1. AFFINITY AND AVIDITY
A. 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 of the antibody as illustrated

2. 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

3. SPECIFICITY AND CROSS REACTIVITY
A. Specificity - Specificity refers to the ability of an individual antibody combining site to react with only one antigenic determinant or the ability of a population of antibody molecules to react with only one antigen. In general, there is a high degree of specificity in Ag-Ab reactions
Cross reactivity - Cross reactivity refers to the ability of an individual antibody combining site to react with more than one antigenic determinant or the ability of a population of antibody molecules to react with more than one antigen. Cross reactions arise because the cross reacting antigen shares an epitope in common with the immunizing antigen or because it has an epitope which is structurally similar to one on the immunizing antigen (multispecificity).

4. TESTS FOR ANTIGEN-ANTIBODY REACTIONS
Agglutination Tests
1. Agglutination/Hemagglutination - When the antigen is particulate the reaction of an antibody with the antigen can be detected by agglutination (clumping) of the antigen
Qualitative agglutination test - Agglutination tests can be used in a qualitative manner to assay for the presence of an antigen or an antibody. The antibody is mixed with the particulate antigen and a positive test is indicated by the agglutination of the particulate antigen.

5. Quantitative agglutination test - Agglutination tests can also be used to quantitate the level of antibodies to particulate antigens. In this test one makes serial dilutions of a sample to be tested for antibody and then adds a fixed number of red blood cells or bacteria or other such particulate antigen and determines the maximum dilution which gives agglutination. The maximum dilution that gives visible agglutination is called the Titre

6. Precipitation tests
1. Radial Immunodiffusion (Mancini) - In radial immunodiffusion antibody is incorporated into the agar gel as it is poured and different dilutions of the antigen are placed in holes punched into the agar. As the antigen diffuses into the gel it reacts with the antibody and when the equivalence point is reached a ring of precipitation is formed as illustrated

7. Immunoelectrophoresis - In immunoelectrophoresis a complex mixture of antigens is placed in a well punched out of an agar gel and the antigens are electrophoresed so that the antigen are separated according to their charge. After electrophoresis a trough is cut in the gel and antibodies are added. As the antibodies diffuse into the agar, precipitin lines are produced in the equivalence zone when an Ag/Ab reaction occurs as illustrated

8. Analytical methods using labeled antigens/antibodies
What is the function of the label?
To provide a means by which the free antigens, or antigen/antibody complexes can be detected
The label does not necessarily distinguish between free and bound antigens

9. Analytical methods using labeled antigens/antibodies
What are desirable properties of labels?
Easily attached to antigen/antibody
Easily measured, with high S/N
Does not interfere with antibody/antigen reaction
Inexpensive/economical/non-toxic

10. Radioisotope labels Advantages Disadvantages Flexibility Sensitivity
Size
Disadvantages
Toxicity
Shelf life
Disposal costs

11. Enzyme labels Advantages Disadvantages Diversity Amplification
Versatility
Disadvantages
Lability
Size
Heterogeneity

12. Fluorescent labels Advantages Disadvantages Size Specificity
Sensitivity
Disadvantages
Hardware
Limited selection
Background

13. Chemiluminescent labels
Advantages
Size
Sensitivity
S/N
Disadvantages
Hardware

14. Chemiluminescent labels

15. Chemiluminescent labels

16. Introduction to Heterogeneous Immunoassay
What is the distinguishing feature of heterogeneous immunoassays?
They require separation of bound and free ligands
Do heterogeneous methods have any advantage(s) over homogeneous methods?
Yes
What are they?
Sensitivity
Specificity

17. Heterogeneous immunoassays
Competitive
Antigen excess
Usually involves labeled competing antigen
RIA is the prototype
Non-competitive
Antibody excess
Usually involves secondary labeled antibody
ELISA is the prototype

18. Enzyme-linked immunosorbent assay
Substrate
2nd antibody E
Specimen S P
Microtiter well E

19. ELISA (variation 1)
Specimen
Labeled antigen E
Microtiter well S P E

20. ELISA (variation 2)
Labeled antibody E
Specimen E
Microtiter well

21. Automated heterogeneous immunoassays
The ELISA can be automated
The separation step is key in the design of automated heterogeneous immunoassays
Approaches to automated separation
immobilized antibodies
capture/filtration
magnetic separation

22. Immobilized antibody methods
Coated tube
Coated bead
Solid phase antibody methods

23. Coated tube methods
Specimen
Labeled antigen
Wash

24. Coated bead methods

25. Microparticle enzyme immunoassay (MEIA)
Labeled antibody E S P E
Glass fiber matrix

26. Magnetic separation methodsFe

27. Magnetic separation methods
Aspirate/Wash Fe

28. Electrochemiluminescence immunoassay (Elecsys™ system)
Flow cell
Oxidized
Reduced Fe

29. Introduction to Homogeneous Immunoassay
What is the distinguishing feature of homogeneous immunoassays?
They do not require separation of bound and free ligands
Do homogeneous methods have any advantage(s) over heterogeneous methods?
Yes
What are they?
Speed
Adaptability

30. Homogeneous immunoassays
Virtually all homogeneous immunoassays are one-site
Virtually all homogeneous immunoassays are competitive
Virtually all homogeneous immunoassays are designed for small antigens
Therapeutic/abused drugs
Steroid/peptide hormones

31. Typical design of a homogeneous immunoassay
No signal
Signal

32. Enzyme-multiplied immunoassay technique (EMIT™)
Developed by Syva Corporation (Palo Alto, CA) in 1970s--now owned by Behring Diagnostics
Offered an alternative to RIA or HPLC for measuring therapeutic drugs
Sparked the widespread use of TDM
Adaptable to virtually any chemistry analyzer
Has both quantitative (TDM) and qualitative (DAU) applications; forensic drug testing is the most common use of the EMIT methods

33. EMIT™ method S
Enzyme
No signal S P
Enzyme S
Signal

34. EMIT™ signal/concentration curve
Signal (enzyme activity)
Antigen concentration
Functional concentration range

35. Fluorescence polarization immunoassay (FPIA)
Developed by Abbott Diagnostics, about the same time as the EMIT was developed by Syva
Roche marketed FPIA methods for the Cobas FARA analyzer, but not have a significant impact on the market
Like the EMIT, the first applications were for therapeutic drugs
Currently the most widely used method for TDM
Requires an Abbott instrument

36. Molecular electronic energy transitions
Singlet A VR
Triplet IC F P
sec
sec E0

37. Polarized radiation z y x
Polarizing
filter

38. Fluorescence polarization
Fluorescein
in
out
( sec)
Orientation of polarized radiation is maintained!

39. Fluorescence polarization
But. . . O H C
Rotational frequency  1010 sec-1
in
out
( sec)
Orientation of polarized radiation is NOT maintained!

40. Fluorescence polarization immunoassay
Polarization maintained
Slow rotation
Rapid rotation
Polarization lost

41. FPIA signal/concentration curve
Signal (I/I)
Antigen concentration
Functional concentration range

42. Cloned enzyme donor immunoassay (CEDIA™)
Developed by Microgenics in 1980s (purchased by BMC, then divested by Roche)
Both TDM and DAU applications are available
Adaptable to any chemistry analyzer
Currently trails EMIT and FPIA applications in market penetration

43. Cloned enzyme donor Spontaneous Monomer (inactive) Active tetramer
Acceptor
Monomer
(inactive)

44. Cloned enzyme donor immunoassay
Acceptor
No activity
Acceptor
Donor
Active enzyme

45. CEDIA™ signal/concentration curve
Signal (enzyme activity)
Antigen concentration
Functional concentration range