title: Expression, purification of metallothionein genes from freshwater crab (Sinopotamon yangtsekiense) and development of an anti-metallothionein ELISA.
abstract: Using the phoA-fusion technology, the recombinant metallothionein (MT) from freshwater crab (Sinopotamon yangtsekiense) has been successfully produced in Escherichia coli. MT purified from the bacterial suspension showed one polypeptide with a molecular weight of 7 kDa by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE). Western-blotting confirmed the polypeptides had a specific reactivity with mouse polyclonal MT anti-serum. Based on the purified MT and MT anti-serum, the reaction parameters for an enzyme-linked immunosorbent assay (ELISA) were developed. The direct coating ELISA showed a higher linear relationship compared to antibody sandwich coating ELISA. The optimal dilution rates of purified MT anti-serum and coating period were shown to be 1:160,000 and 12 hours at 4°C. At 37°C, the appropriate reaction duration of the first antibody and the second antibody were 2 hours and 1 hour, respectively. According to these optimal parameters, the standard linear equation, y = 0.0032x + 0.1769 (R2 = 0.9779, x, y representing MT concentration and OD450 value), was established for the determination of MT concentration with a valid range of 3.9-500 ng/ml. In verification experiments, the mean coefficients of variation of the intra-assay and inter-assay were 3.260% and 3.736%, respectively. According to the result of MT recovery, ELISA with an approaching 100% MT recovery was more reliable and sensitive than the Cd saturation assay. In conclusion, the newly developed ELISA of this study was precise, stable and repeatable, and could be used as a biomarker tool to monitor pollution by heavy metals.
authors.affiliation: Translational Medicine Research Center, Shanxi Medical University, Taiyuan, Shanxi Province, People's Republic of China.
authors.forename: Jian
authors.initials: J
authors.lastname: Yang
authors.affiliation: Institute of Science & Technology of Shanxi, Taiyuan, Shanxi Province, People's Republic of China.
authors.forename: Hui
authors.initials: H
authors.lastname: Sun
authors.affiliation: Translational Medicine Research Center, Shanxi Medical University, Taiyuan, Shanxi Province, People's Republic of China.
authors.forename: Hao
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Translational Medicine Research Center, Shanxi Medical University, Taiyuan, Shanxi Province, People's Republic of China.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhou
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology; D060829:Arthropod Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D003386:Brachyura / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D005618:Fresh Water; D015870:Gene Expression; D043143:Hepatopancreas / Q000378:metabolism; D007106:Immune Sera / Q000276:immunology; D008668:Metallothionein / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism*; D015203:Reproducibility of Results
chemical_list: D000906:Antibodies; D060829:Arthropod Proteins; D007106:Immune Sera; D011994:Recombinant Proteins; D008668:Metallothionein