Investigation of humoral immunity

1. Investigation of humoral immunity Lucie Sedláčková, PhD. Department of Molecular Biology and Cell Pathology, Third Faculty of Medicine lucie.sedlackova@lf3.cuni.cz

2. Humoral immunity components • specific antibodies (Ab) • nonspecific acute-phase proteins complement system • investigation in serum (urine, cerebrospinal fluid, pathological exudates, bronchoalveolar lavage)

3. Physiological levels of serum immunoglobulins (g/l) IgG 7-19 IgA 0.8-4.8 IgM 0.5-3 IgD 0.01-0.2 IgE 0-0.0002

4. Investigation of humoral immunity agglutination turbidimetry and nephelometry enzyme immunoassay (ELISA) electrophoresis, immunoprecipitation, immunoblotting indirect immunofluorescence

5. Agglutination • principle: immune complexes form due to interaction between an antibody (Ab) and particulate antigen (Ag) • antigenic particles (bacteria, Ag on carrier) + patient‘s serum  demonstration of specific Ab • evaluation: qualitative (y/n), quantitative (serum titration)

6. Agglutination – quantitative determination serum dilution by geometrical series of antigen suspension of diluted serum final serum dilution serum titre = reciprocal value of the highest serum dilution, where the reaction is still positive example: patient XY last positive reaction in tube with 1:32 serum dilution → titre = 32

7. Usage of agglutination • infectious serology (Salmonella typhi, Listeria monocytogenes,…) • diagnostics of autoimmune diseases (autoimmune hemolytic anemia – Coombs test) • blood type determination (hemagglutination)

9. Immunoprecipitation techniques • principle: immune complexes form due to interaction between an Ab and soluble Ag (precipitate) Immunoprecipitation curve constant amount of antibody

10. Radial immunodiffusion • principle:Ag and Ab react in agarose gel after diffusion (precipitate ring) • gel with homogenous Ab content + patient‘s serum • diameter of the ring is proportional to Ag amount • usage: IgD class determination

11. Turbidimetry and nephelometry • principle: measurement of immune complexes Ag-Ab amounts in the Ab excess • reaction in liquid phase in cuvette • evaluation: Ag concentration is proportional to - production speed of complexes (kinetic system) - turbidity of complexes („end point“)

12. Turbidimetry • precipitation in solution • measurement of light extraction (precipitate absorption) • standard curve Turbidimetry extraction measurement laser/quartz lamp photodetector cuvette

13. Nephelometry • precipitation in solution • measurement of scattered light • standard curve Scattered light detector Nephelometry Scattered light measurement

14. Usage of turbidimetry and nephelometry • measurement of serum proteins‘ concentration (immunoglobulins, acute-phase proteins, complement components C3, C4, transferrin, albumin,…) • rapid, fully-automated techniques for large quantity of samples

15. Immunoreaction with labelled antibodies • RIA Ab labelled with radioisotope • EIA Ab labelled with enzyme detection of the reaction according to substrate characteristics: spectrophotometry, fluorometry ELISA

16. colored product Enzyme-linked immunosorbent assay (ELISA) enzyme-conjugated antibody antibodies in tested sample substrate for the enzyme

17. Determination of optical density • evaluation: photometry • higher intensity of color reaction, the higher concentration of Ag/Ab OD concentration

18. ELISA KIT

19. ELISA usage • determination of serum concentration of specific antibodies (borreliosis, tetanus, hepatitis), autoantibodies, cytokines • high sensitivity (<1ng/l) • high specifity • reproducibility Photometry substrate washing 2nd labelled antibody washing sample antigen 1st specific antibody (coating)

20. ELISA usage • Determination of IgG against Clostridium tetani • Indication: • unclear anamnesis of previous vaccination • decision about the vaccination way in traumatized patients with unknown vaccination status • humoral immunity investigation - immunodeficiencies

21. IgG (IU/ml) protection recommendation below 0.03 none basic vaccination 0.03-0.1 unsure re-vaccination 0.1-0.5 present re-vaccination 0.6-1.0 sufficient check-up in 2 yrs 1.1-5.0 longterm check-up in 5-10 yrs over 5 very high check-up in 10 yrs Screening of IgG levels against Clostridium tetani basic vaccination: 3x tetanus toxoid (Alteana) re-vaccination: 1x Alteana

