High-affinity rabbit antibodies directed against methotrexate: production, purification, characterization, and...

We describe the production, purification, characterization, and disposition of rabbit polyclonal methotrexate antibodies. These antibodies are prepared for subsequent testing of a drug delivery approach to reduce 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 anti-methotrexate antibodies were sequestered from rabbit serum through the use of a protein-G affinity column which allowed for purification of 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 antibodies have been shown to have high affinity (Keq range: 1.8 x 10(8) to 8.75 x 10(9) M-1) and high selectivity for methotrexate. Preliminary pharmacokinetic studies of the purified antibodies in the rat following a 6 mg/kg intravenous infusion (n = 4) indicate a steady state volume of distribution of 38.0 +/- 11.2 mL kg-1, a systemic clearance of 0.92 +/- 0.67 mL kg-1 h-1 and an elimination half life of 28.9 +/- 7.9 h (mean +/- SD).