BackgroundFor therapeutic monitoring and pharmacokinetic studies of lenalidomide (LND), the potent drug for treatment of multiple myeloma (MM), a specific antibody was required for the development of a sensitive immunoassay system for the accurate determination of LND in plasma.ResultsIn this study, a hapten of LND (N-glutaryl-LND) was synthesized by introducing the glutaryl moiety, as a spacer, into the primary aromatic amine site of the LND molecular structure. The structure of the hapten (G-LND) was confirmed by mass, 1H-NMR, and 13C spectrometric techniques. G-LND was coupled to each of bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) proteins by ethyl-3-(3-dimethylaminopropyl) carbodiimide as a coupling reagent. LND-KLH conjugate was used as an immunogen. Four female 2-3 months old New Zealand white rabbits were immunized with an emulsion of LND-KLH with Freund`s adjuvant. The immune response of the rabbits was monitored by direct enzyme-linked immunosorbent assay (ELISA) using LND-BSA immobilized onto microwell plates as a solid phase. The rabbit that showed the highest antibody titer and affinity to LND was scarified and its sera were collected. The IgG fraction was isolated and purified by affinity chromatography on protein A column. The specificity of the purified antibody for LND was evaluated by indirect competitive ELISA using dexamethasone as a competitor as it is used with LND in a combination therapy.ConclusionsThe high affinity of the antibody (IC50 = 10 ng/mL) will be useful in the development of an immunoassay system for the determination of plasma LND concentrations. Current research is going to optimize the assay conditions and validate the procedures for the routine application in clinical laboratories.
This article describes, for the first time, a highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) for therapeutic monitoring and pharmacokinetic studies of lenalidomide (LND), the potent drug for treatment of multiple myeloma (MM). The assay employed a polyclonal antibody that specifically recognizes LND with high affinity, and LND conjugate of bovine serum albumin (LND-BSA) immobilized onto microplate wells as a solid phase. The assay involved a competitive binding reaction between LND, in plasma sample, and the immobilized LND-BSA for the binding sites on a limited amount of the anti-LND antibody. The assay limit of detection was 0.05 ng/mL and the effective working range at relative standard deviations (RSD) of ≤5% was 0.1-20 ng/mL. Analytical recovery of LND from spiked plasma was 100.98 ± 3.05. The precisions of the assay were satisfactory; RSD was 2.96-6.67% and 4.46-7.14%, for the intra- and inter-assay precision, respectively. The procedure is convenient, and one can analyze ∼200 samples per working day, facilitating the processing of large-number batch of samples in clinical laboratories. The proposed ELISA has a great value in routine analysis of LND for its therapeutic monitoring and pharmacokinetic studies.
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