9th SEMINAR LABORATORY METHODS BASED ON ANTIGEN-ANTIBODY INTERACTIONS II
Dec 23, 2015
9th SEMINARLABORATORY 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)
LaserForward angle light
scatter sensor(FSC)
Side light scatter (SSC) represents granularity
and fluorescence detectors
represents size
BASIC STRUCTURE OF THE FLOW CELL
fluid flow
sheath fluid
forward scatter (FSC)(size)
side scatter (SSC)(granularity)
granulocytes
monocytes
lymphocytes
DENSITY PLOT OF PERIPHERAL BLOOD CELLS AFTER LYISING RED BLOOD CELLS
Different cell types characteristic light scattering
CELL TYPES, DIFFERENTIATION STAGES CAN BE IDENTIFIED USING A COMBINATION OF
CELL SURFACE MARKERSUsed 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+ = 1.6Normal CD4+ T cell count = 600 – 1400/l
AIDS = CD4+ T cell count <200/l
BNKTh Tc
Example: Measurement of CD4+ (helper) and CD8+ (cytotoxic) T cell ratio (e.g. monitoring AIDS progression)
Labeling: FITC labeled anti-CD4 antibody(α-CD4-FITC)PE labeled anti-CD8 antibody (α-CD8-PE)
Lymphocytes in the peripheral blood sample:
Fluorescent microscopy:
IMMUNOPHENOTYPING BY FLOW CYTOMETRY
focu
sed
lase
r bea
m
high velocity flow stream(in cuvette or stream in air)
CD4FITC
CD
8P
E
de
tec
tor
detecting CD4-FITC labeled (TH) cell
signal processing unit
screen
a dot representing aCD4+ CD8- cell
increasing fluorescence
intensity
microscopy:
de
tec
tor
detecting the PE labeled cell(CD8-PE)
CD
8P
E
CD4FITC
signal processing unit
increasing fluorescence intensity
de
tec
tor
detecting an unlabeled cell(e.g. B cell) by autofluorescence
CD
8P
E
CD4FITC
Signal processing unit
increasing fluorescence intensity
microscopy: dim (autofluorescent) cell
quadrantstatistics
CD
8P
E
CD4FITC
Signal processing unit
38%
0%
44%
18%
dot-plot
contour-plot
density-plot
GRAPHICAL REPRESENTATIONS I
GRAPHICAL REPRESENTATIONS IIHistogramm
homogenous cell population has normal distribution (Gaussian)Numeral intensity values: ~ 7 ~ 1300
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
CD14monocytes, macrophages,
some granulocytesLPS 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 adhesionCD62L
(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
antigen excessantibody excess
Increasing antibody amounts
EQUIVALENCEantibody excess antigen excess
Increasing antigen amounts
pre
cip
itat
ed
pro
tein
sp
reci
pit
ated
p
rote
ins
Equivalent amount of Ab and Ag will lead to a visible precipitationExcess in either Ab or Ag will lead to soluble complexes
IMMUNODIFFUSION METHODS I
In gelAntigen
Antibody
Antibody
Antigen
Mancini method / Radial immunodiffusion
2D simple immodiffusion
Applied for quantitative determination of viral antigens or virus specific antibodies in the serum
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
IMMUNODIFFUSION METHODS II
antigen
antibody
antigen
antibody
2D double immunodiffusion
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.
Ro
La
Sm
patient’s serum sample
RNP
Scl-70
Jo1
Ouchterlony method
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 AGGLUTINATIONAggregation is achieved with a secondary antibody recognizing the Fc region
of the primary antibody (mostly IgG) (Rh blood group- D antigen)
PASSIVE AGGLUTINATIONRBCs 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 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.
QUANTITATIVE HAEMAGGLUTINATION TEST
Rh blood group determination Diagnosis of autoimmune hemolytic anemia Cross matching donor and recipient; ABO
and Rh compatibility
Primary AB
Secondary AB
INDIRECT AGGLUTINATION
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.
EFFECTS OF AGGLUTINATION IN VIVO
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
Latex agglutination test for Rheumatoid Factor in Rheumatoid Arthritis
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