Antibodies in oligoclonal immunoglobulins in CSF from patients with acute cerebrovascular disease

Thin-layer polyacrylamide gel isoelectric focusing (PAG IEF) of CSF and serum, and subsequent immunofixation with viral and structural brain components followed by autoradiography revealed in eight out of nine selected patients with oligoclonal CSF IgG and cerebrovascular disease local synthesis within the CNS of antibodies against one or more of the viruses tested: six patients against measles, five against herpes simplex virus type 1, and two against varicella virus. This finding may reflect a polyclonal B cell activation secondary to brain damage and elaboration of certain structural brain components. None of the patients had local synthesis of antibodies against the other viruses tested (mumps, rubella and cytomegalovirus), or against structural brain components (crude saline, lipid-proteolipid, myelin basic protein extracts from human brain and purified bovine myelin basic protein).     

Antibodies to viral and non-viral antigens in subacute sclerosing panencephalitis and multiple sclerosis demonstrated by thin-layer polyacrylamide gel isoelectric focusing,antigen immunofixation and autoradiography

Antibody activity in IgG zones separated by thin-layer polyacrylamide gel isoelectric focusing (PAGIEF) was determined in 3 patients with subacute sclerosing panencephalitis (SSPE), 4 patients with multiple sclerosis (MS) and 4 subjects with psychosomatic disorders, using antigen immunofixation and autoradiography. Viral (measles, herpes simplex type 1, mumps) and non-viral (purified bovine myelin, bovine myelin basic protein, bovine oligodendrocytes, MS and normal human brain extract) were used as antigens. All oligoclonal and some of the polyclonal CSF IgG zones in the patients with SSPE contained measles virus antibodies, as did some of the oligoclonal and polyclonal CSF IgG zones in 3 of the patients with MS. No antibodies were detectable in CSF or serum IgG zones against any of the non-viral antigens tested.     

Viral antibodies in oligoclonal and polyclonal IgG synthesized within the central nervous system over the course of mumps meningitis

The occurrence of viral antibodies in relation to IgG separated by thin-layer polyacrylamide gel isoelectric focusing was studied in CSF and serum from 24 patients with mumps meningitis by immunofixation with viral antigens and autoradiography. Eleven of the patients displayed on the autoradiograms evidence of locally in the central nervous system synthesized mumps virus antibodies which were related to oligoclonal IgG bands in all 5 patients who displayed this CSF abnormality, otherwise to polyclonal IgG bands. Local synthesis of mumps virus antibodies was detectable in 43% of specimens obtained 1–13 days after onset, and in 75% obtained 27–47 days after onset. Only one patient displayed local synthesis of antibodies to other viruses (measles and herpes simplex) which could then be traced to polyclonal IgG bands.     

Antibodies against viruses and structural brain components in oligoclonal IgG obtained from multiple sclerosis brain

Summary Immunoglobulin G (IgG) was isolated from three multiple sclerosis (MS) and two control brains by Protein A Sepharose affinity chromatography and was characterized by thin-layer polyacrylamide gel isoelectric focusing (PAG IEF) and antiserum immunofixation. The three MS brains contained oligoclonal IgG. Immunofixation with measles, herpes simplex, varicella, rubella, mumps and cytomegalovirus as antigens and autoradiography revealed that some of the oligoclonal IgG bands separated by PAG IEF contained antibodies against herpes simplex in one, measles in two and varicella virus in all three MS brains. No antibodies were detected with this technique against structural human (crude saline, lipid-proteolipid, ganglioside, and myelin basic protein extracts of MS and normal human brain) and bovine (purified myelin, myelin basic protein and oligodendrocytes of bovine brain) brain components. The finding of viral antibodies and the absence of antibodies against structural brain proteins in oligoclonal MS brain IgG is similar to that previously recorded in MS cerebrospinal fluid (CSF).

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Zusammenfassung Immunoglobulin G, IgG, wurde aus der Gehirnsubstanz von drei Fällen mit multipler Sklerose (MS) und zwei Kontrollen mittels Affinitätschromatographie mit Protein A-Sepharose isoliert. Das IgG wurde mit isoelektrischer Fokussierung in Polyacrylamidgelplatten (PAG IEF) und anschließender Antiserum-Immunofixation charakterisiert. Bei MS wurde oligoklonales IgG gefunden. Mit Hilfe von Antigen-Immunofixation mit Masern, Herpes simplex, Varizellen, Rubella, Parotitis und Zytomegalievirus und nachfolgender Autoradiographie (radioaktive Zweitantikörper gegen die genannten Antigene) konnte gezeigt werden, daß die oligoklonalen Bande in den drei MS-Gehirnen Antikörper gegen Herpes simplex in einem, Masern in zwei und Varizellen-Virus in allen drei Fällen enthielten. Keine solchen Antikörper wurden entdeckt, wenn die Technik für strukturelle Gehirn-Komponenten vom Menschen (einfache physiologische Salz-, Lipid-Proteolipid-, Gangliosid- und basische Myelo-Protein-Extrakte von MS und normalem Gehirn) und vom Rind (gereinigtes Myelin, basisches Myelo-Protein und Oligo-Dendrozyten aus Gehirnrinde) verwendet wurde.

