Identification of IgG subclasses'' oligoclonal bands in multiple sclerosis CSF

IgG subclasses' oligoclonal bands in unconcentrated CSF from MS patients were detected by isoelectric focusing in agarose gel with subsequent immunoblotting using mouse monoclonal antibodies to human IgG subclasses and double-antibody avidin-biotin-alkaline phosphatase system. All MS CSF showed presence of oligoclonal bands specific to the IgG1 subclass; in addition, several of these samples also had oligoclonal bands specific to IgG3, IgG2, or IgG4, in order of decreasing frequency. Since the CSF of a greater number of MS patients showed oligoclonal bands specific to the IgG1 and IgG3 subclasses, the findings are consistent with those reported in patients with chronic viral infections and autoimmune diseases.     

Identification of light chain type bands in CSF and serum oligoclonal IgG from patients with multiple sclerosis

The light (L) chain types (kappa and lambda) of oligoclonal IgG bands of matching CSF and serum from 10 MS patients were identified in immunofixation after isoelectric focusing in polyacrylamide gel. Each specimen showed 10-15 oligoclonal bands in pH region of 7.5-9.3. In 7 MS CSF and 5 sera a greater number of oligoclonal IgG bands were of kappa (kappa)-type whereas in 3 CSF and 2 sera the majority was of lambda (lambda)-type. In 3 sera a clearcut correlation of bands with either type of L chain was not observed due to diffuse staining background. Only a small number of oligoclonal IgG bands in 7 of 10 CSF and serum pairs had identical isoelectric points and the same type of L chain. The results show that the individual MS patient had oligoclonal IgG bands in serum, differ with respect to number, isoelectric point and L chain type from the oligoclonal IgG profile seen in the patient's CSF.     

Demonstration in children of oligoclonal IgG bands in unconcentrated CSF using agarose isoelectric focusing and immunolabeling

Agarose isoelectric focusing, followed by protein transfer to cellulose nitrate membrane and double-antibody avidin-biotin peroxidase staining (avidin-biotin agarose isoelectric focusing), was used to demonstrate oligoclonal IgG bands in unconcentrated cerebrospinal fluid (CSF) and serum; 161 consecutive pediatric patients, ages 6 months to 16 years with a variety of mainly neurologic disorders, were studied. The procedure was standardized for agarose isoelectric focusing (AIF) using 5 microliter specimens containing 125 ng of IgG. Oligoclonal bands were found in the CSF of 12% of the patients; bands were found simultaneously in the CSF and serum of 10% of the patients, mostly those with nervous system infections, but also those with central nervous system tumors, seizures, or migraine. In about 50% of positive cases, oligoclonal bands constituted the only CSF abnormality, reflecting an abnormal humoral immune response within the CSF-central nervous system compartment. Avidin-biotin AIF can be recommended as an integrated part of routine CSF examinations in children.     

A comparative study of Japanese multiple sclerosis patients with and without oligoclonal IgG bands

The cerebrospinal fluid oligodonal IgG bands (OB) are less frequently observed in Japanese multiple sclerosis (MS) patients compared with Caucasian patients. We studied 40 consecutive Japanese MS patients to investigate the differences in the clinical and magnetic resonance imaging (MRI) features of MS between OB-positive patients and OB-negative ones. Among the 40 patients, 22 (55%) patients were OB-positive by either agarose gel electrophoresis (AGE) or isoelectric focusing (IEF), and 18 (45%) patients were OB-negative by both AGE and IEF. There were differences between the two groups only in the clincal forms of MS, but not in terms of gender, onset age, disease duration, or disease severity. In the OB-negative group, nine (50%) of the patients had the optic-spinal form of MS (OS-MS), but only one patient (4.5%) in the OB-positive group had OS-MS. Although most OB-positive patients showed brain MRI lesions typical of MS, 13 (72%) of the OB-negative patients showed no or few brain MRI lesions and the rest of the OB-negative patients showed atypical MS lesions, such as diffuse white matter lesions or large ring-enhanced lesions. Our results suggest that the majority of OB-negative Japanese MS patents show either no or few brain MRI lesions or atypical brain MRI lesions.     

Detection of oligoclonal IgG bands in unconcentrated CSF by isoelectric focusing in agarose gel and silver staining

We analyzed 102 unconcentrated cerebrospinal fluids from a variety of neurologic diseases for oligoclonal IgG bands by isoelectric focusing in agarose gel followed by staining with silver. Ten to 12 microliter of cerebrospinal fluid containing 0.4-0.8 microgram of IgG was found to be optimum. Cerebrospinal fluid from 38 of 40 patients with clinically definite multiple sclerosis, 6 of 9 with suspected multiple sclerosis and 8 of 53 patients with other neurologic diseases had oligoclonal IgG bands by IEF in agarose followed by immunofixation. The commercial system employed here is a simple sensitive and rapid method for detection of oligoclonal bands in unconcentrated cerebrospinal fluid of patients with multiple sclerosis.     

