Covalently Immobilized Regenerable Immunoaffinity Layer with Orientation-Controlled Antibodies Based on Z-Domain Autodisplay |
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Authors: | Jong-Min Park Mi Yeon Kim Joachim Jose Min Park |
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Affiliation: | 1.Major in Materials Science and Engineering, Hallym University, Chuncheon 24252, Korea; (J.-M.P.); (M.Y.K.);2.Cooperative Course of Nano-Medical Device Engineering, Hallym University, Chuncheon 24252, Korea;3.Integrative Materials Research Institute, Hallym University, Chuncheon 24252, Korea;4.Institute of Pharmaceutical and Medical Chemistry, Westfälische Wilhelms-Universität, 48149 Münster, Germany; |
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Abstract: | A regenerable immunoaffinity layer comprising covalently immobilized orientation-controlled antibodies was developed for use in a surface plasmon resonance (SPR) biosensor. For antibody orientation control, antibody-binding Z-domain-autodisplaying Escherichia coli (E. coli) cells and their outer membrane (OM) were utilized, and a disuccinimidyl crosslinker was employed for covalent antibody binding. To fabricate the regenerable immunoaffinity layer, capture antibodies were bound to autodisplayed Z-domains, and then treated with the crosslinker for chemical fixation to the Z-domains. Various crosslinkers, namely disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS) and poly (ethylene glycol)-ylated bis (sulfosuccinimidyl)suberate (BS(PEG)5), were evaluated, and DSS at a concentration of 500 μM was confirmed to be optimal. The E. coli-cell-based regenerable HRP immunoassay was evaluated employing three sequential HRP treatment and regeneration steps. Then, the Oms of E. coli cells were isolated and layered on a microplate and regenerable OM-based HRP immunoassaying was evaluated. Five HRP immunoassays with four regeneration steps were found to be feasible. This regenerable, covalently immobilized, orientation-controlled OM-based immunoaffinity layer was applied to an SPR biosensor, which was capable of quantifying C-reactive protein (CRP). Five regeneration cycles were repeated using the demonstrated immunoaffinity layer with a signal difference of <10%. |
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Keywords: | autodisplay Z-domains crosslinker immunoaffinity layer covalent immobilization orientation control regeneration SPR biosensor |
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