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[PubMed] [Google Scholar] 13. in soil which have sequences cross-reacting with bovine Cholesteryl oleate neurofilaments and myelin, but there were no antibody elevations against or (4), a common saprophytic microbe found in soil and water supplies (19) which also possesses sequences resembling bovine neurofilaments (Table ?(Table1).1). Furthermore, another common environmental microbe, (4). TABLE 1 Comparison of similar sequences in bovine neurofilaments compared with molecular?sequences (NCIMB 10694) and (NCIMB 9036) were obtained from National Collections of Industrial and Murine Bacteria, Ltd. (Aberdeen, Scotland), and (NCTC 9002) was provided by the Department of Microbiology at King’s College. IgA and total Ig (IgG + IgA + IgM) antibodies were measured by ELISA. Cultures were grown in 2-liter flasks on an orbital shaker for 16 h at 37C for and for 2 days at 30C for and in 200 ml of nutrient broth (Oxoid; 25 g/liter). Flasks were inoculated with 10 ml of the corresponding starter culture and were left shaking at 37C for 6 h. Batch culture cells were harvested by centrifugation 6,000 rpm for 20 min at 4C (Beckman JA-20 rotor, six 250-ml cuvettes). The pellets of cells were then washed three times with 0.15 M phosphate-buffered saline (PBS; pH 7.4) before being finally resuspended in 20 ml of PBS. A stock solution of the suspension was prepared by diluting in 0.05 M carbonate buffer (pH 9.6) to give an ART4 optical density (OD) reading of 0.25 (106 bacterial cells/ml) on the spectrophotometer (Corning Model 258). ELISA. ELISAs were carried out as previously described (20). Briefly, ELISA plates were coated (5 g/well) with neurofilaments prepared from bovine spinal cord (Sigma), myelin basic protein obtained from bovine brain (Sigma), or bacterial suspension (200 l/well) overnight at 4C, and the nonspecific sites were blocked with PBS containing 0.1% Tween and 0.2% ovalbumin (Grade III; Sigma), plates were washed, and a 1/200 dilution of test or control serum was added. The plates were incubated at 37C for 2 h, were washed, and rabbit antibovine alpha-chain-specific horseradish peroxidase conjugate (1/3,000)(Bethyl Laboratories, Ltd.) or rabbit anticow Ig (IgG + IgA + IgM)-horseradish peroxidase conjugate (1/4,000)(Dako Ltd.) was added. The plates were reincubated for 2 h, were washed, and a substrate solution of 0.5 mg of 2,2-azinobis(3-ethylbenzthiazoline-6-sulphonic acid)(ABTS; Sigma) per ml in citrate-phosphate buffer (pH 4.1) containing 0.98 mM H2O2 (Sigma) was added to each well. The reaction was stopped with a 2-mg/ml solution of sodium fluoride (Sigma), the plates were read at 630 nm on a microtiter plate reader (Dynatech MR 600), and results were expressed as OD units +/? standard errors (SE). Each serum sample was tested in duplicate. All studies were blind, in that the tester did not know which were test or control sera. The mean OD units of IgA or total Ig antibodies in serum samples from BSE-positive animals resulting from tests against the two autoantigens and three different microorganisms were compared to the corresponding control groups by using Student’s test. Furthermore, triplicate ELISA studies were carried out in serial doubling dilutions of three selected BSE serum samples which had high, medium, and low reactivities to the respective antigens bovine neurofilaments, bovine myelin, and 0.001), Cholesteryl oleate 30 organically raised cows less than 30 months of age (0.149 0.007; 0.001) (0.078 0.005; 0.001), and 28 organically raised cows greater than 30 months of age (0.157 0.006; 0.001) (0.078 0.005; 0.001). Open in a separate window FIG. 1 IgA antibody titers (bar = mean) for 30 control serum samples from cows aged less than 30 months (A 30m), 28 control serum samples from cows aged more than 30 months (A 30m), and 18 control serum samples from cows not having BSE at postmortem compared to 29 serum samples from cows with BSE at postmortem when tested against bovine neurofilaments (a), bovine myelin (b), and (c). Dashed line represents 95% confidence limits for mean of control as given by A 30M + A 30M ? results of the one-tailed test. Elevated levels of IgA antibodies to whole 0.001), 30 organically raised cows less than 30 months of age (0.409 0.009; 0.001), and 28 organically raised cows greater than 30 months of age (0.432 0.029; 0.001). Absorption of BSE sera with sonicated reduced autoantibodies to bovine myelin and neurofilaments almost to the levels found in control sera (Table ?(Table2),2), although some activity to neurofilaments remained. TABLE 2 Levels of IgA before and after ELISA absorption with Cholesteryl oleate bacteria (mean OD ?SE) of total Ig (IgG + IgA + IgM) were significantly elevated in the sera from animals with BSE (0.99 0.05) (Fig. ?(Fig.2a)2a) compared.