Development and usage of protein microarrays for the quantitative measurement of Panton-Valentine leukocidin

Stieber, Bettina; Monecke, Stefan; Müller, Elke; Baier, Vico; Coombs, Geoffrey W.; Ehricht, Ralf

doi:10.1016/j.mcp.2013.11.003

Cited by 12 publications

(16 citation statements)

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“…In previous experiments, different growth media were tested for strain culturing and detection of proteins [ 21 ],[ 25 ]. Based on these results, strains and isolates were incubated on Columbia Blood agar (Oxoid, Wesel, Germany) at 37°C for 18–24 h. One loop of bacterial material was inoculated into 130 μl phosphate buffered saline (1x PBS) or into 65 μl sodium hydroxide (NaOH; 100mM), respectively, and vortexed.…”

Section: Methodsmentioning

confidence: 99%

“…Monoclonal antibodies (AB) for the targets PBP2a, SAK, HLB, HLA, SEB, SEA, TSST and lukF-PV were generated via phage display [ 47 ] as previously described [ 44 ],[ 21 ],[ 25 ]. SPA antibodies originated from three polyclonal chicken sera (courtesy of Alere Scarborough/Binax).…”

Section: Methodsmentioning

confidence: 99%

“…First, all antibodies were screened to find the optimal ( i . e ., most specific and sensitive) combination of capture and detection antibodies for each target [ 44 ],[ 21 ],[ 25 ]. Then, all selected antibodies were tested in a mixture to find optimal conditions with regard to both, usage and functionality.…”

Section: Methodsmentioning

confidence: 99%

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Direct, Specific and Rapid Detection of Staphylococcal Proteins and Exotoxins Using a Multiplex Antibody Microarray

et al. 2015

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Background S. aureus is a pathogen in humans and animals that harbors a wide variety of virulence factors and resistance genes. This bacterium can cause a wide range of mild to life-threatening diseases. In the latter case, fast diagnostic procedures are important. In routine diagnostic laboratories, several genotypic and phenotypic methods are available to identify S. aureus strains and determine their resistances. However, there is a demand for multiplex routine diagnostic tests to directly detect staphylococcal toxins and proteins.MethodsIn this study, an antibody microarray based assay was established and validated for the rapid detection of staphylococcal markers and exotoxins. The following targets were included: staphylococcal protein A, penicillin binding protein 2a, alpha- and beta-hemolysins, Panton Valentine leukocidin, toxic shock syndrome toxin, enterotoxins A and B as well as staphylokinase. All were detected simultaneously within a single experiment, starting from a clonal culture on standard media. The detection of bound proteins was performed using a new fluorescence reading device for microarrays.Results110 reference strains and clinical isolates were analyzed using this assay, with a DNA microarray for genotypic characterization performed in parallel. The results showed a general high concordance of genotypic and phenotypic data. However, genotypic analysis found the hla gene present in all S. aureus isolates but its expression under given conditions depended on the clonal complex affiliation of the actual isolate.ConclusionsThe multiplex antibody assay described herein allowed a rapid and reliable detection of clinically relevant staphylococcal toxins as well as resistance- and species-specific markers.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Direct, Specific and Rapid Detection of Staphylococcal Proteins and Exotoxins Using a Multiplex Antibody Microarray

et al. 2015

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“…The hybridization signals were processed using the IconoClust software version 3.2r1. All spots were recognized and subsequently normalized automatically by the software according to the equation NI ϭ 1 Ϫ (M/BG), where NI is the normalized intensity, M is the average intensity of the automatically recognized spot, and BG is the intensity of the local background (21)(22)(23). The output range of the signals was from 0 to 1, with 0 being negative and 1 being the maximum possible signal value.…”

