Research paperComparison of two ELISA methods for measuring levels of the phosphorylated neurofilament heavy chain☆
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)
Neurofilament phosphoforms: surrogate markers for axonal injury, degeneration and loss
J. Neurol. Sci.
(2005)- et al.
A specific ELISA for measuring neurofilament heavy chain phosphoforms
J. Immunol. Methods
(2003) - et al.
Hyperphosphorylated neurofilament NF-H is a serum biomarker for axonal injury
Biochem. Biophys. Res. Commun.
(2005) - et al.
Biological markers in CSF and blood for axonal degeneration in multiple sclerosis
Lancet Neurol.
(2005)
Cited by (58)
Small GTPase control of pituitary hormone secretion: Evidence from studies in the goldfish (Carassius auratus) neuroendocrine model
2023, General and Comparative EndocrinologyGPR174 suppression attenuates retinopathy in angiotensin II (Ang II)-treated mice by reducing inflammation via PI3K/AKT signaling
2020, Biomedicine and PharmacotherapyCitation Excerpt :Trypsin-like activity was measured using fluorogenic peptide substrate Ac-RLR-AMC according to the manufacturer’s instructions (Promega, USA). The contents of GPR174 in serum or renal samples were evaluated by ELISA methods [16] using GPR174 polyclonal antibody(AmyJet Scientific Inc, Wuhan, China) according to the manufacturer’s protocols. Eyes were fixed in 4 % paraformaldehyde and then infiltrated with sucrose 25 % (w/v).
Oral administration of the nitroxide radical TEMPOL exhibits immunomodulatory and therapeutic properties in multiple sclerosis models
2017, Brain, Behavior, and ImmunityCitation Excerpt :Secondary antibodies [goat anti-mouse IgG-peroxidase (Jackson ImmunoResearch Laboratories Inc., West Grove, PA), goat anti-mouse IgG1-biotin, IgG2a-biotin, or IgG2c-biotin (Sigma-Aldrich)] and streptavidin-HRP enabled chromogenic conversion of TMB and quantification as for cytokine ELISAs. A sandwich ELISA to detect normal neurofilament H (phosphorylated heavy chain) levels was performed as previously described (Petzold and Shaw, 2007). SMI35R monoclonal antibody (to neurofilament heavy chain, Covance Princeton, NJ)-coated microtitre plates were incubated with 15 μg of spinal cord protein; protein was detected with rabbit polyclonal anti-neurofilament H antibody (N-4142; Sigma) and horseradish peroxidase-conjugated anti-rabbit immunoglobulin.
Biomarkers for CNS Injury and Regeneration
2015, Neural RegenerationIntensive neurorehabilitation and allogeneic stem cells transplantation in canine degenerative myelopathy
2023, Frontiers in Veterinary ScienceThe 2022 Lady Estelle Wolfson lectureship on neurofilaments
2022, Journal of Neurochemistry
- ☆
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.
- 1
Tel.: +1 352 294 0037; fax: +1 352 392 8347.