1. 2. Basic Immunologic Procedures
Terry Kotrla, MS, MT(ASCP)BB
Fall 2005

2. Introduction Detection of antigen/antibody reactions difficult
Sensitization is the binding of a specific antibody to its’ specific antigen
Cannot be visualized
Multitude of laboratory methods have been developed to make this visible

3. Three Distinct Phases of Antigen/Antibody Reactions
Primary Phenomenon – Sensitization
Secondary Phenomenon – Lattice formation
Tertiary Phenomenon – Detected by affect on tissues or cells.

4. Primary phenomenon
Sensitization – binding of antibody to antigen – not visible

5. 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

6. Tertiary Phenomenon
Reaction not visible, detected by affect of reaction on tissues or cells.

7. Phagocytosis

8. Secondary Phenomena Most Frequently Utilized
Precipitation – soluble antibody reacts with soluble antigen
Agglutination – particulate antigens bound together by antibody
Complement Fixation – antibody binding to antigen triggers activation of complement

9. Antigen-Antibody Binding
Affinity
Avidity
Law of Mass Action

10. Affinity
Antibody affinity is the strength of the reaction between a single antigenic determinant and a single combining site on the antibody.
It is the sum of the attractive and repulsive forces operating between the antigenic determinant and the combining site .
Affinity is the equilibrium constant that describes the Ag-Ab reaction as illustrated in Figure 3. Most antibodies have a high affinity for their antigens.

11. Affinity

12. Avidity
Avidity is a measure of the overall strength of binding of an antigen with many antigenic determinants and multivalent antibodies.
Affinity refers to the strength of binding between a single antigenic determinant and an individual antibody combining site whereas avidity refers to the overall strength of binding between multivalent antigens and antibodies.
Avidity is influenced by both the valence of the antibody and the valence of the antigen.
Avidity is more than the sum of the individual affinities.

13. Avidity

14. Law of Mass Action
Governs the reversibility of the antigen-antibody reaction.
Reversible reaction, visible reaction occurs when the rate of binding exceeds the rate of dissociation.

15. Precipitation Curve
Prozone – antibody excess, many antibodies coat all antigen sites- results in false negative
Postzone – antigen excess, antibody coats antigen but cannot get lattice formation, results in false negative
Zone of Equivalence – antigen and antibody present in optimal proportions to bind and give visible reaction

17. Precipitation Curve

18. Precipitation Curve

19. Measurement of Precipitation by Light
Antigen-antibody complexes, when formed at a high rate, will precipitate out of a solution resulting in a turbid or cloudy appearance.
Turbidimetry measures the turbidity or cloudiness of a solution by measuring amount of light directly passing through a solution.
Nephelometry indirect measurement, measures amount of light scattered by the antigen-antibody complexes.

20. Precipitation/Flocculation
When soluble antibody binds to soluble antigen (sensitization) there will come a point where lattice formation will occur resulting in precipitation occurring resulting in a visible reaction
These immune complexes have fallen out of solution. The Ab at the bottom in the illustration at right is still in the soluble phase.

21. Turbidimetry
Measures turbidity or cloudiness of a solution by measuring the amount of light PASSING THROUGH the solution.
Soluble antigen and antibody join and once they join in sufficient amounts precipitate, results in cloudiness.
The more cloudy the solution, the less light can pass through.

22. Nephelometry
Measures SCATTERED light bouncing off antigen-antibody complexes.

23. Nephelometry

24. Passive Immunodiffusion
Reactions in gels
Migrate towards each other and where they meet in optimal proportions form a precipitate.

25. Four Methodologies Single diffusion, single dimension
Single diffusion, double dimension
Double diffusion, single dimension
Double diffusion, double dimension

26. Ouidin Single Diffusion, Single Dimension

27. Oudin Precipitation
Solution of antibody is carefully layered on top of a solution of antigen, such that there is no mixing between the two. 
With time at the interface where the two layers meet, antigen-antibody complexes form a visible precipitate.  The other two tubes are negative controls, containing only antibody or only antigen plus an irrelevant protein in the second layer. 

