A quantitative real-time immuno-PCR approach for detection of staphylococcal enterotoxins
A quantitative real-time immuno-PCR approach for detection of staphylococcal enterotoxins
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DOI：
10.1007/s00109-006-0142-5
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发表时间：
2007-05-01
影响因子：
4.7
通讯作者：
Becker, Karsten
中科院分区：
文献类型：
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作者：
Fischer, Andreas;von Eiff, Christof;Becker, Karsten
细菌中毒是一个重大的公共卫生问题，金黄色葡萄球菌产生的肠毒素是食物中毒的最常见原因之一。葡萄球菌肠毒素作为致热毒素超抗原家族的成员，除了在食物中毒的致病性中发挥作用外，还对免疫系统产生深远的影响。由于经典的诊断生物测定以及常规使用的免疫学方法受到关于灵敏度、特异性和实用性的若干缺点的阻碍，因此需要通过高度灵敏和特异的方法来及时鉴定毒素。为了将酶免疫测定（EIA）的通用性与PCR的扩增能力联合收割机结合，开发了用于检测葡萄球菌肠毒素A和B的定量实时免疫PCR（qRT-iPCR），并与市售EIA进行比较。通过报告DNA与二次检测抗体的共价结合建立了用于预形成的免疫复合物的信号放大的广泛适用的工具。因此，氨基修饰的报告DNA成功偶联到N-琥珀酰亚胺基-S-乙酰基-硫代乙酸酯活化的二次检测抗体。qRT-iPCR能够高度重现地分别检测低至约0.6至6 pg（4至40阿莫尔/μ l）的葡萄球菌肠毒素B和葡萄球菌肠毒素A。总之，qRT-iPCR方法明显克服了细菌毒素的传统免疫学检测方法的灵敏度限制，如本研究中葡萄球菌肠毒素所示。提供通用信号放大的稳定抗体-DNA缀合物的开发提供了通用的以及高度灵敏和特异的工具，用于诊断和研究目的，通常适用于预形成的抗体-抗原复合物。
Bacterial intoxications represent a substantial public health concern with enterotoxins produced by Staphylococcus aureus among the most common causes of food poisoning. In addition to their role in the pathogenicity of food poisoning, staphylococcal enterotoxins have profound effects on the immune system as members of the family of pyrogenic toxin superantigens. As the classical diagnostic bioassays as well as the routinely used immunological methods are hampered by several drawbacks regarding sensitivity, specificity, and practicability, there is a need for the timely identification of toxins by highly sensitive and specific methods. To combine the versatility of an enzyme immunoassay (EIA) with the amplification power of the PCR, a quantitative real-time immuno-PCR (qRT-iPCR) was developed for the detection of staphylococcal enterotoxins A and B and compared to a commercially available EIA. A broadly applicable tool for signal amplification of pre-formed immunocomplexes was established by covalent binding of a reporter DNA to secondary detection antibodies. Therefore, the amino-modified reporter DNA was coupled successfully to N-succinimidyl-S-actyl-thioacetate-activated secondary detection antibodies. The qRT-iPCR was able to detect highly reproducibly as low as approximately 0.6 to 6 pg (4 to 40 amol/mu l) of staphylococcal enterotoxin B and staphylococcal enterotoxin A, respectively. In conclusion, the qRT-iPCR approach was shown to overcome clearly the sensitivity limit of traditional immunological detection procedures for bacterial toxins, as demonstrated in this study for staphylococcal enterotoxins. The development of a stable antibody-DNA conjugate providing a universal signal amplification offers a versatile as well as a highly sensitive and specific tool for diagnostic and research purposes generally applicable for pre-formed antibody-antigen complexes.