金黄色葡萄球菌新型肠毒素I双抗夹心-酶联免疫检测方法的建立

目的：建立简便、灵敏检测金黄色葡萄球菌肠毒素I（staphylococcal enterotoxin I，SEI）的双抗夹心酶联免疫吸附方法（double-antibody sandwich-enzyme linked immunosorbent assay，DAS-ELISA）。方法：利用DAS-ELISA检测程序确定单克隆抗体、抗血清、辣根过氧化物酶（horseradish peroxidase，HRP）标记的羊抗兔IgG（IgG/HRP）的最佳稀释度，再通过检测不同包被缓冲液、封闭时间、抗原包被时间、IgG/HRP作用时间以及四甲基联苯胺（3,3’,5,5’-tetramethylbenzidine，TMB）显色时间条件下的OD450 nm值对实验条件进行优化，最后用灵敏度、批内变异、批间变异和加标回收率指标对方法进行评价。结果：抗SEI单克隆抗体的最佳稀释质量浓度为2.89 mg/L，抗SEI兔血清稀释度1∶2 000，酶标二抗稀释度1∶6 000；1×磷酸缓冲盐溶液（pH 7.4）为最佳包被缓冲液；最佳封闭时间、抗原孵育时间、酶标二抗孵育时间和TMB显色时间分别为60、90、30 min和15 min。该方法的回归方程为y=0.040 9x＋0.042 9，R2=0.993 3；灵敏度为0.5 μg/L，精密度批内变异低于10%，批间变异低于15%，除巴氏杀菌牛乳外，对生理盐水、熟牦牛肉糜、大米饭和超高温瞬时灭菌牛乳回收率达90%以上。结论：本研究建立了一种快速检测SEI的双抗夹心方法。

Development of a Double-Antibody Sandwich Enzyme Linked Immunosorbent Assay for Detection of Staphylococcal Enterotoxin I (SEI)

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).