title: High-affinity rabbit antibodies directed against methotrexate: production, purification, characterization, and pharmacokinetics in the rat.
abstract: 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).
authors.affiliation: Department of Pharmaceutics, State University of New York at Buffalo 14260.
authors.forename: J P
authors.initials: JP
authors.lastname: Balthasar
authors.forename: H L
authors.initials: HL
authors.lastname: Fung
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D000917:Antibody Formation; D000918:Antibody Specificity; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D018796:Immunoconjugates / Q000737:chemistry / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology / Q000302:isolation & purification; D008297:Male; D008727:Methotrexate / Q000276:immunology*; D011817:Rabbits / Q000276:immunology; D011863:Radioimmunoassay; D051381:Rats; D017207:Rats, Sprague-Dawley; D012710:Serum Albumin, Bovine / Q000276:immunology
chemical_list: D000906:Antibodies; D018796:Immunoconjugates; D007074:Immunoglobulin G; D012710:Serum Albumin, Bovine; D008727:Methotrexate