Intrathecal synthesis of free immunoglobulin light chains in multiple sclerosis

Objective – The detection of oligoclonal immunoglobulin free light chains (FLC) in the diagnosis of multiple sclerosis (MS) was compared to IgG isoelectric focusing. Material and methods – Cerebrospinal fluid and serum samples from 69 patients with possible first attacks of MS, 50 patients with clinically definite MS (CDMS), and 118 patients with other neurological diseases (OND) were analyzed. IgG and FLC oligoclonal bands were detected by isoelectric focusing and immunoperoxidase staining. Results – Intrathecal synthesis of IgG, kappa FLC, and lambda FLC oligoclonal bands, respectively, was seen in 92%, 92%, and 86% of MS patients; in 61%, 62%, and 64% of patients with possible first attacks of MS; and in 3%, 3%, and 8% of the patients with OND. In control patients without IgG synthesis intrathecal lambda FLC synthesis was more common than kappa FLC synthesis ( P =0.03). Conclusion – Kappa FLC detection proved as useful as IgG analysis for the laboratory diagnosis of MS whereas the presence of intrathecal lambda FLC synthesis was less specific.     

Immunoglobulin class and light chain type of oligoclonal bands in CSF in multiple sclerosis determined by agarose gel electrophoresis and immunofixation.

Agarose gel electrophoresis and immunofixation of CSF and serum from 39 patients with multiple sclerosis (MS) revealed oligoclonal IgG in the CSF in all cases and oligoclonal IgA and IgM in 1 patient each. IgG kappa bands only were found in 10 patients, while no patient had IgG lambda bands alone. IgG kappa bands predominated in 20 patients and IgG lambda bands in 5, while 4 patients had the same number of IgG kappa and IgG lambda bands. Twenty-seven patients also displayed IgG bands with kappa and lambda present simultaneously. Bands of free lambda chains were found in 7 patients, while free kappa chain bands were not seen. One or 2 faint IgG bands in 4 patients constituted the only serum abnormality. In 4 additional MS patients selected on the basis of normal findings on agarose gel electrophoresis of the CSF, immunofixation did not reveal oligoclonal Ig, while isoelectric focusing showed bands in 1. Immunofixation is recommended for proving the presence of oligoclonal Ig in CSF and for characterizing oligoclonal Ig into classes and types of light chains.     

Detection of oligoclonal free kappa chains in the absence of oligoclonal IgG in the CSF of patients with suspected multiple sclerosis

BACKGROUND: Oligoclonal free kappa bands are present as frequently as oligoclonal IgG bands in the cerebrospinal fluid (CSF) from patients with definite multiple sclerosis (MS) and can even occur in the absence of oligoclonal IgG. As such, they too are markers of an ongoing intrathecal immune process. OBJECTIVES: To determine how frequently oligoclonal free kappa bands are detectable in the CSF from patients with clinical signs and symptoms suggestive of MS in the absence of CSF restricted oligoclonal IgG. METHODS: An immunoaffinity mediated immunoblotting technique specific for free kappa chains was used, after isoelectric focusing of paired CSF and serum samples from 33 patients with clinical signs and symptoms suggestive of MS but without CSF oligoclonal IgG. CSF data were correlated with MRI results in the context of the new diagnostic criteria from McDonald et al. RESULTS: Eighteen CSF samples contained oligoclonal free kappa bands (54%), mainly from patients with motor dysfunction (83%) and optic neuritis (64%). All patients with a positive MRI according to Barkhof's criteria (n = 6) had free kappa bands in their CSF. CONCLUSIONS: (1) Oligoclonal free kappa bands in the CSF are related to the dissemination of MS lesions; (2) such bands should be looked for in oligoclonal IgG negative CSF, and (3) the presence of free kappa bands in the CSF may be a substitute for oligoclonal IgG in the McDonald's criteria for diagnosis of MS.     

