RESUMO
BACKGROUND: On the basis of preliminary evidence from patients with subarachnoid haemorrhage (SAH), axonal degeneration is thought to be an underestimated pathological feature. METHODS: A longitudinal study in 17 patients with aneurysmal SAH. Ventricular CSF was collected daily for up to 14 days. The neurofilament heavy chain(SMI35) (NfH(SMI35), a biomarker for axonal damage) was quantified using a standard ELISA (upper limit of normal 0.73 ng/ml). The primary outcome measure was the Glasgow Outcome Score (GOS) at 3 months. RESULTS: Of 148 samples from patients with SAH, pathologically high NfH levels in the CSF were found in 78 (52.7%) samples, compared with 20 (5%) of 416 samples from the reference population (p<0.0001). A pathological increase in NfH was observed in all patients with a bad outcome (GOS 1-3) compared with 8% of those with a good outcome (GOS 4-5, p<0.0001). This increase typically became significant 7 days after the haemorrhage (p<0.01). The result was confirmed by analysing the individual mean NfH concentrations in the CSF (3.45 v 0.37 ng/ml, p<0.01), and was reinforced by the inverse correlation of NfH in the CSF with the GOS (r = -0.65, p<0.01). Severity of injury was found to be correlated to NfH(SMI35) levels in the CSF (World Federation of Neurological Surgeons, r = 0.63, p<0.01 and Glasgow Coma Score, r = -0.61, p<0.01). CONCLUSION: Patients with SAH thus have secondary axonal degeneration, which may adversely affect their outcome.
Assuntos
Axônios/patologia , Biomarcadores/líquido cefalorraquidiano , Aneurisma Intracraniano/complicações , Proteínas de Neurofilamentos/líquido cefalorraquidiano , Hemorragia Subaracnóidea/complicações , Adulto , Idoso , Feminino , Escala de Resultado de Glasgow , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Prognóstico , Índice de Gravidade de Doença , Hemorragia Subaracnóidea/fisiopatologiaRESUMO
This study evaluated a relationship between nitric oxide (NO) and migraine attacks in order to gain insight into migraine pathomechanism. The study groups consisted of 12 migraineurs and eight controls. All subjects collected morning urine samples for 40 consecutive days. Urinary NO metabolites, nitrite/nitrate (NO(x)) levels were measured with the vanadium-based assay, whilst creatinine (Cr) and neopterin were determined with high-performance liquid chromatography. The mean urinary NO(x)/Cr ratio and number of NO(x) peaks was significantly greater in the migraine group compared with controls (P = 0.01 and P = 0.007, respectively). In the second approach, high NO(x) values were re-assessed in relation to raised neopterin, a marker of systemic infection or inflammation, and were excluded. The excretion of NO(x) persisted being pulsatile, and migraineurs had more peaks compared with controls (P = 0.01). In seven patients, NO(x) peaks coincided with headache days. This was more frequent than expected by random association in four patients (Monte-Carlo simulation; odds ratios: 2.16-7.77; no overlap of 95% CI). In four patients, NO(x) peaks preceded or followed headache days. Although there is a difference in the pattern of urinary NO(x) excretion between control and migraine populations, the variable temporal association of NO(x) peaks and headaches suggests a complex role of NO in this condition.
Assuntos
Transtornos de Enxaqueca/urina , Óxido Nítrico/urina , Biomarcadores/sangue , Creatinina/urina , Monitoramento Ambiental/métodos , Feminino , Humanos , Estudos Longitudinais , Masculino , Neopterina/urina , Valores de ReferênciaRESUMO
Previously described methods for identification of proteins separated in cylindrical polyacrylamide gels have been found to be costly in time and antiserum and difficult to apply to small amounts of protein as are found in cerebrospinal fluid. We describe a method which involves printing of the proteins on the cut surface of the gel onto nitrocellulose paper. The protein bands of the imprint can then be identified using labelled antibodies. We have found this to be economical and quick, and it has permitted sensitive and reliable identification of proteins in unconcentrated cerebrospinal fluid and aqueous humour.
