A quantitative ELISA for bioactive anti-Nogo-A, a promising regenerative molecule for spinal cord injury repair.
The detection and quantification of bioactive anti-Nogo-A mAbs, which is of interest for the treatment of spinal cord injury, has previously been accomplished using cellular or indirect immunoassays. In one such assay the presence of Nogo-A inhibits neurite outgrowth from the PC12 neuronal cell line: pre-treatment with anti-Nogo-A overcomes this inhibition and the concentration of anti-Nogo-A is correlated with the reduction in growth inhibition. In the current work we demonstrate the first anti-Nogo-A sandwich ELISA utilizing a Nogo-A fragment in the role of capture agent and the anti-Nogo-A mAb 11c7 as the soluble analyte. Because the Nogo-A fragment contains the amino acid sequence against which 11c7 was raised, we postulate this combination reproduces the native binding mechanism and results in the detection of bioactive anti-Nogo-A. In support of this hypothesis, we have found good agreement between the inhibitory action of the Nogo-A fragment and myelin proteins used in existing PC12 cell assays. Importantly, unlike the several days required for cellular assays the ELISA is a fast and easy to use method for the detection and quantification of bioactive 11c7 in the range of 500-6000 pg/mL.
Baumann
M Douglas
MD
Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, Ont., Canada M5S 3E5. douglas.baumann@utoronto.ca
Austin
James W
JW
Fehlings
Michael G
MG
Shoichet
Molly S
MS
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D009185:Myelin Proteins / Q000276:immunology*; D016501:Neurites / Q000502:physiology; D000070798:Nogo Proteins; D016716:PC12 Cells; D051381:Rats; D013119:Spinal Cord Injuries
D000911:Antibodies, Monoclonal; D009185:Myelin Proteins; D000070798:Nogo Proteins; C000603197:Rtn4 protein, rat