PMID: 9433458Jan 20, 1998Paper

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

Journal of Immunological Methods
T C Chang, S H Huang

Abstract

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.

Citations

Mar 23, 2005·Trends in Biotechnology·Christof M NiemeyerRon Wacker
Jul 20, 2000·Clinica Chimica Acta; International Journal of Clinical Chemistry·K SugawaraN Watanabe
Sep 13, 2003·Journal of Immunological Methods·Huining LiangSnezna Rogelj
Mar 27, 2007·Journal of Applied Microbiology·J R BranenA L Branen
Feb 27, 2014·Journal of Medical Microbiology·Promod K MehtaGopal K Khuller
May 22, 2008·The Analyst·Michael AdlerChristof M Niemeyer
Jan 12, 2007·Molecular BioSystems·Uwe R Müller
Aug 28, 2004·Clinical Chemistry·Su-Hua Huang, Tsung-Chain Chang
Jan 2, 2021·Analytica Chimica Acta·Mahdis Sadat TabatabaeiMarya Ahmed

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