     

Multiple sclerosis. Electrofocused "bands" of oligoclonal CSF IgG do not carry antibody activity against measles, varicella-zoster or rotaviruses

Electrofocused serum and cerebrospinal fluid (CSF) specimens from patients with multiple sclerosis (MS) were analysed for immunoglobulins (Ig) and for antibodies to measles, varicella-zoster and rotaviruses by an imprint immunofixation method. Patterns of intrathecally synthesized antibodies to the 3 viruses differed from patterns of oligoclonal IgG in the CSF. A variable proportion of virus antibody bands (average 19% for measles antibodies, 8% for varicella-zoster antibodies, 31% for rotavirus antibodies) displayed isoelectric points identical to bands of IgG, but absorption with measles, varicella-zoster and rotavirus antigens produced no change in the bands of IgG and no quantifiable decrease of the CSF IgG. The results confirm previous evidence that the intrathecally synthesized viral antibodies so far demonstrated in MS are not carried by the oligoclonal bands of CSF IgG and account for only a minor fraction of the CSF IgG.     

Mumps meningitis: Specific and non-specific antibody responses in the central nervous system *

Intrathecal production of oligoclonal mumps-specific IgG was demonstrated in nine out of 10 children with mumps meningitis by imprint immunofixation (IIF) of sera and cerebrospinal fluids (CSF) separated by agarose electropheresis and by thin-layer electrofocusing. Four of the patients had intrathecal mumps antibody synthesis demonstrable also by conventional serological tests. Oligoclonal CSF IgG was demonstrable by agarose electrophoresis in four of the patients. A dominance of λ over κ type oligoclonal Ig and mumps antibodies was observed in the CSF of three of these patients. The bulk of the oligoclonal CSF IgG was concluded to represent mumps-specific antibodies on the basis of the IIF as well as virus absorption analysis. Intrathecal production of oligoclonal IgG antibodies to one, two, or three other (measles, rubella, herpes simplex) viruses was demonstrated by IIF in four patients. These antibodies were not associated with the oligoclonal CSF IgG present in three of the patients. It is concluded that a specific intrathecal IgG antibody response is a common feature in children with mumps meningitis. This response sometimes reaches a magnitude that permits detection of oligoclonal IgG in the CSF. In some patients, the specific response appears to be associated with a non-specific activation of cells producing antibodies of other (unrelated) specificity.     

The intrathecal immune response in the early stage of multiple sclerosis

Sequential pairs of cerebrospinal fluid (CSF) and serum samples from 10 patients followed for 2.5-12 years after onset of unilateral optic neuritis (ON) were studied. Eight patients developed definite multiple sclerosis (MS) during the observation period. All patients had normal CSF protein patterns on agar or agarose gel electrophoresis at onset. Six patients developed oligoclonal immunoglobulin (Ig) bands in the CSF during the observation period. Imprint immunofixation of electrofocused specimens disclosed intrathecal synthesis of oligoclonal IgG antibodies to 1 or more of 6 viruses (measles, herpes simplex type 1, varicella-zoster, cytomegalo, mumps, rota) during the observation period in 8 patients. Changes in patterns of intrathecally synthesized viral antibodies, characterized by the appearance of "new" antibody populations and the waxing or waning of others were observed in 6 patients. The results suggest that the early stage of MS in some patients is associated with transient as well as permanent recruitment of B cell clones producing viral antibodies of different specificities.     

Comprehensive viral immunology of multiple sclerosis. III. Analysis of CSF antibodies by radioimmunoassay

The CSF from 279 patients with multiple sclerosis (MS), probable MS, or controls was examined by radioimmunoassay (RIA) for antibodies to measles, rubella, mumps, parainfluenza 1 (Sendai) (strain 6/94), herpes simplex (HSV), varicella, and vaccinia viruses. Significantly more patients with MS than noninflammatory control patients had antibody to measles, rubella and varicella viruses, of which antibody to measles was the most prevalent. The percentage of patients with MS with two or more CSF antibodies was significantly greater than that in the controls. There was no tendency for certain antibodies to be associated. There was a general relationship between presence of CSF antibodies and severity of MS. The data support the hypothesis of local CNS antibody synthesis of several viral antibodies; however, such local synthesis may be a random event, possibly dependent on the number and specificity of peripheral virus antibody-forming lymphocytes available for ingress into the CNS.     

