Development of an Indirect Competitive Enzyme-Linked Immunosorbent Assay Based on the Multiepitope Peptide for the Synchronous Detection of Staphylococcal Enterotoxin A and G Proteins in Milk

Abstract

Staphylococcal food poisoning (SFP), one of the most common foodborne diseases, results from ingestion of staphylococcal enterotoxins (SEs) in foods. In our previous studies, we found that SEA and SEG were two predominant SE proteins produced by milkacquired S. aureus isolates. Here, a tandemly arranged multiepitope peptide (named SEAGepis) was designed with six linear B-cell epitopes derived from SEA or SEG and was heterologously expressed. The SEAGepis-specific antibody was prepared by immunizing rabbit with rSEAGepis. Then, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on rSEAGepis and the corresponding antibody was developed to simultaneously detect SEA and SEG. Under the optimized conditions, the ic-ELISA standard curve for rSEAGepis was constructed in the concentration range of 0.5 to 512 ng/ml, and the average coefficients of variation of intra- and interassay were 4.28 and 5.61% during six standard concentrations. The average half-maximal inhibitory concentration was 5.07 ng/ml, and the limit of detection at a signal-to-noise ratio of 3 was 0.52 ng/ml. The anti-rSEAGepis antibody displayed over 90% cross-reactivity with SEA and SEG but less than 0.5% cross-reactivity with other enterotoxins. Artificially contaminated milk with different concentrations of rSEAGepis, SEA, and SEG was detected by the established ic-ELISA; the recoveries of rSEAGepis, SEA, and SEG were 91.1 to 157.5%, 90.3 to 134.5%, and 89.1 to 117.5%, respectively, with a coefficient of variation below 12%. These results demonstrated that the newly established ic-ELISA possessed high sensitivity, specificity, stability, and accuracy and could potentially be a useful analytical method for synchronous detection of SEA and SEG in milk.

Basis of Virulence in Enterotoxin-Mediated Staphylococcal Food Poisoning

“…Although few studies have examined the physical presence of multiple enterotoxins, it is most likely that multiple SEs contribute to SFP. The expansion of existing multiplex assays (Liang et al, 2015 ; Adhikari et al, 2016 ) would be the most efficient strategy to detect all SEs simultaneously. However, each platform has its advantages and disadvantages (Table 2 ; Wu et al, 2016 for review).…”

Staphylococcus aureus enterotoxin in food of animal origin and staphylococcal food poisoning risk assessment from farm to table

“…Some authors have suggested that the expansion of the existing multiple assays would be the most efficient strategy to detect all SEs. However, some concerns regarding the high risk of cross-reaction have been raised and vigorous testing is required to develop this idea (Liang et al 2015;Adhikari et al 2016;Nia et al 2016).…”

Gaps in the assortment of rapid assays for microorganisms of interest to the dairy industry

“…If enumeration is required and, thus, enrichment cannot be carried out, or where extra matrix cleanup measures in addition to dilution by enrichment are required, physical and chemical matrix clearance methods have been tested for removal and reduction of particulate material, fat and protein. For many platforms such as PCR, isothermal amplification, ELISA, FCM and lateral flow, strategies to remove these components include filtration to remove large particulate material, centrifugation to separate and remove the fat component, treatment with detergents such as SDS or Triton X-100 to solubilize lipids, treatments with EDTA or citrate to solubilize casein micelles, and treatment with proteases such as proteinase K or savinase to digest the protein component ( Bosward, House, Deveridge, Mathews, & Sheehy, 2016 ; Chiao et al, 2013 ; Gunasekera et al, 2000 ; Kumar & Kumar Mondal, 2015 ; Liang et al, 2015 ; Liu et al, 2019 ; Paul et al, 2013 ; Soejima et al, 2012 ). To use bioluminescence-based assays in milk samples, pretreatment of the milk may require the lysing of somatic cells and degradation of the somatic cell ATP, prior to extraction of the bacterial ATP for measurement of microbial contamination without interference from somatic cells.…”