Development of an Immunoassay for Detection of Staphylococcal Enterotoxin-Like J, A Non-Characterized Toxin

Abstract

Staphylococcal enterotoxins (SEs) are the cause of staphylococcal food poisoning (SFP) outbreaks. Recently, many new types of SEs and SE-like toxins have been reported, but it has not been proved whether these new toxins cause food poisoning. To develop an immunoassay for detection of SE-like J (SElJ), a non-characterized toxin in SFP, a mutant SElJ with C-terminus deletion (SElJ∆C) was expressed and purified in an E. coli expression system. Anti-SElJ antibody was produced in rabbits immunized with the SElJ∆C. Western blotting and sandwich enzyme-linked immunosorbent assay (ELISA) detection systems were established and showed that the antibody specifically recognizes SElJ without cross reaction to other SEs tested. The limit of detection for the sandwich ELISA was 0.078 ng/mL, showing high sensitivity. SElJ production in S. aureus was detected by using the sandwich ELISA and showed that selj-horboring isolates produced a large amount of SElJ in the culture supernatants, especially in that of the strain isolated from a food poisoning outbreak in Japan. These results demonstrate that the immunoassay for detection of SElJ is specific and sensitive and is useful for determining the native SElJ production in S. aureus isolated from food poisoning cases.

Quantitative Determination of Staphylococcus aureus Enterotoxins Types A to I and Variants in Dairy Food Products by Multiplex Immuno-LC-MS/MS

“…14 Immunoassays have high sensitivity for detection of SEs at trace levels, but on the downside, antibodies could be influenced by matrix effects in food samples, cross-reactions, and occurrence of sequence variants. 15 For these reasons, implementation of confirmatory methods based on a different technical approach is recommended. 13 As an alternative to immunoassays, bacterial identification approaches involving culture-based counting or polymerase chain reaction (PCR) highlight the presence of a pathogenic strain and SE-coding genes, but none is able to confirm food poisoning outbreaks since they do not provide direct evidence of the presence of toxins.…”

“…Immnunoassays are also commercially available for classical enterotoxins, and ELISA methods were recently reported for detection of novel SEs, e.g., SEG, SEH, or SEI . Immunoassays have high sensitivity for detection of SEs at trace levels, but on the downside, antibodies could be influenced by matrix effects in food samples, cross-reactions, and occurrence of sequence variants . For these reasons, implementation of confirmatory methods based on a different technical approach is recommended .…”

Highly Sensitive and Specific Detection of Staphylococcal Enterotoxins SEA, SEG, SEH, and SEI by Immunoassay

“…That is why a few in-house immunoanalytical methods have been developed and described in the literature to detect one or more of these toxins. They are mainly represented by sandwich ELISA using rabbit polyclonal antibodies, which are reported to detect SEH [ 31 , 32 ], SEG, SEH, and SEI [ 33 ] or SE l J [ 34 ]. The use of such polyclonal antibodies can be considered as a disadvantage for ELISA robustness due to the increased probability of cross-reaction with other SE(s) and even other unrelated proteins (false positives) and higher risk of batch-to-batch variability compared to monoclonal antibodies.…”

“…Finally, a multiplex prototype made of hydrogel immunobiochips has also been designed and reported to detect simultaneously seven enterotoxins, including SEG and SEI [ 38 ], with a demonstrated feasibility. Thanks to these in-house sandwich immunoassays, S. aureus isolates involved in SFP were shown to be able to produce in vitro these “new enterotoxins” such as SEH [ 31 , 32 , 33 ], low levels of SEI [ 33 , 37 ] and SEG [ 33 , 35 ], or even SE l J [ 34 ]. The involvement of SEH in food poisoning outbreaks has since been well established, as this toxin was directly detected in food products responsible for two SFP.…”