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 antigenss. Papain digests of the IgGs were subjected to further HPHT followed by Sephadex G-100 chromatography to yield homogenous 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

Adaptation of the Müller method to allow quantitative characterization of the affinity and cross-reactivity of antibodies by competitive radioimmunoassay

A quantitative expression is derived for the evaluation of antigen-antibody affinity constants from radioimmunoassays for the completely general situation in which antigen and antibody are both multivalent. The theoretical analysis is then extended to encompass quantitative characterization of the competitive inhibition observed in screening tests for cross-reactivity of antibody with structural analogs of the eliciting antigen. These procedures are illustrated with a radioimmunological study of the cross-reactivity of a desipramine-elicited monoclonal antibody with other tricyclic antidepressants. An unexpected finding to emerge from this immunochemical study is the demonstration that a single affinity constant suffices to describe the interaction of desipramine with a polyclonal antibody elicited by this univalent antigen