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Published online before print July 16, 2008, doi:10.1212/01.wnl.0000313038.34337.b1)
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Received October 25, 2007
Accepted February 11, 2008

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

Nguyen thi Man PhD, E. Humphrey PhD, L. T. Lam PhD, H. R. Fuller PhD, T. A. Lynch MPhil, C. A. Sewry PhD, P. R. Goodwin BSc, A. E. MacKenzie PhD, and G. E. Morris DPhil*

From the Wolfson Centre for Inherited Neuromuscular Disease (N.t.M., E.H., L.T.L., H.R.F., T.A.L., C.A.S., G.E.M.), Oswestry, UK; Hallmark Analytical Ventures (P.R.G.), Saltney, Flintshire, UK; Children's Hospital of Eastern Ontario Research Institute (A.E.M.), Ottawa, Canada; and Institute for Science and Technology in Medicine (G.E.M.), Keele University, UK.


* To whom correspondence should be addressed. E-mail: glenn.morris{at}rjah.nhs.uk.

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 x 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.


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