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Investigation of humoral immunity Lucie Sedláčková, PhD. Department of Molecular Biology and Cell Pathology, Third Faculty of Medicine [email protected] Humoral immunity components • specific antibodies (Ab) • nonspecific acute-phase proteins complement system • investigation in serum (urine, cerebrospinal fluid, pathological exudates, bronchoalveolar lavage) 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 Investigation of humoral immunity • agglutination • turbidimetry and nephelometry • enzyme immunoassay (ELISA) • electrophoresis, immunoprecipitation, immunoblotting • indirect immunofluorescence 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) 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 Usage of agglutination • infectious serology (Salmonella typhi, Listeria monocytogenes,…) • diagnostics of autoimmune diseases (autoimmune hemolytic anemia – Coombs test) • blood type determination (hemagglutination) Immunoprecipitation techniques • principle: immune PRECIPITAČNÍ ImmunoprecipitationKŘIVKA curve complexes form due to interaction between an Ab and (precipitate) Y precipitate mass soluble Ag Y ZONE of excess of Ab EQUIVALENCE excess of Ag antigen concentration constant amount of antibody 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 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“) Turbidimetry • precipitation in solution • measurement of light extraction (precipitate absorption) • standard curve Turbidimetry extraction measurement laser/quartz lamp photodetector cuvette Nephelometry • precipitation in solution • measurement of scattered light • standard curve Scattered light detector Nephelometry Scattered light measurement 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 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 Enzyme-linked immunosorbent assay (ELISA) enzyme-conjugated antibody antibodies in tested sample colored product substrate for the enzyme Determination of optical density • evaluation: photometry • higher intensity of color reaction, the higher concentration of Ag/Ab OD concentration ELISA KIT 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) 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 Screening of IgG levels against Clostridium tetani IgG (IU/ml) below 0.03 0.03-0.1 0.1-0.5 0.6-1.0 1.1-5.0 over 5 protection none unsure present sufficient longterm very high recommendation basic vaccination re-vaccination re-vaccination check-up in 2 yrs check-up in 5-10 yrs check-up in 10 yrs basic vaccination: 3x tetanus toxoid (Alteana) re-vaccination: 1x Alteana ELISA usage Antibody level determination against transglutaminase (Anti-tTG = Anti-tissue Transglutaminase Antibodies) • in class IgA Occurance: celiac disease ELISA usage Determination of allergen-specific IgE antibodies • ELISA • UniCAP machine adsorbed allergen on solid phase (CNBr – cellulose) reacts with IgE in patient‘s serum detection with enzyme-labelled secondary Ab against IgE, fluorescence evaluation Electrophoresis • protein separation due to mobility in electric field (according to electric charge and molecular weight) • gel (agarose, polyacrylamide) Serum protein electrophoresis basic orientation in serum protein abundance electrophoreogram cathode anode g globulins Serum protein electrophoresis Immunofixation • serum electrophoresis in several lines • antibodies application (anti-IgG, -IgA, -IgM, -κ, -) • precipitate formation (e.g. immune complex = IgG+anti-IgG) • staining Immunofixation patient A patient A: paraprotein in class IgG (k) healthy control healthy control: negative Usage of immunofixation • paraprotein detection • diagnostics of monoclonal gammapathies (multiple myeloma, macroglobulinemia, CLL, Blymphoma) • paraprotein: synthesized by single clones of myeloma cells (neoplastic proliferation of B lymphocytes) Immunoblotting (Western blot) 1) electrophoresis 2) transfer of proteins from gel to nitrocelullose membrane in electric field 3) immunodetection: + serum sample + labelled secondary Ab (HRP) + substrate – colored reaction Usage of immunoblotting • demonstration of Ab presence against particular Ag (bacteria, autoantigens, …) Borrelia burgdorferi • detection of several Ab against specific Ag at one time • immunodot: commertial artificially-purified or recombinant antigens fixed on the membrane Immunodot 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) Indirect immunofluorescence UV light green light washing specific FITC-labelled anti-human Ig washing specific Ig (antibody from the sample) substrate (healthy tissue) slide ANA antibodies (anti-nuclear) • against nuclear antigens (ds-DNA, histones, nucleolus, mitotic apparatus,…) • substrate: HEp-2 cells nucleolar fluorescence type (autoantibodies against nucleolus) homogeneous fluorescence type (autoantibodies against ds-DNA and histones) Anti-ds DNA antibodies • substrate: Critidium lucilliae (protozoan with kinetoplast containing ds-DNA) • positive sample = kinetoplast and nuclei fluorescence + - Occurance of ANA antibodies • systemic lupus erythematosus, Sjoegren syndrome, sclerodermia, dermatopolymyositis, … patient with SLE ANCA antibodies (antineutrophil cytoplasmic) • against neutrophil granula components (e.g. myeloperoxidase, lactoferrin, proteinase 3, …) • substrate: human neutrophils • occurance: vasculitides ANCA antibodies human neutrophils stained with antibody against myeloperoxidase Gastric parietal cell antibodies • against intrinsic factor (absorbs vitamin B12) • substrate: rat stomach • occurance: atrophic gastritis and pernicious anemia 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) 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% Complement-binding assays • determination of Ab or Ag 1. 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