Optic neuritis: Local synthesis in the central nervous system of oligoclonal antibodies to measles, mumps, rubella, and herpes simplex viruses

Antibodies to measles, mumps, rubella and herpes simplex type 1 viruses in serum and CSF of 10 patients with acute monosymptomatic optic neuritis were characterized by imprint electro-immunofixation (IEIF). Comparisons of the IEIF antibody patterns in serum and CSF indicated that a local synthesis in the central nervous system of oligoclonal virus antibodies took place in five of the 10 patients. Three of the five patients had a local synthesis of antibody to more than one type of virus. Oligoclonal IgG was detected by agarose electrophoresis of the CSF in four of the five patients with a local synthesis of virus antibodies. There was, however, no association between the locally synthesized antibodies and the oligoclonal CSF IgG, indicating that they account for only a minor fraction of the locally synthesized IgG in these patients. In the fifth patient, distinct fractions of locally synthesized virus-specific oligoclonal Ig were detected by the IEIF method, although the agarose electrophoresis showed a normal pattern of CSF IgG.     

Production of viral antibodies in vitro by CSF cells from mumps meningitis and multiple sclerosis patients

Cerebrospinal fluid (CSF) cells from 4 mumps meningitis and 11 multiple sclerosis (MS) patients were cultured in vitro for 7 days with and without pokeweed mitogen (PWM) stimulation. The cells produced varying amounts of IgG without stimulation and no significant increase of IgG synthesis was observed after PWM stimulation. Antibodies against mumps, measles, rubella, herpes simplex, and adeno viruses were measured in the supernatants of the cultures by a sensitive enzyme immunoassay. In the mumps meningitis patients, the largest amount of antibody was against mumps virus but low amounts of antibodies with other specificities were also synthesized by CSF cells of one patient. The most commonly detected specificities in MS patients were against measles and rubella viruses, whereas antibodies against adeno and mumps viruses were detected in only one CSF cell supernatant. No antibodies produced against herpes simplex virus in vitro were detected in any of the supernatants. The amounts of viral antibodies produced in vitro and intrathecally were only partially correlated.     

The intrathecal synthesis of virus-specific oligoclonal IgG in multiple sclerosis

A highly sensitive antigen-mediated capillary blot technique was developed for the detection of virus-specific oligoclonal IgG in paired CSF and serum samples from patients with various neurological diseases. In multiple sclerosis, intrathecal synthesis of oligoclonal antibodies was present against measles (70%), rubella (60%), varicella zoster (40%) and mumps (30%); in most cases (75%), such synthesis involved two or more viruses. In contrast, antibodies against a non-neurotropic virus (cytomegalovirus) were rarely produced in CSF from MS patients (5%). However, this ‘polyspecific’ reaction was not restricted to MS samples but was also observed in neurolupus and in the late phase of infectious diseases of the central nervous system. These anti-viral antibodies could be produced without de novo replication of the corresponding viral genome and are likely mere bystanders of an ongoing immune response.     

Comprehensive viral immunology of multiple sclerosis. II. Analysis of serum and CSF antibodies by standard serologic methods

Sera and CSFs of 85 patients with multiple sclerosis (MS), 49 patients with probable MS, and 165 control patients with other neurologic diseases were assayed for antibodies to rubella, mumps, measles, parainfluenza I (strain 6/94), herpes simplex, cytomegalovirus, varicella-zoster, and vaccinia viruses. Methods included complement fixation (CF), hemagglutination inhibition (HI), and complement-dependent plaque reduction (CPR). Significant differences between the groups with MS and the control groups were higher serum antibody titers to measles virus in the groups with MS, higher proportion of patients with MS with CSF antibodies to measles, rubella, and vaccinia viruses, and greater percentage of patients with MS with more than one CSF viral antibody. Duration and severity of disease in the patients with MS were associated with presence of multiple CSF antibodies. Presence of CSF antibody was positively correlated with the height of the correspnding serum titer, yet a high serum titer did not ensure the presence of CSF antibody. Oligoclonal bands were present in the CSFs of equal proportions of patients with MS with and without CSF viral antibody. Our data support the hypothesis of local antibody synthesis within the CNS. However, we favor the view that preprogrammed antibody-forming lymphocytes enter the CNS and then produce antibody either because of nonspecific polyclonal activation in situ or because of failure of normal regulation.     

