Hypersensitive immunoassay by using <i>Escherichia coli</i> outer membrane with autodisplayed Z-domains

Park, Min,Jose, Joachim,Pyun, Jae-Chul Elsevier 2010 Enzyme and microbial technology Vol.46 No.3

<P><B>Abstract</B></P><P>In this work, the Z-domain of protein A was autodisplayed on the surface of the <I>Escherichia coli</I> outer membrane as a fusion protein of AIDA-1, and then layered on a microplate in order to develop a hypersensitive immunoassay. By using microplates with controlled hydrophilicity the formation of outer membrane layer was carried out, and the driving force for the layer formation was proven to be a hydrophobic interactions. Through the orientation control of the immobilized antibodies by using autodisplayed Z-domains, the limit of detection (LOD) and the sensitivity of this new immunoassay were determined to have improved as much as 30-fold compared to the conventional ELISA. The applicability of the new immunoassay for medical diagnosis was demonstrated by the detection of C-reactive protein (CRP) which is known to be a biomarker protein for inflammatory diseases.</P>

Optimization of a FACS based-immunoassay using E. coli autodisplaying Z-domains

박민,봉지홍,Joachim Jose,강민정,변재철 한국바이오칩학회 2013 BioChip Journal Vol.7 No.2

We developed a fluorescence-activated cell sorter (FACS)-based immunoassay using Escherichia coli with autodisplayed Z-domains as a solid support for detection antibodies as well as a method to optimize the signal-to-noise ratio of fluorescence signals from E. coli. To improve the sensitivity of FACS measurements by eliminating artifactual signals from unrelated small particles in the sample mixture,E. coli with autodisplayed Z-domains was modified to express enhanced green fluorescence protein (eGFP)in the cytosol. By selectively gating the measurement channel for eGFP in the cytosol, we were able to discriminate between green fluorescent E. coli and artifact particles. We confirmed the efficacy of this optimization method for FACS-based immunoassay using several model reactions. Finally, as proof of concept,we used the FACS-based immunoassay and our optimization method for the medical diagnosis of human hepatitis B virus surface antigen.

SPR biosensor based on immobilized E.colicells with autodisplayed Z-domains

이은항,유구,Joachim Jose,강민정,송승민,변재철 한국바이오칩학회 2012 BioChip Journal Vol.6 No.3

The Z-domains of protein A was expressed as a fusion protein at the outer membrane of E.coliby using the autodisplay technology. Because of the specific affinity towards the Fc region of immunoglobulins(IgG’s), the Z-domains have been used for the orientation control of antibodies in order to improve the sensitivity of immunoassays. In this work, the E.coli with autodispalyed Z-domains was immobilized to the SPR biosensor by the charge interaction. The surface modification was carried out by covalent layering of the poly-L-lysine with amino groups to the parylene-H film with formyl groups. And then, the negatively charged E.colicells were immobilized by charge interaction with the positivley charged of poly-L-lysine. The effectiveness of this layer for the immobilization of E.coliwas estimated by counting the number of E.coli cells in comparison with the bare gold surface and the poly-L-lysine coated gold surface. For the test of feasibility of the immobilized E.coli cells to SPR biosensor, the stability of immobilized E.colicells was estimated by treatment of salt solutions at the known concentrations to the immobilized E.colicells which were bound through the charge interaction. From this test, the E.colicells immobilized to the parylene-H film with poly-L-lysine coating were determined to be stable at the salt concentration of human serum. Then, the applicability of the immobilized E.colicells with autodisplayed Z-domains was demonstrated by detection of C-reactive protein(CRP). The effect of orientation control with autodisplayed Z-domains was estimated by comparing the sensivities by immobilization through the physical adsorption and charge interaction to poly-L-lysine coated layer.

Autodisplay of streptavidin

Park, Min,Jose, Joachim,Thö,mmes, Sarah,Kim, Jo-Il,Kang, Min-Jung,Pyun, Jae-Chul Elsevier 2011 Enzyme and microbial technology Vol.48 No.4

<P><B>Abstract</B></P><P>Streptavidin was expressed on the outer membrane of <I>E. coli</I> as a recombinant fusion protein with an autotransporter domain called AIDA-I (adhesin involved in diffuse adherence) using autodisplay technology. The autodisplay of streptavidin was confirmed by SDS-PAGE of the outer membrane proteins, and the number of autodisplayed streptavidin molecules on a single <I>E. coli</I> cell was evaluated with densitometric analysis. The biotin-binding activity of the autodisplayed streptavidin was estimated after treatment with fluorescently labeled biotin by fluorescence microscopy and flow cytometry. The biotin-binding activity of the <I>E. coli</I> with autodisplayed streptavidin was compared with the activity of streptavidin immobilized on magnetic beads. Finally, the outer membrane presenting autodisplayed streptavidin was isolated and layered on a 96-well microplate for an immunoassay.</P>

Immobilization of E.coli with Autodisplayed Z-domains to a Surface-modified for Immunoassays

Gu YOO,Min PARK,Joachim JOSE,Jae-Chul PYUN 한국생물공학회 2011 한국생물공학회 학술대회 Vol.2011 No.10

Development of a wash-free immunoassay using Escherichia coli cells with autodisplayed Z-domains

