Chang T C, Huang S H
Department of Medical Technology, National Cheng Kung University Medical College, Tainan, Taiwan, ROC.
J Immunol Methods. 1997 Oct 13;208(1):35-42. doi: 10.1016/s0022-1759(97)00125-7.
A modified immuno-polymerase chain reaction (immuno-PCR) for the detection of E. coli beta-glucuronidase (GUD) is described. Flexible polycarbonate microtiter plates (Biozyme, Landgraaf) with 96 V-bottomed wells were used throughout all steps including the antigen-antibody reaction and polymerase chain reaction. The plates were coated with anti-GUD antibodies to capture the antigen, which was then detected using biotinylated anti-GUD antibodies. Following this, avidin was used to bridge the biotinylated antibodies and biotinylated lamda phage DNA, which was amplified by PCR to produce a product of 500 nucleotides. Following optimization, the detection limit of the immuno-PCR for GUD was 1 x 10(-17) g/ml (or 5 x 10(-19) g/well); this is equivalent to two GUD molecules in a sample solution of 50 microliters. The method was used to detect GUD in a cell extract of E. coli, and it was found that the enzyme released from a single E. coli cell in a solution of 10 l could be detected. So far, this is the most sensitive method ever published for the detection of an antigen. In addition to high sensitivity, the present protocol is capable of automation.
本文描述了一种用于检测大肠杆菌β-葡萄糖醛酸酶（GUD）的改良免疫聚合酶链反应（免疫PCR）。在包括抗原-抗体反应和聚合酶链反应的所有步骤中，均使用了带有96个V型底孔的柔性聚碳酸酯微量滴定板（Biozyme，兰德格拉夫）。这些板用抗GUD抗体包被以捕获抗原，然后使用生物素化的抗GUD抗体进行检测。在此之后，使用抗生物素蛋白桥接生物素化抗体和生物素化的λ噬菌体DNA，通过PCR扩增产生500个核苷酸的产物。经过优化，GUD免疫PCR的检测限为1×10^(-17) g/ml（或5×10^(-19) g/孔）；这相当于50微升样品溶液中的两个GUD分子。该方法用于检测大肠杆菌细胞提取物中的GUD，发现在10升溶液中单个大肠杆菌细胞释放的酶可以被检测到。到目前为止，这是已发表的检测抗原最灵敏的方法。除了高灵敏度外，本方案还能够实现自动化。
