Poly-protein G-expressing bacteria enhance the sensitivity of immunoassays

Hao, Wen Rui; Chen, Michael; Chen, Yi Jou; Su, Yu-Cheng; Cheng, Chiu Min; Hsueh, Hsiang Yin; Kao, An Pei; Hsieh, Yuan-Chin; Chang, Johny; Tseng, Ming Yang; Chuang, Kai‐Hsiang

doi:10.1038/s41598-017-01022-w

Cited by 6 publications

(7 citation statements)

References 53 publications

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“…In the present study, we constructed the 8pG on the surface of mammalian cells and demonstrated that the antibody-trapping amount of these 8pG cells was higher than that of 1pG cells. A similar phenomenon was seen in our previous studies; specifically, poly-protein G-expressing bacteria non-covalently conjugated to any detection antibodies and had a higher antibody-trapping amount than mono-protein G-expressing bacteria 40 . The data showed that the polymer protein G (8pG) had higher avidity for trapping antibodies than monomer protein G (1pG).…”

Section: Discussionsupporting

confidence: 88%

“…This process is laborious and time consuming. In this study, therefore, we combined the advantages of cell-based and protein G-based microplates to construct the 8pG cell-based microplate, which can provide extensive surface area and more steric space for trapping capture antibody than a flat-bed well 40 . The 8pG cells, which can be seen as self-renewing microparticles, are easy to obtain and can provide the preferred orientation of a capture antibody by trapping the Fc domain.…”

Section: Discussionmentioning

confidence: 99%

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Development of a highly sensitive enzyme-linked immunosorbent assay (ELISA) through use of poly-protein G-expressing cell-based microplates

Chen

Hsieh

et al. 2018

Sci Rep

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The sensitivity of traditional enzyme-linked immunosorbent assays (ELISAs) is limited by the low binding avidity and heterogeneous orientation of capture antibodies coated on polystyrene-based microplates. Here, we developed a highly sensitive ELISA strategy by fixing poly-protein G-expressing cells on microplates to improve the coating amount and displayed orientation of capture antibodies. One or eight repeated fragment crystallisable (Fc) binding domains of protein G are stably expressed on the surface of BALB/c 3T3 cells (termed 1pG cells or 8pG cells), which then act as highly antibody-trapping microparticles. The 8pG cells showed higher antibody-trapping ability than the 1pG cells did. The antibody-coating amount of the 8pG cell-based microplates was 1.5–23 times and 1.2–6.8 times higher than that of traditional polystyrene-based and commercial protein G-based microplates, respectively. The 8pG cell-based microplates were then applied to an anti-IFN-α sandwich ELISA and an anti-CTLA4 competitive ELISA, respectively, and dramatically enhanced their detection sensitivity. Importantly, direct coating unpurified capture antibody produced by mammalian cells did not impair the antigen-capturing function of 8pG cell-based microplates. The 8pG cell-based microplates exhibited a significant improvement in antibody-coating amount and preserved the homogeneous orientation of capture antibodies, making them a potential replacement for traditional microplates in various formats of ELISAs.

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Section: Discussionsupporting

confidence: 88%

Section: Discussionmentioning

confidence: 99%

Development of a highly sensitive enzyme-linked immunosorbent assay (ELISA) through use of poly-protein G-expressing cell-based microplates

Chen

Hsieh

et al. 2018

Sci Rep

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“…No obvious enhancement of absorbance was observed in the wells modified by Anti-IgG k -HRP alone. Therefore, it was confirmed that the PrG increased the sensitivity of the ELISA whereas previous reports have indicated that the presence of the PrG may decrease specificity 70,71 (Fig. 5B).…”

Section: Resultssupporting

confidence: 76%

An investigation into non-covalent functionalization of a single-walled carbon nanotube and a graphene sheet with protein G:A combined experimental and molecular dynamics study

Ebrahim-Habibi

Ghobeh

Mahyari

et al. 2019

Sci Rep

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Investigation of non-covalent interaction of hydrophobic surfaces with the protein G (PrG) is necessary due to their frequent utilization in immunosensors and ELISA. It has been confirmed that surfaces, including carbonous-nanostructures (CNS) could orient proteins for a better activation. Herein, PrG interaction with single-walled carbon nanotube (SWCNT) and graphene (Gra) nanostructures was studied by employing experimental and MD simulation techniques. It is confirmed that the PrG could adequately interact with both SWCNT and Gra and therefore fine dispersion for them was achieved in the media. Results indicated that even though SWCNT was loaded with more content of PrG in comparison with the Gra, the adsorption of the PrG on Gra did not induce significant changes in the IgG tendency. Several orientations of the PrG were adopted in the presence of SWCNT or Gra; however, SWCNT could block the PrG-FcR. Moreover, it was confirmed that SWCNT reduced the α-helical structure content in the PrG. Reduction of α-helical structure of the PrG and improper orientation of the PrG-SWCNT could remarkably decrease the PrG tendency to the Fc of the IgG. Importantly, the Gra could appropriately orient the PrG by both exposing the PrG-FcR and also by blocking the fragment of the PrG that had tendency to interact with Fab in IgG.

