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

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.

Address correspondence and reprint requests to Prof Morris, Wolfson Centre for Inherited Neuromuscular Disease, RJAH Orthopaedic Hospital, Oswestry, SY10 7AG, UK 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.

GLOSSARY: HDAC = histone deacetylase; SMA = spinal muscular atrophy; SMN = survival of motor neurons.

Supplemental data at www.neurology.org

Editorial, page 1752

e-Pub ahead of print on July 16, 2008, at www.neurology.org.

Supported by a research grant from Andrew’s Buddies (FightSMA), USA, and contract number JL-07604-1 SMA03-006 from the SMA Project (National Institute for Neurological Disease and Stroke, USA).

Disclosure: P.R. Goodwin is director and senior scientist of a company that may produce the ELISA kit commercially. None of the other authors has any financial interest in that company nor will they receive any personal financial advantage of any kind from sales. RJAH Orthopaedic Hospital may receive royalty payments from the company.

Received October 25, 2007. Accepted in final form February 11, 2008.

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