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...ns of 150 �l were collected from the bottom of the tube. 60-�l aliquots were transferred to a microtiter plate. Cholinesterase activity was assayed colorimetrically, using acetylcholine as substrate (=-=Ellman et al., 1961-=-). To assay AChE, iso-OMPA (10 �4 M) was included to inhibit BuChE. To assay BuChE, BW284c51 (10 �6 M) was included to inhibit AChE. Alkaline phosphatase (6.1 S) and �-galactosidase (16 S) from E. col...

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...o separate serum from red blood cells. Aliquots of serum (25 ll diluted 1:20) were used for enzymatic analyses. AChE activity was determined in each preparation by Ellman’s spectrophotometric method (=-=Ellman et al., 1961-=-) as previously described (Meneguz et al., 1992; Volpe et al., 1990). The samples were incubated with the substrate in 0.05M sodium phosphate buffer, pH 7.2, containing 0.25mM 5,5#-dithiobis2-nitroben...

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... of Fab« fragments obtained from 12F10 IgGs and coupled to a G4 tetramer of acetylcholinesterase (AchE) (Grassi et al., 1988). After three washes, AchE activity was assessed by adding Ellman reagent (=-=Ellman et al., 1961-=-) and measuring absorbance at 414 nm with an automatic reader (LabSystems). + PrPC immunoprecipitation. Anti-PrP MAbs Pc248 or 4F2 were added to 500 ll (50 mg tissue equivalent) of the different clari...

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...acer Ab are directed against different complementary epitopes. After 18 h of reaction at 4°C, the plates were washed and solid-phase-bound AChE activity was measured using the method of Ellman et al. =-=(12)-=-. Detection limits of 30 and 200 pg/ml were obtained for BLGn and BLGrcm, respectively, with very low or negligible cross-reactivity of the other milk proteins or tryptic fragments of BLG. Cross-react...

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...ich measures the thiobarbituric acid (TBA) reactive substances, at 530 nm, following the methodology described by Satoh (1978). Lipoperoxidation was expressed as TBARS concentration in lmol mg of protein1, using a standard MDA curve. 2.6. Acetylcholinesterase The brains and muscles were homogenized (10 volume) in potassium phosphate buffer (0.1 M, pH 7.5), centrifuged (20 min, 15 000g, 4 C), and the supernatant was removed for analysis of 296 K.A. Modesto, C.B.R. Martinez / Chemosphere 78 (2010) 294–299acetylcholinesterase. Enzyme activity was determined based on the colorimetric method of Ellman et al. (1961) adapted for reading on microplates, according to Alves Costa et al. (2007). The final concentration of the acetylcholine iodide substrate employed was 9 mM, while that of the DTNB color reagent was 0.5 mM for both tissues. Absorbance was determined in a microplate reader at 415 nm and the enzyme activity was expressed in nmol DTNB min1 mg of protein1. The protein concentration in all the samples of all the assays was determined by the method of Lowry et al. (1951), using bovine albumin as standard.2.7. Statistical analysis For each treatment group (CTR and RD), at each period of exposure (6...

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...ermination of inhibitory activity against acetylcholinesterase Acetylcholinesterase inhibition was determined spectrophotometically using acetylthiocholine as substrate by modifying the Ellman method =-=[17]-=-. The reaction was carried in 100 mM sodium phosphate buffer (pH 8.0) at 25 °C. In a typical assay, buffer (140 µL), enzyme preparation (20 mL) and test compound solution (20 µL) were mixed and incuba...