Protein Protocols Protein Protocols

Enzymatic Digestion of MAbs 1049
2.3. Reduction and Alkylation with Cysteine
1. Purified solution of mouse IgM McAb at a concentration of ≥1 mg/mL in PBS.
2. 0.1 M cysteine stock solution in PBS (L-cysteine free base, Sigma number C7755).
3. Borate-buffered saline: 0.015 M sodium borate, 0.15 M sodium chloride, made to pH 8.5
with Sodium hydroxide.
4. A size exclusion column equivalent to 26 × 900 mm Sephacryl S-200 (Pharmacia).
3. Methods
3.1. Preparation of F(ab') 2 from IgG
The various subclasses of mouse IgG vary in their susceptibility to pepsin fragmentation.
IgG 1 is quite resistant to digestion and and it is impossible to fragment IgG 2b to
F(ab') 2 because it breaks down to Fab (see Notes 1–3). The method below is one that
should work in most cases.
1. Dialyze the IgG against acetate buffer, pH 4.0, overnight at 4°C. Use any known amount
of antibody between 1 and 20 mg.
2. Determine the concentration at A 280.
3. Add 0.1 mg/mL pepsin in acetate buffer, pH 4.0, to give an enzyme-to-antibody ratio of
120 (w:w).
4. Incubate for 6 h in a water bath at 37°C.
5. Stop the reaction by adding sufficient Tris base to bring the pH to roughly 8.0 (start by
adding 50 µL Tris, mix, and test the pH with pH paper).
6. Dialyze the mixture against PBS, pH 8.0, overnight.
7. Equilibrate the protein A column with PBS, pH 8.0, and load the dialyzed mixture onto it
1 mL at a time. Collect the unbound fraction that contains the F(ab′) 2 fragments.
8. If further purity is desired, the mixture should be concentrated to a volume of
≤3 mL and added to a precalibrated size exclusion column (26 × 900 mm Sephacryl S-200
or equivalent). At this stage regular PBS can be used to equilibrate the column and elute
the fractions.
9. Collect 2.5-mL fractions over the molecular weight range of F(ab') 2 (110 kDa). The purity
of the product can be assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) in nonreducing conditions and in reducing conditions when a doublet at
25 kDa is seen.
3.2. Preparation of F(ab') 2µ from IgM (see Note 3)
1. Dialyze the IgM against acetate buffer, pH 4.5, overnight at 4°C.
2. Determine the concentration at A 280 (see Chapter 1).
3. Add 0.1 mg/mL pepsin in acetate buffer, pH 4.5, to give an enzyme-to-antibody ratio of
120 (ww).
4. Incubate for between 6 and 12 h in a water bath at 37°C.
5. Stop the reaction by adding sufficient Tris base to bring the pH to roughly 8.0 (start by
adding 50 µL Tris, mix, and test the pH with pH paper).
6. Dialyze the mixture against PBS overnight, or concentrate and change the buffer using a
Centriprep 100 concentrator.
7. Concentrate the mixture to a volume of ≤3 mL and add to a precalibrated size exclusion
column (26 × 900 mm Sephacryl S-200 or equivalent). Elute fractions of between 1 and 2.5 mL.
The molecular weight of F(ab') 2µ is 130 kDa. Assess purity on an 8% polyacrylamide gel
under nonreducing conditions.