Detection of oligoclonal IgG bands in unconcentrated CSF in multiple sclerosis and other neurological diseases by isoelectric focusing on ultrathin-layer polyacrylamide gel immunofixation and silver staining

Isoelectric focusing of proteins (IEF) in ultrathin-layer polyacrylamide gel (0.4 mm, PAG), followed by direct immunofixation with monospecific antiserum and silver staining, is a highly specific, sensitive and simple method for the demonstration of oligoclonal IgG in unconcentrated cerebrospinal fluid (CSF) samples (5-10 microliters). For the present method, the optimal concentrations of IgG in CSF samples are about 0.025-0.030 g/l, corresponding to the applied amount of 125-150 mg. In our testing of this method, oligoclonal IgG bands in CSF specimens were clearly demonstrated in 52 (96%) of 54 patients with clinically established definite diagnosis of multiple sclerosis (MS), in 4 (40%) of 10 patients with infectious diseases of the CNS, and in 9 patients (25%) of 38 with other neurological diseases. Abnormal patterns were also demonstrated in the serum of patients with MS (43%). Intrathecally synthesized IgG was mathematically calculated in 43 (80%) out of 54 patients with MS. This method appears to be a useful alternative for the demonstration of oligoclonal IgG bands in the unconcentrated CSF, especially when questionable or negative results arise by routine electrophoretic technique for oligoclonal bands detection.     

Detection of oligoclonal bands in unconcentrated CSF: isoelectric focusing and silver staining

We have developed a method to detect oligoclonal IgG bands in unconcentrated CSF from patients with MS or guinea pigs with chronic relapsing experimental allergic encephalomyelitis, by isoelectric focusing (IEF) in polyacrylamide gel and silver staining. Five to 10 microliters of CSF was sufficient. The bands were identified as IgG in immunofixation after IEF.     

Cerebrospinal fluid IgG profiles and oligoclonal bands in Chinese patients with multiple sclerosis

OBJECTIVES: To investigate the significance of oligoclonal bands (OCBs) and intrathecal IgG fractions (IgGIF) for the diagnosis of multiple sclerosis (MS) in northern China. MATERIALS AND METHODS: OCBs in cerebrospinal fluid from 30 patients with MS, 34 with other inflammatory neurological diseases (IND) and 22 with non-inflammatory neurological diseases (NIND) were detected using isoelectric focusing. IgGIF was calculated based on corresponding formula. RESULTS: There was no significant difference in the frequencies of positive OCBs and elevated IgGIF between the MS group and the IND group. Compared with NIND, the MS and IND groups had a significantly higher incidence of OCBs and elevated IgGIF. The sensitivity, specificity and positive result likelihood ratio of OCBs for the diagnosis of MS were 63.3%, 74.2% and 2.5 respectively; those of IgGIF were 36.7%, 84.5% and 2.4. CONCLUSIONS: The two parameters, OCBs and IgGIF are of less diagnostic value for MS in China.     

Light chain composition of CSF oligoclonal IgG bands in multiple sclerosis and subacute sclerosing penecephalitis

The light chain composition of MS and SSPE CSF oligoclonal IgG bands was examined using isoelectric focusing and sensitive peroxidase-anteperoxidase (PAP) staining technique specific for gamma heavy chains (γ), kappa light chains (κ), or lambda light chains (λ) and a radioimmunoassay (RIA) for γ, κ, or λ. Many bands in the 7 MS and 4 SSPE CSF examined were monoclonal, staining for either IgG-κ or IgG-λ. By staining, all MS CSF were κ predominant; SSPE CSF were variously κ orλ predominant. RIA confirmed the κ predominance of MS CSF. Three MS and 2 SSPE CSF contained bands staining for λ alone, i.e. free light chains. Analysis of RIA data confirmed these findings in 2 MS cases. The difference in light chain predominance of MS and SSPE CSF may reflect differences in the antigenic target, or the age of patient at the time when band-synthesizing clones are triggered. Six of 7 MS and all 4 SSPE CSF contained oligoclonal bands staining for γ and for both κ and λ, probably representing artifacts of IEF. No predominant immunochemical differences between bands in MS and SSPE were detected.     