Assuntos
Proteínas Sanguíneas/isolamento & purificação , Proteínas do Líquido Cefalorraquidiano/isolamento & purificação , Proteínas/isolamento & purificação , Eletroforese em Gel de Poliacrilamida/métodos , Humanos , Soros ImunesRESUMO
Electrically active axons degenerate in the presence of nitric oxide (NO) in vitro. High CSF NO concentrations have been observed in patients with hemorrhagic brain injury such as subarachnoid hemorrhage (SAH) and intracerebral hemorrhage (ICH). This study investigated the evidence for axonal injury in SAH and ICH and related this to CSF NO levels. In this study, neurofilament phosphoforms (NfH(SMI34), NfH(SMI35), NfH(SMI38), NfH(SMI310)), surrogate markers for axonal injury, and NO metabolites (nitrate, nitrite = NOx) were measured by ELISA in cerebrospinal fluid (CSF) from patients with SAH and ICH and from a group of controls. Injury severity was classified using the Glasgow Coma Scale, and survival was used as the outcome measure. Compared to the control group, a higher proportion of patients with SAH and ICH had elevated NfH(SMI34) levels from day 0 to day 6 (p < 0.001), elevated NfH(SMI35) levels from day 1 to 6 (p < 0.001), and elevated NfH(SMI310) levels at day 0, 1, 4, and 6 (p < 0.001). The NOx levels were higher in the SAH and ICH patients than in the controls (p < 0.05) and distinguished the non-survivors from the survivors (p < 0.05). No direct correlation was found for NOx with any of the NfH phosphoforms. This study provides evidence for primary and secondary axonal injury in patients with SAH and ICH, with non-survivors also having higher NOx levels. CSF NfH phosphoforms might emerge as a putative surrogate marker for monitoring the development for secondary axonal degeneration in neurocritical care and guiding targeted neuroprotective strategies.
Assuntos
Axônios/patologia , Hemorragia Cerebral/líquido cefalorraquidiano , Hemorragia Cerebral/patologia , Hemorragia Subaracnóidea/líquido cefalorraquidiano , Hemorragia Subaracnóidea/patologia , Adulto , Idoso , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Neurofilamentos/líquido cefalorraquidiano , Óxido Nítrico/líquido cefalorraquidiano , Estudos ProspectivosRESUMO
Recent discoveries shed light on the importance of prostaglandin (PG) production in the development of skin cancer. Work by Fischer et al. demonstrates that skin tumor promotion caused by ultraviolet B radiation can be decreased by up to 89% by blocking cyclooxygenase-2 (COX-2) with the drug Celecoxib. A similar study showed that Celecoxib can decrease new tumor formation by 44% in mice that already have tumors. These studies demonstrate the importance of COX-2 and PGs in the development of squamous cell carcinoma. We have explored growth signaling in a model of skin tumor progression. Because changes in PG production have been implicated in skin carcinogenesis, we examined this pathway. We found that malignant cell lines secrete more prostaglandin E(2) (PGE(2)) than the parental cells. We observed increased expression of COX-1 and -2. We also found that these cells express the PGE(2) receptors EP1 and EP4. When the cells are grown in the presence of indomethacin, the growth rate of the malignant cells is decreased. This effect can be reversed by addition of PGE(2) or an EP1 agonist to the medium. Thus, we have shown that skin tumor cells depend in part on PGE(2) signaling through the EP1 prostanoid receptor for their in vitro growth.
Assuntos
Queratinócitos/metabolismo , Receptores de Prostaglandina E/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/metabolismo , Animais , Western Blotting , Divisão Celular/fisiologia , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Primers do DNA/química , Dinoprostona/metabolismo , Técnicas Imunoenzimáticas , Isoenzimas/metabolismo , Queratinócitos/patologia , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Prostaglandina-Endoperóxido Sintases/metabolismo , Receptores de Prostaglandina E Subtipo EP1 , Receptores de Prostaglandina E Subtipo EP4 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/patologia , Células Tumorais CultivadasRESUMO
In a three-way crossover study, 23 patients with hepatic cirrhosis, ascites, and dependent edema received 40 mg/day of furosemide alone and combined with triamterene 50 mg/day and triamterine 100 mg/day. Baseline potassium excretion did not increase when furosemide was given alone, but potassium excretion fell when 50 mg or 100 mg of triamterene was also given. Both doses of triamterene augmented the natriuretic effect of furosemide.
