HIGH-AFFINITY RABBIT ANTIBODIES DIRECTED AGAINST METHOTREXATE - PRODUCTION, PURIFICATION, CHARACTERIZATION, AND PHARMACOKINETICS IN THE RAT

We describe the production, purification, characterization, and dispos ition of rabbit polyclonal methotrexate antibodies. These antibodies a re prepared for subsequent testing of a drug delivery approach to redu ce systemic toxicity upon regional administration of methotrexate. The polyclonal antibodies were raised in New Zealand white female rabbits immunized with a methotrexate-bovine serum albumin conjugate. The ant i-methotrexate antibodies were sequestered from rabbit serum through t he use of a protein-G affinity column which allowed for purification o f up to 100 mg of rabbit IgG in 30-40 min. The extent of purification was demonstrated through SDS-PAGE and calculation of specific binding activity relative to total protein concentration. The purified antibod ies have been shown to have high affinity (K-eq, range: 1.8 x 10(8) to 8.75 x 10(9) M(-1)) and high selectivity for methotrexate. Preliminar y pharmacokinetic studies of the purified antibodies in the rat follow ing a 6 mg/kg intravenous infusion (n = 4) indicate a steady state vol ume of distribution of 38.0 +/- 11.2 mL kg(-1), a systemic clearance o f 0.92 +/- 0.67 mL kg(-1) h(-1) and an elimination half life of 28.9 /- 7.9 h (mean +/- SD).