金黄色葡萄球菌肠毒素P双抗夹心酶联免疫检测方法的建立

Development of a Double-Antibody Sandwich Enzyme Linked Immunosorbent Assay for Detection of Staphylococcal Enterotoxin P

摘要:

本研究目的是建立一种快速检测金黄色葡萄球菌新型肠毒素SEP的双抗夹心酶联免疫方法。通过对单克隆抗体和抗血清质量浓度、辣根过氧化物酶标记的羊抗兔IgG（IgG/HRP）最佳稀释度、不同种类包被缓冲液、封闭时间、抗原包被时间、IgG/HRP作用时间以及四甲基联苯胺（TMB）显色时间等条件进行优化；并采用灵敏度、批内和批间变异和加标回收率等对方法进行评价。结果表明：该方法中抗SEP单克隆抗体的最佳稀释质量浓度为3.28 μg/mL，抗SEI兔血清的质量浓度为3.14 μg/mL，酶标二抗稀释度1:3000，1×磷酸缓冲盐溶液（pH 7.4）为最佳包被缓冲液，封闭时间为1 h，抗原孵育时间为90 min，酶标二抗反应时间为30 min，TMB显色时间15 min。该方法回归方程为y=0.0313x+0.0262，R2=0.9946。灵敏度为1.80 μg/mL，精密度批内变异低于6%，批间变异低于15%，对LB肉汤、牛肉糜和熟米饭回收率达90%以上。

Abstract:

A double-antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) method was developed for detection of a new staphylococcal enterotoxin P (SEP). The experimental conditions were evaluated through the index of sensitivity, intra-assay variation, inter-assay variation and spike recovery by optimizing the concentration of monoclonal antibody and antiserum, the horseradish peroxidase (HRP) labeled goat anti-rabbit IgG (IgG/HRP), different types of buffer, blocking time, antigen incubation time, IgG / HRP incubation time, and TMB chromogenic time. Our results were as follows: the optimal concentration of anti-SEP monoclonal antibody 3.28 μg/mL, the concentration of anti-SEI rabbit serum 3.14 μg/mL, the dilution of enzyme-labeled secondary antibody 1: 3000,the blocking time 1 h, the antigen incubation time 90 min, the secondary antibody incubation time 30 min and the TMB chromogenic time 15 min. And 1 × phosphate buffer solution (pH 7.4) was shown as the best buffer. The regression equation of this method was y=0.0313x+0.0262 with R2of 0.9946. The sensitivity of the developed method was 1.80 μg/mL. The precision within the batch variation was less than 6 %, and inter-assay variation was less than 15 %. The recovery rates for LB broth, beef and cooked rice were more than 90%. Thus, this study successfully established a DAS-ELISA method for rapid detection of newly identified staphylococcal enterotoxin P.