Immunochemical Methods in the Clinical Laboratory

1. Immunochemical Methods in the Clinical Laboratory Roger L. Bertholf, Ph.D., DABCC Chief of Clinical Chemistry & Toxicology, UFHSC/Jacksonville Associate Professor of Pathology, University of Florida College of Medicine

2. Name The Antigen

3. 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.

4. 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.

5. Aminobenzene Sulphonate, a Hapten NH2 NH2 NH2 SO3 SO3 SO3 Ortho Meta Para

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

8. Antibody affinity

9. 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

10. Factors affecting solubility • Size • Charge • Temperature • Solvent ionic strength

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

12. Ag Single radial immunodiffusion

13. r Single radial immunodiffusion

14. Double immunodiffusion Örjan Ouchterlony Developed double immunodiffusion technique in 1948

15. Double immunodiffusion (Ouchterlony)

16. S3 S4 P S2 S5 S1 Quantitative double immunodiffusion

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

18. + - Electroimmunodiffusion

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

20. - -human serum Specimen + Immunoelectrophoresis

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

22.  SPE IgG IgA IgM  Immunofixation electrophoresis

23. 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

24. Light reflection

25. + - - Molecular size and scattering

26. Distribution of scattered radiation

27. Nephelometry vs. Turbidimetry 0°-90°

28. Rate C1 C2 Intensity of scattering Time Rate nephelometry

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

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

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

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

33. Logit plot Logit y Log antigen concentration

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

35. 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

36. 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

37. The birth of immunoassay • Rosalyn Yalow (1921-) and Solomon Berson described the first radioimmunoassay in 1957.

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

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

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

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

42. Chemiluminescent labels

43. Chemiluminescent labels

44. 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

45. 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

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

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

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

49. 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

50. Immobilized antibody methods • Coated tube • Coated bead • Solid phase antibody methods