Monoclonal Antibody-Based Sandwich ELISA for the Detection of Staphylococcal Enterotoxin A

Kuang, Hua; Wang, Wenbing; Xu, Liguang; Ma, Wei; Liu, Liqiang; Wang, Libing; Xu, Chuanlai

doi:10.3390/ijerph10041598

Cited by 70 publications

(48 citation statements)

References 29 publications

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“…The results indicate that the sandwich ELISA with 10-4 HRP is more sensitive than that with 4-2 HRP or 4-11 HRP. This result is consistent with our previous findings that pairs with the highest ratio or absorbance may not guarantee the optimum pair [20]. Therefore, mAb 10 was used as the capture antibody and mAb 4 was used as the detection antibody; mAbs 10 and 4 were designated as the optimum mAb pair.…”

Section: Resultssupporting

confidence: 91%

“…The target cells were selected by indirect ELISA and obtained by limiting dilution. The mAbs were purified by the caprylic acid-ammonium sulfate precipitation method and then conjugated to HRP as described [20]. Antibodies that conjugated to HRP were characterized by direct ELISA.…”

Section: Methodsmentioning

confidence: 99%

“…Immunologic methods such as sandwich ELISA are effective for the detection of proteins and bacteria [18,19,20,21,22]. Suitable antibodies for the detection of β-lactamases by ELISA have been developed.…”

Section: Introductionmentioning

confidence: 99%

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Detection of β-Lactamase Residues in Milk by Sandwich ELISA

Wang

Liu

et al. 2013

IJERPH

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β-Lactamase residues in milk represent a public health risk. The cylinder plate detection method, which is based on bacterial growth, is laborious and time consuming. In this study, 15 monoclonal antibodies (mAbs) were selected against Temoneira (TEM) 1 β-lactamase. A sandwich enzyme-linked immunosorbent assay (ELISA) based on an optimum mAb pair was developed and validated for the detection of β-lactamase. The limit of detection and linear dynamic range of the method were 4.17 ng/mL and 5.5–100 ng/mL, respectively. β-Lactamase recovery in pure milk was 96.82–103.13%. The intra- and inter-assay coefficients of variation were 6.21–7.38% and 12.96–13.74%, respectively. Our developed sandwich ELISA can be used as a rapid detection method of β-lactamase in milk.

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Section: Resultssupporting

confidence: 91%

Section: Methodsmentioning

confidence: 99%

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Detection of β-Lactamase Residues in Milk by Sandwich ELISA

Wang

Liu

et al. 2013

IJERPH

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“…Numerous techniques are currently available for detection of SEs in food samples, such as ELISAs (Chiao et al 2013;Kuang et al 2013) and PCR-based methods (Jeyasekaran et al 2011). Commercially, many antibody-based kits are available for detection of SEs with good levels of sensitivity.…”

Section: Introductionmentioning

confidence: 99%

Use of biotin-labeled IgY overcomes protein A interference in immunoassays involving Staphylococcus aureus antigens

et al. 2015

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Staphylococcal protein A (SpA) is an immunoglobulin-binding protein secreted by all Staphylococcus aureus strains and is responsible for high false positives during immunoassays. Avian IgY were reported to eliminate SpA interference when used as a capture antibody in a double antibody sandwich format. But this procedure requires generating two antibodies in different animals for capture and revealing antibodies. In the present study, a simple enzymelinked immunosorbent assay (ELISA) was developed and evaluated for detection of staphylococcal enterotoxin B without any interference by SpA. Biotin-labeled IgY were used for probing for staphylococcal enterotoxin B (SEB) so that streptavidin-horseradish peroxidase (HRP) could be used for color development instead of anti-chicken IgG-HRP, which interferes in the assay by binding to SpA. Western blotting, dot ELISA, and polymerase chain reaction (PCR) were performed to validate the results of the biotin IgY ELISA to show that the assay was free from SpA interference. Large numbers of S. aureus strains were screened for SEB production. Sensitivity of the assay was~10 ng/ml of SEB in spiked milk samples. The ELISA was specific to SEB, with no crossreactivity to other closely related enterotoxins (SEA, SEC, SED, SEE). The presently described ELISA is highly effective in eliminating SpA interference and this method does not require the need for two different antibodies, as in the sandwich ELISA.

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“…The sequence of doses was 10, 10, and 5μg recombinant SEA [17]. Ent A were emulsified with an equal volume of freund's complete adjuvant and injected Intraperitoneal for the first and incomplete adjuvents for the second two subsequent intramuscular injections at weekly intervals.…”

Section: Production Of Polyclonal Antibodymentioning

confidence: 99%

Cloning, Sequencing and Production of Recombinant Polyclonal Antibodies against Egyptian Staphylococcal Enterotoxin A

El-Din¹,

el-naga²,

El-Fouly³

et al. 2017

AJMR

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Staphylococcus aureus (S.aureus) is the most common pathogen found in hospitals, community-acquired infections and colonizes up to 50% of humans, including mucous membranes and damaged skin. Some strains produce enterotoxins (Ent) as staphylococcal enterotoxin A (SEA) which is involved in 75% of food poisoning outbreaks. Few methods are sensitive and specific enough to confirm the diagnosis of staphylococcal food poisoning. In this study, a segment of Ent A-ORF with molecular weight 774 bp was amplified, cloned, sequenced and aligned with published Ent A-ORFs. Ent A-ORF was subcloned into bacterial expression vector (GST-PGEX4T1 vector) and expressed as a fusion protein with GST-tagged protein. A band size of 27 kDa of purified Ent A protein was cleaved from GST protein by thrombin. The expressed protein of Ent A was identified by strong reaction with commercialized polyclonal antibodies against Ent A. Antiserum against Egyptian recombinant Ent A protein was produced by immunization of Balb/c mice, the produced recombinant polyclonal antibodies had a titre of 1:2500 in direct ELISA. The produced recombinant polyclonal antibody was evaluated and reacted specifically in Western immunoblotting analysis and ELISA test. Finally from the obtained results, the produced recombinant protein of Ent A can be used successfully for production of recombinant polyclonal antibodies and used in large-scale detection of staphylococcal enterotoxin A.

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Monoclonal Antibody-Based Sandwich ELISA for the Detection of Staphylococcal Enterotoxin A

Cited by 70 publications

References 29 publications

Detection of β-Lactamase Residues in Milk by Sandwich ELISA

Detection of β-Lactamase Residues in Milk by Sandwich ELISA

Use of biotin-labeled IgY overcomes protein A interference in immunoassays involving Staphylococcus aureus antigens

Cloning, Sequencing and Production of Recombinant Polyclonal Antibodies against Egyptian Staphylococcal Enterotoxin A

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