Cortisol metabolism by 11β-hydroxysteroid dehydrogenase as a novel target in the treatment of inflammation- or immune-mediated bone loss: Comment on the article by Makrygiannakis et al
✍ Scribed by Thomas Wilckens; Ariane Volkmann
- Book ID
- 101651741
- Publisher
- John Wiley and Sons
- Year
- 2006
- Tongue
- English
- Weight
- 43 KB
- Volume
- 56
- Category
- Article
- ISSN
- 0004-3591
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✦ Synopsis
BSA binding activities. It appears that all sera with anti-HSA also contain antibodies to BSA (4). We therefore reexamined whether CSF and sera from SLE patients contain antibodies to HSA, using the method described by Yoshio et al.
Wells of a 96-well microtiter plate (Falcon Pro-Bind; Becton Dickinson, Lincoln Park, NJ or Nunc-immuno module F8 Maxisorp; Nunc, Roskilde, Denmark) were coated with HSA (Miles, Elkhart, IN) or HSA conjugated (at a 1:1 weight ratio) with highly purified synthetic DWEYSVWLSN peptide (purity Ͼ95%) (HSA-NR2 peptide), at 20 g/ml in phosphate buffered saline (PBS), overnight at 4°C. The wells were blocked with Block Ace (Dainippon, Osaka, Japan) for Falcon Pro-Bind plates or with PBS containing 1% BSA (Miles) for Nunc Maxisorp plates, for 2 hours at room temperature. Before being added to the wells, serum and CSF samples were diluted 1:200 and 1:2, respectively, in PBS containing 1% BSA. After incubation at 37°C for 1 hour, bound IgG anti-NR2 glutamate receptor antibody was detected with peroxidaseconjugated F(abЈ) 2 fragments of goat anti-human IgG (Cappel, Cochranville, PA). Binding activity was expressed as optical density at 492 nm (OD 492 ) as measured in a 2-wavelength microplate photometer (MTP-450; Corona Electric, Ibaraki, Japan).
As seen in Table 1, all 16 samples exhibited positive binding to HSA-NR2 peptide in Falcon Pro-Bind plates (Yoshio and colleagues' method) as well as in Nunc Maxisorp plates. However, 8 of the 16 samples showed higher binding activity (OD 492 ) to HSA alone than to HSA-NR2, indicating that those samples would yield false-positive results for IgG anti-NR2 glutamate receptor antibodies unless nonspecific binding to HSA alone were subtracted. In addition, the levels of binding activity obtained with Falcon Pro-Bind plates were ϳ20-30% of those with Nunc Maxisorp plates, even though peroxidase-conjugated F(abЈ) 2 goat anti-human IgG was used at 1:5,000 in the former plates and at 1:10,000 in the latter. Therefore, it would be preferable to use Nunc Maxicorp plates. Nonetheless, these results confirm that ϳ50% of serum and CSF samples contain antibodies to HSA (and presumably to BSA as well, since it was previously shown that all sera positive for anti-HSA contained antibodies to BSA [4]).
These findings raise serious concern about the specificity of the ELISA used by Yoshio et al. It is highly likely that the presence of anti-BSA antibodies would have significantly influenced their results and conclusions. Therefore, their conclusion that IgG anti-NR2 glutamate receptor antibodies in CSF may cause focal neurologic damage such as seizure disorders, aseptic meningitis, and transverse myelopathy is not supported.