Biomedical Engineering Reference
In-Depth Information
µ
Alternatively, add 20
g pepsin (from Soln. C) per milligram IgG to
Soln. B. Incubate rabbit IgG at 37
◦
C for 4 h, add 1/10 volume Soln. D
and dialyze against TBS or PBS. Apply the dialysate on a Protein A
column and collect the passage containing F(ab
)2 fragments.
The preparation of F(ab
)2 fragments from monoclonal (mouse)
IgG is similar, but check for optimal cleavage time and use a Protein-
Gcolumn.
References
Kürzinger K (1993) Enzymatic and chemical modifications: antibody frag-
ments. In: Masseyeff RF, Albert WH, Staines NA (eds.) Methods of
immunological analysis. VCH, Weinheim, p 383
4.6.2 Fab' Fragments (Rabbit)
A
550 mM Tr i s , 5 mM EDTA, pH 8.2
Solutions/Reagents
/
B , 5mg
ml in A
2-mercaptoethanol
iodoacetamide
PBS
Dialyze F(ab
)2 fragments against Soln. A. Add 2-mercaptoethanol
to a final concentration of 0.2 M (15
µ
l/ml). Incubate at RT for
10 min,coolinanicebath,andaddiodoacetamidetoafinalcon-
centration of 0.3 M(55.5 mg
/
ml). Incubate on ice for 1 h and dialyze
against PBS or desalt on a Sephadex G-25 column, equilibrated with
PBS (Fab
fragments in the void volume).
4.6.3 Fab Fragments (Rabbit)
A
100 mM sodium acetate buffer, pH 5.5
Solutions/Reagents
/
B , 5mg
ml in A
C1M cysteine
D 0mM EDTA
papain
µ
Add 1/20 volume Soln. C, 1/20 volume Soln. D and 10
g papain/mg
IgGtoSoln.B.Incubateat37
◦
C for 8 - 12 h, and then add 13.8 mg
per milliliter assay volume of iodoacetamide. Allow to react at
RT for 30 min, then dialyze against PBS or separate on a Protein-
A column (Fab fragments in the passage).
4.7 H
EIDELBERGER
Curve (Precipitin Curve)
If two molecules having at least two binding sites for each other
will interact, you will find a concentration range characterized by
forming large aggregates. These aggregates are easy to precipitate.
