title: 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.
authors.affiliation: College of Animal Science and Technology, Anhui Agricultural University, 130 Changjiang Avenue, Hefei 230036, Anhui, People's Republic of China.
authors.forename: Mingyan
authors.initials: M
authors.lastname: Liang
authors.affiliation: College of Animal Science and Technology, Anhui Agricultural University, 130 Changjiang Avenue, Hefei 230036, Anhui, People's Republic of China.
authors.forename: Tingting
authors.initials: T
authors.lastname: Zhang
authors.affiliation: College of Animal Science and Technology, Anhui Agricultural University, 130 Changjiang Avenue, Hefei 230036, Anhui, People's Republic of China.
authors.forename: Xuelan
authors.initials: X
authors.lastname: Liu
authors.affiliation: College of Animal Science and Technology, Anhui Agricultural University, 130 Changjiang Avenue, Hefei 230036, Anhui, People's Republic of China.
authors.forename: Yanan
authors.initials: Y
authors.lastname: Fan
authors.affiliation: Center of Animal Epidemic Disease Prevention and Control, 460 Liming Avenue, Wuhu 241000, Anhui, People's Republic of China.
authors.forename: Shenglin
authors.initials: S
authors.lastname: Xia
authors.affiliation: College of Animal Science and Technology, Anhui Agricultural University, 130 Changjiang Avenue, Hefei 230036, Anhui, People's Republic of China.
authors.forename: Yiqing
authors.initials: Y
authors.lastname: Xiang
authors.affiliation: College of Animal Science and Technology, Anhui Agricultural University, 130 Changjiang Avenue, Hefei 230036, Anhui, People's Republic of China.
authors.forename: Ziqi
authors.initials: Z
authors.lastname: Liu
authors.affiliation: College of Animal Science and Technology, Anhui Agricultural University, 130 Changjiang Avenue, Hefei 230036, Anhui, People's Republic of China. lijinnian2000@163.com.
authors.forename: Li
authors.initials: L
authors.lastname: Jinnian
mesh_terms: D000818:Animals; D060326:Chemistry Techniques, Synthetic / Q000379:methods; D003429:Cross Reactions; D003611:Dairy Products / Q000382:microbiology; D004768:Enterotoxins / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes / Q000032:analysis*; D005516:Food Microbiology / Q000379:methods; D008892:Milk / Q000737:chemistry* / Q000382:microbiology; D010455:Peptides / Q000032:analysis*; D011817:Rabbits; D012680:Sensitivity and Specificity; D059629:Signal-To-Noise Ratio; D013202:Staphylococcal Food Poisoning; D013211:Staphylococcus aureus / Q000737:chemistry; D018089:Superantigens / Q000302:isolation & purification*
chemical_list: D004768:Enterotoxins; D000939:Epitopes; D010455:Peptides; D018089:Superantigens; C419397:enterotoxin G, staphylococcal; C026066:enterotoxin A, Staphylococcal