A two-site ELISA can quantify upregulation of SMN protein by drugs for spinal muscular atrophy

thi Man, Nguyen; Humphrey, E.; Lam, L. T.; Fuller, H. R.; Lynch, T. A.; Sewry, C. A.; Goodwin, P. R.; MacKenzie, A. E.; Morris, G. E.

Abstract

Objectives: Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by loss of lower motor neurons during early or postnatal development. Severity is variable and is inversely related to the levels of survival of motor neurons (SMN) protein. The aim of this study was to produce a two-site ELISA capable of measuring both the low, basal levels of SMN protein in cell cultures from patients with severe SMA and small increases in these levels after treatment of cells with drugs.
Methods: A monoclonal antibody against recombinant SMN, MANSMA1, was selected for capture of SMN onto microtiter plates. A selected rabbit antiserum against refolded recombinant SMN was used for detection of the captured SMN.
Results: The ratio of SMN levels in control fibroblasts to levels in SMA fibroblasts was greater than 3.0, consistent with Western blot data. The limit of detection was 0.13 ng/mL and SMN could be measured in human NT-2 neuronal precursor cells grown in 96-well culture plates (3 × 104 cells per well). Increases in SMN levels of 50% were demonstrable by ELISA after 24 hours treatment of 105 SMA fibroblasts with valproate or phenylbutyrate.
Conclusion: A rapid and specific two-site, 96-well ELISA assay, available in kit format, can now quantify the effects of drugs on survival of motor neurons protein levels in cell cultures.
GLOSSARY: HDAC = histone deacetylase; SMA = spinal muscular atrophy; SMN = survival of motor neurons.

Citation

thi Man, N., Humphrey, E., Lam, L. T., Fuller, H. R., Lynch, T. A., Sewry, C. A., …Morris, G. E. (2008). A two-site ELISA can quantify upregulation of SMN protein by drugs for spinal muscular atrophy. Neurology, 71(22), 1757-1763. https://doi.org/10.1212/01.wnl.0000313038.34337.b1

• Journal Article Type: Article
• Online Publication Date: Nov 25, 2008
• Publication Date: Nov 25, 2008
• Deposit Date: Jun 7, 2024
• Journal: Neurology
• Print ISSN: 0028-3878
• Electronic ISSN: 1526-632X
• Publisher: American Academy of Neurology
• Peer Reviewed: Peer Reviewed
• Volume: 71
• Issue: 22
• Pages: 1757-1763
• DOI: https://doi.org/10.1212/01.wnl.0000313038.34337.b1
• Public URL: https://keele-repository.worktribe.com/output/847219
• Publisher URL: https://www.neurology.org/doi/10.1212/01.wnl.0000313038.34337.b1