Biomedical Engineering Reference
In-Depth Information
References
Schwarzkopf C, Thiele B (1996) ALTEX 13 Suppl. 16:35
Staak C, Schwarzkopf C, Behn I, Hommel U, Hlinak A, Schade R, Erhard M
(2001) In: Schade R, Behn I, Erhard M, Hlinak H, Staak C (eds.) Chicken
egg yolk antibodies, production and application - IgY-technology.
Springer, Berlin, p 65
4.6 Antibody Fragmentation
Since antibodies of different species and different subclasses are
variably cleaved by pepsin and papain, a test run is recommended
to check incubation time and cleavage conditions. Take samples
of different incubation time, freeze rapidly, and monitor the frag-
mentation by SDS-PAGE at the end of the experiment. Apply the
samples both with and without DDT.
4.6.1 F(ab')2 Fragments from IgG
A
100 mM sodium acetate buffer, pH 4.5
Solutions/Reagents
/
B
ml in Soln. A
C2M Tris pH 8.8
D 0mM Tr i s , 0 . 1 5 M NaCl, pH 7.6
pepsin
, 5mg
/
Prepare a series of different concentrations of IgG (1 - 5 mg
ml)
Test of cleavage
conditions
µ
from Soln. A and aliquots of Soln. B. Add 5
g pepsin per mil-
ligram IgG and incubate at 37
◦
C for 15 - 24 h. Stop proteolysis by
addition of 1/10 volume Soln. C. Check by SDS PAGE for optimal
IgG concentration and cleavage time (unreduced F(ab
)2: 110 kD;
reduced F(ab
)2: doublet at 25 kD; unreduced Fc: 25 kD; reduced
Fc: somewhat below reduced F(ab
)2).
Run the preparative cleavage with up to 10 mg IgG using the
optimal conditions found in the previous experiment.
Separate the F(ab
)2 fragments by GPC on Sephadex G-100
from degradation products. The F(ab
)2 fragments elute in the
void volume.
References
Harlow E, Lane D (1988) Antibodies: a laboratory manual. Cold Spring
Harbor, p 630
Kerr MA, Thorpe R (eds.) (1994) Labfax immunochemistry. Academic
Press, Oxoford, p 105
A
100 mM sodium citrate buffer, pH 3.7
Solutions/Reagents
/
B
,
0mg
ml in Soln. A
/
C1mg
ml pepsin in Soln. A
D2M Tr i s b a s e
