Abstract:

Objective: To establish a simple and sensitive double-antibody sandwich enzyme linked immunosorbent
assay (DAS-ELISA) for the detection of a newly identified Staphylococcal aureus enterotoxin, SEI. Methods: Different
combinations of coating antibody, polyclonal antibody and goat anti-rabbit IgG/HRP were tested through square matrix
titration. The experimental conditions were optimized such as buffer, blocking time, antigen incubation time, goat antirabbit
IgG/HRP incubation time, and chromogenic time of 3,3’,5,5’-tetramethylbenzidine (TMB). Further, the developed
method was analyzed and evaluated by sensitivity, intra/inter-batch coefficients of variation and recovery of spiked samples.
Results: The optimum experimental conditions were determined as follows: anti-SEI monoclonal antibody concentration,
2.89 mg/L; dilution ratio of polyclonal antibody, 1:2 000; and dilution ratio of goat anti-rabbit IgG/HRP, 1:6 000,
respectively. Moreover, 1 × PBS (pH 7.4) buffer solution was the optimal coating buffer, and the optimal blocking time,
antigen incubation time, goat anti-rabbit IgG/HRP incubation time, and TMB chromogenic time were 60, 90, 30, and
15 min, respectively. The equation between SEI concentration and optical density at 450 nm (OD450 nm) was fitted as follows:
y = 0.040 9 x + 0.042 9 (R2 = 0.993 3). The sensitivity of the developed method was 0.5 μg/L, with intra-batch coefficient of
variation < 10% and inter-batch variation < 15%. The recoveries for spiked saline, minced yak meat, steamed rice and UHT
milk were all above 90%, except for pasteurized milk. Conclusion: This study has established a DAS-ELISA method for
detecting newly identified staphylococcal enterotoxin I (SEI).

Key words: DAS-ELISA, Staphylococcus aureus, staphylococcal enterotoxin I (SEI), detection method