Identification of twelve Aeromonas spp. species with monoclonal antibody-based immunoassay in water and fish samples

Wang, Wenbin; Liu, Dandan; Gao, Yuwei; Sang, Yunong; Liang, Xianwei; Liu, Jianxin; Shi, Jinyan; Guo, Lei; Pan, Saikun

doi:10.1016/j.aquaculture.2019.734646

Cited by 9 publications

(7 citation statements)

References 47 publications

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“…Wang et al ( 2020 ) revealed that the newly identified mAb 2G12 had good specificity for recognizing ECO157 and it bound with a high abundance protein of approximately 35 kDa. In addition, mAb 2G12 bound with an approximately 70 kDa protein of ECO157 CICC 21,530.…”

Section: Discussionsupporting

confidence: 86%

“…WB was conducted following the protocols described by Wang et al ( 2020 ). To better fit the TE 70 semi-dry transfer system (14 cm16 cm), the 2-DE gel (18 cm16 cm) was cut into four parts (9 cm9 cm1.5 mm2, two 9 cm7 cm1.5 mm2) and transferred onto PVDF membrane (9 cm9 cm1.5 mm2, two 9 cm7 cm1.5 mm2) individually.…”

Section: Methodsmentioning

confidence: 99%

“…In Fig. 2C-E, "FGLRPSLAYLQSKGKNL-GVINGRNYD" is 286-311aa and is indicated with red signal; "GNKLDLYGKVD" is 29-39aa and the signal is labeled with yellow; "NFMQQRGNGFATYRNTD" is 140-156aa and is shown by pink signal; "RAETYTGGLKYD" is 234-245aa and its signal is in orange; "VGSFDYGRNYGVVYD" is 106-120aa and shown with cyan color signal; "NQFTRDAGINTD" is 345-356aa and indicated with in green signal; "ANNIYLAAQYTQTYNATRVGSLG" is 246-268aa and its signal is blue; "YKINLLDDNQFTRD" is 337-350aa and labeled with green signals ◂ Discussion Wang et al (2020) revealed that the newly identified mAb 2G12 had good specificity for recognizing ECO157 and it bound with a high abundance protein of approximately 35 kDa. In addition, mAb 2G12 bound with an approximately 70 kDa protein of ECO157 CICC 21,530.…”

Section: The Availability Of Epitopes Under Different Simulated Stressful Conditionsmentioning

confidence: 99%

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Identification of a specific surface epitope of OmpC for Escherichia coli O157:H7 with protein topology facilitated affinity mass spectrometry

Wang¹,

Zhou²,

Sang³

et al. 2021

Appl Microbiol Biotechnol

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The goal of this work was to identify the target protein and epitope of a previously reported Escherichia coli O157:H7 (ECO157)–specific monoclonal antibody (mAb) 2G12. mAb 2G12 has shown high specificity for the recovery and detection of ECO157. To achieve this goal, the target protein was first separated by two-dimensional gel electrophoresis (2-DE) and located by Western blot (WB). The protein spots were identified to be the outer membrane protein (Omp) C by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF–MS). After that, the target protein was purified by immunoaffinity chromatography (IAC) and subjected to in situ enzymatic cleavage of the vulnerable peptides. Eight eluted peptides of OmpC identified by liquid chromatography–tandem mass spectrometry (LC–MS/MS) were further mapped onto the homologous protein structure of E. coli OmpC (2IXX). The topology of OmpC showed that three peptides had extracellular loops. Epitope mapping with overlapping peptide library and sequence homology analysis revealed that the epitope consisted of a specific peptide, “LGVING,” and an adjacent conservative peptide, “TQTYNATRVGSLG.” Both peptides loop around the overall structure of the epitope. To test the availability of the epitope when ECO157 was grown under different osmolarity, pH, and nutrition levels, the binding efficacy of mAb 2G12 with ECO157 grown in these conditions was evaluated. Results further demonstrated the good stability of this epitope under potential stressful environmental conditions. In summary, this study revealed that mAb 2G12 targeted one specific and one conservative extracellular loop (peptide) of the OmpC present on ECO157, and the epitope was stable and accessible on ECO157 cells grown in different environment. Key points • OmpC is the target of a recently identified ECO157-specific mAb 2G12. • Eight peptides were identified from the OmpC by using LC–MS/MS. • The specificity of mAb 2G12 is mainly determined by the “LGVING” peptide.

