10 4
FL4 -->
%+ 48 peak 501 mcf 79
A CLASSIC CASE OF NON- SPECIFIC BINDING
RPCI LFC
WHAT IF IT IS OFF SCALE? DO I NEED TO DILUTE?
900
0 10
0 10
10 1
10 2
10 3
10 4
FL4 -->
%+ 70
mcf 82
10 1
10 2
10 3
10 4
FL4 -->
%+ 70
mcf 79
10 1
10 2
10 3
10 4
FL4 -->
%+ 70
mcf 65
10 1
10 2
10 3
10 4
FL4 -->
%+ 68
mcf 51
Resolution = 128
100
LESS THAN 1% BOUND
80
percent free
60
40
107
10-8
10-9
10-10
Affinity Constant (L/M)
VIRTUALLY ALL BOUND
10-11
ANTIBODY BINDING TO MEMBRANE ANTIGENS
1000 10
MEASURED BINDING
AMOUNT BOUND (ng/ml)
1 0.1 0.01 0.001
SPECIFIC BINDING Ka = 5 x 108
NON-SPECIFIC BINDING Ka = 5 x 104
0.0001
0.01
0.1
1
10
ANTIBODY (mg/ml)
100
1000
ANTIBODY BINDING TO INTRACELLULAR ANTIGENS
Non Specific:
binding is low affinity and not saturable
RPCI LFC
ANTIBODY PURITY
10000
1000
Figure A
Figure B
1000 100
100
10 10
MCF MCF
1 1
10
102
103
104
105
106
107
1 1
IgM IgG1
IgA
IgG2a
IgG2b
IgD IgG2c
IgE
IgG3
THE LAW OF MASS ACTION
Ab + Ep Kf AbEp Kd
RATE f = Kf x[Ab] [Ep] RATE d = Kdx[AbEp] Kf range is usually ~ 106 Kd range is usually ~ 10-3 Ka = Kf/Kd = 106/10-3 = 109
1000
ANTIBODY BINDING TO INTRACELLULAR ANTIGENS HIGH AFFINITY ANTIBODY
10 2
MEASURED BINDING
10
AMOUNT BOUND ( ng /ml)
1 0.1 0.01 0.001
SPECIFIC BINDING Ka = 5 x 10 9
170
150
130
Number
110
Number
Number
0 10 30 50 70 90 110 130 150 170 190 210 230 250
110 130 150 170 190 210
Number
90
70
0 10 20 30 40 50 60 70 80 90
90
70
50
50
30
30
Another reason to determine the titer is to verify that it does not exhibit a non-specific binding component at the suppliers recommended amount. Depending on the supplier, the recommended amount to use may be the wrong amount to use.
OSWE L
RP L ARK
Laboratory
Cancer Institute
of
Flow
Cytometry
IMMUNOPHENOTYPING
Los Alamos 2001
lab
OSWE L
RP L ARK
Laboratory
Cancer Institute
of
Flow
Cytometry
CC Stewart & SJ Stewart stewarts@
ANTIBODY BINDING TO CELLS
RPCI LFC
ANTIBODY STRUCTURE
Light Chain
Kappa or Lambda
s
Fab
s
s s
papain pepsin
Fc
s s
Fab
s
s
Heavy Chain
1000 EPITOPES/ANTIGEN 2 LIGHT CHAINS 9 HEAVY CHAINS 20,000 CLONES
CELLS IN A PROPERLY TITERED ANTIBODY
RPCI LFC
WHAT TITER DO YOU CHOOSE?
Recommended amount
R5
1/4 dilution
R5
1/16 dilution
R5
1/64 dilution
R5
110 130 150 170 190 210
WHAT TITER DO YOU USE?
Recommended
amount
R2
1/4 dilution
R2
190
1/16 dilution
R2
190
1/64 dilution
R2
170
170
150
150
110 130 150 170 190 210
110 130 150 170 190 210
RPCI LFC
Ka=1011
Ka=1010 Ka=109 Ka=108 Ka=107
1 x 10-11 1 x 10-10 1 x 10-9 1 x 10-8 1 x 10-7
EFFECT OF AFFINITY CONSTANT ON ANTIBODY BINDING AT 50% SATURATION
AT EQUILIBRIUM RATE f = RATE d = 0 and
[AE]=Ka[Ab][Ep]/(1+Ka[Ab])
WAYS ANTIBODIES BIND TO CELLS Specific:
Fab to epitope Fc to Fc receptor binding is high affinity and saturable
1000
100
MEASURED BINDING
AMOUNT BOUND (ng/ml)
10
1
SPECIFIC BINDING
Ka = 5 x 108
0.1
NON-SPECIFIC BINDING
Ka = 5 x 104
0.01
0.001
0.001
0.01
0.1
1
10
ANTIBODY (mg/ml)
100
10 3
10 4
FL1-Height -->
%+ 26 mcf 62
10 1
10 2
10 3
10 4
FL1-Height -->
%+ 17 mcf 66
10 1
10 2
10 3
10 4
FL1-Height -->
%+ 16 mcf 64
Resolution = 128
THE BEST ISOTYPE CONTROL IS THE NEGATIVE
R5
140
120
120
130
110
110
Number
0 10 20 30 40 50 60 70 80 90
0 10 20 30 40 50 60 70 80 90
Number
0 10 20 30 40 50 60 70 80 90 100
Number
0 10 20 30 40 50 60 70 80 90 100
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
Thus, the only time you would not have to titer the antibody is if it is being used as a single reagent or, if a combination, the supplier has provided appropriate documentation of its performance characteristics on relevant surrogate targets.