MR peri‐CSF lesions and CSF oligoclonal bands in Italian multiple sclerosis patients

Objectives – We investigated the association between brain lesion distribution and the presence of oligoclonal IgG bands (OCBs) in Italian multiple sclerosis (MS) patients. Materials and methods – We retrospectively selected brain magnetic resonance imaging (MRI) uniformly performed in 56 relapsing patients (41 patients OCB positive). Results – Brain lesions in periventricular areas occurred in 92.86% of the patients (100% OCB+ and 73.33% OCB?) (P = 0.004), but we did not find a significant difference for their median volume (P = 0.553) and median number (P = 0.606) between the two groups. Parenchymal lesions occurred in 76.8% of the patients with a similar distribution (P = 1.00) and no significant difference in the median volume (P = 0.818) and number (P = 0.643) between the two groups. Conclusions – The present study on cohort of Italian MS patients demonstrated a lack of correlation between lesion distribution and OCBs, suggesting that B cells producing them could be localized both in meningeal niches and cerebral parenchyma.     

CSF protein examinations with thin-layer isoelectric focusing in multiple sclerosis.

Thin-layer IEF, due to its extremely high resolving capacity, has been found to be quite valuable for CSF protein examinations, one important advantage of the technique being its excellent capacity for separation of immunoglobulins. The CSF and serum proteins of 230 patients with clinically verified or probable MS and 20 subjects with optic neuritis were examined with thin-layer IEF and the findings were compared with clinical data and results of other CSF examinations. All but 3 of the MS patients and about two thirds of the subjects with optic neuritis inhibited one or combinations of different CSF protein aberrations in the acidic and alkaline range. Oligoclonal bands and/or regional increases of Ig fractions, changes compatible with intrathecal Ig synthesis, were detected in respectively 95 and 80% of patients with clinically verified and probable MS and 30% of subjects with optic neuritis. Other aberrant CSF protein fractions (including transferrin, the taufraction and gamma-trace protein) were found in about half of the cases; some of these fractions had the highest occurrence in patients with the most extensive Ig abnormalities. The diverse CSF protein aberrations seemed to be influenced by the duration and course of the disorder as well as the probable sites of lesions; further factors might be the release of decomposition products from destroyed tissues, the genetically determined reactivity of the individual and the presence of possible agents.     

Absence of oligoclonal IgA in CSF and serum of multiple sclerosis patients

CSF and serum from patients with MS and other neurologic diseases were analyzed by isoelectric focusing (IEF) in polyacrylamide gel and immunofixation with specific anti human IgA sera. The IgA band patterns seen in pH 4.5-6.0 region were rather diffuse and lacked oligoclonal feature.     

Discontinuous distribution of IgG oligoclonal bands in cerebrospinal fluid from multiple sclerosis patients

To test the effect of sampling on the detection of immunoglobulin (Ig) cerebrospinal fluid (CSF) abnormalities, we analyzed the first and last 1 ml fraction of 10 ml obtained during a single CSF removal from 27 multiple sclerosis (MS) patients and six patients with other neurological diseases. IgG index, hyperbolic function, and IgG synthesis rate decreased between the first and the last CSF aliquot. Discordant results were found in 4/27 (15%) MS patients. In 2/27 (7.5%) clinically definite MS patients, the number of CSF oligoclonal bands (OCB) decreased between the first and the last fraction. In one of the two patients, the three OCB visualized in the first fraction were not found in the last. We conclude that fractionated sampling may partially account for the absence of OCB in the CSF of some definite MS patients.     

Discontinuos distribution of IgG oligoclonal bands in cerebrospinal fluid from multiple sclerosis patients

To test the effect of sampling on the detection of immunoglobulin (Ig) cerebrospinal fluid (CSF) abnormalities, we analyzed the first and last 1 ml fraction of 10 ml obtained during a single CSF removal from 27 multiple sclerosis (MS) patients and six patients with other neurological diseases. IgG index, hyperbolic function, and IgG synthesis rate decreased between the first and the last CSF aliquot. Discordant results were found in (15%) MS patients. In (7.5%) clinically definite MS patients, the number of CSF oligoclonal bands (OCB) decreased between the first and the last fraction. In one of the two patients, the three OCB visualized in the first fraction were not found in the last. We conclude that fractionated sampling may partially account for the absence of OCB in the CSF of some definite MS patients.     

