Highly specific polyclonal rabbit antimetallothionein antibodies are isolated and characterized. The possibility of using these antibodies in immunohistochenical assay was verified on normal and tumor mammary tissues, the common metallothionein-positive models.Key Words: metallothioneins; polyclonal antibodies; specificity; immunohistochem&try Metallothioneins (MT) is a family of inducible cytoplasmic proteins (6.5 kD) consisting of 61 amino acid residues, of them 20 strictly arranged cystein residues are responsible for unique metal-binding properties [7]. MT-positive cells were immunochemically detected in various organs and tumors [1,5,13]. It was shown that MT participate in the storage and metabolism of essential metals (Zn, Cu, Mn, etc.) and elimination of toxic metals (Cd, Pb, Hg, etc.) [2]. MT possess antioxidant activity and protect cells and organisms from ionizing and UV radiation, chemical agents, and other inductors of free radical processes [2,11].In the present study we describe isolation of rabbit polyclonal anti-MT antibodies and analysis of their specificity by enzyme-linked immunosorbent assay (ELISA) and demonstrate the possibility of using these antibodies for immunolocalization of MT in histological sections.
MATERIALS AND METHODSRandom-bred male rats weighing 180+20 g were subcutaneously injected with CdCI 2 (0.2 ml, Chemapol) in doses of 1 mg/kg on day 1 and 3 mg/kg during the subsequent 3 days. One day after the last Medial Radiological Center, Russian Academy of Medical Sciences, Obninsk injection, the liver was removed and homogenized, and MT fraction was precipitated with alcohol and lyophilized [4]. The MT fraction was purified on a Sephadex G-75 preparative G-75 colunm (Phan'nacia), the MT-peak fractions were pooled, lyophilized, dissolved and desalted on a Sephadex G-10 column (Pharmacia). MT was measured spectrophotometrically (Philips) (e220=48,200 M-~cm -~ at pH 1) [14].MT aggregates prepared by incubation with an excess of glutaraldehyde (Serva) [6] were used for immunization. The immunogen consisting of insoluble MT aggregates was resuspended by passing through a syringe and dialyzed against 20-fold volume of 0.01 M boron-borate buffer, pH 8.5 at 4~ for 24 h and stored at -40~ Four Chinchilla rabbits weighing 2-2.5 kg were subcutaneously injected with 2 ml immunogen in Freund's complete adjuvant (1:1, Calbiochem, multipoint injection regimen). The doses of MT for the 1st, 2nd (after 30 days), and 3rd (after 90 days) were 1.5, 2, and 3.1 mg per rabbit, respectively. Blood was sampled on days 44, 60, and 100 after the first immunization.The antigen was adsorbed on polystyrene plates (Nunc). To this end, 0.1 ml/well (0.2 ~tg/ml) MT in 0.1 M carbonate buffer (pH 9.6) was incubated in the plates at 4~ for 18 h. At all stages of ELISA, free components were removed by 3-fold washout with a buffer containing 0.01 M phosphate buffer saline (pH 7.2), 0.85% NaC1, 0.05% Triton X-100