Oligoclonal immunoglobulin bands in cerebrospinal fluid of patients with Alzheimer''s disease and vascular dementia

We examined serum and cerebrospinal fluid (CSF) of 16 patients with Alzheimer's disease (AD), 28 patients with vascular dementia (VD), their age-matched controls and multiple sclerosis (MS) patients in order to evaluate the humoral immune response within the central nervous system both quantitatively and qualitatively. Intra-blood-brain barrier (BBB) protein synthesis was calculated by CSF IgG index. The presence of oligoclonal banding (OCB) was investigated with agarose isoelectric focusing (IEF) followed by immunoblotting with antihuman IgG. No patient with AD and only 4 patients with VD had slightly elevated IgG indexes, and no statistically significant differences in the indexes were found between the two groups. No bands were found in the CSF of AD patients but 3 VD patients had OCB in both serum and CSF. One VD patient had bands in serum but no bands in CSF. No kappa or lambda free light chains were found in those demented patients with demonstrable bands in the CSF and serum. No OCB were found in control sera and CSF. For comparison, the majority of patients with MS had OCB in CSF. Thus, no consistent increase of intrathecal protein synthesis was found in patients with AD and VD. Methodological differences explain at least part of the conflicting results published earlier.     

Intrathecal synthesis of free immunoglobulin light chains and IgM in initial multiple sclerosis

We studied the intrathecal synthesis of free kappa, free lambda immunoglobulin light chains and of IgM in 33 consecutive patients with possible or probable MS at the time of their first diagnosis. Nineteen patients presented free kappa or lambda light chain bands in CSF after agarose isoelectric focusing, protein transfer to nitrocellulose and immunostaining with specific antisera. Nineteen patients had increased CSF levels of free kappa light chains as measured with a competitive ELISA. Fourteen had an increased IgM index, as evaluated with a sandwich ELISA. Twenty-six patients displayed CSF oligoclonal IgG bands in CSF and 25 had cerebral magnetic resonance imaging lesions suggestive of MS. The local production of free immunoglobulin light chains and IgM is often detected in the CSF of patients with early MS.     

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.     

Multiple sclerosis: clinical and laboratorial correlation

The clinical and demographic characteristics of 86 Brazilian patients with clinically definite multiple sclerosis (MS) were compared to the cerebrospinal fluid (CSF) findings. The disease course was relapsing-remitting in 71% and chronic progressive in 29% of the cases. The IgG index was increased in 76% in the chronic progressive status and 46% and 49% during the bout and remission, respectively (p < 0.005). Only 36% of the MS patients using corticosteroids had increased IgG index, in comparison to the 64% of the patients without immunosupressive treatment. Oligoclonal IgG bands were detected in the CSF of 77% and 88% of the MS corticosteroids users and non-users, respectively. The quantitative study of intrathecal synthesis of IgG contributes to demonstrate the immunological differences between the two forms of MS, the relapsing-remitting and the chronic progressive. The treatment with corticosteroids decreases quantitatively the intrathecal synthesis of IgG but not the presence of oligoclonal bands.     

Predominance of oligoclonal IgG type lambda in CSF in aseptic meningitis.

Oligoclonal immunoglobulins (Ig) demonstrble as bands by agarose-gel electrophoresis of CSF were characterized in ten patients with aseptic meningitis. Oligoclonal CSF IgG was found in all patients, IgM was found in two. The oligoclonal Ig was more often of lambda light-chain type than of kappa type. In four patients, oligoclonal Ig bands contained kappa and lambda light chains simultaneously. In two patients, bands consisting of free lambda light chains were found. On repeated investigations more than one year later in three cases, the oligoclonal IgG was of the same light-chain type. The immune response within the CNS in aseptic meningitis seems to be dominated by synthesis of IgG with lambda light chains.     

