Isoelectric Focusing of Cerebrospinal Fluid Immunoglobulin G: An Annotated Update

Keir, Geoffrey; Luxton, Richard; Thompson, Edward J.

doi:10.1177/000456329002700504

Cited by 132 publications

(76 citation statements)

References 3 publications

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“…20% of the samples (Table 1). When 10 immunoblots were scanned, the CV was higher (300%) as would be expected since the same volume of CSF was applied 4 irrespective of the total protein, but only nine of the total of 432 had to be repeated, i.e. 2% of the total number of samples.…”

Section: Resultsmentioning

confidence: 91%

“…Finally, to compare the effectiveness of the two different approaches in the detection of OCBs, 16 paired samples of CSF/serum positive for OCBs were selected in London according to the 'gold standard' method, 4 and sent to California to be tested using both the commercial kits, one based on silver and the other on EAC/HRP. The results from the silver technique are shown in Figures 3(a) and (b).…”

Section: Resultsmentioning

confidence: 99%

“…from 2 to 120 ng IgG). 4 Unfortunately, silver staining has a much more narrow linear range. As a separate issue, it is important to realise that the problem of band recognition is not because of inadequate sensitivity, since when using a different enzyme with a different substrate, namely alkaline phosphatase with nitroblue tetrazolium (Sadaba), 5 most CSF samples have to be routinely diluted given the high sensitivity of this particular method.…”

Section: Discussionmentioning

confidence: 99%

“…(2) Separation in agarose gel followed by nitrocellulose blotting, then specific immunodetection of IgG heavy chains (Helena Biosciences, Beaumont, TX, USA) based on the method by Keir et al 4 again performed at Specialty Labs. Individual gels were scanned in California to produce a digital image with 256 tones of grey.…”

Section: Methodsmentioning

confidence: 99%

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Discrepancies between qualitative and quantitative analysis of cerebrospinal fluid IgG: densitometric comparison of nephelometry-silver versus immuno-enzymes

Keir

Barrio²,

Thompson

2008

Ann Clin Biochem

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Background: Detection of local synthesis of IgG within the central nervous system is important for the diagnosis of brain inflammatory diseases such as multiple sclerosis. This is typically done by comparing the amounts of IgG in serum and parallel cerebrospinal fluid (CSF). Although there have been well-described problems with qualitative versus quantitative measurements of abnormal IgG, such as in myeloma paraproteins, similar difficulties are also found with CSF IgG. Methods: Traditional quantitative analysis of IgG by rate nephelometry was followed by separation of the IgG using isoelectric focusing and then either silver stain or immunofixation. Finally, quantitative analysis was performed by scanning densitometry using public domain software downloaded from the National Institutes of Health. Results: We report here the major discrepancies that can occur with CSF IgG between the silver stain versus the IgG stain. Conclusions: We concur with the earlier recommendation that qualitative separation followed by densitometric estimation of enzyme-linked immunofixation is also more useful than simple quantitative nephelometric analysis followed by silver staining in the detection of local synthesis of IgG, analogous to the earlier work on paraproteins.

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Section: Resultsmentioning

confidence: 91%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Discrepancies between qualitative and quantitative analysis of cerebrospinal fluid IgG: densitometric comparison of nephelometry-silver versus immuno-enzymes

Keir

Barrio²,

Thompson

2008

Ann Clin Biochem

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“…IgM was analyzed both in native form and after reduction with 1% 2-mercaptoethanol for 15 min before focusing in order to optimize resolution. After focusing, Igs were transferred to nitrocellulose and immunostained with class-specific antibodies using a double antibody method as previously described (34,37).…”

Section: Methodsmentioning

confidence: 99%

A somatically mutated human antiganglioside IgM antibody that induces experimental neuropathy in mice is encoded by the variable region heavy chain gene, V1-18.

Willison

O’Hanlon²,

Paterson³

et al. 1996

J. Clin. Invest.

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IgM paraproteins associated with autoimmune peripheral neuropathy and anti-Pr cold agglutinins react with sialic acid epitopes present on disialylated gangliosides including GD1b, GT1b, GQ1b, and GD3. A causal relationship between the paraprotein and the neuropathy has never been proven experimentally. From peripheral blood B cells of an affected patient, we have cloned a human hybridoma secreting an antidisialosyl IgM mAb, termed Ha1, that shows identical structural and functional characteristics to its serum counterpart. Variable region analysis shows Ha1 is encoded by the same V H 1 family heavy chain gene, V1-18, as the only other known anti-Pr antibody sequence and is somatically mutated, suggesting that is arose in vivo in response to antigenic stimulation. In the rodent peripheral nervous system, Ha1 immunolocalizes to dorsal root ganglia, motor nerve terminals, muscle spindles, myelinated axons, and nodes of Ranvier. After intraperitoneal injection of affinity-purified antibody into mice for 10 d, electrophysiological recordings from the phrenic nerve-hemidiaphragm preparation demonstrated impairment of nerve excitability and a reduction in quantal release of neurotransmitter. These data unequivocally establish that an antidisialosyl antibody can exert pathophysiological effects on the peripheral nervous system and strongly support the view that the antibody contributes to the associated human disease. ( J. Clin. Invest. 1996. 97:1155-1164.)

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Recommended Standard of Cerebrospinal Fluid Analysis in the Diagnosis of Multiple Sclerosis

Thompson²,

et al. 2005

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Isoelectric Focusing of Cerebrospinal Fluid Immunoglobulin G: An Annotated Update

Abstract: SUMMARY. A revised agarose isoelectric focusing method for detecting oligoclonal IgG in unconcentrated cerebrospinal fluid is presented. The technique is shown to be robust and reproducible and suitable for the detection of intrathecal IgG synthesis.

Cited by 132 publications

References 3 publications

Discrepancies between qualitative and quantitative analysis of cerebrospinal fluid IgG: densitometric comparison of nephelometry-silver versus immuno-enzymes

Discrepancies between qualitative and quantitative analysis of cerebrospinal fluid IgG: densitometric comparison of nephelometry-silver versus immuno-enzymes

A somatically mutated human antiganglioside IgM antibody that induces experimental neuropathy in mice is encoded by the variable region heavy chain gene, V1-18.

Recommended Standard of Cerebrospinal Fluid Analysis in the Diagnosis of Multiple Sclerosis

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