Mudili Venkataramana, Makam Shivakiran S, Sundararaj Naveen, Siddaiah Chandranayaka, Gupta Vijai Kumar, Rao Putcha V Lakshmana
DRDO-BU-CLS, Bharathiar University Campus, Coimbatore, Tamil Nadu- 641046, India.
DOS in Biotechnology, University of Mysore, Manasagangotri, Mysore, India.
Sci Rep. 2015 Oct 19;5:15151. doi: 10.1038/srep15151.
In the present study, we introduce a novel hybrid sandwich-ALISA employing chicken IgY and ssDNA aptamers for the detection of staphylococcal enterotoxin B (SEB). Cloning, expression and purification of the full length recombinant SEB was carried out. Anti-SEB IgY antibodies generated by immunizing white leg-horn chickens with purified recombinant SEB protein and were purified from the immunized egg yolk. Simultaneously, ssDNA aptamers specific to the toxin were prepared by SELEX method on microtiter well plates. The sensitivity levels of both probe molecules i.e., IgY and ssDNA aptamers were evaluated. We observed that the aptamer at 250 ngmL(-1) concentration could detect the target antigen at 50 ngmL(-1) and the IgY antibodies at 250 ngmL(-1), could able to detect 100 ngmL(-1) antigen. We further combined both the probes to prepare a hybrid sandwich aptamer linked immune sorbent assay (ALISA) wherein the IgY as capturing molecule and biotinylated aptamer as revealing probe. Limit of detection (LOD) for the developed method was determined as 50 ngmL(-1). Further, developed method was evaluated with artificially SEB spiked milk and natural samples and obtained results were validated with PCR. In conclusion, developed ALISA method may provide cost-effective and robust detection of SEB from food and environmental samples.
在本研究中，我们介绍了一种新型的混合夹心酶联免疫吸附测定法（ALISA），该方法采用鸡抗体重链（IgY）和单链DNA适配体来检测葡萄球菌肠毒素B（SEB）。我们进行了全长重组SEB的克隆、表达和纯化。用纯化的重组SEB蛋白免疫白来航鸡产生抗SEB IgY抗体，并从免疫蛋黄中纯化该抗体。同时，通过在微量滴定板上的指数富集配体系统进化（SELEX）方法制备了对该毒素具有特异性的单链DNA适配体。评估了两种探针分子即IgY和单链DNA适配体的灵敏度水平。我们观察到，浓度为250 ng/mL的适配体能够检测浓度为50 ng/mL的靶抗原，而浓度为250 ng/mL的IgY抗体能够检测100 ng/mL的抗原。我们进一步将两种探针结合起来，制备了一种杂交夹心适配体连接免疫吸附测定法（ALISA），其中IgY作为捕获分子，生物素化适配体作为检测探针。所开发方法的检测限（LOD）确定为50 ng/mL。此外，用人工添加SEB的牛奶和天然样品对所开发的方法进行了评估，并用聚合酶链反应（PCR）对获得的结果进行了验证。总之，所开发的ALISA方法可为从食品和环境样品中经济高效且可靠地检测SEB提供方法。
