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