Unconcentrated Cerebrospinal Fluid Research Articles

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.

CNS tumours: oligoclonal immunoglobulin D in cerebrospinal fluid and serum.

Oligoclonal immunoglobulin D bands were detected by isoelectric focusing in 7 out of 25 unconcentrated cerebrospinal fluid (CSF) samples obtained from patients with tumours of the central nervous system (CNS). The tumours were confirmed by clinical and histological findings. Two patients with CNS malignancy had intrathecal synthesis of oligoclonal bands both IgD and IgG. Four patients with a variety of CNS tumours had a systemic IgD immune response but no oligoclonal IgG bands. One patient with the most malignant tumour histology had a systemic IgD response as well as local synthesis of IgG. The study reveals several new aspects regarding CNS tumours: they are immunologically active and are capable of invoking oligoclonal immunoglobulin production both within the CNS and systemically. Multiple immunoglobulin activation can be found in malignant CNS tumours, and systemic IgD production may occur independently from IgG synthesis and may represent an immune response to a neoantigen produced in the CNS compartment.

Direct quantification of CSF α-synuclein by ELISA and first cross-sectional study in patients with neurodegeneration

Because accumulation of α-synuclein (αS) in the brain is a hallmark of Parkinson disease (PD) and related disorders, we examined its occurrence in human cerebrospinal fluid (CSF). Following affinity enrichment and trypsin digestion of CSF collected from a neurologically healthy donor, we identified several αS-derived peptides by mass spectrometry. The concentration of αS amounted to <0.001% of the CSF proteome. We then built, validated and optimized a sandwich-type, enzyme-linked immunoadsorbent assay (ELISA) to measure total αS levels in unconcentrated CSF. In a cross-sectional study of 100 living donors, we examined cell-free CSF samples from subjects clinically diagnosed with advanced PD, dementia with Lewy bodies (DLB), Alzheimer disease (AD), and a group of non-neurodegenerative disease controls (NCO). In these four groups the CSF αS concentrations ranged from 0.8 to 16.2 pg/μl. Mean CSF αS values were lower in donors with a primary synucleinopathy (PD, DLB: n=57) than in the other two groups (AD, NCO: n=35; p=0.025). By contrast, living Creutzfeldt–Jakob disease patients showed markedly elevated CSF αS levels (n=8; mean, 300 pg/μl; p<0.001). Our results unequivocally confirm the presence of αS in adult human CSF. In a first feasibility study employing a novel ELISA, we found relatively low CSF αS concentrations in subjects with parkinsonism linked to synucleinopathy, PD and DLB. In definite prion disease cases, we recorded a marked rise in total CSF αS resulting from rapid cell death. Our results will likely aid future biomarker explorations in neurodegenerative conditions and facilitate target validation studies.

Association of platelet-derived soluble glycoprotein VI in plasma with Alzheimer’s disease

Accumulating evidence from epidemiological, clinical and experimental studies suggests that vascular risk factors and angiopathic mechanisms are involved in the pathogenesis of Alzheimer’s disease (AD). Platelets could be the missing link between AD and the vasculature. Soluble glycoprotein VI (sGPVI) and β-thromboglobulin (β-TG) plasma and cerebrospinal fluid (CSF) levels as markers of platelet activity were measured in 30 AD patients and 20 age-matched healthy elderly controls by ELISA. The severity of dementia was assessed by mini-mental state examination (MMSE). We found in AD patients significantly decreased sGPVI plasma levels (0.55 ± 0.18 ng/ml) as compared to healthy controls (0.75 ± 0.43 ng/ml; p = 0.033). In AD patients, sGPVI levels were positively correlated with β-TG plasma levels ( r = 0.244, p = 0.05) and with cognitive status as measured by MMSE score ( r = 0.271; p = 0.048). In unconcentrated CSF samples, levels of β-TG and sGPVI were below the detection limit of the assays in AD patients and healthy controls. Our results suggest an association of sGPVI with the pathogenesis of AD. These findings encourage future research into whether sGPVI plasma levels may reflect or even mediate neuroprotective mechanisms in AD.

BDNF serum and CSF concentrations in Alzheimer’s disease, normal pressure hydrocephalus and healthy controls

Alzheimer’s disease (AD) and normal pressure hydrocephalus (NPH) are common forms of dementia in the elderly. Recent findings have suggested an involvement of brain-derived neurotrophic factor (BDNF) in the pathogenesis of AD. BDNF is an endogenous protein involved in the maintenance of neuronal function, synaptic plasticity and structural integrity in the adult brain. BDNF serum and cerebrospinal fluid (CSF) concentrations were assessed by a sensitive ELISA in 27 AD patients in comparison to 9 NPH patients and 28 age-matched healthy controls (10 CSF samples). We found a significant decrease of BDNF serum concentration in AD (18.6 ng/ml) and NPH patients (18.1 ng/ml) as compared to healthy controls (21.3 ng/ml; p = 0.041/ p = 0.017). BDNF serum concentrations did not correlate with CSF levels, age or MMSE scores both in AD and NPH patients. In unconcentrated CSF samples, BDNF could be detected in AD patients in 8/27 cases (29.6%; mean of 4.6 pg/ml), in NPH patients in 1/9 cases (11.1%; mean of 6.4 pg/ml) and in the control subjects in 5/10 cases (50%; mean of 1.6 pg/ml) with no significant differences as regards mean concentration and frequency of detectable BDNF in CSF. The decrease of BDNF serum levels in AD and NPH may reflect a lack of trophic support and thus contribute to progressive degeneration in both diseases. In contrast to serum, CSF seems to be no useful source to determine BDNF in AD or NPH because of too low concentrations. Further examinations have to follow to elucidate the potential sources and the meaning of reduced BDNF levels in the blood in AD and NPH.

Place de l'immunofixation des IgG du LCR dans le diagnostic de sclérose en plaques (SEP)

We presented the analytical and diagnostic performances of immunofixation for detection of oligoclonal IgG bands in native unconcentrated cerebrospinal fluid (CSF) and diluted serum samples analysed in the same run on Hydragel 6 CSF Sebia on Hydrasys ® Sebia, an alternative to the manual technique of IgG isoelectric focusing (IEF). Pairs of CSF and serum of 124 patients studied over a 2 year period were classified in: 29 definite multiple sclerosis (MS) 35 possible MS, 11 inflammatory neurological diseases other than MS, and 49 non-inflammatory diseases of the central nervous system. Pairs of CSF and serum of 98 patients were tested by isoelectric focusing for control of intrathecal IgG synthesis. The analytical performances of the two techniques to reveal IgG oligoclonal bands are practically identical. The sensivity of immunofixation is 96%, of isoelectric focusing is 93%. Their specificity is 93% and 96%, respectively. The positive predictive value to establish diagnosis of MS is 90% with immunofixation and 92% with isoelectric focusing. The negative predictive value to exclude diagnosis is 97% with immunofixation and 96% with isoelectric focusing. The interpretation of IgG immunofixation is in practice more difficult than IgG isoelectric focusing, the procedure is easy and quick to perform.

An improved, ultrasensitive method for the detection of IgM oligoclonal bands in cerebrospinal fluid

A new, 10-fold more sensitive method, based on an improved immunofixation technique, has been devised to detect oligoclonal IgM bands in unconcentrated cerebrospinal fluid (CSF). Using agarose gel electrophoresis, 5 μl of an unconcentrated sample containing oligoclonal bands was separated and blotted on to a polyvinyldifluoride membrane. To visualise the pattern, a peroxidase-labelled double-antibody technique was used. No prior concentration of CSF was needed and the process required only 5 h. The technique may prove very useful in diagnosing an early intrathecal immune response.

A zone immunoelectrophoresis assay method for quantification of apolipoprotein D in human cerebrospinal fluid

A zone immunoelectrophoresis assay (ZIA) has been developed for the quantification of apolipoprotein D (apo D) in human unconcentrated cerebrospinal fluid (CSF). The apo D concentrations of samples of the serum, plasma and CSF were directly proportional to the migration distances of the corresponding zones of immunoprecipitates developed during electrophoresis in glass capillaries filled with antibody-containing agarose gel. A linear standard curve, between about 1 and 12 mg of apo D 1 was obtained using a commerical serum preparation, Seronorm ®, as apo D standard. The coefficients of variation of the ZIA were below 8% ( n = 5 × 6) and 10% ( n = 8) for within-run and between-run reproducibility, respectively. Quantification experiments with disulfide-reducing agent, mixtures of CSF and urine as well as frozen and stored CSF samples indicated parallelism between the precipitate-forming immunologic reactions of apo D in different sample matrices when performed with ZIA. Application of this method to quantify apo D of CSF and plasma samples from 51 normal healthy men aged 16–72 years yielded means ± SD of 5.3 ± 1.5 mg/l and 128.4 ± 22.7 mg/l, respectively. No correlation was found between the CSF and plasma apo D concentrations.

