Immunoassay of P2 protein in cerebrospinal fluid in neurological disorders.

Cerebrospinal fluid samples were obtained at lumbar puncture from 53 patients with a wide variety of neurological disorders. Cerebrospinal fluid samples were tested for the presence of P2 protein, a constituent of myelin, with an enzyme linked immunosorbent assay technique using a specific polyclonal antibody. High concentrations of P2 in the cerebrospinal fluid paralleled a raised IgG index (clearance ratio), the presence of oligoclonal bands, as well as raised white cell counts or depressed albumin:IgG ratios. Twenty one patients had been diagnosed as having definite or probable multiple sclerosis and the remaining 32 had other conditions. Of the 13 patients with high positive P2, 12 (92%) were in the multiple sclerosis category; of the 40 patients with low (12) or undetectable (28) P2 concentrations, only nine (23%) were diagnosed as having multiple sclerosis. In this patient population the presence of high immunoreactive P2 concentrations in cerebrospinal fluid was closely associated with evidence of intrathecal immunoglobulin synthesis and with the clinical diagnosis of multiple sclerosis. On this basis it is suggested that immunoassay of P2 concentration in the cerebrospinal fluid may be of potential value in the investigation of patients with demyelinating disorders.     

Cerebrospinal fluid activity of tissue plasminogen activator in patients with neurological diseases.

AIM: To study cerebrospinal fluid (CSF) activity of tissue plasminogen activator (tPA) in patients with neurological diseases. METHODS: CSF tPA and urokinase (uPA) activities were studied using an immunocapture assay and zymography in 44 patients with neurological disease and 20 reference subjects. The patient group comprised three patients with meningitis, 21 with encephalitis, nine with acute lymphoblastic (n = 7) and myeloid (n = 2) leukaemia, seven with multiple sclerosis, three with facial paresis, and one with polyradiculitis. RESULTS: Raised tPA activities were observed in patients with multiple sclerosis, leukaemia and encephalitis. In contrast, there were no differences in the mean activities of tPA in patients with meningitis or other diseases compared with the reference subjects. The highest tPA activities were found in patients with multiple sclerosis. The mean activity in patients with leukaemia was higher than in those with meningitis and polyradiculitis, but not encephalitis and facial paresis. Although the CSF tPA activity correlated positively with age in reference subjects, no correlation was observed in patients. Samples were qualitatively screened for both tPA and uPA activity by zymography and positive samples were quantitated. Some of the samples had quantifiable levels of uPA activity: three of seven multiple sclerosis samples, 10 of 21 samples from patients with encephalitis and five of nine leukaemic samples. The highest activities were recorded in patients with leukaemia. uPA was not detected in the CSF of the patients with meningitis, facial paresis or polyradiculitis. CONCLUSIONS: Plasminogen activator activity can be measured reliably in CSF and the assessment of tPA activity may be useful for studying the pathogenesis of neurological diseases.     

Cytoimmunological profile of cerebrospinal fluid in diagnosis of multiple sclerosis

The purpose of this paper is to report cerebrospinal fluid (CSF) findings in multiple sclerosis (MS) from our laboratory, to discuss the implications of CSF abnormalities in terms of diagnosis. Paired CSF-serum samples from of 1533 on 3893 patients with suspected neurological diseases over a 10 year period were analysed by routine laboratory microscopy and assays of immunoglobulin G by isoelectric focusing for the detection of intrathecal oligoclonal IgG. Patients were grouped further into four headings according to their disorders: MS (625 cases), definite (246 cases) probable (123 cases) and possible (256 cases) according to Poser, others inflammatory neurological diseases (91 cases), various non-inflammatory neurological disorders (732 cases) and uncertain neurological disorders (85 cases). Definite MS group (16%) was compared to non-inflammatory neurological disorders (48%). Important signs for activity of multiple sclerosis are observed. Cell counts were 10/microl in 71% (N < or =2/microl). Inflammatory cytology is observed after concentration and cytocentrifugation on slides with activated B-lymphocytes, lymphoplasmocytes and/or plasmocytes (76%), total protein concentration is increased in 37% (N < 0.40g/l), CSF/serum albumin quotient with age dependent references for the blood-CSF barrier dysfunction is increased in 26% (N < 0.65 x 10(-2)), IgG index for intrathecal synthesis of IgG is increased in 69% (N < 0.70), sensitive detection of oligoclonal IgG restricted to CSF by isoelectric focusing is positive in 91% (86-96%) with a specificity of 96% (93-99%).     

Rapid diagnosis of tuberculous meningitis with a dot enzyme immunoassay to detect antibody in cerebrospinal fluid

A simple dot enzyme immunoassay (Dot-EIA) was carried out to detect antibody toMycobacterium tuberculosis antigen 5 in cerebrospinal fluid (CSF) specimens from 40 patients with a clinical diagnosis of tuberculous meningitis (TBM). The assay gave a positive reaction in all ten patients with culture proven TBM. In 30 culture negative patients with TBM, the assay was positive at a titre of 1:16 in 18 patients. In 40 patients with non-tuberculous neurological diseases (control group) the assay was negative at a titre of 1:16. The Dot-EIA had an overall sensitivity of 70 % and a specificity of 100 % in the diagnosis of TBM. This assay could be used as a rapid screening test to establish the diagnosis of TBM, particularly in patients in whom bacteriological investigations forMycobacterium tuberculosis in CSF specimens are negative.     

Detection of specific IgG and IgM antibodies to Toxoplasma gondii with a commercially available enzyme immunoassay kit system

A total of 138 serum samples submitted for toxoplasma serology have been examined by enzyme immunoassay using kits produced by Labsystems Oy for the detection of specific antibodies of the IgG and IgM class. Results were compared with the dye test, an indirect haemagglutination test, and an indirect immunofluorescence test for specific IgM. The enzyme immunoassay was less sensitive than the dye test, but by running both IgG and IgM enzyme immunoassays, 92.4% sensitivity was achieved. The specificity of the enzyme immunoassay was good, with only one dye test negative serum giving a positive (but weak) IgG enzyme immunoassay reaction. Thirty serum samples from patients with no evidence of exposure to Toxoplasma gondii gave negative results in the IgM enzyme immunoassay. Enzyme immunoassay results were expressed in enzyme immunoassay units, as a percentage value of a standard serum. This convention will be of value in the direct comparison of assay systems and in the application of quality control procedures.     

In vivo activated T lymphocytes in the peripheral blood and cerebrospinal fluid of patients with multiple sclerosis

We found an increase in peripheral-blood lymphocytes bearing the T-cell-specific activation antigen Ta1 in 20 of 35 patients with progressive multiple sclerosis, 4 of 18 patients with stable or improving multiple sclerosis, 1 of 17 patients with other neurologic diseases, and 1 of 14 normal controls (P less than 0.0002, Fisher's exact test). No increases in two other markers of T-cell activation, T113 and the interleukin-2 receptor, were found. In the cerebrospinal fluid, patients with progressive multiple sclerosis (pleocytosis, 3.9 +/- 1.6 cells per cubic millimeter) had 42 +/- 3.0 per cent Ta1+ cells. In contrast, patients with other inflammatory central nervous system diseases (36 +/- 13 cells per cubic millimeter) had 9.6 +/- 1.8 per cent Ta1+ cells (P less than 0.01). In patients with other neurologic diseases without inflammation (0.7 +/- 0.16 cells per cubic millimeter), the percentage of Ta1+ cells was equivalent to that in patients with multiple sclerosis (39 +/- 5.4 per cent), although the absolute number was lower. There was a positive correlation between the presence of Ta1+ cells in the spinal fluid and blood of patients with other neurologic diseases, but not patients with multiple sclerosis. Less than 1 per cent of lymphocytes from the spinal fluid of patients with multiple sclerosis expressed interleukin-2 receptors, as compared with 9.8 per cent of cells from subjects with other inflammatory neurologic diseases (P less than 0.01). These results suggest that the T cells in the spinal fluid of patients with multiple sclerosis may be activated by a different mechanism or in a different temporal sequence from that in patients with other nervous system diseases. Furthermore, the increase in Ta1+ cells in the peripheral blood of patients with multiple sclerosis demonstrates systemic immune activation in the disease; monitoring such cells may provide an objective measure of abnormal immunologic activity.     

Multiplexed AtheNA multi-lyte immunoassay for ANA screening in autoimmune diseases

BACKGROUND: Multiplexed assays using fluorescence microspheres is an exciting technology with multiple applications including the detection of antinuclear autoantibodies (ANA) and autoantibody profiles. It is a rapid, sensitive and automatic method for simultaneous quantitative detection of several autoantibodies. The aim of our study was to determinate ANA and other autoantibodies to the nine extractable nuclear antigens by the AtheNA Multi-Lyte ANA system and compare the results achieved by this method to the routinely used enzyme immunoassay. METHODS: Four hundred eighteen serum samples were tested utililizing the multiplexed method: 96 healthy donors, 86 requested ANA specimens obtained from routine lab, and 236 samples from patients with known autoimmune diseases (43-scleroderma, 113-systemic lupus erythematosus, 38-Sjogren's syndrome, and 42 rheumatoid arthritis). The ANA and antibodies to nine different analytes (SS/A, SS/B, Sm, RNP, Jo-1, Scl-70, dsDNA, Centromere B and Histone) were tested. RESULTS: ANA screening by AtheNA system revealed high concordance of 99 and 97.7% with the enzyme immunoassay test in samples obtained from healthy donors and ANA requested samples, respectively. Evaluation of autoimmune disease-related samples for ANA by AtheNA technology also confirmed a high rate of concordance of 92-97.7% and correlated with the enzyme immunoassay. Positive discrepant results were found for Scl-70 specificity in 12.7% of SLE specimens by AtheNA technology, while all tested sera were negative for this antibody by enzyme immunoassay. Negative discrepant results were observed by the AtheNA system for anti-dsDNA. The sera (15 randomly obtained samples from SLE patients) were positive for anti-dsDNA in 50% of samples in Farr assay and 55% in enzyme immunoassay, respectively. CONCLUSION: We suggest that the AtheNA technology may be a useful diagnostic tool for ANA screening. Additional investigations are required to compare an analytic performance between AtheNA and routine methods in determination of the individual autoantibody profile.     

Determination of patient herpes simplex virus immune status by latex agglutination.

A rapid latex agglutination (LA) test was compared with complement fixation and enzyme immunoassay for the determination of herpes simplex virus immune status in sera of patients. Of 173 samples, 88% gave concordant results in all three assays (115 positive and 37 negative). The LA and complement fixation tests agreed 92% of the time, and the LA agreed with the enzyme immunoassay 94% of the time. LA is a simple and rapid test which can be used for determining the herpes simplex virus immune status in sera of patients.     

Detection of Chlamydia trachomatis in genital specimens by the Chlamydiazyme test.

Cotton swabs were used to collect two specimens each from 416 patients (206 males, 210 females) attending a sexually transmitted disease clinic. The first swab was transported in Specimen Storage Reagent and extracted in Specimen Dilution Buffer for enzyme immunoassay by Chlamydiazyme (Abbott Laboratories); the second swab was extracted into 2SP and inoculated into McCoy cell cultures. In the first phase of the study (215 patients: 111 males, 114 females) enzyme immunoassay results were positive (optical density greater than or equal to 0.1) in 30 of 35 instances in which Chlamydia trachomatis was isolated (sensitivity, 86%). Of 18 false-positive enzyme immunoassay results, 15 (83%) were cervical swabs (specificity, 90%). In a phase II study, using a modified Chlamydiazyme kit, 201 patients were tested (95 males, 106 females). Of 41 chlamydial isolates, 8 were not detected by the Chlamydiazyme test (sensitivity, 81%). Only three positive Chlamydiazyme test results could not be confirmed by culture (specificity, 98%). Overall, Chlamydiazyme assay provided a rapid (4 h), sensitive, and specific assay for the detection of chlamydial antigens.     

