1. Antigen – Antibody Reactions or Serological Reactions

2. Antigen-Antibody interactions
Characterized as:
Non-covalent interaction (similar to “lock and key” fit of enzyme-substrate)
Do not lead to irreversible alteration of Ag or Ab
This exact and specific interaction has led to many immunological assays that are used to:
detect Ag or Ab
diagnose disease
measure magnitude of humoral IR
identify molecules of biological and medical interest

3. Introduction
Ag – Ab reactions are one of the most specific noncovalent biochemical reactions known
The forces that hold the reactants together are:
- van der Waals forces
- Electrostatic forces
- Hydrophobic forces
They can be represented by the simple formula:
Ag + Ab ↔ AgAb
The reaction is driven to the right but it is reversible

5. Strength of Reaction
The strength of the reaction (how far it is driven to the right) is referred to as affinity
Antibody affinity
- A quantitative measure of binding strength
- Combined strength of the noncovalent
interactions between a binding site on an Ab &
monovalent Ag
- Affinity varies broadly among immunoglobulins

6. Strength of Reaction Antibody avidity
- Avidity is often used to describe the collective affinity of multiple binding sites on an antibody molecule
- 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 )

7. CROSS REACTIVITY
Antibody elicited by one Ag can cross-react with a related Ag.
Occurs if two different Ags share identical or very similar epitope
1- Vaccinia virus and smallpox virus
2- Rabies & JE vaccine
3- Streptococcus pyogenes infection: heart &
Kidney damage following infection
4- Original antigenic sin.
5- Bacterial Ag and sugars on RBC

8. STAGES OF Ag - Ab REACTIONS
Primary reactions Vs secondary reactions: Small Ag - Ab complexes Vs large complexes (The Lattice hypothesis)
Development of macroscopic manifestations reactions (e.g. immunoprecipitation)
Ag – Ab reactions involving IgM are confined to the blood stream, while those of lower molecular weight (IgG and IgE) can leave the vasculature and enter tissues
Time required is hours to days for precipitin formation leading to irreversible immunoprecipitates

9. LATTICE THEORY
Lattice formation (visible Ag - Ab aggregates) occurs when:
Ag is multivalent (contains more than 2 identical epitopes)
Cross-linking of Ags by specific Abs (2 or more antigen-binding sites)
Molar ratios of epitopes and antigen-binding sites are optimal (zone of equivalence)

10. Zone of
equivalence

11. LATTICE THEORY Zones of lattice formation
Far Ag excess (no ppt. formed; free Ag in supernatant) -- “postzone”
Ag excess (sub-optimal ppt.; free Ag in spnt.)
Zone of equivalence (maximum ppt.; no Ag or Ab in spnt.)
Ab excess (sub-optimal ppt; Ab in spnt.)
Far Ab excess (no ppt; Ab in spnt.) -- “prozone”

12. ZONES OF PRECIPITIN FORMATION
Precipitin Curve

13. METHODS THAT DETECT Ag- Ab REACTIONS
Primary Reactions:
- Immunofluorescence (IF)
- Radioimmunoassay (RIA)
- Enzyme immunoassay (EIA)
- Immunonephelometry (measures picogram to
nanogram quantities of analyte)
Secondary Reactions
- Agglutination Techniques
- Precipitation Techniques ± Electrophoresis

15. Precipitation
Precipitation can take place in capillary tubes, test tubes, and in gel

16. Precipitation in gel - Double diffusion - Single (radial) diffusion
- Combination of diffusion in gel and
electrophoresis

17. SINGLE VS. DOUBLE DIFFUSION
Single diffusion
Supporting medium (gel) contains one reactant at a uniform concentration
Only the unknowns move through the medium
Double diffusion
Gel is inert (contains no reactants)
Both Ag and Ab travel through the medium

