Design of immunogens: the effect of bifunctional chelator on immunological response to chelated copper

In this study, Cu2+ was coupled with carrier proteins via three different macrocyclic BFCs: p-SCN-Bn-DOTA, p-SCN-Bn-NOTA, and p-SCN-Bn-TETA. The stability in plasma and the immunogenicity of three copper immunoconjugates were compared. The chelators other than p-SCN-Bn-DOTA were very stable in plasma, with <9% dissociation of Cu2+ over 96 h. The immune response varied depending on the choice of chelator; notably, antisera from the Cu2+-NOTA-KLH conjugate demonstrated the best reactivity toward chelated Cu2+. p-SCN-Bn-NOTA, which showed significant advantages over the other chelators, was used for antibody production. The efficiency of immune-positive hybridoma production was satisfactory, and the resultant monoclonal antibodies (McAbs) 4B7 showed sensitivity (half-maximal inhibitory concentration (IC50) of 8.9 ng/mL) to chelated Cu2+, with a working range from 1.21 to 48.9 ng/mL. The recovery of Cu2+ from water samples was 85.7–108%, and the intra- and inter-assay coefficients of variation were 4.0–10.1% and 7.1–11.4%, respectively. Compared with previously reported McAb specific to Cu2+, DF4, the sensitivity of the newly developed assay was improved 100-fold. The results of this study indicate the utility of NOTA for the efficient generation of highly sensitive McAbs against Cu2+.
Source: Journal of Pharmaceutical and Biomedical Analysis - Category: Drugs & Pharmacology Source Type: research