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

著者 Zhu X , Miao X , Qin X , Zhu X
J Pharm Biomed Anal.2019 Jun 03 ; 174():263-269.
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To produce specific antibodies for the detection and quantification of copper ions, bifunctional chelators (BFCs) are commonly applied in the preparation of copper conjugates. However, some copper-chelator complexes exhibit limited stability under in vivo conditions. In this study, Cu 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 Cu over 96 h. The immune response varied depending on the choice of chelator; notably, antisera from the Cu-NOTA-KLH conjugate demonstrated the best reactivity toward chelated Cu. 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 (IC) of 8.9 ng/mL) to chelated Cu, with a working range from 1.21 to 48.9 ng/mL. The recovery of Cu 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 Cu, 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 Cu.
PMID: 31181489 [PubMed - as supplied by publisher]
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