Rapid analysis of immunoglobulin isoelectric focusing patterns with cellulose nitrate sheets and immunoperoxidase staining

Glynn, Paul; Gilbert, Heather; Newcombe, Jia; Cuzner, M. L.

doi:10.1016/0022-1759(82)90264-2

Cited by 40 publications

(8 citation statements)

References 10 publications

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“…Peroxidase-labelled antibody to rat immunoglobulin (Miles-Yeda) was used at a dilution of I: 1,000. Immunostaining was done as described previously by Glynn et al (1982).…”

Section: Polyacrylamide Gel Electrophoresis (Page)mentioning

confidence: 99%

Basic Protein Dissociating from Myelin Membranes at Physiological Ionic Strength and pH Is Cleaved into Three Major Fragments

Glynn

Chantry

Groome

et al. 1987

Journal of Neurochemistry

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Experiments were performed with isolated human myelin membrane preparations to analyse factors that may modulate association of myelin basic protein (MBP) with the membranes and could contribute to demyelinating processes. Transfer of membranes (5 mg protein ml-1) at 37 degrees C and pH 7.4 from a hypotonic medium, in which they were relatively stable, to one of physiological ionic strength produced three major effects: (1) initial dissociation of MBP from the membranes by a nonenzymatic process that was doubled in the presence of millimolar Ca2+/Mg2+; (2) within 10 min, the appearance in the medium of three major MBP fragments (14.4, 10.3, and 8.4 kilodaltons); and (3) progressive acidification of dissociated MBP and its fragments, probably due to deamidation. Between 1 and 6 h a steady state was apparent in which protein equivalent to 15% of the MBP originally bound to the membranes was found in the medium. The three major MBP fragments formed two-thirds of this solubilised material and appeared metabolically stable for 24 h. The kinetics of peptide formation suggested that dissociated, rather than membrane-bound, MBP was cleaved by myelin-associated neutral proteases. Two-dimensional electrophoresis and immunoblotting using two monoclonal antibodies indicated that proteolysis occurred in the vicinity of residues 35 and 75. Evidence was also obtained for removal of C-terminal arginines and relatively rapid deamidation in the C-terminal half of MBP. These modifications of MBP might also occur if extracellular fluid gained access to the compacted cytoplasmic space of the myelin sheath.

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“…Peroxidase-labelled antibody to rat immunoglobulin (Miles-Yeda) was used at a dilution of I: 1,000. Immunostaining was done as described previously by Glynn et al (1982).…”

Section: Polyacrylamide Gel Electrophoresis (Page)mentioning

confidence: 99%

Basic Protein Dissociating from Myelin Membranes at Physiological Ionic Strength and pH Is Cleaved into Three Major Fragments

Glynn

Chantry

Groome

et al. 1987

Journal of Neurochemistry

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“…Immediately after focusing, all proteins were transferred to and immobilized on sheets of nitrocellulose. 24 Proteins reacting with various antisera were then visualized by use of peroxidase-labelled secondary antibodies and the enzyme substrate 3-amino-9-ethylcarbazole.…”

Section: Immunochemical Techniquesmentioning

confidence: 99%

Immunohistochemical characterization of the amyloid deposits and quantitation of pertinent cerebrospinal fluid proteins in hereditary cerebral hemorrhage with amyloidosis.

Löfberg¹,

Grubb²,

Nilsson³

et al. 1987

Stroke

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“…Several papers have previously been published on the role of IEF in the analysis of CSF especially related to MS diagnosis (5,(8)(9)(10)13). Here, we have described a modified protocol which presents evident advantages in comparison with others (9,10,13).…”

Section: Discussionmentioning

confidence: 99%

“…In some of these conditions, it can be particularly useful to determine that synthesis of IgG occurs within the CNS. During the last few years, effort has been focused on the perfection of the determination of OB by increasing the resolution and improving the detection methods (7)(8)(9). Unfortunately, the IgG index represents an insensitive test, which is not efficient, especially for the diagnosis of MS (6).…”

Section: Introductionmentioning

confidence: 99%

Identification of IgG-specific Oligoclonal Banding in Serum and Cerebrospinal Fluid by Isoelectric Focusing: Description of a Simplified Method for the Diagnosis of Neurological Disorders

Marchetti

Gutiérrez²,

Vélia³

et al. 1999

CCLM

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Clinical laboratory procedures using cerebrospinal fluid (CSF) to assist in the diagnosis of multiple sclerosis (MS) are based essentially on the determination of oligoclonal bands (OB) in CSF and not in serum. To date, the techniques using isoelectric focusing (IEF) have represented one of the most efficient methods for detecting supernumerary bands in CSF but unfortunately were not always compatible with a routine use. Here, we describe a revised method of IEF suitable for the diagnosis of neurological disorders. The kit-based technique was simplified and the present procedure allowed an easier execution, reduced the overall analysis time and permitted an uncomplicated intepretation of oligoclonal profiles. Moreover, the technique developed was very sensitive and specific for the diagnosis of MS diagnosis. In conclusion, the IEF protocol described in this report is suitable for OB detection in any laboratory.

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Rapid analysis of immunoglobulin isoelectric focusing patterns with cellulose nitrate sheets and immunoperoxidase staining

Cited by 40 publications

References 10 publications

Basic Protein Dissociating from Myelin Membranes at Physiological Ionic Strength and pH Is Cleaved into Three Major Fragments

Basic Protein Dissociating from Myelin Membranes at Physiological Ionic Strength and pH Is Cleaved into Three Major Fragments

Immunohistochemical characterization of the amyloid deposits and quantitation of pertinent cerebrospinal fluid proteins in hereditary cerebral hemorrhage with amyloidosis.

Identification of IgG-specific Oligoclonal Banding in Serum and Cerebrospinal Fluid by Isoelectric Focusing: Description of a Simplified Method for the Diagnosis of Neurological Disorders

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