Assuntos
Furosemida/uso terapêutico , Cirrose Hepática/tratamento farmacológico , Potássio/urina , Triantereno/uso terapêutico , Cloretos/urina , Ensaios Clínicos como Assunto , Quimioterapia Combinada , Eletrólitos/sangue , Feminino , Humanos , Masculino , Sódio/urinaRESUMO
Oligoclonal immunoglobulin D (IgD) bands were observed in 20 of 110 CSF samples investigated by our method for isoelectric focusing (IEF) of IgD in unconcentrated CSF. These were mostly from demyelinating disorders, viral CNS infections, and CNS tumors. The patterns of expression of IgD were similar to that of IgG, except in cases with tumors where oligoclonal IgD may occur independently of IgG. The study shows for the first time the presence of oligoclonal IgD in CSF from neurologic patients.
Assuntos
Imunoglobulina D/líquido cefalorraquidiano , Imunoglobulinas/líquido cefalorraquidiano , Doenças do Sistema Nervoso/líquido cefalorraquidiano , Humanos , Focalização Isoelétrica , Doenças do Sistema Nervoso/sangue , Bandas OligoclonaisRESUMO
BACKGROUND: The pathogenesis of fatigue in patients with MS is poorly understood. OBJECTIVE: To test the hypothesis that fatigue in MS is related to inflammatory disease activity as measured by systemic markers of inflammation. METHODS: Fatigue as assessed by the Fatigue Questionnaire Scale (FQS) and Krupp's Fatigue Severity Scale (KFSS) was correlated with several inflammatory markers in 38 patients with MS (16 relapsing-remitting [RR; 7 of whom had benign MS), 9 secondary progressive [SP], 13 primary progressive [PP]). The markers included daily urinary neopterin excretion, a marker of interferon-gamma-activated macrophage activity, and serum C-reactive protein (CRP) and soluble intercellular adhesion molecule-1 (sICAM-1) levels. Urinary neopterin excretion was measured daily for 2 weeks. RESULTS: No correlation was found between urinary neopterin excretion, CRP, or sICAM-1 and the fatigue scores. However, patients with a raised serum CRP level had higher KFSS, but not FQS, scores than patients with normal CRP levels (KFSS, 50 +/- 8 vs 41 +/- 14, p = 0.05; FQS, 13 +/- 4 vs 11 +/- 5, p = NS). When assessed using the FQS, patients with RR and SP MS were more fatigued than patients with PP MS (RR = 12.5 [4 to 23] vs SP = 13 [8 to 18] vs PP = 9 [7 to 14], p = 0.02). The patients with benign MS were as fatigued as patients with nonbenign disease. CONCLUSION: The pathogenesis of fatigue in MS is complex and does not appear to be directly related to systemic markers of inflammatory disease activity. Interestingly, patients with PP MS were less fatigued than patients with RR disease.
Assuntos
Fadiga/metabolismo , Fadiga/patologia , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Adulto , Idoso , Análise de Variância , Biomarcadores , Proteína C-Reativa/biossíntese , Distribuição de Qui-Quadrado , Fadiga/psicologia , Feminino , Humanos , Inflamação/metabolismo , Inflamação/patologia , Inflamação/psicologia , Molécula 1 de Adesão Intercelular/biossíntese , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/psicologia , Neopterina/biossínteseRESUMO
BACKGROUND: Treatment with human i.v. immunoglobulin (IVIg) modifies the course of Guillain-Barré syndrome (GBS), but its specific mode of action is unknown. Cellular interactions mediated through the release of cytokines play a role in the pathogenesis of GBS and may be regulated by IVIg therapy. OBJECTIVE: To delineate possible immunoregulatory mechanisms of IVIg in patients with GBS. METHODS: Circulating levels of the proinflammatory cytokines, tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta, were assayed in 21 patients with GBS before and serially after IVIg therapy. Comparisons were made with serum concentration of the anti-inflammatory cytokines, soluble TNF-alpha receptor and IL-10. Serial measurements were also performed in 12 untreated patients with relatively mild disease and 7 patients treated by plasma exchange. RESULTS: Circulating levels of TNF-alpha and IL-1beta decreased after treatment with IVIg but remained relatively high in untreated patients and in those treated by plasma exchange. Clinical improvement in patients treated with IVIg was associated with a reduction in unbound TNF-alpha during the acute phase of the illness. Circulating levels of anti-inflammatory cytokines were not affected by IVIg treatment. CONCLUSION: Data presented here suggest a novel mechanism of action of IVIg that involves selective modulation of circulating proinflammatory cytokines.
Assuntos
Citocinas/sangue , Imunoglobulinas Intravenosas/administração & dosagem , Polirradiculoneuropatia/tratamento farmacológico , Adulto , Seguimentos , Humanos , Inflamação/tratamento farmacológico , