Detection and quantification of Panton–Valentine leukocidin in Staphylococcus aureus cultures by ELISA and Western blotting: Diethylpyrocarbonate inhibits binding of protein A to IgG

Nguyen, Hien M.; Rocha, Miguel A.; Chintalacharuvu, Koteswara R.; Beenhouwer, David O.

doi:10.1016/j.jim.2010.03.005

Cited by 17 publications

(10 citation statements)

References 15 publications

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“…Equal amount of protein from each isolate was resolved on 12% acrylamide gels and transferred to nitrocellulose membrane using semidry transfer technique. Membrane was blocked using 5% skim milk powder and 5 mM DEPC in PBS pH 6.0 for 20 minutes at room temperature, washed thrice with PBS pH 7.4 containing 0.1% tween 20 (PBST), followed by overnight incubation at 4°C with 1∶10,000 dilution of rabbit anti SEA antibody (Sigma Aldrich, India) in PBS containing 0.05% tween 20 [45]. Membranes were washed thrice with PBST followed by incubation with 1∶20,000 dilution of Anti rabbit IgG-HRP conjugate (Sigma Aldrich) in PBS containing 0.05% tween 20.…”

Section: Methodsmentioning

confidence: 99%

Genome Sequencing Unveils a Novel Sea Enterotoxin-Carrying PVL Phage in Staphylococcus aureus ST772 from India

Prabhakara¹,

Khedkar

Shambat

et al. 2013

PLoS ONE

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Staphylococcus aureus is a major human pathogen, first recognized as a leading cause of hospital-acquired infections. Community-associated S. aureus (CA-SA) pose a greater threat due to increase in severity of infection and disease among children and healthy adults. CA-SA strains in India are genetically diverse, among which is the sequence type (ST) 772, which has now spread to Australia, Europe and Japan. Towards understanding the genetic characteristics of ST772, we obtained draft genome sequences of five relevant clinical isolates and studied the properties of their PVL-carrying prophages, whose presence is a defining hallmark of CA-SA. We show that this is a novel prophage, which carries the structural genes of the hlb-carrying prophage and includes the sea enterotoxin. This architecture probably emerged early within the ST772 lineage, at least in India. The sea gene, unique to ST772 PVL, despite having promoter sequence characteristics typical of low expression, appears to be highly expressed during early phase of growth in laboratory conditions. We speculate that this might be a consequence of its novel sequence context. The crippled nature of the hlb-converting prophage in ST772 suggests that widespread mobility of the sea enterotoxin might be a selective force behind its ‘transfer’ to the PVL prophage. Wild type ST772 strains induced strong proliferative responses as well as high cytotoxic activity against neutrophils, likely mediated by superantigen SEA and the PVL toxin respectively. Both proliferation and cytotoxicity were markedly reduced in a cured ST772 strain indicating the impact of the phage on virulence. The presence of SEA alongside the genes for the immune system-modulating PVL toxin may contribute to the success and virulence of ST772.

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Section: Methodsmentioning

confidence: 99%

Genome Sequencing Unveils a Novel Sea Enterotoxin-Carrying PVL Phage in Staphylococcus aureus ST772 from India

Prabhakara¹,

Khedkar

Shambat

et al. 2013

PLoS ONE

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“…However, this method requires antibodies from two animal sources. Several other methods were reported to reduce SpA interference in single antibody ELISAs by employing several modifications, such as incubation in diethylpyrocarbonate (DEPC; Nguyen et al 2010), biotinylation of rabbit IgG to mask the protein A binding site (Hahn et al 1986), or removal of protein A from the sample by immobilized IgG (Hjelm et al 1972). However, we have observed that most of these methods are not reliable, given the reduction in sensitivity by DEPC treatment and persistent cross-reaction of protein A with biotin-labeled IgG.…”

Section: Discussionmentioning

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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“…One important point to be considered is that Staphylococcus aureus produces protein A (6), which reacts with the Fc region of IgG, potentially causing false-positive results in immunoassay systems (18,20). We used the monoclonal antibodies to IgG1 and IgM for the present ICT.…”

Section: Discussionmentioning

confidence: 99%

“…The VOL. 18,2011 SIMPLE DETECTION OF PBP2Ј 249 chromatograph was developed for 10 min, and the reaction products at the test line and the control line were observed macroscopically. The cross-reactivity test was carried out using the strains used for indirect ELISA, except for the MRSA strains.…”

Section: Expression and Purification Of His-tagged Recombinant Pbp2 (mentioning

confidence: 99%

Development of an Immunochromatographic Strip for Simple Detection of Penicillin-Binding Protein 2′

Matsui

Hanaki

Inoue

et al. 2011

Clin Vaccine Immunol

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Infections with methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulasenegative Staphylococcus (MR-CNS) are a serious problem in hospitals because these bacteria produce penicillin-binding protein 2 (PBP2 or PBP2a), which shows low affinity to ␤-lactam antibiotics. Furthermore, the bacteria show resistance to a variety of antibiotics. Identification of these pathogens has been carried out mainly by the oxacillin susceptibility test, which takes several days to produce a reliable result. We developed a simple immunochromatographic test that enabled the detection of PBP2 within about 20 min. Anti-PBP2 monoclonal antibodies were produced by a hybridoma of recombinant PBP2 (rPBP2)-immunized mouse spleen cells and myeloma cells. The monoclonal antibodies reacted only with PBP2 of whole-cell extracts and showed no detectable cross-reactivity with extracts from other bacterial species tested so far. One of the monoclonal antibodies was conjugated with gold colloid particles, which react with PBP2, and another antibody was immobilized on a nitrocellulose membrane, which captures the PBP2-gold colloid particle complex on a nitrocellulose strip. This strip was able to detect 1.0 ng of rPBP2 or 2.8 ؋ 10 5 to 1.7 ؋ 10 7 CFU of MRSA cells. The cross-reactivity test using 15 bacterial species and a Candida albicans strain showed no detectable false-positive results. The accuracy of this method in the detection of MRSA and MR-CNS appeared to be 100%, compared with the results obtained by PCR amplification of the PBP2 gene, mecA. This newly developed immunochromatographic test can be used for simple and accurate detection of PBP2-producing cells in clinical laboratories.

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Detection and quantification of Panton–Valentine leukocidin in Staphylococcus aureus cultures by ELISA and Western blotting: Diethylpyrocarbonate inhibits binding of protein A to IgG

Cited by 17 publications

References 15 publications

Genome Sequencing Unveils a Novel Sea Enterotoxin-Carrying PVL Phage in Staphylococcus aureus ST772 from India

Genome Sequencing Unveils a Novel Sea Enterotoxin-Carrying PVL Phage in Staphylococcus aureus ST772 from India

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

Development of an Immunochromatographic Strip for Simple Detection of Penicillin-Binding Protein 2′

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