22. ELISA usage Antibody level determination against transglutaminase (Anti-tTG = Anti-tissue Transglutaminase Antibodies) • in class IgA Occurance: celiac disease

23. ELISA usage Determination of allergen-specific IgE antibodies • ELISA • UniCAP machine adsorbed allergen on solid phase (CNBr –cellulose) reactswith IgE in patient‘s serum detection with enzyme-labelled secondary Ab against IgE, fluorescence evaluation

24. Electrophoresis • protein separation due to mobility in electric field (according to electric charge and molecular weight) • gel (agarose, polyacrylamide)

25. Serum protein electrophoresis basic orientation in serum protein abundance electrophoreogram cathode anode g globulins

26. Serum protein electrophoresis

27. Immunofixation • serum electrophoresis in several lines • antibodies application • (anti-IgG, -IgA, -IgM, -κ, -) • precipitate formation • (e.g. immune complex = IgG+anti-IgG) • staining

28. Immunofixation patient A healthy control patient A: paraprotein in class IgG (k) healthy control: negative

29. Usage of immunofixation • paraprotein detection • diagnostics of monoclonal gammapathies (multiple myeloma, macroglobulinemia, CLL, B-lymphoma) • paraprotein: synthesized by single clones of myeloma cells (neoplastic proliferation of B lymphocytes)

30. Immunoblotting (Western blot) • electrophoresis • transfer of proteins from gel to nitrocelullose membranein electric field • immunodetection: + serum sample + labelled secondary Ab (HRP) + substrate – colored reaction

31. Usage of immunoblotting • demonstration of Ab presence against particular Ag (bacteria, autoantigens, …) Borrelia burgdorferi • detection of several Ab against specific Agat one time • immunodot:commertial artificially-purified or recombinant antigens fixed on the membrane

32. Immunodot

33. Autoantibodies • targeted against body‘s own cells • physiologically in low concentration,  with age • used in diagnostics of autoimmune diseases • non-organ-specific (e.g. against nuclei, mitochondria,…) • organ-specific (e.g. against pancreatic antigens)

34. Indirect immunofluorescence green light UV light washing specific FITC-labelled anti-human Ig washing specific Ig (antibody from the sample) substrate (healthy tissue) slide

35. ANA antibodies (anti-nuclear) • against nuclear antigens (ds-DNA, histones, nucleolus, mitotic apparatus,…) • substrate: HEp-2 cells homogeneous fluorescence type (autoantibodies against ds-DNA and histones) nucleolar fluorescence type (autoantibodies against nucleolus)

36. Anti-ds DNA antibodies • substrate: Critidium lucilliae (protozoan with kinetoplast containing ds-DNA) • positive sample = kinetoplast and nuclei fluorescence - +

37. Occurance of ANA antibodies • systemic lupus erythematosus, Sjoegren syndrome, sclerodermia, dermatopolymyositis, … patient with SLE

38. ANCA antibodies (antineutrophil cytoplasmic) • against neutrophil granula components (e.g. myeloperoxidase, lactoferrin, proteinase 3, …) • substrate: human neutrophils • occurance: vasculitides

39. ANCA antibodies human neutrophils stained with antibody against myeloperoxidase

40. Gastric parietal cell antibodies • against intrinsic factor (absorbs vitamin B12) • substrate: rat stomach • occurance: atrophic gastritis and pernicious anemia

41. Investigation of complement • nephelometry – measurement of concentration C3 and C4 indication: suspected genetic deficiency, pathogenetic role of immune complexes C1 inhibitor indication: suspected deficiency (dg. of hereditary angioedema)

42. Functional assays for complement • CH50 hemolytic assay Principle: tested serum + sheep erythrocytes with bound Ab → complement activation, ery lysis Evaluation: • spectrophotometry –absorbance measurement of released hemoglobin, proportional to number of lysed ery • reciprocal value of serum dilution required to produce hemolysis of 50%

43. Complement-binding assays • determination of Ab or Ag • step – Ag and Ab reaction in presence of known C amount activation (fixation) and C consumption 2. step – hemolytic activity determination of activated (available) C Evaluation: • the highest serum dilution with C activity (Ab determination) • Ag concentration