title: Optimal conditions for the preparation of Fab and F(ab')2 fragments from monoclonal IgG of different rat IgG subclasses.
abstract: 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.
mesh_terms: 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
chemical_list: D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D010206:Papain