Quantifying HIV-1 RNA using the polymerase chain reaction on cerebrospinal fluid and serum of seropositive individuals with and without neurologic abnormalities.

We quantified HIV-1 RNA levels (copies per milliliter) in cerebrospinal fluid (CSF) and serum from subjects at various stages of HIV-1 disease and determined the relationship of RNA levels to clinical and neurologic disease status (HND) and to laboratory values. Ninety-seven HIV-1-seropositive men without CNS opportunistic infections, tumors, or neurosyphilis and 13 high-risk seronegative controls were included in the study. Each individual underwent a structured interview and physical and neurologic examinations, followed by standardized collection of blood and CSF. A custom-designed, fully automated polymerase chain reaction (PCR) system was used to perform a minimum of four separate amplifications per specimen, using two HIV-1 gag primer pairs. Southern blotting followed by hybridization with product-specific probes was used for post-PCR detection. The number of copies per milliliter was determined by relating unknowns to a built-in dilution-series standard curve using an image analysis system. HIV-1 RNA was detectable in 96% of the sera, 78% of the concentrated CSF samples, and 54% of the unconcentrated CSF samples. Serum RNA levels were significantly higher than in CSF. Serum RNA levels were significantly inversely correlated with CD4+ cell counts (p = -0.34; p = 0.03): i.e., higher RNA levels in seropositive subjects were associated with lower numbers of CD4+ cells. Serum RNA levels correlated positively with number of AIDS-related symptoms, dysfunction scores for total neurological examination, mental status score, cranial nerve score, and CNS motor signs score. Serum RNA levels did not correlate significantly with length of time on zidovudine therapy, intrathecal IgG synthesis rate, or albumin leakage. RNA levels in CSF significantly correlated only with intrathecal IgG synthesis rate and with serum RNA levels. These results confirm that serum levels of HIV-1 RNA correlate with HND and inversely correlate with CD4 counts, demonstrating that HND occurs predominantly in late stages of HIV-1 disease, although HIV-1 RNA can be detected in CSF from a majority of HIV-1-seropositive individuals at all stages of disease, which suggests that there can be early penetration of HIV into the CNS. However, HND can occur in the absence of high levels of CSF HIV-1 RNA. We also found that the concentration of HIV-1 in CSF is correlated with intrathecal IgG synthesis rate.

A high-sensitivity immuno-chemiluminescence technique for detection of oligoclonal IgG and IgM in unconcentrated cerebrospinal fluid.

A method is described for detection of oligoclonal IgG and IgM in unconcentrated cerebrospinal fluid (CSF). The proteins were separated by agarose gel electrophoresis and transferred to cellulose nitrate membranes. A double antibody technique with a peroxidase conjugated secondary antibody was used to detect the protein. Bound secondary antibody was visualized by autoradiography using chemiluminescence (ECL) reagent. The procedure presented here allows examination of oligoclonal bands of IgG and IgM in less than 6h. The detection limit for a single band was approximately 10pg and the results were in complete agreement with those achieved by isoelectric focusing (Phast System) and subsequent immunofixation. In 9 of the samples oligoclonal IgM was also detected.

Oligoclonal IgG in cerebrospinal fluid detected by isoelectric focusing using PhastSystem™

Hansson L-O, Link H, Sandlund L, Einarsson R. Oligonal IgG in cerebrospinal fluid detected by isoelectric focusing using PhastSystem. Scand J Clin Lab Invest 1993; 53: 487-492.Identification of oligoclonal immunoglobulin G (IgG) in cerebrospinal fluid (CSF) is one of the major laboratory signs in multiple sclerosis (MS). Oligoclonal IgG can also be seen in other immunostimulating central nervous system diseases, e.g. meningitis caused by Borrelia spirochetes. A convenient isoelectric focusing (IEF) method combined with polyethyleneglycol-enhanced (PEG) immunofixation and silverstaining using PhastSystem™ for detection of oligoclonal IgG bands in serum and unconcentrated spinal fluid was developed. Serum and cerebrospinal fluid samples earlier investigated using agarose IEF combined with immunoblotting and avidin-biotin amplified double-antibody peroxidase staining (= A-IEF) were blindly re-analysed using PhastGel IEF 3-9 modified with addition of Pharmalyte 8-10.5 followed by immunofixation and silver staining (= PhastlEF).PhastSystem IEF using PhastGel had the same capability to separate and visualize oligoclonal IgG bands as the conventional agarose IEF. However, PhastlEF was more convenient and less time-consuming than A-IEF. PhastlEF combined with immunofixation and silver staining using application of standardized amounts of IgG for both the serum and CSF samples (40-60 ng IgG /lane is fast, well-standardized and reproducible. The method is sensitive and uses only a minute volume of unconcentrated cerebrospinal fluid for the investigation of CSF IgG pattern.

Oligoclonal IgG in cerebrospinal fluid detected by isoelectric focusing using PhastSystem.

Identification of oligoclonal immunoglobulin G (IgG) in cerebrospinal fluid (CSF) is one of the major laboratory signs in multiple sclerosis (MS). Oligoclonal IgG can also be seen in other immunostimulating central nervous system diseases, e.g. meningitis caused by Borrelia spirochetes. A convenient isoelectric focusing (IEF) method combined with polyethyleneglycol-enhanced (PEG) immunofixation and silverstaining using PhastSystem for detection of oligoclonal IgG bands in serum and unconcentrated spinal fluid was developed. Serum and cerebrospinal fluid samples earlier investigated using agarose IEF combined with immunoblotting and avidin-biotin amplified double-antibody peroxidase staining (= A-IEF) were blindly re-analysed using PhastGel IEF 3-9 modified with addition of Pharmalyte 8-10.5 followed by immunofixation and silver staining (= PhastIEF). PhastSystem IEF using PhastGel had the same capability to separate and visualize oligoclonal IgG bands as the conventional agarose IEF. However, PhastIEF was more convenient and less time-consuming than A-IEF. PhastIEF combined with immunofixation and silver staining using application of standardized amounts of IgG for both the serum and CSF samples (40-60 ng IgG/lane is fast, well-standardized and reproducible. The method is sensitive and uses only a minute volume of unconcentrated cerebrospinal fluid for the investigation of CSF IgG pattern.

A new qualitative method for detecting IgD in unconcentrated cerebrospinal fluid

We report a method which is capable of demonstrating the isoelectric focusing (IEF) pattern of immunoglobulin D in unconcentrated cerebrospinal fluid (CSF) samples containing as little as 0.1–0.5 ng of total IgD. The method used was an immuno-sandwich technique, with alkaline phosphatase enzyme amplification. Oligoclonal and polyclonal IgD patterns were seen in CSF samples. No cross-reactivity with other immunoglobulins (IgG, IgA and IgM) was detected.

Isoelectric Focusing of Cerebrospinal Fluid Immunoglobulin G: An Annotated Update

A revised agarose isoelectric focusing method for detecting oligoclonal IgG in unconcentrated cerebrospinal fluid is presented. The technique is shown to be robust and reproducible and suitable for the detection of intrathecal IgG synthesis.

The separation and detection of alkaline oligoclonal IgG bands in cerebrospinal fluid using immobilised pH gradients

A method is described for the separation and detection of highly alkaline IgG bands in unconcentrated cerebrospinal fluid (CSF). These bands are frequently found in the cerebrospinal fluid of patients with inflammatory diseases of the central nervous system, particularly in the case of multiple sclerosis, and their detection is an important aid in clinical diagnosis. An isoelectric focusing technique using an immobilised pH gradient in polyacrylamide gel has been developed over the pH range 7-10, producing a linear and stable pH gradient with excellent resolution. After electrofocusing, the protein patterns were blotted onto polyvinylidene difluoride membranes and visualised using anti-human IgG followed by an enzyme-labelled second antibody. Blotting could be carried out by capillary diffusion for up to 16 h duration without any loss in resolution. Using this method, highly alkaline intrathecal IgG bands were found in the cerebrospinal fluid of all of the 14 multiple sclerosis patients. There were also 2 patients with alkaline IgG bands in their cerebrospinal fluid who were not diagnosed as multiple sclerosis. By contrast, no alkaline IgG bands with an isoelectric point (pI) greater than 8.6 were found in any of the serum samples studied (n = 50) from patients with various neurological disorders including multiple sclerosis.

