Research PaperAn ELISA for glial fibrillary acidic protein
Section snippets
Capture antibodies
Mouse monoclonal IgG1 anti-GFAP antibodies were purchased from Sternberg Monoclonals (Sternberg Monoclonals, Lutherville, MD). SMI21 is a mouse monoclonal IgG1 raised against human brain microvessels. No cross-reactivity with other intermediate filaments or tissues was observed. Astrocytes, Bergmann glia in human, monkeys and dogs were GFAPSMI21 positive. In contrast, rat, rabbit and mouse GFAP was not recognised by SMI21. SMI22 is the mouse monoclonal cocktail of all three Bigner–Eng clones
Selection of the capture antibody
The standard curves for the GFAP capture antibodies (GFAPSMI21, GFAPSMI22, GFAPSMI24, GFAPSMI26) were run on the same plate to minimise the inter-assay variation. In order to determine which antibody combination was best in terms of a low detection limit, the signal-to-noise ratio (absorbance value of the lowest standard divided by the absorbance of the blank reading) for each standard curve was calculated. The signal-to-noise ratio for SMI26 was about threefold (Fig. 1A) and the analytical
Discussion
The sandwich ELISA presented here is simple and based on commercially available antibodies. The sensitivity of the assay is 5 pg/ml, the upper reference limit 9 pg/ml and the standard curve ranges from 0 to 200 pg/ml.
A previously described 4- and a more sensitive 5-layer assay used in-house monoclonal antibodies (Albrechtsen et al., 1984b) and a standard curve derived from human brain homogenate Albrechtsen et al., 1984a, Albrechtsen and Bock, 1985. Albrechtsen et al. reported a control group
Acknowledgements
This study was supported by the Multiple Sclerosis Society of Great Britain and Northern Ireland and the BR Kirk Fund of the Institute of Neurology.
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