9th SEMINAR
LABORATORY METHODS BASED ON
ANTIGEN-ANTIBODY INTERACTIONS II
THE SENSITIVITY OF IMMUNOASSAYS
Sensitive methods:
• precise
• expensive
• usually used for verification
Less sensitive methods:
• give semiquantitative results
• cheap
• usually used for screening
FLOW CYTOMETRY
An immunofluorescent method that is similar to
immunohistochemistry but gives rather
quantitative than qualitative results
 Investigation of cells travelling in a flow with high velocity
 Detects light scatter of particles and fluorescence intensity of the labeled
cells
Enormous number of cells can be investigated in short period of time
ADVANTAGES OF FLOW CYTOMETRY
 Most cells can be easily suspended and labeled by fluorescent cell surface
marker-specific antibodies
 The cells’ light scatter and immunofluorescent properties can be analyzed
statistically
 Rare cell populations can be identified and examined (e.g. antigen specific
lymphocytes)
 The method provides both qualitative and quantitative data: presence of antigen
and the expression levels of these antigens
Changes in the expression of certain molecules can be followed after different
treatments (e.g. cell activation, disease progression)
BASIC STRUCTURE OF THE FLOW CELL
fluid flow
sheath fluid
Laser
Forward angle light
scatter sensor
(FSC)
represents size
Side light scatter (SSC)
represents granularity
and fluorescence detectors
DENSITY PLOT OF PERIPHERAL BLOOD
CELLS AFTER LYISING RED BLOOD CELLS
Different cell types  characteristic light scattering
granulocytes
side scatter (SSC)
(granularity)
monocytes
lymphocytes
forward scatter
(FSC)
(size)
CELL TYPES, DIFFERENTIATION STAGES CAN
BE IDENTIFIED USING A COMBINATION OF
CELL SURFACE MARKERS
Used in diagnostics:
 ratio of different cell types
 altered expression of cell surface markers
Examples:
 Inflammatory processes – increased neutrophil numbers
 increase of CD5+ B cells – typical for some B cell leukemias
 HIV progression – decrease of CD4+ T cell count
In normal case CD4+ : CD8+ = 1.6
Normal CD4+ T cell count = 600 – 1400/l
AIDS = CD4+ T cell count <200/l
IMMUNOPHENOTYPING BY FLOW
CYTOMETRY
Example: Measurement of CD4+ (helper) and CD8+ (cytotoxic) T cell ratio
(e.g. monitoring AIDS progression)
Labeling:
Lymphocytes in the
peripheral blood sample:
Fluorescent
microscopy:
FITC labeled anti-CD4 antibody(α-CD4-FITC)
PE labeled anti-CD8 antibody (α-CD8-PE)
Th
NK
Tc
B
detecting CD4-FITC
labeled (TH) cell
high velocity flow stream
(in cuvette or stream in air)
detector
signal
processing unit
CD8
PE
screen
increasing
fluorescence
intensity
a dot representing a
CD4+ CD8- cell
CD4
FITC
microscopy:
detecting the PE labeled cell
(CD8-PE)
CD8
PE
detector
signal
processing unit
increasing
fluorescence
intensity
CD4
FITC
detecting an unlabeled cell
(e.g. B cell) by autofluorescence
CD8
PE
detector
Signal
processing unit
increasing
fluorescence
intensity
microscopy:
dim (autofluorescent) cell
CD4
FITC
Signal
processing unit
0%
44%
38%
CD8
PE
18%
quadrant
statistics
CD4
FITC
GRAPHICAL REPRESENTATIONS I
dot-plot
contourplot
densityplot
GRAPHICAL REPRESENTATIONS II
Histogramm
Numeral intensity values:
~7
~ 1300
homogenous cell population has
normal distribution (Gaussian)
CD antigen
cell type
function
ligand
CD3
T cells
TCR signaling
-
CD4
helper T cells, (monocytes, pDC)
T cell co-receptor,
(HIV receptor)
MHC-II, HIV
CD5
T cells, (B cell subset: B1)
adhesion, activation signals
CD72
CD8
cytotoxic T cells, (NK,  T cells)
T cell co-receptor
MHC-I
CD14
monocytes, macrophages,
some granulocytes
LPS binding
LPS, LBP
CD19
B cells
part of CR2,
B cell co-receptor
C3d, C3b
CD28
T cells
co-stimulatory signals
to T cells
(B7-1, B7-2)
CD80, CD86
CD34
hematopoietic progenitor cell
adhesion
CD62L
(L-selectin)
CD56
NK cell, (T and B cell subset)
homoadhesion
(N-CAM isoform)
CD80, CD86
(B7-1, -2)
APC: DC, B, monocyte,
macrophage
co-stimulatory signals
CD28, CD152
THE SENSITIVITY OF IMMUNOASSAYS
Sensitive methods:
• precise
• expensive
• usually used for verification
Less sensitive methods:
• give semiquantitative results
• cheap
• usually used for screening
