A modified immuno-polymerase chain reaction for the detection of b-glucuronidase from Escherichia coli

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题名:	A modified immuno-polymerase chain reaction for the detection of b-glucuronidase from Escherichia coli
作者:	Tsung C. Chang;Su H. Huang
贡献者:	Department of Biotechnology
关键词:	Immuno-PCR;β-glucuronidase;Escherichia coli
日期:	1997-10
上传时间:	2010-03-15 08:12:06 (UTC+0)
出版者:	Asia University
摘要:	A modified immuno-polymerase chain reaction (immuno-PCR) for the detection of E. coli β-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 1×10?17 g/ml (or 5×10?19 g/well); this is equivalent to two GUD molecules in a sample solution of 50 μl. 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.
關聯:	Journal of Immunological Methods 208(1):35-42
显示于类别:	[生物科技學系] 期刊論文

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