28. Radial Immunodiffusion

29. Standard Curve

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

31. Ouchterlony Gel Diffusion
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.

32. Ouchterlony Immunodiffusion

33. Ouchterlony - 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.

34. Ouchterlony – Partial Identity
FIGURE 2: If a solution with antigens X and Y is placed in well 1, a solution with antigen X only is placed in well 2, and antiserum containing antibodies specific for both X and Y is placed in well 3, a reaction similar to that appearing in Fig. 2 will occur. Notice that there is a spur reaction towards the XY well. This indicates that the two antigenic materials in wells 1 and 2 are related, but that the material in well 1 possesses an antigenic specificity not possessed by the material in well 2. Such a reaction with spur formation indicates partial identity

35. Ouchterlony – Non-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 as in Fig. 3

36. Ouchterlony-Interpret
Determine which interpretation fits for samples 1, 2 and 3.

37. Electrophoretic Techniques
Immunodiffusion can be combined with electrical current to speed things up.

38. Rocket Immunoelectrophoresis
Antigen is electrophoresed into gel containing antibody. The distance from the starting well to the front of the rocket shaped arc is related to antigen concentration.

39. Rocket Electrophoresis

40. Immunoelectrophoresis

41. Immunoelectrophoresis
Two-dimensional immunoelectrophoresis. Antigens are separated on the basis of electrophoretic mobility. The second separation is run at right angles to the first which drives the antigens into the antiserum-containing gel to form precipitin peaks; the area under the peak is related to the concentration of antigen.

42. Immunoelectrophoresis-Antivenom
Each antibody molecule can bind two separate sites on an antigen molecule (venom toxin), consequently antibodies have the ability to cross link many antigen molecules simultaneously.    This cross-linking causes the antibody antigen-complex to become insoluble and precipitate out from the solution.
The immunoelectrophoresis technique makes use of this capability of the antibodies to form giant insoluble complexes with their respective antigens.    The antigen-antibody precipitate which forms can be visualised by specific staining techniques, or quantified by various means.

43. 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.

44. Immunofixation Electrophoresis

45. Immunofixation Electrophoresis

46. Enhancement of Agglutination
Additive to neutralize charge
Viscosity
Treatment with enzymes
Agitation and centrifugation
Temperature pH

47. Direct Agglutination Antigen found naturally on particle.
Blood Grouping is an example, antigen on cell

48. ABO Blood Grouping

49. Passive Agglutination
Employs particles that are coated with antigens, ie , RBCs, polystyrene latex, bentonite or charcoal.

50. Reverse Passive Agglutination
Antibody attached to carrier particle instead of antigen.
Serologic Typing of Shigella: Positive Test

51. Agglutination Inhibition
Based on competition between particulate and soluble antigens for limited antibody combining sites.
Patient sample added to reagent antibody specific for antigen being tested, if antigen is present it binds to reagent antibody.
Reagent particles (latex or RBCs) coated with the same antigen are added, if antigen was present in the sample all reagent antibody binds to it so no antibody is present to react with antigens coating the particles

52. Agglutination Inhibition
In row A wells 1-8 are positive.

53. Coagglutination
Name given to systems using bacteria as the inert particles to which antibody is attached.

54. Labeled Immunoassays
Some antigen/antibody reactions not detected by precipitation or agglutination.
Measured indirectly using a labeled reactant.
Referred to as receptor-ligand assays.
Ligand is the substance to be measured and is defined as a molecule that binds to
nother molecule of a complementary configuration, usually it binds to the
substance the test is trying to detect.
The receptor is what binds the specific target molecule.

55. Labeled Immunoassays Ligand – substance to be measured.
Receptor – binds the specific target molecule
Sandwich technique
Next slide - example

56. “Sandwich” Technique ELISA

57. Labels Used to detect reaction which has occurred.
Radioactive, fluorescent, chemiluminescent and enzymes.
Competitive or non-competitive
Heterogenous or homogeneous

58. Standards or Calibrators
Substance of exact known concentration.
Usually run for each new lot number
Based on results create standard curve.
Standard curve used to “read” results or built into machine to provide results.

