Synthesis of hapten and preparation of specific polyclonal antibody with high affinity for lenalidomide, the potent drug for treatment of multiple myeloma

Abstract

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.

Development of Monoclonal Hybridoma Cell Lines and Extracting Antibody Against Fummonisin B1

“…Therefore, the antigen for detection must be prepared for screening of the specific antibodies. In this study, the classical glutaraldehyde method was applied to couple FB 1 and keyhole limpet hemocyanin (KLH), which is a common carrier protein with a high molecular weight and good immunogenicity, to produce a macromolecule with both the toxin structure and the immunogenicity [13]. Furthermore, to avoid cross-reaction of the obtained antibody with KLH after immunizing animals, FB 1 was also coupled with BSA as the detection antigen to preliminarily assess coupling by detecting the serum titer in mice and screen the target antibody.…”

Research on the Mechanism of Action of a Citrinin and Anti-Citrinin Antibody Based on Mimotope X27

“…In order to verify the correctness of the docking model, X27 (28)(29)(30)(31)(32) was constructed through the five amino acids 28-32 in the WT, mutated to T28AF29AN30AR31AY32A, and the binding activity of the mutant was identified by indirect competitive ELISA. As a result, the wild-type phage had a greater binding ability of the anti-CIT monoclonal antibody than X27 (28)(29)(30)(31)(32) at the same titer. Moreover, the X27 (28-32) phages basically lost the binding ability to CIT MAb (monoclonal antibody) when both the X27 and X27 (28-32) phages were diluted by more than 400 times (Figure 3A).…”

Development of an Enzyme-Linked Immunosorbent Assay and Immunoaffinity Column Chromatography for Saikosaponin d Using an Anti-Saikosaponin d Monoclonal Antibody

“…The immune responses of the animals were monitored using the procedures described by Darwish et al [32,33]. Tail blood samples from all mice were collected after the third immunization.…”