Non-specific peroxidase (donor: H2O2-oxidoreductase, EC 1.11.1.7) activity in multiple sclerosis

The non-specific peroxidase (donor: H2O2-oxidoreductase, EC 1.11.1.7) activity of red blood cells in patients with multiple sclerosis, patients with other neurological diseases, and healthy control individuals was investigated. To this end, a simple method was developed. No significant difference was found in the non-specific peroxidase activity of red blood cells from patients with multiple sclerosis and controls.     

Improved serodiagnosis of tuberculosis using two assay test.

An antigen capture immunoassay was developed for the detection of mycobacterial antigens in sera from patients with tuberculosis. The assay was evaluated together with an antibody measuring enzyme immunoassay in a clinical trial for serodiagnosis of tuberculosis. Sensitivity of the antibody assay for active pulmonary tuberculosis, including relapsed infections, was 75%, and specificity with other lung diseases was 97%. Sensitivity for extrapulmonary tuberculosis was 84.5% and specificity 84%. Sensitivity of the antigen assay for active tuberculosis was 45% with no false positive reactions. Combination of the results from the two assays increased total sensitivity to 96.5% with a positive predictive value of 0.81 and a negative value of 0.98. The two assay test was relatively simple to perform and offered improved serological diagnosis of tuberculosis over a single antibody test.     

Evaluation of enzyme immunoassay (Chlamydiazyme) for detecting Chlamydia trachomatis in genital tract specimens.

An enzyme immunoassay (Chlamydiazyme) for detecting Chlamydia trachomatis was evaluated on genital specimens from 96 men and 272 women attending a clinic for sexually transmitted diseases (STD clinic). Compared with a direct immunofluorescence test for chlamydial elementary bodies, the enzyme immunoassay had a sensitivity of 58% on specimens from men, a specificity of 90%, a positive predictive value of 93%, and a negative predictive value of 88%; the assay had a sensitivity of 67% on specimens from women, a specificity of 89%, a positive predictive value of 63% and a negative predictive value of 90%. Immunofluorescence provided the most stringent test for the performance of the enzyme immunoassay as values were improved a little when a cell culture procedure was used for comparison. Further evidence for the lack of sensitivity was the detection of elementary bodies, sometimes in large numbers, in the enzyme immunoassay buffer of 13 of 19 specimens that had given a negative enzyme immunoassay result and the finding in comparative titrations of four laboratory strains that the enzyme immunoassay was at least 100-fold less able to detect chlamydiae than either immunofluorescence or the cell culture procedure. Lack of specificity may be associated with the finding that the enzyme immunoassay antibody reacted with strains of Acinetobacter calcoaceticus, Escherichia coli, Gardnerella vaginalis, Neisseria gonorrhoeae and group B streptococci. The enzyme immunoassay was not considered to be sufficiently sensitive, specific, or reproducible for routine use.     

Multiplexed AtheNA multi-lyte immunoassay for ANA screening in autoimmune diseases

Background: Multiplexed assays using fluorescence microspheres is an exciting technology with multiple applications including the detection of antinuclear autoantibodies (ANA) and autoantibody profiles. It is a rapid, sensitive and automatic method for simultaneous quantitative detection of several autoantibodies. The aim of our study was to determinate ANA and other autoantibodies to the nine extractable nuclear antigens by the AtheNA Multi-Lyte ANA system and compare the results achieved by this method to the routinely used enzyme immunoassay.

Methods: Four hundred eighteen serum samples were tested utililizing the multiplexed method: 96 healthy donors, 86 requested ANA specimens obtained from routine lab, and 236 samples from patients with known autoimmune diseases (43-scleroderma, 113-systemic lupus erythematosus, 38-Sjogren's syndrome, and 42 rheumatoid arthritis). The ANA and antibodies to nine different analytes (SS/A, SS/B, Sm, RNP, Jo-1, Scl-70, dsDNA, Centromere B and Histone) were tested.