The cerebrospinal fluid in multiple sclerosis. Quantitative assessment of intrathecal immunoglobulin synthesis by empirical formulae

Intrathecal synthesis of IgG occurs in more than 90% of patients with clinically definite multiple sclerosis. The prevalence and significance of intrathecal synthesis of IgA and IgM are, however, less thoroughly characterized. We estimated intrathecal synthesis of IgG, IgA and IgM with various empirical formulae. The concentrations of albumin, IgG, IgA and IgM and the presence of IgG oligoclonal bands were determined in CSF and serum from 350 patients, including 97 with clinically definite multiple sclerosis. Intrathecal synthesis of IgG oligoclonal bands was detected in 95% of patients with multiple sclerosis (95% confidence interval 88–98%). The IgG-index, an extended IgG-index, and a hyperbolic IgG formula performed approximately equally in identifying patients with MS, but they were all inferior to the detection of IgG oligoclonal bands. In quantitative measurements, the extended immunoglobulin indices appeared to perform well; studies comparing the extended IgA- and IgM-indices to qualitative analyses (electrophoresis or isoelectric focusing) are, however, needed to confirm this. Detection of intrathecal synthesis of IgA of IgM was of little value in the diagnosis of multiple sclerosis.     

Oligoclonal free kappa and lambda bands in the cerebrospinal fluid of patients with multiple sclerosis and other neurological diseases. An immunoaffinity-mediated capillary blot study.

We describe an affinity-mediated capillary blotting technique for the detection of free kappa or lambda light chains in native cerebrospinal fluid (CSF) after isoelectric focusing in agarose gel. Interferences by light chains bound to immunoglobulins were carefully excluded. An absolute amount of 20-50 ng of free kappa or lambda Bence-Jones proteins were detectable by this method, under the form of several discrete bands with isoelectric points between 5 and 8.5. No free light chains were observed in CSF and sera from patients without neurological disorders (n = 26). Such bands were present in most CSF samples in the case of central nervous system (CNS) infections, except in aseptic meningitis. In a group of 48 multiple sclerosis (MS) patients, 44 (92%) displayed oligoclonal free kappa bands restricted to the CSF; oligoclonal IgG bands were observed in 40 cases, and oligoclonal free lambda bands in 33. In this group, the presence of CSF free light chain bands was highly correlated with their absolute levels (p less than 0.001). In other neurological diseases (n = 44), oligoclonal free kappa and free lambda bands were detected much more rarely, in seven (16%) and four (9%) cases respectively. Surprisingly, the CSF from three unrelated patients with Huntington's disease (out of five tested) contained both oligoclonal IgG and free kappa bands.     

Cerebrospinal fluid immunoelectrophoresis in multiple sclerosis

In 95 patients with definite multiple sclerosis (MS) cerebrospinal fluid (CSF) cytomorphology, protein electrophoresis and immunoelectrophoresis using antisera to human serum, Fab fragments of immunoglobulin G(IgG) and to kappa and lambda light chains and the CSF IgG concentration were examined. In the CSF diagnosis of MS, the set of the examinations used is considered to be superior to any individual laboratory technique currently applied in the diagnostic process of MS. In the set of the CSF examinations, CSF immunoelectrophoresis revealed abnormal findings in patients with MS in whom presence of bands (oligoclonal gammopathy) in the gamma-globulin field of the CSF electropherogram and/or increased concentration of CSF IgG were not demonstrated.     

Multiple sclerosis in Brazil. Analysis of cerebrospinal fluid by standard methods

The demonstration of intrathecal IgG synthesis has been used as an important laboratory parameter to support the diagnosis of multiple sclerosis (MS). The Committee for European Concerted Action for Multiple Sclerosis has recommended a protocol for the assessment of intrathecal IgG synthesis. We applied this methodology to determine the cerebrospinal (CSF) profile of 128 Brazilian patients with MS. We detected hypercytosis lower than 35 cells/mm3 in 97%, protein lower than 80 mg/dl in 99%, normal blood-CSF barrier function in 76%, increased IgG local production around 53% and oligoclonal IgG bands by isoelectric focusing in 85% of the definite MS patients. The diagnostic accuracy of the quantitative analysis was lower than the qualitative. The detection of oligoclonal bands was especially important in the cases of normal quantitative assays of IgG. In addition, we found a lower frequency of inflammatory reaction in CSF in our MS cases, in comparison to some European studies.     