Oligoclonal IgG bands in Japanese patients with multiple sclerosis. A comparative study between isoelectric focusing with IgG immunofixation and high-resolution agarose gel electrophoresis

A low prevalence of oligoclonal IgG bands (OB) has been reported as a unique feature of Japanese multiple sclerosis (MS) as compared with Western MS. We compared the frequency of OB between isoelectric focusing (IEF) and agarose gel electrophoresis (AGE) in 59 Japanese patients with clinically definite MS [39 with conventional form of MS (CMS) and 20 with optic-spinal form of MS (OSMS)]. The frequency of IEF-OB in total MS was 54%, whereas that of AGE-OB was only 17%. When OSMS was excluded, the frequency increased to 77% by IEF, whereas it remained 23% by AGE. Our study strongly suggests that IEF is highly effective for detecting OB in Japanese MS patients.     

Detection of IgG oligoclonal bands in unconcentrated CSF by means of agarose isoelectric focusing, double immunofixation peroxidase staining and avidin-biotin amplification

To detect immunoglobulin G (IgG) oligoclonal bands in unconcentrated cerebrospinal fluid (CSF) we used a recently developed method combining agarose isoelectric focusing (IEF) and double immunofixation peroxidase staining with Avidin-Biotin amplification. We studied 65 CSF and serum paired specimens from normals, multiple sclerosis (MS), other neurological diseases (OND) and benign monoclonal gammopathies (BMG). We found that the oligoclonal IgG pattern can be demonstrated after IEF of 15 l of CSF specimens with an IgG concentration of 15 mg/L. In 98% of CSF from patients with clinically definite MS a sharp oligoclonal band pattern was detected. The reliability and the sensitivity of this powerful technique is compared to agarose IEF of concentrated CSF, followed by Coomassie Brilliant Blue staining.This method constitutes a real improvement in the detection of CSF IgG oligoclonal bands because it avoids CSF concentration and allows the detection of IgG bands only.

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Sommario Abbiamo adottato la focalizzazione isoelettrica in agarosio seguita da doppia immunofissazione, amplificazione con Avidina-Biotina e colorazione con la perossidasi per evidenziare le bande oligoclonali nel liquido cerebrospinale (LCS) non concentrato. Sono stati studiati 65 campioni di LCS e del rispettivo siero prelevati da soggetti normali, con Sclerosi Multipla (SM), altre malattie neurologiche e gammopatie monoclonali benigne. Abbiamo riscontrato che le bande oligoclonali possono essere evidenziate in soli 15 l di LCS non concentrato con una concentrazione di 15 mg/L.Le bande oligoclonali sono chiaramente presenti nel 98% di campioni di LCS prelevati da pazienti affetti da SM clinicamente accertata. Nel nostro studio abbiamo paragonato l'attendibilità e la sensibilità di questa metodica a quella della focalizzazione isoelettrica in agarosio di LCS concentrato, seguita da colorazione con Coomassie Brilliant Blue. Tale metodica rappresenta un reale miglioramento nell'evidenziare delle bande oligoclonali in quanto evita la concentrazione del LCS e consente l'identificazione esclusivamente di bande IgG.

     

CSF oligoclonal bands, MRI, and the diagnosis of multiple sclerosis

In this retrospective study, the results from investigations (MRI, evoked potentials, alkaline oligoclonal bands [OBs] in CSF) in 94 patients with clinical suspicion of demyelinative disease were evaluated to assess their impact on diagnosis. Forty-three patients were diagnosed as having definite MS, 10 probable MS, and 9 possible MS. MRI findings strongly suggestive of MS were evident in 52/62 (84%) patients, while 47/62 (76%) patients demonstrated OBs in their CSF. In 63% of patients both abnormalities were present. Patients with no OBs in their CSF were on the average older, were more often male, had experienced their first symptoms at a later age, and suffered more often from the chronic-progressive form of the disease than those with a positive CSF finding.     

寡克隆区带和IgG指数在多发性硬化中的诊断意义

目的研究寡克隆区带（OCBs）和IgG指数（IgGI）对多发性硬化（MS）诊断的敏感性及其影响因素。方法用等电聚焦结合银染色法检测30例MS、40例神经系统炎性疾病（NID）和22例神经系统非炎性疾病（NNID）患者CSF中OCBs，并计算IgG I。结果MS组和NID组比较OCBs阳性率、IgG I异常率均无显著性差异（P〉0．05）；MS组、NID组与NNID组比较。差异均有显著性（P〈0．05）；传统型MS和脊髓型MS比较，差异均无显著性（P〉0．05）。OCBs对MS诊断的敏感性、特异性和阳性结果似然比分别为63．3％、77．7％和2．8；IgG I分别为40．0％、76．7％和1．7。结论本地区MSOCBs阳性率和IgG I异常率较低，可能与遗传背景、疾病类型和药物应用有关，OCBs和IgG I对MS诊断具有相对特异性。