Development and Validation of Enzyme‐Linked Immunosorbent Assays for Quantification of Anti‐Methotrexate IgG and Fab in Mouse and Rat Plasma

Abstract

This laboratory is investigating the use of anti‐methotrexate IgG (AMI) and anti‐methotrexate Fab fragments (AMF) within an inverse targeting strategy that is designed to enhance the pharmacokinetic selectivity of intraperitoneal (i.p.) chemotherapy. The goal of this study was to develop enzyme‐linked immunosorbent assays (ELISAs) to determine concentrations of AMI and AMF in mouse and rat plasma. An antigen‐specific ELISA was developed for AMI and AMF in mouse and rat plasma. The assay was validated with respect to precision and accuracy by evaluating the recovery of AMI and AMF from mouse and rat plasma samples. Preliminary pharmacokinetic studies of AMI and AMF were performed in Sprague‐Dawley rats and Swiss Webster mice. The animals were instrumented with a jugular vein cannula and administered AMI or AMF, 15 mg kg−1 via the cannula. Plasma samples were taken at various time points and analyzed using the ELISA, and the observed concentration vs. time profiles were subjected to non‐compartmental pharmacokinetic analyses. Standard curves for the ELISAs were found to be linear over concentration ranges of 0–250 and 0–350 ng mL−1 for AMI and AMF, respectively. Intra‐assay and inter‐assay recovery of AMI and AMF from plasma samples were found to be within 15% of theoretical values. Preliminary pharmacokinetic investigations of AMI allowed estimation of AMI clearance to be 0.017 mL kg−1 min−1 in the rat and 0.043 mL kg−1 min−1 in the mouse. AMF clearance was estimated to be 0.038 and 1.93 mL kg−1 min−1 in the mouse and rat, respectively. In conclusion, ELISAs have been developed and validated for quantitation of AMI and AMF in rat and mouse plasma. The assays will allow further investigations of AMI and AMF pharmacokinetics.