Biological Index for Cadmium Exposure: Assay of Metallothionein by Competitive ELISA / 대한산업의학회지

ABSTRACT

Metallothionein (MT) is a cadmium binding protein that played major' roles in protective mechanism for cadmium toxicity. In the present study, competitive ELISA was established to assay the MT expression utilizing monospecific antibody which was generated against MT-L A total of 10 Sprague-Dawley rats was injected with CdCl2 for two weeks to induce MT. The cytosolic fraction of rat liver was obtained by differential centrifugation. Two major MT isozymes (MT-I & MT-II) at ca. 10 kDa were purified by Sephadex G-75 gel filtration followed by DEAE-Trisacryl-M anion exchange column chromatogra-phy, respectively. Two rabbits were immunized 4 times consecutively with the conjugate of purified MT-L The sera were collected by heart puncture. When immunoblot was carried out, the immunized rabbit sera (anti-MT-I) exhibited specific immunoreactive band at MT-I while showed any cross reactions neither with MT-II nor with other cytosolic proteins.. By chequerboard titration using the monospecific antibody, the competitive ELISA was established. The dose-dependent relationship was observed between anti-MT-I antibody and the amount of MT in biological samples (r(2)=O.9980). These results suggested strongly that competitive ELISA is a simple, rapid and reproducible method for screening cadmium exposure.