Direct production of Fv-fragments from a family of monoclonal IgGs papain digestion.
Fv fragments of four monoclonal antibodies specific for morphine binding have been produced from their divalent IgG forms by papain digestion using the classic procedure for Fab formation. The binding characteristics of one of the Fv fragments have been determined relative to the intact antibody by equilibrium dialysis. Its dissociation constant is a factor of five lower than the IgG. Previous work had resulted in the sequences of each the chains for the four Fv fragments. The light chains are all from the highly homologous lambda subclass while the gamma heavy chains are closely related except for their CDR regions. In this work optical molar extinction coefficients are predicted from amino acid sequences for each of the fragments. It is found that they differ significantly from each other and from the commonly used value for intact IgG. Detailed comparisons between our results and those reported previously on the molecular masses of Fv-derived light and heavy chains and hapten-Fv dissociation constants are given based on analytical gel electrophoresis and electroblotting experiments using dye and immunovisualization techniques. Isoelectric focusing experiments have been performed and the pIs obtained are compared to those predicted theoretically from the chain sequences. Gel filtration column chromatography, acrylamide gel electrophoresis and equilibrium dialysis experiments are consistent with significant aggregation of the Fv fragments in neutral solution with accompanying inactivation of the binding site. Comparison of sequences for the Fv light and heavy chains are made with those which have been proposed to be important for chain dimer association and for canonical hypervariable regions. This methods of Fv production is not regarded as a general one. However, it may be an approach which is general to lambda chain containing antibodies.
Ornatowska
M
M
Department of Biochemistry, University of Connecticut Health Center Farmington 06032.
Glasel
J A
JA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D000915:Antibody Affinity; D002846:Chromatography, Affinity; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry* / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000378:metabolism; D066298:In Vitro Techniques; D051379:Mice; D008970:Molecular Weight; D009020:Morphine / Q000276:immunology / Q000378:metabolism; D010206:Papain; D013057:Spectrum Analysis
D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region; D009020:Morphine; D010206:Papain