An ultra-specific and sensitive sandwich ELISA for imatinib using two anti-imatinib antibodies

The development of an immunoassay for a low-molecular-weight drug first requires the identification of specific antibodies that do not cross-react with the drug's metabolites. If two antibodies can simultaneously recognize the entire structure of the drug, we can then utilize them to establish an ultra-specific sandwich ELISA, free from interference due to the metabolic products of the drug. This paper reports an ultra-specific and sensitive sandwich ELISA for determination of the tyrosine kinase inhibitor imatinib using two anti-imatinib antibodies. The anti-imatinib antibodies were obtained by two partial structures of imatinib as haptens (2-(5-amino-2-methylanilino)-4-(3-pyridyl)pyrimidine and 4-{(4-methyl-1-piperazinyl)-methyl}-benzoate). Under optimized conditions, this sandwich ELISA shows a linear detection range from 64 pg mL-1 to 8 ng mL-1, and a limit of detection of approximately 64 pg mL-1 for 100-μL samples. The ELISA is specific to imatinib and while there was no cross-reactivity with the major metabolite N-desmethyl-imatinib, slight cross-reactivity was found with metabolite pyridine-N-oxide-imatinib. This assay demonstrated significantly lower cross-reactivity with metabolites than competitive ELISAs. Using this assay, drug levels were easily measured in rat blood after oral administration of imatinib via a single dose of 30 mg kg-1 or 100 mg kg-1. The levels in rat serum measured by this ELISA were comparable with those measured by HPLC, and there was a strong correlation between the values determined by the two methods (y = 0.983x + 0.081, R2 = 0.948). Thus, we have successfully developed the first specific and sensitive sandwich ELISA for imatinib using two anti-imatinib antibodies. This sandwich ELISA will be a valuable tool for therapeutic drug monitoring and pharmacokinetic studies of imatinib.