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Applications of Immunochemical Methods in the Clinical Laboratory

Applications of Immunochemical Methods in the Clinical Laboratory. Roger L. Bertholf, Ph.D. Associate Professor of Pathology University of Florida College of Medicine. The University of Florida. University of Florida Health Science Center in Gainesville. The University of Florida.

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Applications of Immunochemical Methods in the Clinical Laboratory

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  1. Applications of Immunochemical Methods in the Clinical Laboratory Roger L. Bertholf, Ph.D. Associate Professor of Pathology University of Florida College of Medicine

  2. The University of Florida

  3. University of Florida Health Science Center in Gainesville

  4. The University of Florida

  5. University of Florida Health Science Center/Jacksonville

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

  7. 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

  8. Types of labels • Radioactive • Enzyme • Fluorescent • Chemiluminescent

  9. 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 Heterogeneous immunoassays

  10. The birth of immunoassay • Rosalyn Yalow and Solomon Berson developed the first radioimmunoassay in 1957

  11. Labeled antigen Specimen Wash Coated tube methods

  12. Coated bead methods

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

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

  15. Fe Fe Fe Fe Fe Fe Fe Fe Fe Magnetic separation methods

  16. Fe Fe Fe Fe Fe Aspirate/Wash Magnetic separation methods

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

  18. ASCEND (Biosite Triage™)

  19. Wash ASCEND

  20. Developer ASCEND

  21. 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

  22. No signal Signal Typical design of a homogeneous immunoassay

  23. 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

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

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

  26. Fluorescence polarization immunoassay (FPIA) • Developed by Abbott Diagnostics, about the same time as the EMIT was developed by Syva • Like the EMIT, the first applications were for therapeutic drugs • Currently the most widely used method for TDM • Requires an Abbott instrument

  27. Singlet E4 E3 E2 Triplet VR E1 IC A F 10-6-10-9 sec P 10-4-10 sec E0 Molecular electronic energy transitions

  28. z x Polarizing filter y Polarized radiation

  29. in out (10-6-10-9 sec) Fluorescein Fluorescence polarization Orientation of polarized radiation is maintained!

  30. H O O O H O C O in out (10-6-10-9 sec) Rotational frequency  1010 sec-1 Fluorescence polarization But. . . Orientation of polarized radiation is NOT maintained!

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

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

  33. 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

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

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

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

  37. No signal Signal Fluorescence excitation transfer immunoassay

  38. Oxidized Reduced Electrochemical differential polarographic immunoassay

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

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

  41. Early theories of antibody formation • Paul Ehrlich (1854-1915) proposed that antigen combined with pre-existing side-chains on cell surfaces. • Ehrlich’s theory was the basis for the “genetic theory” of antibody specificity.

  42. The “Template” theory of antibody formation • Karl Landsteiner (1868-1943) was most famous for his discovery of the A/B/O blood groups and the Rh factor. • Established that antigenic specificity was based on recognition of specific molecular structures; he called these “haptens”; formed the basis for the “template” theory of antibody formation.

  43. History of molecular imprinting • Linus Pauling (1901-1994) first suggested the possibility of artificial antibodies in 1940 • Imparted antigen specificity on native globulin by denaturation and incubation with antigen.

  44. OH NH3+ N H 2 C l N O O O- O- Fundamentals of antigen/antibody interaction CH2-CH2-CH2-CH3

  45. O O H H C C N N H H 3 3 N N N N O O N N CH3 CH3 Molecular imprinting (Step 1) Methacrylic acid + Porogen

  46. O O H H C C N N H H 3 3 N N N N O O N N CH3 CH3 Molecular imprinting (Step 2)

  47. O O H H C C N N H H 3 3 N N N N O O N N CH3 CH3 Molecular imprinting (Step 3) Cross-linking monomer Initiating reagent

  48. Molecular imprinting (Step 4)

  49. In vivo preparation Limited stability Variable specificity General applicability In vitro preparation Unlimited stability Predictable specificity Limited applicability Comparison of MIPs and antibodies Antibodies MIPs

  50. Immunoassays using MIPs • Therapeutic Drugs: Theophylline, Diazepam, Morphine, Propranolol, Yohimbine (2-adrenoceptor antagonist) • Hormones: Cortisol, Corticosterone • Neuropeptides: Leu5-enkephalin • Other: Atrazine, Methyl--glucoside

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