investigation of humoral immunity lucie sedláčková, phd. department of molecular biology and cell...
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Investigation of humoral Investigation of humoral immunityimmunity
Lucie Sedláčková, PhD.
Department of Molecular Biology and Cell
Pathology, Third Faculty of Medicine
Humoral immunity Humoral immunity componentscomponents
• specificantibodies (Ab)
• nonspecificacute-phase proteinscomplement system
• investigation in serum (urine, cerebrospinal fluid, pathological exudates, bronchoalveolar lavage)
IgG 7-19 IgA 0.8-4.8
IgM 0.5-3 IgD 0.01-0.2 IgE 0-0.0002
Physiological levels of Physiological levels of serum immunoglobulins serum immunoglobulins
(g/l)(g/l)
Investigation of humoral Investigation of humoral immunityimmunity
• 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
determinationserum 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
complexes form
due to interaction
between an Ab
and soluble Ag
(precipitate)
Immunoprecipitation curve
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
laser/quartz lampcuvette
extraction measurement
photodetector
Nephelometry
• precipitation in solution
• measurement of scattered light
• standard curve
Nephelometry
Scattered light detector
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
• RIAAb labelled with radioisotope
• EIAAb labelled with enzymedetection of the reaction according to substrate characteristics:
spectrophotometry, fluorometry ELISA
Enzyme-linked immunosorbent assay (ELISA)
colored product
antibodies in tested sample
enzyme-conjugated antibody
substrate for the enzyme
Determination of optical density
• evaluation: photometry
• higher intensity of color reaction, the
higher concentration of Ag/Ab
concentration
OD
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
sample antigen
1st specific antibody (coating)
2nd labelled antibody
washing
washing
ELISA usage
Determination of IgG against Determination of IgG against Clostridium Clostridium
tetani 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 Screening of IgG levels against Clostridium tetaniagainst Clostridium tetani
basic vaccination: 3x tetanus toxoid (Alteana)
re-vaccination: 1x Alteana
IgG (IU/ml) protection recommendationbelow 0.03 none basic vaccination0.03-0.1 unsure re-vaccination0.1-0.5 present re-vaccination0.6-1.0 sufficient check-up in 2 yrs1.1-5.0 longterm check-up in 5-10 yrsover 5 very high check-up in 10 yrs
ELISA usage
Antibody level determination against Antibody level determination against transglutaminasetransglutaminase
(Anti-tTG = Anti-tissue Transglutaminase Antibodies)
• in class IgA
Occurance: celiac disease
ELISA usageDetermination of allergen-specific IgE Determination of allergen-specific IgE
antibodiesantibodies• 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, fluorescencefluorescence 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
cathode
anode
electrophoreogram
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
healthy control
healthy control: negative
patient A: paraprotein in class IgG ()
Usage of immunofixation
• paraprotein detection
• diagnostics of monoclonal gammapathies
(multiple myelomamultiple myeloma, macroglobulinemia,
CLL, B-lymphoma)
• 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
slide
substrate (healthy tissue)
specific Ig (antibody from the sample)
washing
washing
specific FITC-labelled anti-human Ig
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 C4indication: suspected genetic deficiency,
pathogenetic role of immune complexes
C1 inhibitorindication: 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 CC amountactivation (fixation) and C C consumption
2. step – hemolytic activity determination of activated (available) CC
Evaluation:• the highest serum dilution with CC activity
(Ab determination)• Ag concentration