Enzyme-immunoassay for the determination of metallothionein in human urine: application to environmental monitoring

Abstract

The objectives of this study were to develop an enzyme immunoassay for metallothioneins in human urine using a polyclonal antiserum and to demonstrate a possible relationship between the level of this biomarker and heavy metal exposure. The antiserum was raised in sheep against horse metallothionein conjugated to carboxylated bovine serum albumin. The antibody was used to construct a two-step competitive ELISA procedure. Human urine was treated with activated charcoal powder to remove traces of metallothioneins and known amounts of pure metallothioneins were added to provide standards for a standard curve. Metallothionein levels were measured in two groups of children living in areas of mild and high environmental pollution due mainly to heavy metals. A comparison was made between the biomarker levels and the levels of cadmium and lead in urine samples in the two groups. A group of children from a non-polluted area acted as controls. The results show that the detected levels of metallothioneins appear to correspond to levels of the two heavy metals studied and that there was an apparent relationship to the environmental exposure. Thus according to results of this study the increase in the metallothionein excretion seems to provide an indication of previous of exposure to metals. The ELISA procedure is sensitive and robust and can be used to screen large numbers of samples and is more rapid than the physical procedures currently used for analysis of these proteins. The assay can therefore be used as an additional tool for screening at-risk populations where either environmental or occupational exposure to divalent heavy metals is suspected.

Analytical Methods for Metallothionein Detection

“…Direct, indirect, competitive, sandwich and multiplex types of the assays have been used. ELISA has been applied for MT determination is number of samples including hepatic cell lines [35], Sertoli cells of rats exposed to cadmium [36][37][38] and urine of children living in polluted environment [39]. The investigation of relationships between MT level and tumor diseases has shown down regulation of MTs in thyroid carcinoma [40] and MT overexpression in oral squamous cell carcinoma [41], in hepatocellular carcinoma [42] and also in laryngeal carcinoma [43].…”

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

“…Investigations performed by Nordberg et al (1982) using radioimmunoassay showed that MT concentrations in plasma and urine ranged between 2-11.3 ng/g of protein and 2-155.2 ng/g of protein, respectively. MT levels were measured by a two-step competitive ELISA procedure in urine of two groups of children living in areas of mild and high environmental pollution due mainly to heavy metals (Swierzcek et al, 2004). The MT concentrations in that study ranged between 3-30 µg/g of creatinine.…”

Metallothionein as Potential Biomarker of Cadmium Exposure in Persian Sturgeon (Acipenser persicus)

“…Considering heavy metal detoxification significance of MTs, these proteins can serve as biomarker of heavy metal pollution of the environment [12][13][14][15]. On the other hand, a comparison between the content of heavy metals and determined MTs could be very useful not only from ecotoxicological aspects but also from biochemical aspects due to the better understanding of different functions of MTs in an organism [16].…”