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.     

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.

     

Antinuclear antibodies positivity is not rare during multiple sclerosis and is associated with relapsing status and IgG oligoclonal bands positivity

IntroductionAs an immune-mediated disease of the central nervous system, multifaceted aspects of a humoral immune response are widely described during multiple sclerosis (MS). However, the prevalence of different auto-antibodies, such as antinuclear antibodies (ANA), during MS is very variable and their clinical relevance remains controversial. Our aim was to evaluate the prevalence and clinical correlations of ANA positivity in South Tunisian MS patients.Material and methodsWe performed ANA screening using indirect immunofluorescence (IIF) on HEp-2 cells (Biosystems®) in 82 MS patients. For ANA positive samples (titer ≥1/160), anti-ds-DNA detection (IIF on Crithidia luciliae (Biosystems®)) and extractable nuclear antigen typing (immunodot (Euroimmun®)) were performed.ResultsANA were positive in 35/82 MS patients (42.7%). The titer was ≥ 1/320 in 16/35 patients. The antigenic specificity of ANA was identified in 7/35 patients. None of the patients had extra-neurological manifestations. No correlation was found between ANA and age, gender, MS course, disease duration, disability, annual relapse rate nor IgG index. ANA positivity was more frequent in patients with IgG oligoclonal bands (OCB) (47.1%) than in patients without IgG OCB (16,6%) (p = 0.049). Regarding disease activity, ANA positivity was significantly more frequent in patients with relapse (52.6%) than in patients in remission (25.9%) (p = 0.031).ConclusionOur results showed that ANA positivity in MS disease is not rare. This positivity was not associated with clinical expression of any connective tissue disease. ANA occurrence in MS was associated with IgG OCB+ profile and relapsing status, probably reflecting an ongoing immune dysregulation.     

寡克隆带和IgG鞘内合成率对多发性硬化的诊断价值

目的探讨寡克隆带(OCBs)和IgG鞘内合成率(IgGSyn)对多发性硬化(MS)诊断的敏感性、特异性,以及定性和定量指标的相关性。方法选取30例MS(MS组)、40例神经系统炎性疾病(NID组)和22例神经系统非炎性疾病(NNID组)患者,应用速率散射比浊法测定血清和脑脊液(CSF)中免疫球蛋白G(IgG)、白蛋白(Alb)水平,等电聚焦结合银染色法检测CSF中OCBs,计算IgGSyn,并对其敏感性、特异性和阳性结果似然比(PRLR)进行分析。结果OCBs阳性率和IgGSyn异常率MS组与NID组比较差异无显著性;MS组、NID组与NNID组比较差异有极显著性(均P<0.01)。MS组和NID组中,OCBs阳性者与阴性者IgGSyn值差异无显著性。对MS诊断的敏感性、特异性和PRLR,OCBs分别为63.3%、77.7%和2.8;IgGSyn为46.7%、75.2%和1.9。结论OCBs和IgGSyn检测结果的不完全一致性提示中枢神经系统内存在不同的体液免疫反应机制,综合分析OCBs和IgGSyn,对MS诊断具有参考价值。     

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.     

多发性硬化脑脊液寡克隆区带及24小时鞘内IgG合成率检测的比较

采取垂直平板聚丙烯酰胺凝胶电泳法、火箭免疫电泳法对30例多发性硬化病人进行脑脊液寡克隆区带及24小时鞘内IgG合成率的检测,结果发现寡克隆区带、合成率与多发性硬化的发病年龄、病程、病程进展类型、病残程度无统计学上的相关性,寡克隆区带阳性率46.7％,合成率增高占60％,两者合计异常率可达73.3％,提示我国多发性硬化寡克隆区带阳性率、合成率增高均较国外低,因而在我国对多发性硬化的实验室诊断两者同时检测更有意义。     

Analysis of Chlamydia pneumoniae-specific oligoclonal bands in multiple sclerosis and other neurologic diseases

OBJECTIVE: Paired serum and cerebrospinal fluid (CSF) specimens were investigated for Chlamydia pneumoniae-specific oligoclonal bands (OCBs) to determine band specificity in multiple sclerosis (MS). MATERIAL AND METHODS: Serum and CSF samples were collected from patients with relapsing-remitting MS (n = 56), other inflammatory (n = 18) or non-inflammatory (n = 15) neurologic diseases, and from 10 healthy controls. OCBs were determined with affinity immunoblotting of C. pneumoniae-specific IgG onto antigen-coated nitrocellulose paper after protein separation with agarose isoelectric focusing. RESULTS: Chlamydia pneumoniae-specific OCBs were present in 5 of 56 patients with MS, and in 3 of 18 patients with other inflammatory neurologic diseases. CONCLUSIONS: The intrathecal production of C. pneumoniae-specific oligoclonal IgG occurs in a minority of patients with MS. This intrathecal anti-C. pneumoniae reactivity is likely part of a polyspecific humoral immune response in MS.     