Results: ANA screening by AtheNA system revealed high concordance of 99 and 97.7% with the enzyme immunoassay test in samples obtained from healthy donors and ANA requested samples, respectively. Evaluation of autoimmune disease-related samples for ANA by AtheNA technology also confirmed a high rate of concordance of 92–97.7% and correlated with the enzyme immunoassay. Positive discrepant results were found for Scl-70 specificity in 12.7% of SLE specimens by AtheNA technology, while all tested sera were negative for this antibody by enzyme immunoassay. Negative discrepant results were observed by the AtheNA system for anti-dsDNA. The sera (15 randomly obtained samples from SLE patients) were positive for anti-dsDNA in 50% of samples in Farr assay and 55% in enzyme immunoassay, respectively.

Conclusion: We suggest that the AtheNA technology may be a useful diagnostic tool for ANA screening. Additional investigations are required to compare an analytic performance between AtheNA and routine methods in determination of the individual autoantibody profile.     

Beta 2-microglobulin decrease in cerebrospinal fluid from parkinsonian patients

A sandwich enzyme immunoassay (EIA) was established by using purified beta 2-microglobulin (beta 2-MG) as a standard protein and a polyclonal antibody raised against human beta 2-MG. The EIA was applied for the measurement of beta 2-MG levels in human cerebrospinal fluid (CSF) from parkinsonian patients and control patients devoid of neurological diseases. beta 2-MG contents in CSF of the control group and the parkinsonian group were 1.81 +/- 0.11 micrograms/ml CSF and 0.63 +/- 0.09 microgram/ml CSF, respectively. Thus, beta 2-MG content in CSF was reduced in parkinsonian patients to less than 35% of the control value (P less than 0.005). We had previously reported that the activity and content of dopamine beta-hydroxylase (DBH) were decreased in CSF from parkinsonian patients. A significant positive correlation (r = 0.87) was observed between the beta 2-MG content and DBH activity for CSF from 45 patients. These results suggest a probable link between an immunological change and the changes in catecholaminergic neurons in Parkinson's disease.     

多发性硬化患者抗髓鞘碱性蛋白抗体的检测及其意义

目的 检测多发性硬化(MS)患者抗髓鞘碱性蛋白(MBP)抗体并探讨其意义.方法 采用ELISA方法测定56例多发性硬化患者急性期血清和脑脊液配对标本的抗髓鞘碱性蛋白抗体,30例其它神经疾病(OND)患者及36例正常人(NC)为对照.结果 以OND组患者OD值为标准,测得MS患者脑脊液抗MBP抗体,其阳性率为26.8%,与OND组比较有显著性意义;以NC组OD值为对照,MS患者血清抗MBP抗体阳性率为78.6%,OND组抗MBP抗体阳性率为50%,与NC组均有显著性意义,MS患者与OND组患者抗MBP抗体阳性率比较亦有显著性意义.结论 多发性硬化患者脑脊液和血液中均可检测到抗MBP抗体,可为临床诊断和治疗提供指导.     

The distribution of interleukin-2 receptor bearing lymphocytes in multiple sclerosis: evidence for a key role of activated lymphocytes.

The identification of T cells in the brain using monoclonal antibodies has suggested a role for T cells in the pathogenesis of multiple sclerosis (MS). In the present study the monoclonal antibody anti-Tac, shown to react with interleukin-2 (IL-2) receptors expressed on activated T cells, was used to determine levels of recently activated T cells in blood, cerebrospinal fluid (CSF) and brain sections from MS patients at different stages of disease. The CSF of MS patients contained much higher numbers of IL-2 receptor positive lymphocytes (up to 67%) than blood cells from the same patients, or the CSF of patients with non-inflammatory neurological diseases. In histological sections of the brain of MS patients with active disease, perivascular lymphocytes expressing IL-2 receptors were detected, as were lymphocytes containing IL-2. In contrast, these were absent in brain sections from patients with chronic MS, secondary demyelination or from normal controls. These observations in CSF and brain suggest that in multiple sclerosis, T-cell activation is occurring within the CNS and not in peripheral lymphoid tissue.     

