. Direct Coombs Test / Direct Antiglobin test
Indirect Coombs Test / Indirect Antglobin test

52. Applications
These include detection of anti-rhesus factor (Rh) antibodies. Antibodies to the Rh factor generally do not agglutinate red blood cells. Thus, red cells from Rh+ children born to Rh- mothers, who have anti-Rh antibodies, may be coated with these antibodies. To check for this, a direct Coombs test is performed. To see if the mother has anti-Rh antibodies in her serum an Indirect Coombs test is performed.  52

53. Antigen-Antibody Reactions In Vivo
THE GOOD
Neutralization of viruses and toxins
Opsonization of pathogens
Complement lysis of bacteria
Prevention of bacterial adherence
The Bad
Autoimmunity - myasthenia gravis
Transfusion reactions
The Ugly
Allergy

54. Monoclonal vs. Polyclonal
Selection is based on application, time and money

55. IMMUNOPRECIPITATION

56. Immune Testing

57. Assays Based on Complement
Lattice formation not required

58. Complement Fixation Phosphate Buffer containing Ca+ & Mg+
Inactivated Patient Serum
Rabbit Complement.
SRBC
IgM Anti SRBC

59. Complement Fixation Y Methodology Ag No Ag
Ag mixed with test serum to be assayed for Ab
Standard amount of complement is added
Erythrocytes coated with Abs is added
Amount of erythrocyte lysis is determined Ag
No Ag Ag Y
Patient’s
serum Ag Y

60. +veC
veC
P.S CC BC

61. Specimens for Routine Tests

62. Sensitivity of Various Immunoassays
ASSAY Sensitivity(mg Ab/ml)
Precipitation reaction in fluid
Precipitation reaction in Gel (DD)
Precipitation reaction in Gel (IEP)
Precipitation reaction in Gel (RID)
Precipitation reaction in Gel Rocket IE 2
Agglutination
Direct
Passive
Inhibition
Radioimmunoassay
ELISA <
ELISA using Chemiluminescence <
Immunoflourescence
Flowcytometry ,006