Determination of metallothionein in biological fluids using enzyme-linked immunoassay with commercial antibody.

Abstract

Metallothionein (MT) is a low molecular weight cysteine-rich protein with a number of roles in the pro/antioxidant balance and homeostasis of essential metals, such as zinc and copper, and in the detoxification of heavy metals, such as cadmium and mercury. Until now, detection of metallothionein in biological fluids remained difficult because of a lack of a broadly reactive commercial test. Meaningful comparison of the values of metallothionein concentrations reported by different authors using their specific isolation procedures and different conditions of enzyme-linked immunoassay is difficult due to the absence of a reference material for metallothionein. Therefore in the present study, we describe a quantitative assay for metallothionein in biological fluids such as plasma and urine performed by a direct enzyme-linked immunoassay using a commercially available monoclonal mouse anti-metallothionein clone E9 antibody and commercial standards of metallothionein from rabbit liver and a custom preparation of metallothionein from human liver. The sensitivity of the assay for the standard containing two isoforms MT-I and MT-II from human liver was 140 pg/well. The reactivity of the commercial standards and standards containing two isoforms MT-I and MT-II isolated from human liver in our laboratory with a commercial monoclonal mouse anti-metallothionein clone E9 antibody were similar. This suggests that the described ELISA test can be useful for determination of metallothionein concentration in biological fluids. The concentrations of metallothionein in human plasma, erythrocyte lysate and in urine of smoking and non-smoking healthy volunteers are reported. Tobacco smoking increases the extracellular metallothionein concentration (plasma and urine) but does not affect the intracellular concentration (erythrocyte lysate).

Serum Metallothioneins in Childhood Tumours — A Potential Prognostic Marker

“…In the sample (mean age 27 years), MT level was 0.52 ± 0.2 μM [25]. Milnerowicz and Bizoń found similar results in healthy adult volunteers using enzyme-linked immunosorbent assay [26] and Singh and Hanson found lower MT levels in 50 normal children (aged 2–11 years) by immunoassay (approx. 2 mg/L) [27].…”

Stereoselectivity of the Hydrogen‐Atom Transfer in Benzophenone–Tyrosine Dyads: An Intramolecular Kinetic Solvent Effect

“…MT, containing two isoforms of MT I and MT II, was purified from human liver as described previously 11. The study protocol was approved by Local Bioethics Committee of Wroclaw Medical University (KB No: 165/1999).…”

“…The direct methods, including flow cytometry 9, radioimmunoassay 10, and enzyme‐linked immunosorbent assay 11, use specific antibodies directed against the MT molecules. These techniques are very specific but isoform‐specific antibodies are needed to differentiate isoforms 12.…”

Bioaccumulation patterns, element partitioning and biochemical performance of Venerupis corrugata from a low contaminated system

“…Concentration of proteins was calculated using Quantity One program software (Bio-Rad). After separation of proteins each band was cut, and the extraction was done according to Milnerowicz and Bizo n (2010). Confirmation of MTs was done through quantification of thiols groups, according to Moron et al (1979).…”