A Sensitive Immunodetection Assay Using Antibodies Specific to Staphylococcal Enterotoxin B Produced by Baculovirus Expression

Jang, Ju-Hong; Kim, Sungsik; Kim, Seul-Gi; Lee, Jaemin; Lee, Dong-Gwang; Jang, Jaeduck; Jeong, Young‐Su; Song, Dong-Hyun; Min, Jeong‐Ki; Park, Jong‐Gil; Lee, Moo‐Seung; Han, Baek‐Soo; Son, Joung Sik; Lee, Jangwook; Lee, Nam-Kyung

doi:10.3390/bios12100787

Cited by 5 publications

(4 citation statements)

References 51 publications

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“…An amine-reactive 2 nd generation (AR2G) biosensor (ForteBio, USA) was used to immobilize recombinant PirA or PirB as described previously ( Jang et al., 2022 ). The biosensors were quenched with 1 M ethanolamine–HCl (pH 8.5) for 300 s, and a zero baseline was obtained with PBS for 120 s. Kinetic analysis was performed using the Octet K2 system (ForteBio) by determining the association (K on ) and dissociation (K off ) of anti-PirA and anti-PirB antibodies for 600 s. The K D values of each antibody were calculated based on K on and K off using data analysis software (HT 12.0; ForteBio).…”

Section: Methodsmentioning

confidence: 99%

“…After blocking with MPBS for 1 h, a mixture of recombinant PirA and PirB at 1:1 ratio (each 100 nM) was serially diluted in PBS and added to each well, followed by incubation for 2 h at room temperature. Anti-PirB IgG was biotinylated for detection as previously described ( Jang et al., 2022 ). After washing the plate with PBST three times, 100 μl of biotinylated anti-PirB IgG (15 μg/ml) was added to each well and incubated for 1 hr at room temperature.…”

Section: Methodsmentioning

confidence: 99%

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Novel sandwich immunoassay detects a shrimp AHPND-causing binary PirABVp toxin produced by Vibrio parahaemolyticus

Jeon,

Han,

Lee

et al. 2023

Front. Cell. Infect. Microbiol.

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IntroductionThe binary PirA/PirB toxin expressed by Vibrio parahaemolyticus (PirABVp) is a virulent complex that causes acute hepatopancreatic necrosis disease (AHPND) in shrimps, affecting the global shrimp farming industry. AHPND is currently diagnosed by detecting pirA and pirB genes by PCR; however, several V. parahaemolyticus strains do not produce the two toxins as proteins. Thus, an immunoassay using antibodies may be the most effective tool for detecting toxin molecules. In this study, we report a sandwich ELISA-based immunoassay for the detection of PirABVp.MethodsWe utilized a single-chain variable fragment (scFv) antibody library to select scFvs against the PirA or PirB subunits. Phage display panning rounds were conducted to screen and identify scFv antibodies directed against each recombinant toxin subunit. Selected scFvs were converted into IgGs to develop a sandwich immunoassay to detect recombinant and bacterial PirABVp.ResultsAntibodies produced as IgG forms showed sub-nanomolar to nanomolar affinities (KD), and a pair of anti-PirA antibody as a capture and anti-PirB antibody as a detector showed a limit of detection of 201.7 ng/mL for recombinant PirABVp. The developed immunoassay detected PirABVp in the protein lysates of AHPND-causing V. parahaemolyticus (VpAHPND) and showed a significant detectability in moribund or dead shrimp infected with a VpAHPND virulent strain compared to that in non-infected shrimp.DiscussionThese results indicate that the developed immunoassay is a reliable method for diagnosing AHPND by detecting PirABVp at the protein level and could be further utilized to accurately determine the virulence of extant or newly identified VpAHPND in the global shrimp culture industry.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Novel sandwich immunoassay detects a shrimp AHPND-causing binary PirABVp toxin produced by Vibrio parahaemolyticus

Jeon,

Han,

Lee

et al. 2023

Front. Cell. Infect. Microbiol.

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“…Approximately 80% of S. aureus strains clinically isolated are enterotoxin producers [4], and staphylococcal enterotoxin B (SEB) is the main toxin produced by this bacterium. SEB is a superantigen (SAg) that activates T-cells in a non-specific manner, causing an exaggerated and potentially harmful immune response [3][4][5][6][7].…”

