1. Immunochemical Methods in the Clinical Laboratory
Roger L. Bertholf, Ph.D., DABCC
Mark A. Bowman, Ph.D., MT(ASCP)

2. The University of Florida

3. University of Florida Health Science Centers in Gainesville and Jacksonville

4. The University of Iowa

5. University of Iowa College of Medicine
                                                                                      
           

6. Florida vs. Iowa
                                                               

7. The American Society of Clinical Pathologists
Marie Bass, MT(ASCP)
Manager, ASCP Workshops for Laboratory Professionals
Kathleen Dramisino, MT(ASCP)
Workshop coordinator
Tommie Ware
A/V and materials support

8. Classification of immunochemical methods
Particle methods
Precipitation
Immunodiffusion
Immunoelectrophoresis
Light scattering
Nephelometry
Turbidimetry
Label methods
Non-competitive
One-site
Two-site
Competitive
Heterogeneous
Homogeneous

9. Properties of the antibody-antigen bond
Non-covalent
Reversible
Intermolecular forces
Coulombic interactions (hydrogen bonds)
Hydrophobic interactions
van der Waals (London) forces
Clonal variation

10. Antibody affinity

11. Precipitation of antibody/antigen complexes
Detection of the antibody/antigen complex depends on precipitation
No label is involved
Many precipitation methods are qualitative, but there are quantitative applications, too

12. Factors affecting solubility
Size
Charge
Temperature
Solvent ionic strength

13. The precipitin reaction
etc.
Precipitate
Zone of equivalence
Antibody/Antigen

14. Single radial immunodiffusionAg

15. Single radial immunodiffusion

16. Electroimmunodiffusion
Why would we want to combine immunodiffusion with electrophoresis?
SPEED
Specificity
Carl-Bertil Laurell (Lund University, Sweden)
Laurell Technique (coagulation factors)
“Rocket electrophoresis”

17. Electroimmunodiffusion+ -

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

19. Immunoelectrophoresis+ -
-human serum
Specimen

20. Immunoelectrophoresis+ - P C P C P C     

21. Immunofixation electrophoresis
Immunochemical Methods (handout addendum)
Immunofixation electrophoresis
SPE
IgG
IgA
IgM  
Bertholf and Bowman

22. Particle methods involving soluble complexes
The key physical property is still size
Measurement is based on how the large antibody/antigen complexes interact with light
The fundamental principle upon which the measurement is made is light scattering
Two analytical methods are based on light scattering: Nephelometry and Turbidimetry

23. Light reflection

24. Molecular size and scattering- +

25. Distribution of scattered radiation

26. Nephelometry vs. Turbidimetry
0°-90°

27. Rate nephelometry
Intensity of scattering
Time
Rate C1 C2

28. Additional considerations for quantitative competitive binding immunoassays
Response curve
Hook effect

29. Competitive immunoassay response curve
%Bound vs. log concentration
%Bound label
Antigen concentration

30. Log antigen concentration
Logistic equation a
%Bound label c
Slope = b d
Log antigen concentration

31. Log antigen concentration
Logit transformation a
%Bound label d
Log antigen concentration

32. Log antigen concentration
Logit plot
Logit y
Log antigen concentration

33. High dose “hook” effect
%Bound antigen
Antigen concentration

34. 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

35. 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

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

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

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

39. Chemiluminescent labels
Advantages
Size
Sensitivity
S/N
Disadvantages
Hardware ?

40. Chemiluminescent labels

41. Chemiluminescent labels

42. 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

43. 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

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

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

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

47. Human anti-animal antibodies
Humans exposed to animals can produce antibodies to animal immunoglobulins
Heterophilic antibodies
Anti-isotypic
Anti-idiotypic
Human anti-mouse antibodies (HAMA) are most common
Anti-animal antibodies can cross-link capture and detection reagent antibodies

48. 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

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

50. Coated tube methods
Specimen
Labeled antigen
Wash

51. Coated bead methods

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

53. Magnetic separation methodsFe

54. Magnetic separation methods
Aspirate/Wash Fe

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

56. ASCEND (Biosite Triage™)

57. ASCEND
Wash

58. ASCEND
Developer

59. Solid phase light scattering immunoassay

60. 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

61. 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

62. Typical design of a homogeneous immunoassay
No signal
Signal

63. 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

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

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

66. 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

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

68. Polarized radiation z y x
Polarizing
filter

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

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

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

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

73. 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

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

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

76. CEDIA™ signal/concentration curve
Immunochemical Methods (handout addendum)
CEDIA™ signal/concentration curve
Functional concentration range
Signal (enzyme activity)
Antigen concentration
Bertholf and Bowman

77. Other approaches to homogeneous immunoassay
Fluorescence methods
Electrochemical methods
Enzyme methods
Enzyme channeling immunoassay

78. Substrate-labeled fluorescence immunoassay
Enzyme
No signal S
Fluorescence
Enzyme S
Signal

79. Fluorescence excitation transfer immunoassay
No signal
Signal

80. Electrochemical differential polarographic immunoassay
Oxidized
Reduced

81. Prosthetic group immunoassay
Enzyme
No signal S P
Enzyme P
Signal

82. Enzyme channeling immunoassay
Substrate
Product 1 E1
Product 2 E2

83. Artificial antibodies
Immunoglobulins have a limited shelf life
Always require refrigeration
Denaturation affects affinity, avidity
Can we create more stable “artificial” antibodies?
Molecular recognition molecules
Molecular imprinting

84. Molecular imprinting

85. Immunochemical Methods (handout addendum)
A final thought. . .
“In science one tries to tell people, in such a way as to be understood by everyone, something that no one ever knew before. But in poetry, it's the exact opposite.”
Paul Adrien Maurice Dirac ( )
Bertholf and Bowman