Determination of metallothionein-3 by a competitive enzyme-linked immunosorbent assay in experimental animals

Abstract

-An easy and specific enzyme-linked immunoassay (ELISA) for the determination of metallothinein-3 (MT-3) in experimental animals for the research of heavy metal and chemical toxicity has not been reported yet. Therefore, we have developed a competitive ELISA, using a specific monoclonal antibody raised against human recombinant MT-3 (rMT-3). The epitope mapping of the antibody was conducted using mouse, rat, and human MT-3s and peptide fragments of human MT-3. MT-1/2, MT-3 knock-out (KO) mice and human brain and liver were used for the evaluation of the ELISA. A pretreatment method of the tissue homogenates was also examined. The antibody used for the ELISA had the same cross-reactivity with MT-3 in humans and experimental animals. The human MT-3 NH 2 terminal peptide (Fr. 1-17) was the demonstrated epitope of this antibody. The reactivity of this ELISA in brain homogenate of MT-3 KO mouse was significantly low compared with the wild type and MT-1/2 KO mice. The lowest detection limit of the ELISA was 10 ng/ml and over 80% of the spiked rMT-3 was recovered in the brain homogenate. The assay linearity was intact with a 5-fold dilution in the brain homogenate. The inter-and intra-assay CV was 6.5%, respectively. An effective pretreatment procedure of the tissue homogenate was also established for this MT-3 ELISA. In conclusion, this competitive ELISA is an easy and specific method for measuring the brain MT-3 level in experimental animals.

Microalgal Metallothioneins and Phytochelatins and Their Potential Use in Bioremediation

“…Metallothioneins can also be determined using immunoassays, Western blot, and mRNA sequencing. The enzyme-linked immunosorbent assays (ELISA) is the most common immunochemical method to detect MTs, and is used in both humans and experimental animals (Saito et al, 2013;Nakazato et al, 2014). Sequencing of mRNA has been used to determine the basal expression (Kim et al, 2002) of MTs or variations in their expression levels after exposure to metals (Wu et al, 2008) or for medical purposes (Nagamine et al, 2005).…”