Section: Methodsmentioning

confidence: 99%

Development of a Rapid Microarray-Based DNA Subtyping Assay for the Alleles of Shiga Toxins 1 and 2 of Escherichia coli

et al. 2014

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In this study, we developed a new rapid, economic, and automated microarray-based genotyping test for the standardized subtyping of Shiga toxins 1 and 2 of Escherichia coli. The microarrays from Alere Technologies can be used in two different formats, the ArrayTube and the ArrayStrip (which enables high-throughput testing in a 96-well format). One microarray chip harbors all the gene sequences necessary to distinguish between all Stx subtypes, facilitating the identification of single and multiple subtypes within a single isolate in one experiment. Specific software was developed to automatically analyze all data obtained from the microarray. The assay was validated with 21 Shiga toxin-producing E. coli (STEC) reference strains that were previously tested by the complete set of conventional subtyping PCRs. The microarray results showed 100% concordance with the PCR results. Essentially identical results were detected when the standard DNA extraction method was replaced by a time-saving heat lysis protocol. For further validation of the microarray, we identified the Stx subtypes or combinations of the subtypes in 446 STEC field isolates of human and animal origin. In summary, this oligonucleotide array represents an excellent diagnostic tool that provides some advantages over standard PCR-based subtyping. The number of the spotted probes on the microarrays can be increased by additional probes, such as for novel alleles, species markers, or resistance genes, should the need arise.

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“…This device measures the intensity of staining from every spot on the microarray with a value between 0 (no signal, white spot) and 1 (maximum signal, black spot) as previously described [5]. Signal intensities between 0.1 and 0.7 are within the dynamic range of the test, a value below 0.1 cannot be assumed to show a correct antigenantibody binding and a value above 0.7 indicates a color saturation of the spot [11]. Disruptive factors such as protein residues, scratches, dust or lint that are visible on the image, can result in an invalid measurement of one or several spots by the Iconoclust software (Abbot (Alere Technologies GmbH)) on the reading device.…”

Section: Microarray Test Proceduresmentioning

confidence: 99%

Use of meat juice and blood serum with a miniaturised protein microarray assay to develop a multi-parameter IgG screening test with high sample throughput potential for slaughtering pigs

et al. 2020

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Background: Serological screening of pig herds at the abattoir is considered a potential tool to improve meat inspection procedures and herd health management. Therefore, we previously reported the feasibility of a miniaturised protein microarray as a new serological IgG screening test for zoonotic agents and production diseases in pigs. The present study investigates whether the protein microarray-based assay is applicable for high sample throughput using either blood serum or meat juice.Material and methods: Microarrays with 12 different antigens were produced by Abbott (formerly Alere Technologies GmbH) Jena, Germany in a previously offered 'ArrayTube' platform and in an 'ArrayStrip' platform for large-scale use. A test protocol for the use of meat juice on both microarray platforms was developed. Agreement between serum and meat juice was analysed with 88 paired samples from three German abattoirs. Serum was diluted 1:50 and meat juice 1:2. ELISA results for all tested antigens from a preceding study were used as reference test to perform Receiver Operating Characteristic analysis for both test specimens on both microarray platforms.Results: High area under curve values (AUC > 0.7) were calculated for the analysis of T. gondii (0.87), Y. enterocolitica (0.97), Mycoplasma hyopneumoniae (0.84) and Actinobacillus pleuropneumoniae (0.71) with serum as the test specimen and for T. gondii (0.99), Y. enterocolitica (0.94), PRRSV (0.88), A. pleuropneumoniae (0.78) and Salmonella spp. (0.72) with meat juice as the test specimen on the ArrayStrip platform. Cohens kappa values of 0.92 for T. gondii and 0.82 for Y. enterocolitica were obtained for the comparison between serum and meat juice. When applying the new method in two further laboratories, kappa values between 0.63 and 0.94 were achieved between the laboratories for these two pathogens.(Continued on next page) Conclusion: Further development of a miniaturised pig-specific IgG protein microarray assay showed that meat juice can be used on microarray platforms. Two out of twelve tested antigens (T. gondii, Y. enterocolitica) showed high test accuracy on the ArrayTube and the ArrayStrip platform with both sample materials.

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Development and usage of protein microarrays for the quantitative measurement of Panton-Valentine leukocidin

Cited by 12 publications

References 44 publications

Direct, Specific and Rapid Detection of Staphylococcal Proteins and Exotoxins Using a Multiplex Antibody Microarray

Direct, Specific and Rapid Detection of Staphylococcal Proteins and Exotoxins Using a Multiplex Antibody Microarray

Development of a Rapid Microarray-Based DNA Subtyping Assay for the Alleles of Shiga Toxins 1 and 2 of Escherichia coli

Use of meat juice and blood serum with a miniaturised protein microarray assay to develop a multi-parameter IgG screening test with high sample throughput potential for slaughtering pigs

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