Development of an enzyme-linked immunosorbent assay for metallothionein-I and -II in plasma of humans and experimental animals

Background An easy and specific enzyme-linked immunoassay (ELISA) for the determination of metallothinein-1 (MT-1) and 2 (MT-2) simultaneously in serum and other biological specimens in humans and experimental animals has not been developed yet. Methods We developed a competitive ELISA, a specific polyclonal antibody against rat MT-2. The epitope mapping of the antibody was conducted using MTs in mouse, rat, rabbit, human and the fragment peptides of human MT-2. MT1/2 and MT-3 knock-out mice and cadmium treated mice were used for the evaluation of the ELISA. Pretreatment method of serum was examined to deplete blocking factors for this assay. Results The antibody used for this ELISA had the same cross-reactivity with MT in humans and experimental animals. NH 2 terminal peptide of MT with acetylated methionine was proved to be the epitope of this antibody. The reactivity of this ELISA system with liver, kidney and brain in MT1/2 knock-out mice was significantly low, but was normal in MT-3 knock-out mouse. The lowest detection limit of this ELISA was 0.6 ng/ml and the added MT-1 was fully recovered from serum. The mean MT concentration in our preliminary study was 23 ± 4.6 ng/ml in human serum. Cadmium treatment to mice induced significantly higher amount of MT in serum, liver, kidney and spleen as reported previously by different established methods. Conclusion The proposed competitive ELISA is an easy and specific method for practical use, determining total MT-1 and -2 simultaneously in serum and other biological specimens of human and experimental animals.