title: A plasmid containing the human metallothionein II gene can function as an antibody-assisted electrophoretic biosensor for heavy metals.
abstract: Different forms of heavy metals affect biochemical systems in characteristic ways that cannot be detected with typical metal analysis methods like atomic absorption spectrometry. Further, using living systems to analyze interaction of heavy metals with biochemical systems can be laborious and unreliable. To generate a reliable easy-to-use biologically-based biosensor system, the entire human metallothionein-II (MT-II) gene was incorporated into a plasmid (pUC57-MT) easily replicated in Escherichia coli. In this system, a commercial polyclonal antibody raised against human metal-responsive transcription factor-1 protein (MTF-1 protein) could modify the electrophoretic migration patterns (i.e. cause specific decreases in agarose gel electrophoretic mobility) of the plasmid in the presence or absence of heavy metals other than zinc (Zn). In the study here, heavy metals, MTF-1 protein, and polyclonal anti-MTF-1 antibody were used to assess pUC57-MT plasmid antibody-assisted electrophoretic mobility. Anti-MTF-1 antibody bound both MTF-1 protein and pUC57-MT plasmid in a non-competitive fashion such that it could be used to differentiate specific heavy metal binding. The results showed that antibody-inhibited plasmid migration was heavy metal level-dependent. Zinc caused a unique mobility shift pattern opposite to that of other metals tested, i.e. Zn blocked the antibody ability to inhibit plasmid migration, despite a greatly increased affinity for DNA by the antibody when Zn was present. The Zn effect was reversed/modified by adding MTF-1 protein. Additionally, antibody inhibition of plasmid mobility was resistant to heat pre-treatment and trypsinization, indicating absence of residual DNA extraction-resistant bacterial DNA binding proteins. DNA binding by anti-DNA antibodies may be commonly enhanced by xenobiotic heavy metals and elevated levels of Zn, thus making them potentially effective tools for assessment of heavy metal bioavailability in aqueous solutions and fluid obtained from metal implant sites.
authors.affiliation: a United States Air Force School of Aerospace Medicine, Advanced Technology and Genetics Center, Wright-Patterson Air Force Base , OH , USA.
authors.forename: Dennis C
authors.initials: DC
authors.lastname: Wooten
authors.affiliation: a United States Air Force School of Aerospace Medicine, Advanced Technology and Genetics Center, Wright-Patterson Air Force Base , OH , USA.
authors.forename: Clarise R
authors.initials: CR
authors.lastname: Starr
authors.affiliation: a United States Air Force School of Aerospace Medicine, Advanced Technology and Genetics Center, Wright-Patterson Air Force Base , OH , USA.
authors.forename: Wanda J
authors.initials: WJ
authors.lastname: Lyon
mesh_terms: D000906:Antibodies / Q000378:metabolism*; D015374:Biosensing Techniques / Q000706:statistics & numerical data*; D004268:DNA-Binding Proteins / Q000276:immunology; D017266:Dental Prosthesis / Q000009:adverse effects; D054750:Ecotoxicology; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics*; D005818:Genetic Engineering; D006801:Humans; D008668:Metallothionein / Q000235:genetics*; D019216:Metals, Heavy / Q000009:adverse effects; D010957:Plasmids / Q000235:genetics*; D014157:Transcription Factors / Q000276:immunology; D000097809:Transcription Factor MTF-1
chemical_list: D000906:Antibodies; D004268:DNA-Binding Proteins; D008668:Metallothionein; D019216:Metals, Heavy; D014157:Transcription Factors; D000097809:Transcription Factor MTF-1
keywords: Anti-DNA; MRE; MTF-1; anti-MTF-1; metal; metallothionein; plasmid; xenobiotic