Immunoglobulin M in the cerebrospinal fluid: an indicator of recent immunological stimulation.

Using a sensitive new method for detecting oligoclonal immunoglobulin M (IgM) in unconcentrated cerebrospinal fluid (CSF), the intrathecal synthesis of IgM was correlated with that of free light chains in patients with multiple sclerosis, infections of the central nervous system and other neurological conditions. A strong correlation was found between the presence of oligoclonal IgM and that of free light chains in the CSF from patients with multiple sclerosis and infections of the central nervous system. No oligoclonal IgM response was observed in patients with non-inflammatory or non-infectious neurological conditions. This implies that oligoclonal IgM has the same diagnostic significance as free light chains.

Glutaraldehyde-enhanced immunofixation: a sensitive new method for detecting oligoclonal immunoglobulin M

A new, specific and economical method with enhanced sensitivity for rapid detection of IgM oligoclonal bands in unconcentrated cerebrospinal fluid (CSF) is presented. It is based on the use of c glutaraldehyde during immunofixation of electrophoresed specimens and utilizes peroxidase-conjugates F(ab') 2 fragment of anti-IgM antibody to avoid cross-reactivity with other CSF gamma globulins. It is able to detect 1 ng of IgM banded protein in electrophoresed CSF. Using this method, oligoclonal IgM was mainly detected in inflammatory and infectious conditions of the central nervous system.

Human immunodeficiency virus infection: affinity-mediated immunoblot detects intrathecal synthesis of oligoclonal IgG specific for individual viral proteins.

An affinity-mediated immunoblot (AMI) is introduced, able to identify virus-specific, oligoclonal IgG in unconcentrated cerebrospinal fluid (CSF) of patients infected by HIV. CSF specimens are isoelectrically focused in agarose gels and blotted to nitrocellulose (NC) strips coated with E. coli recombinant HIV-specific proteins. Virus-specific antibody clones transferred to the filter are detected by solid-phase enzyme immunoassay (EIA). Only 0.5 micrograms IgG is sufficient to demonstrate the clonal distribution of antibodies specific for an individual HIV protein. Intrathecal synthesis of oligoclonal antibodies is seen by AMI, comparing equal quantities of serum- and CSF-derived IgG. Because of its excellent sensitivity and specificity, AMI detects autochthonous HIV-specific IgG synthesis even when comparative EIA fails. Because multiple specimens can be processed within a few hours by AMI, this technique is recommendable for routine diagnosis.

An immunobioluminescence assay for gamma-gamma enolase activity in human serum and cerebrospinal fluid

A solid phase immunobioluminescence assay for the measurement of gamma-gamma enolase activity in human serum and cerebrospinal fluid is described. The assay combines the specificity of the antiserum with the specificity of measuring enolase activity, thus making the assay highly specific for gamma-gamma enolase. The sensitivity of the method allows determinations in unconcentrated cerebrospinal fluid samples. The new immunobioluminescence assay has been compared with a radioimmunoassay method. The correlation is good both for serum and CSF samples. However, different correlation lines are found for serum and CSF.

Blood-brain barrier damage restricts the reliability of quantitative formulae and isoelectric focusing in detecting intrathecally synthesized IgG

The demonstration of intrathecally synthesized IgG constitutes one of the most important aids in the diagnosis of multiple sclerosis (MS). This can be done either by the application of empirical formulae or by analytical electrophoretic migration of IgG. However, all methods are influenced by the presence of blood-brain barrier (BBB) damage, as confirmed by the present study. We compared the results achieved by the application of 3 formulae (IgG index, intra-BBB IgG synthesis rate, IgG hyperbolic function) to those by isoelectric focusing (IEF) of unconcentrated cerebrospinal fluid (CSF). The most reliable method to detect intrathecal IgG production was IEF followed by specific immunofixation; even so, this method was subjected to pitfalls due to BBB damage. All formulae gave incorrect and misleading results, especially in case of BBB damage. Therefore, this limiting factor should be considered when formulae are used for clinical purposes.