FORMATION OF IMMUNECOMPLEXES
Immune complexes are rapidly formed in solutions. In a steady-state
equilibrium, determined by the dissociation constant (Kd), they may
form precipitates by secondary interactions
PRECIPITATION OF IMMUNE COMPLEXES
EQUIVALENCE
antigen excess
precipitated
proteins
antibody excess
precipitated
proteins
Increasing antigen amounts
antigen excess
antibody excess
Increasing antibody amounts
Equivalent amount of Ab and Ag will lead to a visible precipitation
Excess in either Ab or Ag will lead to soluble complexes
IMMUNODIFFUSION METHODS I
Mancini method / Radial immunodiffusion
2D simple immodiffusion
Antigen
In gel
Antibody
Antigen
Antibody
The size of the precipitation ring is relative to the concentration of the
antigen; higher concentrations of antigens will diffuse further until
they reach the equilibrium
Applied for quantitative determination of viral antigens or virus
specific antibodies in the serum
IMMUNODIFFUSION METHODS II
Ouchterlony method
2D double immunodiffusion
La
Sm
Ro
antigen
antibody
RNP
antigen
antibody
Jo1
Scl-70
patient’s serum sample
Gold standard method for detection of Extractable Nuclear Antigen (ENA)-specific
autoantibodies
ENAs: e.g. Ro, La, Sm, RNP, Scl-70, Jo1
Autoantigens located in the nuclei of cells. In case of systemic autoimmune disorders
autoreactive B-cells specific to these antigens get activated and continuously produce antibodies
against their targets.
AGGLUTINATION REACTIONS
 Agglutination: The clumping of cells such as bacteria or red
blood cells in the presence of antibodies.
 It differs from precipitation only in size – here the antigen
carrying substances are cells, or colloidal structures.
 Hemagglutination: When the particles involved are RBCs.
TYPES AND USES OF
AGGLUTINATION REACTIONS
 DIRECT AGGLUTINATION
Aggregation followed by antibody binding (mostly IgM)
(AB0 blood group antigens)
 INDIRECT AGGLUTINATION
Aggregation is achieved with a secondary antibody recognizing
the Fc region of the primary antibody (mostly IgG)
(Rh blood group- D antigen)
 PASSIVE AGGLUTINATION
RBCs or latex particles (latex beads) to which haptens or protein antigens
artificially are coupled, aggregate after the addition of the specific
antibodies
(RF- Rheumatoid Factor detection)
DIRECT AGGLUTINATION
A, B or both AB antigens on the
surface of RBCs will bind to the
appropriate anti-A, Anti-B antibodies
and agglutinate
 AB0 blood group typing
 Detecting the presence of an antigen
DONORS AND RECIPIENTS FOR BLOOD TRANSFUSION
Only theoretically, recipients must receive their own type of blood!
QUANTITATIVE HAEMAGGLUTINATION TEST
Quantitative detection of antibodies (antibody-titer) specific to a given antigen.
The antigen is present on the surface of RBCs / bacteria, or fixed to the
surface of RBCs / latex beads.
INDIRECT AGGLUTINATION
 Rh blood group determination
 Diagnosis of autoimmune hemolytic anemia
 Cross matching donor and recipient; ABO
and Rh compatibility
Primary AB
Secondary AB
EFFECTS OF AGGLUTINATION IN
VIVO
ABO incompatibility
intravascular hemolysis
(complement mediated hemolysis)
Rh incompatibility
hemolytic disease of the newborn
(erythroblastosis fetalis)
(opsonisation of red blood cells,
which are then phagocytosed by
macrophages and granulocytes)
Rh prophylaxis
Administration of Anti-D IgG vaccine
within 72 hours after birth of the first
Rh-incompatible fetus prevents
production of maternal antibodies.
PATHOLOGICAL CONSEQUENCES OF
PLACENTAL TRANSPORT OF IgG
(hemolytic disease of the newborn)
anti-Rh
Erythropoiesis
in lung
Erythroblasts in
bone marrow
Both Coombs tests are indirect
agglutinations because the usage of
secondary antibodies
Direct Coombs test – e.g. detection of
autoimmune hemolitic anemia
Indirect Coombs test – e.g. detection of
minor blood group incompatibility
PASSIVE AGGLUTINATION
Fixating antigens on the
surface of latex beads /
RBCs and mixing them with
the patient’s serum
Rheuma factor is self
IgG/IgM reactive IgM
produced by autoreactive
plasma cells
 Latex agglutination test for Rheumatoid Factor in Rheumatoid Arthritis