IgG ratios and oligoclonal IgG in multiple sclerosis and other neurological disorders

The findings are reported of various CSF abnormalities, including IgG indices and oligoclonal IgG, in 160 patients with multiple sclerosis of differing diagnostic certainty and 146 patients with other neurological disorders.

An abnormal IgG index, defined as the ratio of IgG/albumin in CSF to that in serum, has been found in 77.7% of definite MS cases, falling to a figure of 32.1% in the single lesion group. A tendency, reported previously, for IgG levels to be higher in disabled patients, particularly those with a short history or early onset, has been confirmed.

Oligoclonal IgG, on the other hand, has been found in 56% of definite MS cases, less frequently than in most other reported series. Analysis of the literature suggests considerable variability in the finding of oligoclonal IgG in other than definite MS, and in other neurological disorders. The possibility that subjective factors are partly responsible for this variability, rather than discrepancies in patient selection requires consideration, and suggests that CSF electrophoresis and IgG estimations are complementary aids in the diagnosis of multiple sclerosis.

Differences have been expressed regarding the relationship of oligoclonal IgG to clinical parameters of the disease. Further sequential analysis of the development and variability of the oligoclonal pattern in MS is required.     

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。     

CFS Oligoclonal bands in MS and other neurological diseases detected by agarose isoelectric focusing and electrophoresis

Agarose gel isoelectric focusing (IEF) and electrophoresis (EF) were compared for detection of CSF oligoclonal bands in multiple sclerosis (MS) and other neurological diseases. The CSF IgG/albumin ratio, the IgG-index and the IgG synthesis per day in the CNS were also considered. IEF was highly sensitive, revealing oligoclonal bands in 100% of CSF from 40 clinically definite MS, while EF had a sensitivity of 70%. In 90 patients with other neurological diseases, IEF revealed oligoclonal bands in 25.5%, EF in 11.1%. The IgG-index was the most sensitive of the quantitative parameters.

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Sommario La focalizzazione isoelettrica in agarosio e l'elettroforesi del liquor permettono di evidenziare bande oligoclonali in pazienti affetti da sclerosi multipla e da altre malattie neurologiche. Sono stati pure valutati il rapporto IgG/albumina liquorale, l'indice di Link e la sintesi intratecale giornaliera di IgG secondo la formula di Tourtellotte. La tecnica della focalizzazione si è dimostrata altamente sensibile, evidenziando bande oligoclonali liquorali nel 100% di 40 sclerosi multiple clinicamente definite, mentre l'elettroforesi ha dimostrato una positività soltanto nel 70% dei casi. In 90 casi comprendenti varie malattie neurologiche, la focalizzazione ha rilevato bande oligoclonali nel 25.5%, l'elettroforesi nell'11.1%. Il parametro quantitativo più sensibile è risultato l'indice di Link.

     

Agarose isoelectric focusing, antiserum immunofixation and silver staining for detection of oligoclonal bands in unconcentrated cerebrospinal fluid

Unconcentrated cerebrospinal fluid (CSF) and corresponding serum from 30 patients with multiple sclerosis (MS), 30 with other neurological disease and 30 controls suffering from tension headache or psychoneurosis, were examined for oligoclonal IgG bands by initial separation employing agarose isoelectric focusing (AIF) followed by a modified procedure of immunofixation with monospecific antiserum and silver staining. This method is specific for demonstration of IgG and has a limit for detection of 0.4 microgram of IgG. Comparing the results with those obtained by AIF followed by capillary blotting to nitrocellulose membrane, double antibody peroxidase labeling and avidin-biotin amplification, both methods revealed similar frequencies of positive findings for oligoclonal IgG bands in the three patient groups. AIF followed by antiserum immunofixation and silver staining is a simple, sensitive and specific method for detection of oligoclonal IgG in unconcentrated CSF.     

Correlation between the IgG index, oligoclonal bands in CSF, and the diagnosis of demyelinating diseases

Intrathecal immunoglobulin synthesis can be assessed by different approaches. It can be measured quantitatively by the IgG index or qualitatively by isoelectric focusing (IEF) to detect oligoclonal bands (OCB). In this study we investigated if there is a correlation between the frequency of OCB and the IgG index and if the IgG index predicts the diagnosis of a demyelinating CNS disease (DMD). We found a positive correlation between the IgG index and the frequency of OCB as well as the probability of DMD. We conclude that quantitative assessment of CSF-IgG can be useful because it is easier and quicker to perform than IEF but cannot replace IEF in general because this is the most sensitive method to detect abnormal IgG in CSF.     

