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Immunochemical Methods in the Clinical Laboratory

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Presentation on theme: "Immunochemical Methods in the Clinical Laboratory"— Presentation transcript:

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 immunodiffusion

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

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 methods

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

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