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“…Shieh et al demonstrated that capture ELISA (using a mouse anti-GNNV monoclonal antibody for capture and a rabbit anti-GNNV polyclonal antibody for detection) represents a specific, sensitive tool for GNNV detection [30], but additional costs are incurred for capture antibodies. Furthermore, Hao et al presented a poly-protein G-expressing bacterium (with the AIDA surface expression system) as an antibody-binding microparticle to enhance the sensitivity of immunoassays [51]. Our results reveal that BL21/lpp-Mx-based capture ELISA was more efficient than indirect ELISA at binding GNNV and that it might be a more cost-effective method for GNNV detection in aquaculture.…”

Section: Discussionmentioning

confidence: 69%

Developing a Virus-Binding Bacterium Expressing Mx Protein on the Bacterial Surface to Prevent Grouper Nervous Necrosis Virus Infection

Lin¹,

Chen²,

Cheng³

2021

J. Microbiol. Biotechnol.

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Viral nervous necrosis disease can cause mass mortality among groupers (Epinephelus coioides) and is a leading cause of economic loss in the aquaculture industry in Taiwan. Infected fish exhibit an abnormal swimming and swinging pattern and develop vacuoles within the brain and retina, ultimately leading to death. This pathology is caused by grouper nervous necrosis virus (GNNV), an RNA-containing piscine betanodavirus (Nodaviridae). It can spread through vertical transmission, contact with contaminated water, the food chain, or contact with contaminated aquacultural equipment. GNNV-infected fish constitute a major transmission vector for the disease [1][2][3][4][5][6]. Traditional methods of disease control in the aquaculture industry involve sterilization techniques, including ozone, ultraviolet radiation, iodine, and chlorine dioxide treatments [1,4,[7][8][9][10]. However, at low doses, these treatments are ineffective in controlling GNNV infection; at relatively high doses, these treatments may harm surrounding aquatic animals, probiotics, and even humans. Therefore, developing new methods for safely removing GNNV from water and preventing further viral outbreaks is essential.A method to bind and remove the virus from water could inhibit virus spread. Virus-binding proteins (VBPs) can efficiently recognize and bind to viruses, consequently inhibiting virus-host cell interactions and even viral replication. These proteins are present on the cell surface and are associated with channels through which the virus enters the cell, such as severe-acute respiratory syndrome-coronavirus (SARS-CoV) receptor angiotensinconverting enzyme 2 (ACE2) [11]. VBP production may be induced by adaptive immune responses against viral infections, such as responses involving therapeutic neutralizing antibodies (NAbs) against SARS-CoV-2 [13], or involved in innate immune responses against viral infection, such as those involving high-mannose-binding Grouper nervous necrosis virus (GNNV) infection causes mass grouper mortality, leading to substantial economic loss in Taiwan. Traditional methods of controlling GNNV infections involve the challenge of controlling disinfectant doses; low doses are ineffective, whereas high doses may cause environmental damage. Identifying potential methods to safely control GNNV infection to prevent viral outbreaks is essential. We engineered a virus-binding bacterium expressing a myxovirus resistance (Mx) protein on its surface for GNNV removal from phosphate-buffered saline (PBS), thus increasing the survival of grouper fin (GF-1) cells. We fused the grouper Mx protein (which recognizes and binds to the coat protein of GNNV) to the C-terminus of outer membrane lipoprotein A (lpp-Mx) and to the N-terminus of a bacterial autotransporter adhesin (Mx-AIDA); these constructs were expressed on the surfaces of Escherichia coli BL21 (BL21/lpp-Mx and BL21/Mx-AIDA). We examined bacterial surface expression capacity and GNNV binding activity through enzyme-linked immunosorbent assay; we also evaluated the GNNV re...

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Poly-protein G-expressing bacteria enhance the sensitivity of immunoassays

Cited by 6 publications

References 53 publications

Development of a highly sensitive enzyme-linked immunosorbent assay (ELISA) through use of poly-protein G-expressing cell-based microplates

Development of a highly sensitive enzyme-linked immunosorbent assay (ELISA) through use of poly-protein G-expressing cell-based microplates

An investigation into non-covalent functionalization of a single-walled carbon nanotube and a graphene sheet with protein G:A combined experimental and molecular dynamics study

Developing a Virus-Binding Bacterium Expressing Mx Protein on the Bacterial Surface to Prevent Grouper Nervous Necrosis Virus Infection

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