카드뮴폭로에 대한 생물학적 지표 Competitive ELISA에 의한 Metallothionein 정량

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 estab- lished to assay the MT expression utilizing monospecific antibody which was generated against MT-I. A total of 10 Sprague-Dawley rats was injected with CdCla for two weeks to induce MT. The cytosolic fraction of rat liver was obtained by differential centrifuga- tion. 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-I. The sera were collected by heart puncture. When immunoblot. was car ried out, the immunized rabbit sera (anti-MT-1) exhibited specific immunoreactive band at MT-I while showed any cross reactions neither with MT-II nor with other cytosolic pro teins. By chequerboard titration using the monospecific antibody, the competitive ELISA was established. The dose-dependent relationship was observed between anti-MT-I anti- body and the amount of MT in biological samples (r=0.9980). These results suggested strongly that competitive ELISA is a simple, rapid and reproducible method for screening cadmium exposure.