Cerebrospinal fluid oligoclonal bands and progression of disability in multiple sclerosis

Antibody-mediated inflammation is believed to contribute to tissue injury in multiple sclerosis (MS). The majority of patients with MS have oligoclonal bands (OCB), corresponding to antibodies against a variety of antigens, in their cerebrospinal fluid (CSF). The relation of CSF OCB and disease progression in MS is uncertain. To investigate whether there is a relation between CSF OCB and a more aggressive disease course of MS, 143 patients with definite MS according to the Poser diagnostic criteria and CSF analysis at time of diagnosis were followed over a period of 5 years. There were no differences in presence or number of CSF OCB between patients with significant worsening of disability and stable patients. There were no differences in presence or number of CSF OCB between patients with stable relapsing-remitting MS and patients developing secondary progression during follow-up. The presence or number of CSF OCB does not seem to influence early disease progression in MS.     

寡克隆区带和IgG指数对多发性硬化的诊断价值

目的　探讨寡克隆区带 (OCB)和IgG指数对多发性硬化 (MS)诊断的敏感性及特异性。方法　收集 4 8例MS、6 8例神经系统炎性疾病 (NID)及 110例非炎性疾病 (NNID) 3组患者的脑脊液 (CSF)和血清标本 ,分别进行OCB的检测 (等电聚焦 )和IgG指数的计算。并对其阳性结果似然比 (PRLR)进行分析。结果　MS组与NID组比较 ,CSF中OCB阳性率和IgG指数异常率的差异均没有显著性 (均P >0 0 5 ) ;但MS组、NID组与NNID组比较 ,差异均有极显著性 (均P <0 0 0 0 1)。MS组CSF中OCB和IgG指数的敏感性分别为 39 6 %、6 0 4 % ;特异性分别为 80 3%、72 1% ;PRLR分别为 2 0、2 2。当用于判断有无IgG鞘内合成时 ,特异性分别为 97 2 %、92 7% ;PRLR分别为 13 5、7 3。结论　CSF中OCB阳性和IgG指数升高强烈提示有中枢神经系统局部IgG合成 ,对MS有一定的辅助诊断价值     

Optic neuritis: oligoclonal bands increase the risk of multiple sclerosis

ABSTRACT- In 1974 we examined 30 patients 0.5–14 (mean 5) years after acute unilateral optic neuritis (ON), when no clinical signs of multiple sclerosis (MS) were discernable. 11 of the patients had oligoclonal bands in the cerebrospinal fluid (CSF). Re-examination after an additional 6 years revealed that 9 of the 11 ON patients with oligoclonal bands (but only 1 of the 19 without this CSF abnormality) had developed MS. The occurrence of oligoclonal bands in CSF in a patient with ON is - within the limits of the present observation time - accompanied by a significantly increased risk of the future development of MS. Recurrent ON also occurred significantly more often in those ON patients who later developed MS.     

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.     

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.     

Study of the IgG oligoclonal bands in cerebral spinal fluid in multiple sclerosis by electroimmunofixation

22 MS CSFs and 20 control CSFs were studied using an electroimmunofixation (EIF) technique on Cellulose Acetate strips. The oligoclonal gammaglobulin bands on MS CSFs were composed almost exclusively of complete IgG molecules. In a few MS CSFs bands composed of free heavy chains and of free light chains were observed. The bands were very often (93 percent) found to be homogeneous as far as the light chain type was concerned. In such homogeneous bands the light chain type most frequently observed was Kappa (59 percent). In 5 MS cases only Kappa type bands were detected, while a CSF with Lambda type bands only was never observed. On control CSFs, clear cut IgG oligoclonal bands were observed only in a case of subacute sclerosing panencephalitis (SSPE).

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Sommario 22 liquor da pazienti con Sclerosi Multipla (SM) e 20 di controllo sono stati studiati usando l'elettroimmunofissazione su striscie di acetato di cellulosa. Le bande oligoclonali dei liquor con SM sono risultate composte quasi esclusivamente da molecole complete di IgG. In alcuni casi di liquor da SM sono state osservate bande composte da catene leggere o da catene pesanti libere. Le bande sono state trovate quasi sempre (93% dei casi) omogenee riguardo alla camposizione in catene leggere. In tali bande omogenee il tipo di catena leggera osservato con maggiore frequenza è stato il tipo Kappa (59%). In 5 casi di SM sono state osservate solo bande con catene di tipo Kappa, mentre casi con bande solo di tipo Lambda non sono mai stati osservati. Net liquor di controllo, chiare bande oligoclonali composte da IgG sono state osservate solo nel caso di panencefalite sclerosante subacuta.

     

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.