Research paper
Comparison of two ELISA methods for measuring levels of the phosphorylated neurofilament heavy chain

https://doi.org/10.1016/j.jim.2006.09.021Get rights and content

Abstract

Background

Recent studies suggest that the quantification of neurofilament subunits in cerebrospinal fluid (CSF), blood and amniotic fluid may reflect neuroaxonal damage and be of high clinical value. The present study aims to cross-validate two different independently developed ELISA techniques for the quantification of the phosphorylated axonal forms of the neurofilament heavy chain (pNfH).

Methods

The London ELISA method is based on barbitone buffer and the commercially available SMI35 capture antibody. The Gainesville method uses Tris-buffered saline (TBS) and an affinity purified chicken polyclonal capture antibody (C-pNfH). Coded CSF from 50 patients with neurological diseases were analyzed in duplicate by both laboratories, each using both ELISA methods, but with each lab using their own detection antibody, tertiary antibody and chromogen. Methods were compared using Bland–Altman plots. Correlation and regression analyses were used to allow for transformation of values between both methods.

Results

The Bland–Altman plots demonstrated that 96% of all samples fell into the narrow 95% limits of agreement (0.04 units of OD). There was a high correlation (Spearman R = 0.92, p < 0.0001 and Pearson R = 0.98, p < 0.0001) between the Gainesville (Y) and the London (X) method with Y = 0.132 + 1.104  X. The previously determined upper reference limit of 0.73 mg/l (London method) corresponds to 0.94 mg/l for the Gainesville method. CSF pNfH levels above the reference limit were observed in patients with encephalitis, encephalomyelitis, hydrocephalus, subarachnoid haemorrhage, spino-muscular atrophy, stroke and cancer with both methods agreeing in all cases.

Conclusion

The two assays are in excellent agreement, suggesting that pNfH, which has a number of unusual protein chemical features, may be the biomarker of choice for the routine and robust detection of axonal injury and degeneration in both research and clinical contexts.

Introduction

Neurofilaments in the mature CNS are composed of several subunit proteins, namely the triplet of NfL, NfM, NfH and α-internexin/Nf66 and in some cases also peripherin (Shaw, 1998, Petzold, 2005). Since neurofilament subunits are among the most abundant proteins of the nervous system and are particularly concentrated in axons, it is expected that they would be released in relatively large amounts from damaged neurons and particularly their axons. Accordingly, detection of these subunit proteins in cerebrospinal fluid (CSF), blood and other bodily fluids might provide a convenient means to detect and possibly measure damage to the axonal compartment (Teunissen et al., 2005, Petzold, 2005).

Recently the laboratories of the present authors have independently described pNfH capture ELISAs. This report aims to rigorously compare these two methods.

Section snippets

Materials and methods

This study focuses on two 4-layer sandwich ELISA systems (Petzold et al., 2003, Shaw et al., 2005). The first of these assays, developed in London, uses SMI35 in the capture role. This antibody binds to the highest molecular weight and some of the lower molecular weight phosphoforms of NfH on Western blots, but not the completely dephosphorylated form (Petzold et al., 2003). The second assay was developed independently in Gainesville and uses an affinity purified polyclonal antibody which

Results

Parallelism between calibrant and CSF for the Gainesville method was studied by measuring doubling dilutions of native CSF samples with NfHC-pNfH concentrations between 2.3 and 87 mg/l (n = 8) and buffer spiked with NfH concentrations between 2.5 and 100 mg/l (n = 3). The absorbance dilution was normalized to the highest value (100%). The parallel relationship of the linear regression lines is demonstrated in Fig. 1B. Parallelism for the London method has already been shown (see Fig. 4 in (Petzold

Discussion

The main finding of this study was the excellent agreement between the London and Gainesville assays as shown by the Bland–Altman plot (Fig. 2A). The high degree of agreement and the narrow 95% limit of agreement suggest that these two methods reveal comparable results for the measurement of pNfH from the human CSF. Additionally there was a strong correlation of the two methods (RP = 0.98, RS = 0.92, p < 0.0001). The slope is close to 1.00, showing that the proportional analytical error is 1.04%.

Acknowledgments

We would like to thank Dr. Geoffrey Keir for comments on the manuscript and Mr. Yong Wang and Ms. Donna Grant for excellent technical assistance.

References (8)

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Gerry Shaw holds equity in EnCor Biotechnology Inc., a company commercializing some of the antibodies and potentially the ELISA procedure used in this study, and may benefit by receiving royalties or equity growth.

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