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Section: Discussionsupporting

confidence: 86%

Section: Methodsmentioning

confidence: 99%

Section: The Availability Of Epitopes Under Different Simulated Stressful Conditionsmentioning

confidence: 99%

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Identification of a specific surface epitope of OmpC for Escherichia coli O157:H7 with protein topology facilitated affinity mass spectrometry

Wang¹,

Zhou²,

Sang³

et al. 2021

Appl Microbiol Biotechnol

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“…The affinity constants (Ka) of the mAbs were measured by indirect ELISA and were calculated as

Ka = \frac{n ‐ 1}{2 \times n {(false[{Ab}^{'} false] t ‐ false[Ab false] t)}^{'}}

, (Wang et al . 2020a). In this equation, [Ab] t and [Ab’] t are the antibody concentrations corresponding to the half the maximum absorbance of each titre curve at different coating concentration of the tested live strain (7 log 10 CFU per ml, 6 log 10 CFU per ml, 5 log 10 CFU per ml for ECO157 strains; 8 log 10 CFU per ml, 7·52 log 10 CFU per ml, 7 log 10 CFU per ml for untargeted strains), while n is the ratio of the coating concentration for the two curves.…”

Section: Methodsmentioning

confidence: 99%

Identification of novel monoclonal antibodies targeting the outer membrane protein C and lipopolysaccharides for Escherichia coli O157:H7 detection

Wang¹,

Sang²,

Liu³

et al. 2020

J Appl Microbiol

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Aims To identify and evaluate the application of two novel monoclonal antibody (mAb) 2G12 against outer membrane protein (Omp) C and mAb 12B1 targeting the O chain of the lipopolysaccharides (LPS) of Escherichia coli O157:H7 (ECO157). Methods and Results The sensitivity and specificity of these two antibodies were evaluated with eight ECO157 strains and 68 untargeted strains. mAb 2G12 and 12B1 had no detectable binding with any of the non‐O157 strains at 6·0 log10 CFU per ml, while its high specificity and affinity remained with all ECO157 strains. When a higher level (8·0 log10 CFU per ml) was tested, 2G12 and 12B1 did not react with 82·35 and 97·06% of the non‐O157 strains respectively. Based on the pair of two antibodies, the sandwich enzyme‐linked immunosorbent assay detected 100% (8/8) of ECO157 strains and none of the non‐ECO157 strains. The detection limit of ECO157 strains in pure culture were 4·2 ± 0·2 log10 CFU per ml. When the developed test was applied to artificially inoculated beef samples, the detection limit was 6·0 log10 CFU per gram without enrichment and 1·0 log10 CFU per gram after 12 h of enrichment. Conclusions The two novel antibodies identified in this study served as great candidates for the recovery, and detection of ECO157 from different environmental and food samples. Significance and Impact of the Study ECO157‐specific detection was improved by a combination of the novel OmpC mAb and LPS mAb with defined target antigen and good specificity.

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“…Common methods used for the detection of microbiological hazards include culture-based methods [3], immunological methods such as enzyme-linked immunosorbent assays (ELISA) [4,5], polymerase chain reaction (PCR) [6,7], and enzyme-based (ß-D-galactosidase or ß-glucuronidase assays) methods [8]. Although some of the methods like ELISA or PCR have good sensitivity and a short response time, these methods are limited by some drawbacks such as false positives and negatives as well as by their inability to distinguish between live and death microorganisms [9,10].…”

Section: Introductionmentioning

confidence: 99%

Specific detection of Escherichia coli using a phage-assisted ß-galactosidase assay

Hosseini

Mas

2023

Preprint

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Fast and reliable detection of microbial contaminants in food, water and environmental samples is critical for an efficient public health management. Most available methods provide good results although many of them have a number of drawbacks ranging from low sensitivity to the need of sophisticated equipment, the use of expensive reagents or the participation of highly skilled personnel. This work describes an easy to implement method for the detection of E. coli in liquid samples using a robust non-specific ß-galactosidase assay made highly selective through the use of a specific T4 lytic phage as a permeabilization reagent. The assay is performed in 96 well plates using MUG (4-methylumberlliferyl-ß-D-galactopyranoside) as the enzyme substrate and has a total length of 90 minutes. The method is able to detect 75 cells of E. coli. Under the conditions of the assay this corresponds to a concentration of 1.49·103 cells·mL− 1 of sample. For the analysis of field samples, we produced an extended version of the assay that incorporates preconcentration and preincubation steps with a total running length of 7.5 hours. When tested with field samples and compared with Colilert-18 the method performed well, with a limit of detection of 96 cells·100 mL− 1.

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Identification of twelve Aeromonas spp. species with monoclonal antibody-based immunoassay in water and fish samples

Cited by 9 publications

References 47 publications

Identification of a specific surface epitope of OmpC for Escherichia coli O157:H7 with protein topology facilitated affinity mass spectrometry

Identification of a specific surface epitope of OmpC for Escherichia coli O157:H7 with protein topology facilitated affinity mass spectrometry

Identification of novel monoclonal antibodies targeting the outer membrane protein C and lipopolysaccharides for Escherichia coli O157:H7 detection

Specific detection of Escherichia coli using a phage-assisted ß-galactosidase assay

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