Section: Introductionmentioning

confidence: 99%

The SEB1741 Aptamer Is an Efficient Tool for Blocking CD4+ T Cell Activation Induced by Staphylococcal Enterotoxin B

et al. 2023

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Staphylococcal enterotoxin B (SEB) is a protein produced by Staphylococcus aureus, which is toxic to humans. It is well known for its ability to stimulate the exacerbated activation of proinflammatory CD4+ T cells (Th1 profile), and in vitro studies have been conducted to understand its mechanism of action and its potential use as an immune therapy. However, the efficiency of the SEB1741 aptamer in blocking SEB has not been experimentally demonstrated. Methods: Enrichment CD4+ T cells were stimulated with SEB, and as a blocker, we used the SEB1741 aptamer, which was previously synthesised by an “in silico” analysis, showing high affinity and specificity to SEB. The efficiency of the SEB1741 aptamer in blocking CD4+ T cell activation was compared with that of an anti-SEB monoclonal antibody. Flow cytometry and Bio-Plex were used to evaluate the T-cell function. Results: In vitro, SEB induced the activation of CD4+ T cells and favoured a Th1 profile; however, the SEB1741 aptamer was highly efficient in decreasing the frequency of CD4+ T cells positive to ki-67 and CD69 cells, this means that proliferation and activation of CD4+ T cells was decreased. Moreover, the production of interleukin 2 (IL-2) and interferon-gamma (IFN-γ) was affected, suggesting that the Th1 profile is not present when the SEB1441 aptamer is used. Thus, the SEB1741 function was similar to that of anti-SEB. Conclusions: The SEB1741 aptamer is a valuable tool for blocking CD4+ T cell activation and the subsequent release of proinflammatory cytokines by SEB stimulation.

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“…Several conventional methods have been established for detecting SEB, including chemiluminescent immunoassay [ 8 ], electrochemiluminescence immunoassay [ 9 ], fluorescent immunoassay [ 10 ], enzyme-linked immunosorbent assay [ 11 ], and lateral flow assay [ 12 ]. Although these methods have played important roles in the past decades for quantitative analysis of SEB, their sensitivity still cannot meet the requirements of warning of SEB contamination at the early stage.…”

Section: Introductionmentioning

confidence: 99%

Development of a Gold Nanoparticle-Linked Immunosorbent Assay of Staphylococcal Enterotoxin B Detection with Extremely High Sensitivity by Determination of Gold Atom Content Using Graphite Furnace Atomic Absorption Spectrometry

Song

Liu

et al. 2023

Pharmaceutics

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Highly sensitive staphylococcal enterotoxin B (SEB) assay is of great importance for the prevention of toxic diseases caused by SEB. In this study, we present a gold nanoparticle (AuNP)-linked immunosorbent assay (ALISA) for detecting SEB in a sandwich format using a pair of SEB specific monoclonal antibodies (mAbs) performed in microplates. First, the detection mAb was labeled with AuNPs of different particle sizes (15, 40 and 60 nm). Then the sandwich immunosorbent assay for SEB detection was performed routinely in a microplate except for using AuNPs-labeled detection mAb. Next, the AuNPs adsorbed on the microplate were dissolved with aqua regia and the content of gold atoms was determined by graphite furnace atomic absorption spectrometry (GFAAS). Finally, a standard curve was drawn of the gold atomic content against the corresponding SEB concentration. The detection time of ALISA was about 2.5 h. AuNPs at 60 nm showed the highest sensitivity with an actual measured limit of detection (LOD) of 0.125 pg/mL and a dynamic range of 0.125–32 pg/mL. AuNPs at 40 nm had an actual measured LOD of 0.5 pg/mL and a dynamic range of 0.5 to 128 pg/mL. AuNPs at 15 nm had an actual measured LOD of 5 pg/mL, with a dynamic range of 5–1280 pg/mL. With detection mAb labeled with AuNPs at 60 nm, ALISA’s intra- and interassay coefficient variations (CV) at three concentrations (2, 8, and 20 pg/mL) were all lower than 12% and the average recovery level was ranged from 92.7% to 95.0%, indicating a high precision and accuracy of the ALISA method. Moreover, the ALISA method could be successfully applied to the detection of various food, environmental, and biological samples. Therefore, the successful establishment of the ALISA method for SEB detection might provide a powerful tool for food hygiene supervision, environmental management, and anti-terrorism procedures and this method might achieve detection and high-throughput analysis automatically in the near future, even though GFAAS testing remains costly at present.

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A Sensitive Immunodetection Assay Using Antibodies Specific to Staphylococcal Enterotoxin B Produced by Baculovirus Expression

Cited by 5 publications

References 51 publications

Novel sandwich immunoassay detects a shrimp AHPND-causing binary PirABVp toxin produced by Vibrio parahaemolyticus

Novel sandwich immunoassay detects a shrimp AHPND-causing binary PirABVp toxin produced by Vibrio parahaemolyticus

The SEB1741 Aptamer Is an Efficient Tool for Blocking CD4+ T Cell Activation Induced by Staphylococcal Enterotoxin B

Development of a Gold Nanoparticle-Linked Immunosorbent Assay of Staphylococcal Enterotoxin B Detection with Extremely High Sensitivity by Determination of Gold Atom Content Using Graphite Furnace Atomic Absorption Spectrometry

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