Viral and bacterial antibody responses in multiple sclerosis

An imprint electroimmunofixation method (IEIF) was used to characterize antibodies to eight viral antigens (measles, mumps, rubella, herpes simplex type 1, varicella-zoster, vaccinia, cytomegalovirus, adenovirus) and four bacterial antigens (β-hemolytic streptococcus, Hemophilus influenzae type B, Escherichia coli, enterococcus) in serum and cerebrospinal fluid (CSF) of 12 patients with multiple sclerosis (MS). Twelve patients matched for age and sex served as controls. Evidence for intrathecal synthesis of oligoclonal antibodies to one or more antigens was found in all 12 MS patients and in 1 of the controls. In the MS group, antibodies to viruses with neurotropic properties were more frequently associated with local synthesis than antibodies to other viruses and bacteria. The types and number of locally synthesized antibodies showed no correlation with disease duration and severity. The antibodies were not associated with oligoclonal CSF IgG and appear to account for only a minor fraction of the locally synthesized CSF IgG in MS.     

Assessment of cerebrospinal fluid immunoglobulin patterns after isoelectric focusing. Use of kappa and lambda light chain immunoperoxidase staining

Assessment of the presence of immunoglobulin (Ig) of restricted heterogeneity i.e. oligoclonal Ig in cerebrospinal fluid subjected to isoelectric focusing (IEF) may be difficult because of the tendency of the technique to subdivide even normal polyclonal IgG into bands and zones. Focused Ig stained by the immunoperoxidase technique for kappa and lambda light chains shows correspondence between the two patterns in the case of normal polyclonal Ig, but a marked discrepancy between kappa and lambda patterns in disorders associated with oligoclonal Ig, and kappa and lambda immunofixation makes assessment of agarose IEF separations of IgG more reliable.     

Measles and rubella virus antibodies in patients with multiple sclerosis. A longitudinal study of serum and CSF specimens by radioimmunoassay.

A longitudinal study on rubella, measles, and respiratory syncytial virus antibodies in serial serum and CSF specimens from 20 multiple sclerosis (MS) patients was performed, using solid-phase radioimmunoassay. Albumin and immunoglobulin G (IgG) levels were also measured to check the integrity of the blood-brain barrier and the intrathecal IgG production. All the patients had local IgG production in their CNA. A local antibody production against one or more of the viruses studied was evident in 15 patients. Fluctuations in the intrathecal viral antibody synthesis were evident in eight patients. No correlation was found between these changes and the clinical course of the disease. The results suggest that the intrathecal antibody synthesis in MS is only partially against any given virus, and in most patients the bulk of the oligoclonal CSF antibodies is against antigens other than those studied here.     

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.     

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 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.     

DNA and RNA antibodies in serum and CSF of multiple sclerosis and subacute sclerosing panencephalitis patients.

DNA and RNA (nucleic) antibodies were found in the CSF of 18 patients with multiple sclerosis (MS) (out of 45), 11 with subacute sclerosing panencephalitis (SSPE) (out of 12) and 9 controls (out of 30). Viral (measles and rubella, by HAI) antibodies were present in all SSPE, 23 MS and 11 control patients. A clear correlation exists between (1) CSF immunological patterns, (2) oligoclonal aspect, (3) simultaneous presence of nucleic and viral antibodies, suggesting the local synthesis of both. This is confirmed by the comparison of the ratios IgG/antibodies in serum and CSF: the CSF ratio may be higher for nucleic and/or viral antibodies in SSPE and MS patients. Thus nucleic antibodies seem to be related to a persistent active infection within the central nervous system.     

Measles virus antibodies in serum and cerebrospinal fluid in patients with multiple sclerosis and other neurological disorders,with special reference to measles antibody synthesis within the central nervous system

Measles virus hemagglutination-inhibiting (HI) and gel precipitating (GP) antibodies were determined in sera and cerebrospinal fluids (CSF) from 65 patients with multiple sclerosis (MS) and 65 patients with other neurological diseases. The serological results were correlated to content of immunoglobulin-G (IgG) and electrophoretic patterns of sera and CSF.Measles GP antibodies, identified as directed against measles virus ribonucleoprotein antigens, were detected in sera and in CSF from a significantly higher proportion of MS than of non-MS patients. No significant difference between the 2 groups of patients was found for measles HI antibodies.Reduced serum/CSF HI and/or GP antibody ratios were found in about one half of the MS patients and in 2 patients with chronic myelopathy. All patients with reduced antibody ratios had evidence of IgG synthesis within the central nervous system (CNS), as inferred from oligoclonal IgG patterns of the CSF. Reduced ratios of measles GP antibodies were 3 times as common as reduced ratios of HI antibodies. Immuno-electrophoretic assays indicated that the CSF GP antibodies were electrophoretically restricted in a number of MS patients.The results indicate that measles virus may be an active immunogen within the CNS in many MS patients and in some patients with chronic myelopathy, giving rise to an oligoclonal IgG antibody response.