Research Paper
An ELISA for glial fibrillary acidic protein

https://doi.org/10.1016/j.jim.2004.01.015Get rights and content

Abstract

Glial fibrillary acidic protein (GFAP) is the major intermediate filament protein of the astrocyte, and body fluid levels of GFAP are an important tool for estimating astrogliosis and astrocytic activation in vivo. This paper presents a new sandwich ELISA allowing quantification of GFAPSMI26 from the cerebrospinal fluid (CSF). The sensitivity of the GFAPSMI26 ELISA is 5 pg/ml with a recovery of 94% and a mean within- and between-batch precision of 6% and 10%, respectively.

The upper reference value for CSF GFAPSMI26 levels (9 pg/ml) was defined as the 95% cumulative frequency from 315 CSF samples. Based on this cut-off, a significantly higher proportion of patients with subarachnoid hemorrhage (100%), traumatic brain injury (100%), dementia (76%) and normal pressure hydrocephalus (85%) had pathologically elevated CSF GFAPSMI26 levels compared to patients with peripheral nervous system disorders (0%).

In a critical review of the literature, we compare the analytical and clinical sensitivity of previous GFAP ELISA methods with particular reference to patients with dementia.

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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