High sensitivity of free lambda and free kappa light chains for detection of intrathecal immunoglobulin synthesis in cerebrospinal fluid

Background – So far, an inflammation of the central nervous system (CNS) is diagnosed by immunoglobulin measurement in cerebrospinal fluid (CSF) and serum as well as by determination of the oligoclonal bands. With the free kappa and lambda light chains, new markers to diagnose intrathecal synthesis are available. Methods – In addition to routine diagnostic tests and the assessment of standard parameters, free immunoglobulin light chains were measured in the CSF of patients with neurological disorders. Results – A significant agreement was found between an increase in free kappa light chain CSF serum quotients and results of the currently widely applied method of oligoclonal band measurement for the detection of intrathecal immunoglobulin synthesis. A sensitivity of 95% and 100% specificity for free kappa light chain concentrations at a cut‐off of 0.41 mg/l was determined for free kappa light chains compared with oligoclonal bands. However, the free lambda light chains in 20 out of the 110 investigated samples were characterized by inconsistent behaviour. These otherwise unremarkable samples yielded increased CSF quotients, leading to the assumption that free lambda light chains represent a highly sensitive measure of intrathecal immunologlobulin synthesis. Thirteen of the 20 samples described above were obtained from patients with cerebral infarction, 4 samples derived from patients with cerebral paresis (primarily facial paresis), one sample was from a patient with multisystem atrophy and two were obtained from patients with migraine and neuralgia. Conclusion – These findings suggest that the high sensitivity of lambda light chains for the detection intrathecal immunoglobulin synthesis may be of benefit in establishing clinical diagnoses.     

Intrathecal IgG synthesis and autoantibody-secreting cells in multiple sclerosis

We studied intrathecal IgG synthesis and autoantibody-secreting cells in 148 patients with possible onset symptoms of MS (POSMS) or clinically definite MS (CDMS). In POSMS intrathecal synthesis of IgG oligoclonal bands and abnormalities on T2-weighted magnetic resonance imaging were associated but the former were more prevalent. The cerebrospinal fluid (CSF) leukocyte count and the number of anti-protelipid protein antibody-secreting cells in cerebrospinal fluid (CSF) correlated with disease activity in POSMS. Intrathecal IgG synthesis levels and the number of anti-myelin basic protein antibody-secreting cells in CSF correlated with disease activity in CDMS. Our results support recent reports of pathogenetic heterogeneity and a pathogenetic role of the antibody response in MS.     

The early stage of multiple sclerosis in the light of molecular and immunological studies

The studied material consisted of patients in the first and second year from the onset of multiple sclerosis. In majority of the studied 18 cases the junctional repertoire of TCR in blood lymphocytes of patients in early phase of MS was restricted demonstrating the mono- or oligoclonal character of rearrangement in the spectrum V delta 1-J delta 1, V delta 5-J delta 1 and V delta 3-J delta 1 in contrast to overwhelming polyclonal picture in the control group. In majority of the cases oligoclonal bands were detected and IgG index was above normal level indicating on intrathecal IgG synthesis. The comparison of humoral immunological markers in the early phase of MS with the control group revealed several higher values in patients, but only concerning TNF alpha level in serum, IgG in CSF and IgG index the differences were statistically significant. After treatment with Prednisone the decrease of all studied markers was established, but significant only of free kappa chains to creatinine ratio in urine. The obtained results indicate, that the early phase of MS is characterised by the profound shift of gamma/delta TCR receptors in direction of mono- or oligoclonal bands in CSF, what may be explained by the oligoclonal expansion of certain B and T cells due to stimulation by an antigen related to MS pathogen.     

Qualitative evidence of anti-Yo-specific intrathecal antibody synthesis in patients with paraneoplastic cerebellar degeneration

We investigated the presence of anti-Yo-specific oligoclonal antibody bands in cerebrospinal fluid (CSF) and serum samples of 9 patients with anti-Yo syndrome and 11 controls. Isoelectric focusing combined with affinity blotting, revealed anti-Yo-specific intrathecal antibody synthesis in all patients with anti-Yo syndrome: Four patients had positive anti-Yo-specific oligoclonal IgG bands in CSF which were not demonstrable in their sera; five CSF/serum pairs showed additional, more intensive, oligoclonal bands in CSF compared to the corresponding serum. Interestingly, four patients with absence of oligoclonal bands of total IgG in CSF revealed positive anti-Yo-specific oligoclonal bands in the same sample. This speaks for a higher sensitivity of detection of oligoclonal bands using an affinity blot loaded with Yo-specific antigen compared to an affinity blot coated with anti-human IgG used for the detection of oligoclonal bands of total IgG. In conclusion, the presence of anti-Yo-specific oligoclonal IgG bands in CSF which were absent, or less strong, in patients sera provides qualitative evidence of anti-Yo-specific IgG synthesis by intrathecal B-cell clones. These results could be of interest in detection of intrathecal-specific IgG synthesis in nervous system infectious diseases provided that the target antigen is known.     