18. The region of
equivalence

19. RADIAL IMMUNODIFFUSION
Ab uniformly distributed in gel; Ag diffuses outward from a well (single diffusion)
Ag- Ab complexes form as concentric rings around the well at zone of equivalence
At a set time, ring diameters are measured
[Ag] is directly proportional to the ring d2
Unknown value is determined by comparing to a 3-standard curve

20. RADIAL IMMUNODIFFUSION
Samples
Standards
Precipitin Rings
A B C
a b c
Standard Curve

21. RADIAL IMMUNODIFFUSION
Fahey method (kinetic)
Read at 18 hours
Plot [std] vs. ring diameter on semi-log paper
Mancini method (endpoint)
Read at 48 or 72 hours
Plot [std] vs. ring diameter squared on graph paper
Results reliable only if the ring size is within the range of the standards; if greater than highest std, dilute and repeat test
Used to measure IgM, IgG, C4,C3,transferrin, CRP, others

23. OUCHTERLONY DOUBLE DIFFUSION
Ag & Ab placed in wells cut into an agarose gel (both reactants diffuse)
Precipitin line (or arc) indicates Ab has specificity for Ag
Position of precipitin between wells depends on MW and concentration of reactants
3 possible patterns of reaction: identity, non-identity, partial identity

25. OUCHTERLONY DOUBLE DIFFUSION
Ouchterlony Plates Precipitin Patterns

28. ELECTROIMMUNOASSAY (ROCKET)
Electrophoresis hastens movement of Ag (placed in wells) through Ab -imbedded gel (single diffusion)
Selected pH (8.6) keeps Abs at their isoelectric point; they will not move
Rocket-shaped precipitin bands will form at zone of equivalence (changes as reactants move)
[Ag] proportional to length of rocket
Unknowns compared to standards

29. ELECTROIMMUNOASSAY (ROCKET)

30. ELECTROIMMUNOASSAY (ROCKET)
May be used to quantitate plasma proteins such as coagulation factors, alpha- fetoprotein, C3, C4, CRP, haptoglobin
Compared with RID:
faster
similar sensitivity
requires electrophoretic equipment and more technological finesse
Largely replaced by immunonephelometry

31. IMMUNONEPHELOMETRY Ag + Ab  AgAb  microscopic Ag - Ab complexes
Microcomplexes cause light moving through the suspending solution to scatter
Nephelometer detects light scattered at a 90o angle
Amount of light scattered at 90o is proportional to Ag - Ab complexes formed
Sensitive and quantitative technique used for measurement of many serum proteins

32. IMMUNOELECTROPHORESIS (IEP)
Electrophoresis and double diffusion
2 stages
Proteins separated by electrophoresis
Antiserum placed in trough parallel to separated proteins; all reactants diffuse in all directions
Precipitin forms at zones of equivalence
Trough may be filled with simple or complex antisera yielding simple to complex patterns

33. Immunoelectrophoresis of normal human serum.33
Immunoelectrophoresis of normal human serum.

36. IMMUNOELECTROPHORESIS (IEP)
Qualitative to semi-quantitative
Serum, urine, or CSF may be analyzed
Complex patterns may be difficult to interpret
Useful to detect:
missing proteins
abnormal proteins
normal proteins in abnormal concentrations
Used to evaluate conditions such as multiple myeloma
Largely replaced by immunofixation

37. IMMUNOFIXATION ELECTROPHORESIS (IFE)
Proteins that were separated by electrophoresis are exposed to Ab directly, instead of through diffusion
Steps:
Electrophoresis of protein mixture in gel (use serum or urine samples)
Paper strips imbedded with specific Ab are “blotted” onto gel; Ags transfer to paper and bind to Abs
Strips washed (unbound material washes away)
Strips stained to reveal precipitin bands

38. IFE

39. IMMUNOFIXATION ELECTROPHORESIS (IFE)
Used to detect the presence of Igs in conditions like multiple myeloma
Fairly sensitive - Ab is highly specific, electrophoresis leaves Ag isolated and accessible
Faster and easier to interpret than IEP
Only 1 Ab may be used per strip