Pyun, Jae-Chul,Jose, Joachim,Park, Min Royal Society of Chemistry 2017 The Analyst Vol.142 No.10

<P><I>Escherichia coli</I> cells that autodisplay Z-domains have been used to improve the sensitivity and limit of detection (LOD) of immunoassays by controlling antibody orientation. In this study, a rapid, wash-free immunoassay was developed using <I>E. coli</I> cells with autodisplayed Z-domains and flow cytometry. The fluorescence signal from a single <I>E. coli</I> cell could be identified and background noise from non-bound detection antibodies was minimal. Flow cytometric measurement was demonstrated to be more effective than microscopy or fluorescence photometry. The concentrations of the capture and detection antibodies were optimized by testing various concentrations; the incubation time was optimized in the same way. The wash-free immunoassay was used to quantify the C-reactive protein (CRP) and the results were compared with those of an established assay method. The results of the wash-free immunoassay were significantly correlated with the results of the washing method, but showed an improved LOD and dynamic range. Thus, the wash-free immunoassay based on <I>E. coli</I> cells with autodisplayed Z-domains and flow cytometry developed here was confirmed to be feasible for use in medical diagnosis.</P>

Immobilization of E.coli with autodisplayed Z-domains to a surface-modified magnetic bead for immunoassays

Ju-Kyung Lee,Gu Yoo,Min Park,Joachim Jose,Jae-Chul Pyun 한국생물공학회 2011 한국생물공학회 학술대회 Vol.2011 No.10

Functional Recovery Not Correlated with Axon Regeneration through Olfactory Ensheathing Cell- Seeded Scaffolds in a Model of Acute Spinal Cord Injury

Haktan Altinova,Sven Möllers,Ronald Deumens,Jose Gerardo-Nava,Tobias Führmann,Sabien Geraldine Antonia van Neerven,Ahmet Bozkurt,Christian Andreas Mueller,Hans Joachim Hoff,Ingo Heschel,Joachim Weis,G 한국조직공학과 재생의학회 2016 조직공학과 재생의학 Vol.13 No.5

The implantation of bioengineered scaffolds into lesion-induced gaps of the spinal cord is a promising strategy for promoting functional tissue repair because it can be combined with other intervention strategies. Our previous investigations showed that functional improvement following the implantation of a longitudinally microstructured collagen scaffold into unilateral mid-cervical spinal cord resection injuries of adult Lewis rats was associated with only poor axon regeneration within the scaffold. In an attempt to improve graft-host integration as well as functional recovery, scaffolds were seeded with highly enriched populations of syngeneic, olfactory bulb-derived ensheathing cells (OECs) prior to implantation into the same lesion model. Regenerating neurofilament-positive axons closely followed the trajectory of the donor OECs, as well as that of the migrating host cells within the scaffold. However, there was only a trend for increased numbers of regenerating axons above that supported by non-seeded scaffolds or in the untreated lesions. Nonetheless, significant functional recovery in skilled forelimb motor function was observed following the implantation of both seeded and non-seeded scaffolds which could not be correlated to the extent of axon regeneration within the scaffold. Mechanisms other than simple bridging of axon regeneration across the lesion must be responsible for the improved motor function.

SPR biosensor based on immobilized E.coli cells with autodisplayed Z-domains

Eun-Hang Lee,Gu Yoo,Min Park,Joachim Jose,Jae-Chul Pyun 한국생물공학회 2011 한국생물공학회 학술대회 Vol.2011 No.10

Orientation and density control of proteins on solid matters by outer membrane coating: Analytical and diagnostic applications

Park, Min,Pyun, Jae-Chul,Jose, Joachim Elsevier 2018 Journal of pharmaceutical and biomedical analysis Vol.147 No.-

<P><B>Abstract</B></P> <P>Autodisplay is an expression system for the display of recombinant proteins on the outer membrane (OM) of gram negative bacteria and has been developed for translocation studies, whole cell biocatalysis, bioremediation, inhibitor screening, and enzyme refolding. Recently, affinity proteins such as IgG-binding Z-domains and biotin-binding streptavidin have been autodisplayed on the OM of <I>Escherichia coli</I> for analytical and biomedical applications. The secretion mechanism of the autodisplay system was used and orientation and density control of these affinity proteins were determined. Affinity protein-autodisplaying <I>E. coli</I> cells have been used to coat solid supports in immunoassays. For this purpose, the OM of autodisplayed <I>E. coli</I> cells was separated and isolated by the aid of detergents. The structure of the resulting OM liposomes as well as their physico-chemical parameters, were analyzed. OM liposomes were used subsequently for coating various solid matters including microplates and biosensor transducer surfaces and the formation of OM layers were monitored. OM layer formation on solid matters was shown to increase the sensitivity of immunoassays and biosensors. In this review, analytical and diagnostic applications are described in particular concerning orientation and density control of autodisplayed affinity proteins.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A lack of orientation control exists when solid matters are coated with biomolecules. </LI> <LI> This leads to a severe reduction in sensitivity and the limit of detection. </LI> <LI> Autodisplay leads to an oriented expression of proteins on the OM. </LI> <LI> OM liposomes can be prepared and used for solid matter coating. </LI> <LI> Orientation and density control obtained thereby leads to an increase in sensitivity and LOD. </LI> </UL> </P>