Qualitative evidence of Ri specific IgG-synthesis in the cerebrospinal fluid from patients with paraneoplastic neurological syndromes

The presence of Ri-specific oligoclonal IgG bands in the CSF was investigated in five patients with paraneoplastic anti-Ri associated neurological syndromes (PNS) and six controls. In 4/5 CSF samples reactivity of IgG bands with recombinant Ri antigen was found using isoelectrofocusing combined with affinity blotting; in one patient with absence of oligoclonal bands of total IgG in CSF Ri-specific oligoclonal bands were detected with the same sample, indicating a higher sensitivity of Ri-specific affinity blotting as compared to affinity blotting with anti-human IgG antibodies. Our results confirm previous studies demonstrating IgG synthesis against onconeuronal antigens by intrathecal B-cell clones in PNS and extend this observation to patients with anti-Ri syndrome. The pathogenic relevance of these antibodies, however, is further challenged by the finding that specific intrathecal IgG synthesis might not be a prerequisite of CNS involvement, because it was missed in one of our patients.     

Oligoclonal IgG bands in cerebrospinal fluid and serum during asymptomatic human immunodeficiency virus infection

Serum and cerebrospinal fluid (CSF) samples from asymptomatic patients seropositive for human immunodeficiency virus (HIV) showed frequent evidence of intrathecal IgG synthesis and oligoclonal IgG bands, with different isoelectric focusing patterns in serum and CSF; 2 of 7 had a CSF pleocytosis. The results suggest frequent, early, chronic central nervous system infection following HIV infection.     

Immunoglobulins within the central nervous system in primary Sjögren's syndrome

Cerebrospinal fluid (CSF) and sera from 17 patients with primary Sjögren's syndrome (PSS) with or without clinical evidence of nervous system involvement were studied. Intrathecal IgG synthesis as measured by oligoclonal IgG bands on agarose isoelectric focusing or elevated IgG index in CSF was found in 6 of 8 patients with clinical nervous system involvement but also in 5 of 9 patients without clinical nervous system involvement. Elevated IgM-index in CSF was found in 7 of 8 patients with clinical nervous system involvement and in 6 of 9 patients without clinical nervous system involvement. By immunoblotting, CSF IgG-antibodies against myelin basic protein (MBP) were found in 3 of 12 patients with multiple sclerosis (MS), but in none of the patients with PSS or in the 12 controls. Intrathecal anti-viral IgG-antibodies, as measured by immunoblotting against measles, mumps, varicella or herpes simplex, were found in 8 of 17 patients with PSS, and in 7 of 12 patients with MS, but were not detected in the controls. Our observations support the concept that the central nervous system (CNS) is included in the multiple immunological phenomena of PSS. Interestingly, in some PSS patients intrathecal IgG synthesis occurred without overt clinical nervous system involvement and thus the clinical significance of intrathecal IgG synthesis in PSS is uncertain. The similarities with MS regarding intrathecal antiviral antibody production may be interpreted as the result of polyclonal B-cell activation.     

IgG synthesis rate in evaluation of multiple sclerosis in a community hospital

We obtained CSF and serum from 21 patients with definite MS in a community hospital. Ninety percent of patients had an elevated IgG synthesis rate, comparable with the sensitivity of oligoclonal bands (0.95) or the IgG index (0.90) and better than the IgG% (0.47) in definite MS. We found that no area hospital routinely employs the IgG synthesis rate. The low-sensitivity IgG% and the dimensionless IgG index can be abandoned. The linear IgG synthesis rate and oligoclonal band scanning should be the CSF tests of choice.