Optimal conditions for the preparation of Fab and F(ab')2 fragments from monoclonal IgG of different rat IgG subclasses.
The optimal conditions for the preparation of Fab and F(ab')2 fragments from monoclonal rat IgG of different subclasses are described. Digestion of IgG for 2-4 h at 37 degrees C with 1% (w/w) papaïn at pH 7.0 in the presence of 0.01 M cysteine leads to almost complete cleavage into Fab and Fc fragments. Fab fragments are isolated by sequential chromatography on Ultrogel AcA 44 and on DEAE-cellulose columns. In the case of IgG2c subclass, Fab fragment may be directly isolated by chromatography on Protein A-Sepharose. Production of F(ab')2 fragments from rat IgG1 and IgG2a is obtained with best yield by treatment at acid pH (pH 2.8) before incubation with 1% (w/w) pepsin at pH 4.5 for 4 h at 37 degrees C. For monoclonal IgG2b the best procedure is incubation with S. aureus V8 protease at pH 7.8 (4 h at 37 degrees C with an E/S ratio of 1/30 (w/w]. The best yield of F(ab')2 from monoclonal IgG2c is obtained by incubation for 4 h at 37 degrees C with 1% (w/w) pepsin. F(ab')2 fragments (or the F(ab)2-like fragment released by digestion of IgG2b with S. aureus V8 protease) are isolated by gel filtration on Ultrogel AcA 44.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D007122:Immunoelectrophoresis; D007140:Immunoglobulin Fab Fragments / Q000302:isolation & purification*; D007074:Immunoglobulin G / Q000302:isolation & purification*; D008970:Molecular Weight; D010206:Papain; D051381:Rats
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D010206:Papain