Large scale production and purification of paraquat and desipramine monoclonal antibodies and their Fab fragments.
We describe the rapid, large scale purification of Fab fragments from mouse monoclonal antibodies. Antibodies against two clinically important and often fatal toxins, paraquat and desipramine, were isolated from mouse ascites fluid by preparative high performance hydroxylapatite (HPHT) or ion exchange (DEAE) high performance liquid chromatography. A competitive inhibition ELISA was used to determine the cross-reactivity of the antibody with analogs of the antigens. Papain digests of the IgGs were subjected to further HPHT followed by Sephadex G-100 chromatography to yield homogeneous Fab fragment preparations. The high purity of these preparations, demonstrated by SDS polyacrylamide gel electrophoresis, has only been achieved previously by affinity chromatography. Intrinsic association constants for the intact IgG and the Fab fragment--antigen interactions, determined by competitive inhibition ELISA, were similar. This indicates that antigen-binding activity was conserved during the production and purification of the Fab fragments.
Bowles
M
M
Department of Medicine, University of Queensland, Princess Alexandra Hospital, Brisbane, Australia.
Johnston
S C
SC
Schoof
D D
DD
Pentel
P R
PR
Pond
S M
SM
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003891:Desipramine / Q000276:immunology*; D017886:Durapatite; D006882:Hydroxyapatites; D007140:Immunoglobulin Fab Fragments / Q000302:isolation & purification*; D007074:Immunoglobulin G / Q000302:isolation & purification; D051379:Mice; D010269:Paraquat / Q000276:immunology*
D000911:Antibodies, Monoclonal; D006882:Hydroxyapatites; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D017886:Durapatite; D010269:Paraquat; D003891:Desipramine