Covalently Immobilized Regenerable Immunoaffinity Layer with Orientation-Controlled Antibodies Based on Z-Domain Autodisplay

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%.

The Conjugation Strategy Affects Antibody Orientation and Targeting Properties of Nanocarriers

Antibody-modified drug delivery systems in the nano-range have the ability to overcome current challenges for treating diseases due to their high specificity towards the targeted body region. However, no antibody-bound...

A Regenerative Immunoaffinity Layer Based on the Outer Membrane of Z-Domains Autodisplaying E. coli for Immunoassays and Immunosensors

Through orientation control of antibodies, Z-domains autodisplaying Escherichia coli outer cell membrane (OM) may be utilized to improve the sensitivity and limit of detection (LOD) of immunoassays and immunosensors. A regenerative immunoaffinity layer based on Z-domains autodisplaying E. coli OM was developed for the surface plasmon resonance (SPR) biosensor. Regeneration conditions for the Z-domains autodisplaying E. coli OM-based immunoassays and immunosensors were optimized by varying pH and detergent concentration. An E. coli cell-based HRP immunoassay was tested and validated in three sequential regenerative immunoassays under optimal conditions. The OM of Z-domains autodisplaying E. coli was isolated and coated on the two-dimensional substrate (microplate). The OM-based HRP immunoassay was tested and validated in four regenerative immunoassays. This regenerative OM layer was applied to the SPR biosensor. Z-domains autodisplaying OM layered onto the gold surface of SPR biosensors was developed, and the OM-based regenerative immunoaffinity layer with orientation control was tested using CRP analyte. The SPR biosensor regenerative immunoaffinity layer demonstrated that CRP biosensing was repeated for five regeneration cycles with less than 2% signal difference. Therefore, the newly developed regenerative immunoaffinity layer with antibody orientation control may improve biosensing sensitivity and reduce the cost of medical diagnosis.

Direct immunoassays and their performance – theoretical modelling of the effects of antibody orientation and associated kinetics

Mathematical and computational modelling are used to quantify immunoassay signals for various immobilized antibody orientations, antigen sizes and kinetic parameters, in order to determine optimal surface coverage.