40. WESTERN BLOTTING Similar to IFE but the unknown is Ab rather than Ag
Steps:
Separation of complex antigenic material (eg., viral proteins) by electrophoresis
Separated components transferred from gel to nitrocellulose paper by “blotting”
Unknown (or control) sera (which may have Abs) incubated with paper strips; Ag - Ab complexes ppt. at site of transfer
Strips washed; staining reveals complexes

41. WESTERN BLOTTING

42. The Western blot procedure.42

43. FLOCCULATION
Immunoprecipitation (or agglutination) of insoluble particles
Characterized by very sharp pro- and postzones
No precipitin formed in zones of Ab or Ag excess, only in zone of equivalence
Clinically important examples, VDRL and RPR tests (screening tests for syphilis)

44. FLOCCULATION VS. IMMUNOPRECIPITATION

45. VDRL (Venereal Disease Research Lab.) test
Flocculation Tests
VDRL (Venereal Disease Research Lab.) test
RPR (Rapid Plasma Reagin) test

46. Agglutination Titer Zeta potential Types of Agglutination
- Direct agglutination or hemagglutination
- Indirect (passive) agglutination or hemagglutination
- Agglutination or hemagglutination inhibition
The Coombs test
- Direct
- Indirect

47. Agglutination Reactions

48. Agglutination Qualitative slide agglutination
- identification of bacteria with antisera directed against
O, H, K antigens

49. Agglutination
Latex agglutination
Coagglutination

50. Agglutination Tube agglutination tests:
- Gruber-Widal: typhoid fever (S. typhi)
- Weil-Felix: typhus (Rickettsia)
- Wright: brucellosis
Identify and titrate antibodies in the patient’s
serum.
Titre: is defined as the reciprocal of the
highest dilution of serum showing agglutination.

51. 1:200
1:400
1:100
Titer

53. Agglutination inhibition

54. Hemagglutination Inhibition Test
To Detect Antibodies (Rubella)
- Serum (Ab)+ HA +RBCs= No Hemagglutination
= Positive Test
- Serum (No Ab)+ HA + RBCs =Hemagglutination =Negative Test
To Detect Antigen (HBsAg)
- Serum (HBsAg) +Anti HBsAG + HBsAg coated RBCs = No Hemagglutination = Positive Test
- Serum (No HBsAg)+ Anti HBsAG + HBsAg coated RBCs = Hemagglutination =Negative Test

55. Use of Labels in Ag – Ab Reactions
Immunoassays
- Radioimmunoassay (RIA)
- Enzyme Immunoassys (EIA)
Immunofluorescence (IF)
- Direct IF
- Indirect IF
Flow cytometry and Cell Sorting (FACS)

58. Immunologic Tests
4) Radioimmunoassay (RIA)– a very sensitive test; used for measuring hormones, serum proteins, drugs, etc. at low concentrations (≤ 0.001ug/ml) measures “competitive binding” of radiolabelled Ag + unlabelled (test) Ag to high affinity Ab

59. ELISA

60. ELISA tests
Depend on enzyme conjugated to 2 Ab reacting with a specific substrate to produce a color reaction.
Variations of ELISA’s: Allows for qualitative or quantitative testing. Each one can be used for qualitative detection of Ag or Ab
Also, a standard curve based on known concentrations of Ag/Ab can be prepared and an unknown concentration can be determined
Indirect ELISA
Sandwich ELISA
Competitive ELISA

62. Direct and indirect Immunofluorescence

63. Immunoprecipitation
Provides a quick and sensitive test for finding proteins/Ag’s especially in low concentrations
Binds Ab to synthetic bead support  centrifuged
Or 2° Ab with bead or magnetic bead and collect by magnetism

65. Distribution of selected markers on some leukemia
cell types → Immunophenotyping using
“flow cytometry & mAb”

66. Sensitivity of various immunoassays