Técnicas de Imunologia
Citometria de Fluxo
Slides adaptados de uma colecção
amavelmente cedida por
Alexandre Salvador (BioAtlântico)
Prof.Doutor José Cabeda
Técnicas de Imunologia
Marcação celular com Anticorpos
em Citometria de Fluxo
Prof.Doutor José Cabeda
Técnicas de Imunologia
THE LAW OF MASS ACTION
Ab + Ep
Kf
AbEp
Kr
RATE f = Kf X[Ab] [Ep] RATE r = Kr X[AbEp]
Kf range is usually ~ 106
Kr range is usually ~ 10-3
Ka = Kf/Kr = 106/10-3 = 109
AT EQUILIBRIUM RATE f = RATE r = 0
and
[AE]=Ka[Ab][Ep]/(1+Ka[Ab])
Prof. Doutor José Cabeda
Técnicas de Imunologia
WAYS ANTIBODIES BIND TO CELLS
Specific:
Fab to epitope
Fc to Fc receptor
binding is high affinity and saturable
Non Specific:
binding is low affinity and not saturable
Prof. Doutor José Cabeda
Técnicas de Imunologia
Specific Activity is the concentration
of bindable antibody to its epitope
divided by the protein concentration.
SA =
[F(ab') 2]
(protein)
Prof. Doutor José Cabeda
Técnicas de Imunologia
Reasons Antibodies
do not bind to cells:
•overconjugation
•not purified
•degradation of binding site
•aggregation
Prof. Doutor José Cabeda
Técnicas de Imunologia
STORING OF ANTIBODIES :
Proteases destroy antibodies in:
• ascitic fluid
• serum
• bacteria
Use sodium azide
Use highly purified albumin or gelatin
as carrier
Purify antibodies immediately
Prof. Doutor José Cabeda
Técnicas de Imunologia
Ka=1011
Ka=1010
Ka=109
Ka=108
Ka=107
1 x 10-11
1 x 10-10
1 x 10-9
Prof. Doutor José Cabeda
1 x 10-8
1 x 10-7
Técnicas de Imunologia
EFFECT OF AFFINITY CONSTANT ON
ANTIBODY BINDING
percent free
100
80
60
40
107
10-8
10-9
1010
-
10-11
Affinity Constant (L/M)
Prof. Doutor José Cabeda
Técnicas de Imunologia
ANTIBODY BINDING TO INTRACELLULAR ANTIGENS
AMOUNT BOUND ( ng /ml)
HIGH AFFINITY ANTIBODY
10 2
MEASURED BINDING
10
1
SPECIFIC BINDING
0.1
Ka = 5 x 10
9
0.01
NON-SPECIFIC BINDING
Ka = 5 x 10 4
0.001
0.0001
0.001
0.01
0.1
1
10
100
1000
ANTIBODY ( m g/ml)
Técnicas de Imunologia
Prof. Doutor José Cabeda
TITERING ANTIBODIES
Prof.Doutor José Cabeda
Técnicas de Imunologia
MEMBRANE EPITOPES
Prof.Doutor José Cabeda
Técnicas de Imunologia
3 µg
s/n = 2.5
1 µg
s/n = 2.1
0.3 µg
s/n = 2.4
0.1 µg
s/n = 4.1
0.03 µg
s/n = 4.8
0.01 µg
s/n = 4.6
0.001 µg
s/n = 3.2
auto
3
0.003 µg
s/n = 3.5
Prof. Doutor José Cabeda
Técnicas de Imunologia
Prof. Doutor José Cabeda
Técnicas de Imunologia
MEMBRANE ANTIGENS
5
Signal to Noise
TITER
4
3
2
0
1
2
3
4
5
Dilution
6
Prof. Doutor José Cabeda
7
8
Técnicas de Imunologia
MEMBRANE ANTIGENS
2,2
non-specific and
specific binding
2
titer
1,8
r
a
t
i
o
1,6
1,4
1,2
1
0,017 0,051 0,2
0,5
1
4
12
37
111
333 1000 3000
ng/test
Prof. Doutor José Cabeda
Técnicas de Imunologia
NUMBER OF CELLS
Verification of Specific Binding
CD4 FITC
Prof. Doutor José Cabeda
Técnicas de Imunologia
INTRACELLULAR EPITOPES
Prof.Doutor José Cabeda
Técnicas de Imunologia
In the beginning there is a high extracellular antibody
concentration.
Prof. Doutor José Cabeda
Técnicas de Imunologia
Prof. Doutor José Cabeda
Técnicas de Imunologia
Antibodies diffuse relatively fast into the cell down the
electrochemical gradient and bind specifically and nonspecifically.
Prof. Doutor José Cabeda
Técnicas de Imunologia
Prof. Doutor José Cabeda
Técnicas de Imunologia
BARRIERS TO INTRACELLULAR STAINING
• immobilized epitope decreases antibody binding affinity
• molecular crowding limits diffusion
• fixation induced epitope degradation
• fixation induced epitope obstruction
Prof. Doutor José Cabeda
Técnicas de Imunologia
BARRIERS TO SPECIFIC BINDING
• molecular crowding increases non-specific binding
• NSB reduces effective antibody concentration
• washing efficiency reduced due to slow off-rate
• multiple targets specificity with different epitope affinities
(e.g. multiple bands on a western blot)
Prof. Doutor José Cabeda
Técnicas de Imunologia
So, what is the answer? Only one. Antibodies of high affinity so
that there is a low free antibody concentration to begin with!
Prof. Doutor José Cabeda
Técnicas de Imunologia
INTRACELLULAR ANTIGENS
isotype
control
number
antibody
101
102
103
104
101
102
103
104
101
102
103
104
cytokeratin
1 µg MCF
Ab 278
IC 5.8
.3 µg MCF
Ab 100
IC 3.6
Prof. Doutor José Cabeda
.01 µg MCF
Ab 25.7
IC 2.6
Técnicas de Imunologia
BLOCKING IS IMPORTANT
Prof. Doutor José Cabeda
Técnicas de Imunologia
DIRECT IMMUNOFLUORESCENCE STAINING
NO BLOCKING
Primary Antibody:
murine monoclonal
antibody
Fab
Fab
Fc
%+=75
Fab
Fc
Fc
FcR
A
epitope
B
C
D
Fab
Fab
Fc
Fc
Prof. Doutor José Cabeda
Técnicas de Imunologia
ISOTYPE CONTROL- myeloma protein
%+=50
%+=75%-50=25%
AUTOFLUORESCENCE CONTROL
Prof. Doutor José Cabeda
%+=0
Técnicas de Imunologia
BLOCKING WITH GOAT IgG
goat IgG
FcR
A
epitope
B
C
D
Fab
Fab
Fc
Fc
%+=50
Prof. Doutor José Cabeda
Técnicas de Imunologia