Screening test for rheumatic diseases: a combined enzyme immunoassay of rheumatoid factors and antibodies to DNA and extractable nuclear antigens.

Three hundred and one sera from patients with rheumatic and other diseases were investigated using a simple enzyme immunoassay for screening of rheumatoid factors and antinuclear antibodies. The assay had a sensitivity of 77% for systemic lupus erythematosus, 90% for the primary sicca syndrome, and 89% for rheumatoid arthritis. Only 13% of sera from patients with chronic non-rheumatic diseases were positive. The test was further evaluated in a group of patients with suspected rheumatic disease who were followed up for six to 12 months. The test was positive in 16 of 17 sera from patients with connective tissue diseases but in only seven of 36 sera (19%) from patients with non-inflammatory joint diseases. None of the four patients with reactive arthritis was positive by this test. The sensitivity of the assay was comparable with that of the agglutination and immunofluorescence tests for rheumatoid factors and antinuclear factors. For the screening of rheumatoid factor and antinuclear antibodies this kind of test panel offers a simple alternative to the conventional tests for small clinical laboratories and for those in which the autoantibody tests could be automated, as the assay can be performed in one working day and only one dilution of serum is needed to obtain a quantitative result.     

Disproportionate elevation of the immunoglobulin G1 concentration in cerebrospinal fluids of patients with multiple sclerosis

We determined immunoglobulin G (IgG) subclass concentrations and studied their distributions in the cerebrospinal fluids of patients suffering from multiple sclerosis, other inflammatory neurological diseases, and non-inflammatory diseases of the nervous system in comparison with a control group. In addition, the four subclass concentrations were measured in serum specimens of the multiple sclerosis and control groups. These data were correlated with the extent of local IgG synthesis in the subarachnoid spaces of the patients belonging to the different groups. We found a selective elevation of the IgG1 subclass in the cerebrospinal fluids of multiple sclerosis patients, and there was only a very small overlap of the IgG1 ranges of the multiple sclerosis and control groups. No major differences were detected between the IgG subclass distributions in different courses of multiple sclerosis nor between multiple sclerosis and control sera. The group with non-inflammatory diseases showed a uniform elevation of all four subclasses and a greater overlap with the normal range. This latter feature was combined with an elevated IgG1 concentration in the group with other inflammatory diseases. It is concluded that locally synthesized IgG in the cerebrospinal fluids of multiple sclerosis patients consists mainly of IgG1.     

Immunoglobulin M immunoblot for diagnosis of Borrelia burgdorferi infection in patients with acute facial palsy.

We used immunoblotting to improve the specificity of the serologic diagnosis of Lyme borreliosis in cases of acute facial palsy. Twelve of 15 patients (80%) with suspected Lyme borreliosis, versus 0 of 10 controls, were positive by immunoglobulin M immunoblotting of acute-phase sera and 3 were negative, including 2 with borderline enzyme immunoassay results. Immunoglobulin M immunoblotting is a useful test to confirm Borrelia burgdorferi infection in patients with acute facial palsy and a positive enzyme immunoassay result.     

A Symbol Digit Modalities Test version suitable for functional MRI studies

The Symbol Digit Modalities Test is an easy test used to assess cognitive impairment in a wide range of neurological diseases, like multiple sclerosis. We adapted the oral version of this cognitive task making it suitable for functional Magnetic Resonance Imaging studies. Symbol Digit Modalities Test performance was associated with increased brain activity in frontal and parietal areas involved in selective attention and working memory functions. These may provide the basis for future studies assessing potential abnormal cortical activations in multiple sclerosis patients and other clinical populations.