59. Competitive Binding Add known labeled antigen Add unknown antigen
Will compete with each other for sites on bound antibody molecule.
Must wash off unreacted substances.
Type of label on known antigen will determine method of detection.

60. Competitive Binding

61. Radioimmunoassay

62. Radioimmunassay (RIA)
Competitive binding
Uses Iodine 125 (I 125) as label
Radioactive label competes with patient for sites
High radioactivity, small amount of patient substance
Low radioactivity high amount of patient substance
Textbook page 159 figure 11-2

64. Radioimmunassay

65. Radioimmunoassay Competitive
A plasma (or urine) sample that contains ADH is mixed with the antibodies.
A known amount of "standard" ADH that has been tagged with radioactivity is added to the antibody mixture.
The "standard" ADH is something that has already been measured and that can be used to compare the unknown quantity of natural ADH.
A radioactive tag is added to the standard ADH so that it can be distinguished from the natural ADH that is in the plasma.
As the antibodies, the standard radioactive ADH, and the plasma are mixed, one main requirement must be met: there must be too little antibody to bind completely to both the radioactive hormone and the natural hormone in the fluid to be assayed.
Therefore, the natural hormone in the plasma and the radioactive standard hormone compete for the binding sites on the antibody (next slide)

66. Radioimmunoassay Competitive

67. Immunoradiometric Assay (IRMA)
Labeled antibody plus patient antigen
Solid phase antigen added to bind excess antibody
Spin down to separate
Labeled antibody/antigen remain in solution.
Measure radioactivity
Textbook page 160 fig 11-3

68. Enzyme Immunoassay Advantages Reason for choosing enzyme as label
Enzymes used
Two classifications:
Heterogenous – separation of reactants must be done
Homogeneous – no separation step
Two types:
Competitive – known and unknown compete
Noncompetitive – only unknown reacts

69. Enzyme Immunoassay http://tinyurl.com/ckdaz

70. Heterogenous Enzyme Assays
Competitive ELISA
Noncompetetive ELISA
Immunoenzymometric Assay
Sandwich or capture assays

71. Competitive ELISA Unknown antigen competes with labeled known antigen
Enzyme produces color reaction

72. Noncompetitive ELISA Referred to as “Indirect” ELISA
Antigen bound to solid phase
Add patient, antibody will bind if present
Add known labeled antibody
Measure enzyme label
Textbook page 161 figure 11-4
Disadvantage more manipulation of test

73. Immunoenzymometric Assay
Noncompetitive ELISA
Detects unknown antigen by means of excess labeled antibody
Textbook page 162 figure 11-5

74. Sandwich or Capture Assays
Used for antigens with multiple epitopes, ie, HCG
Antibody to one epitope fluid, antibody to second epitope fixed.
Enzyme label used to detect reaction
Textbook page 163 figure 11-6

75. Homogeneous Enzyme Assay
Labeled reagent antigen and patient sample added.
Compete for bound antibody
Antibody binds to patient antigen causing inactivation of enzyme
Competitive Assay
Enzyme activity proportional to concentration of patient antigen

76. Fluorescence Immunoassay

77. Markers Fluorophores or fluorochromes
Ability to absorb energy and emit light
Two most commonly used:
Fluorescein – green
Tetramethylrhodamine – red

78. Fluorescence

79. Fluorescence Antibodies and bacteria are fixed on a glass-plate.
The surplus i.e. non-bounded antibodies are washed out, antibody-bacteria-complexes ("sandwiches") remain.
The "sandwich" becomes visible by adding fluorescent anti bovine immunoglobulin which can be seen as green light in the fluorescence microscope.

80. Fluorescent Immunoassay
Direct immunofluorescence
Tagged antibody added to unknown antigen fixed to slide
Indirect immunofluorescence
Patient plus known fixed antigen
Add tagged anti-antibody
Fluorescence

82. Positive

83. Heterogeneous Fluorescent Immunoassays

84. Homogenous Assays

85. Fluorescence Polarization Immunoassay

86. References http://www.nsbri.org/HumanPhysSpace/focus4/sf-hormonal.html
ELISA
ELISA
molecular diagnostics

87. References (Continued)