Limits...
Organization and ELISA-Based Results of the First Proficiency Testing to Evaluate the Ability of European Union Laboratories to Detect Staphylococcal Enterotoxin Type B (SEB) in Buffer and Milk

View Article: PubMed Central - PubMed

ABSTRACT

The aim of this work was to organize the first proficiency test (PT) dedicated to staphylococcal enterotoxin B (SEB) detection in milk and buffer solutions. This paper describes the organization of the PT trial according to EN ISO 17043 requirements. Characterization of the SEB stock solution was performed using SDS-PAGE and SE-specific ELISA, and amino acid analysis was used to assign its protein concentration. The solution was then used to prepare six PT materials (four milk and two buffer batches) at a ng/g toxin level, which included one blank and one SEA-containing milk as specificity control. Suitable material homogeneity and stability were assessed using screening and quantitative ELISAs. Among the methods used by the participants, ELISA-based methods demonstrated their efficiency for the detection of SEB in both simple and complex matrices. The results serve as a basis for further improving the detection capabilities in expert laboratories and can therefore be considered as a contribution to biopreparedness.

No MeSH data available.


SDS-PAGE and silver staining of the SEB solution used for spiking.1: SEB solution (Toxin Technology, Sarasota, FL, USA, Ref BT202 batch number: 91709B); 2: Precision Plus Protein all blue prestained proteins standards (Biorad, Marnes la coquette, France).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5037494&req=5

toxins-08-00268-f001: SDS-PAGE and silver staining of the SEB solution used for spiking.1: SEB solution (Toxin Technology, Sarasota, FL, USA, Ref BT202 batch number: 91709B); 2: Precision Plus Protein all blue prestained proteins standards (Biorad, Marnes la coquette, France).

Mentions: Due to the lack of an available reference material for SEB, there was a crucial need to characterize and verify the purity of the commercially obtained toxin before organizing the PT trial. In this paper, we demonstrated that the SEA and SEB toxins purchased from Toxin Technology (Sarasato, FL, USA) were sufficiently pure to be used for sample preparation. This was accomplished by performing SDS-PAGE with silver staining and SEA-SEE specific ELISAs (Figure 1). Results obtained highlighted that the SEB solution used was not as pure as expected considering the quality control certificate provided by the manufacturer but toxins SEA, SEC, and SED have not been quantified by quantitative ELISA method used. However, the results confirmed that the purity was sufficiently high to use LC-ID-MS/MS amino acid analysis (AAA) for establishing a protein quantity value from the SEB solution, and that this solution was fit-for-purpose to be used as spike for the preparation of suitable matrix PT materials.


Organization and ELISA-Based Results of the First Proficiency Testing to Evaluate the Ability of European Union Laboratories to Detect Staphylococcal Enterotoxin Type B (SEB) in Buffer and Milk
SDS-PAGE and silver staining of the SEB solution used for spiking.1: SEB solution (Toxin Technology, Sarasota, FL, USA, Ref BT202 batch number: 91709B); 2: Precision Plus Protein all blue prestained proteins standards (Biorad, Marnes la coquette, France).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037494&req=5

toxins-08-00268-f001: SDS-PAGE and silver staining of the SEB solution used for spiking.1: SEB solution (Toxin Technology, Sarasota, FL, USA, Ref BT202 batch number: 91709B); 2: Precision Plus Protein all blue prestained proteins standards (Biorad, Marnes la coquette, France).
Mentions: Due to the lack of an available reference material for SEB, there was a crucial need to characterize and verify the purity of the commercially obtained toxin before organizing the PT trial. In this paper, we demonstrated that the SEA and SEB toxins purchased from Toxin Technology (Sarasato, FL, USA) were sufficiently pure to be used for sample preparation. This was accomplished by performing SDS-PAGE with silver staining and SEA-SEE specific ELISAs (Figure 1). Results obtained highlighted that the SEB solution used was not as pure as expected considering the quality control certificate provided by the manufacturer but toxins SEA, SEC, and SED have not been quantified by quantitative ELISA method used. However, the results confirmed that the purity was sufficiently high to use LC-ID-MS/MS amino acid analysis (AAA) for establishing a protein quantity value from the SEB solution, and that this solution was fit-for-purpose to be used as spike for the preparation of suitable matrix PT materials.

View Article: PubMed Central - PubMed

ABSTRACT

The aim of this work was to organize the first proficiency test (PT) dedicated to staphylococcal enterotoxin B (SEB) detection in milk and buffer solutions. This paper describes the organization of the PT trial according to EN ISO 17043 requirements. Characterization of the SEB stock solution was performed using SDS-PAGE and SE-specific ELISA, and amino acid analysis was used to assign its protein concentration. The solution was then used to prepare six PT materials (four milk and two buffer batches) at a ng/g toxin level, which included one blank and one SEA-containing milk as specificity control. Suitable material homogeneity and stability were assessed using screening and quantitative ELISAs. Among the methods used by the participants, ELISA-based methods demonstrated their efficiency for the detection of SEB in both simple and complex matrices. The results serve as a basis for further improving the detection capabilities in expert laboratories and can therefore be considered as a contribution to biopreparedness.

No MeSH data available.