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ARTICLES: J. William Lindsey, Michael P. Crawford, and Landon M. Hatfield Soluble Nogo-A in CSF is not a useful biomarker for multiple sclerosis Neurology 2008; 71: 35-37 [Abstract] [Full text] [PDF]

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Soluble Nogo-A in CSF is not a useful biomarker for multiple sclerosis

Krzysztof Selmaj, Anna Jurewicz, Mariola Matysiak, Cedric S. Raine   (22 September 2008)

Reply from the author

J. William Lindsey   (22 September 2008)

Soluble Nogo-A in CSF is not a useful biomarker for multiple sclerosis 22 September 2008
Krzysztof Selmaj, Medical University of Lodz, Department of Neurology 22 Kopcinskiego str, 90-153 Lodz, Poland, Anna Jurewicz, Mariola Matysiak, Cedric S. Raine Send Correspondence to journal: Re: Soluble Nogo-A in CSF is not a useful biomarker for multiple sclerosis kselmaj{at}afazja.am.lodz.pl Krzysztof Selmaj, et al.	We read Lindsey et al.’s article [1] with interest where the authors refer to our findings on the same subject. [2] They repeated our experiments and concluded with a different interpretation that staining with anti-Nogo A Ab in CSF of MS patients is non-specific. In reviewing their and our data, we believe that our interpretation is still correct. Firstly, we abolished anti-Nogo A staining with preabsorption of anti-Nogo A Ab with a specific Nogo A blocking peptide (sc-11032P). We believe that this is the most reliable criterion for proving that binding of an antibody is specific. In addition, Lindsey et al. found non-specific staining with a very high concentration (1:5000) of secondary antibody. We used a lower concentration of secondary Ab (1:20,000) showing no staining of CSF gels not exposed to anti-Nogo A Ab. Specificity of antibody staining is determined by antibody concentration and non-specific binding is common with higher concentrations, a known phenomenon in antibody binding assays. We are satisfied that Lindsey et al. were unable to detect staining using a secondary Ab at a concentration of 1:20,000. Thirdly, in response to the authors' claim that anti-Nogo A Ab staining is non-specific, we obtained similar results with two different primary Abs directed against the Nogo A molecule. We also disagree that Nogo A staining resulted from the higher concentration of IgG in the CSF of MS patients. We also had patients in our control group with very high IgG content (tbc, bacterial and viral encephalomeningitis), and we did not see staining with anti-Nogo A in these conditions. We also concentrated the CSF of MS patients and controls by 10 times and were still unable to see any increase in anti-Nogo A Ab binding. Regarding the loss of anti-Nogo A binding in CSF samples subjected to a G-protein absorbance column and in gels with the omission of DDT, we would like to hypothesize that the Nogo A fragment we detected might be complexed with its naturally- occurring Abs. Such Abs have been shown to occur in the CSF of MS patients. [3] To account for this, we showed that a filtration cut of CSF below 30 kD completely removed anti-Nogo A staining at the level of 20kD. However, subsequent gel staining with the fraction > 30 kD under denaturated conditions revealed anti-Nogo A staining of CSF once more. We believe that this provides convincing evidence for the presence of a Nogo A fragment in a complexed form with its natural antibodies in the CSF. References 1. Lindsey JW, Crawford MP, Hatfield LM. Soluble Nogo-A in CSF is not a useful biomarker for multiple sclerosis. Neurology. 2008;71:35-37. 2. Jurewicz A, Matysiak M, Raine CS, Selmaj K. Soluble Nogo-A, an inhibitor of axonal regeneration, as a biomarker for multiple sclerosis. Neurology. 2007;68;283-287. 3. Reindl M, Khantane S, Ehling R, et al. CESerum and cerebrospinal fluid antibodies to Nogo-A in patients with multiple sclerosis and acute neurological disorders. J Neuroimmunol. 2003;145:139-147. Disclosure: The authors report no disclosures.
Reply from the author 22 September 2008
J. William Lindsey, University of Texas-Houston, 6431 Fannin, Suite 7.044, Houston, TX 77030 Send Correspondence to journal: Re: Reply from the author john.w.lindsey{at}uth.tmc.edu J. William Lindsey	We appreciate the comments of Dr. Selmaj et al. regarding our recent article. [1] We did not do any experiments with the Nogo-A blocking peptide. Since we identified a 25 kD band with the secondary antibody alone, peptide blocking experiments did not seem worthwhile. The concentration of secondary antibody we used, 1:5000, is within the recommended range of 1:500 to 1:10,000 given by the manufacturer and 1:5000 is the recommended starting concentration. The concentration of 1:20,000 used by Jurewicz et al. gave light and poorly reproducible signal in our experiments. [2] We found similar results with a different secondary antibody, so we would predict similar results regardless of the primary antibody. Some of the CSF specimens in the study of Jurewicz et al. may have had high IgG concentrations, but they state in their methods that the protein concentration of each CSF was measured and 3 mcg of protein was used for each. [2] The actual amount of IgG loaded for each specimen would depend on the ratio of IgG to other proteins, and would tend to be higher in MS. The results of the experiments with filtration with a 30 kD cutoff described by Dr. Selmaj’s are consistent with either antibody light chains or antibody-bound Nogo-A as the source of the signal. The results of the experiments without DTT are more consistent with our interpretation, since denaturation with SDS should release antibody-bound Nogo-A but not disulfide-bonded light chains. We stand by our experimental results and our interpretation. Non-specific binding of antibody is a constant concern with Western blots, particularly in areas corresponding to the major protein bands present in that sample. Definitive demonstration that Nogo-A is present in MS CSF will require confirmatory evidence from an independent method such as immunoprecipitation or mass spectroscopy. Disclosure: The authors report no disclosures.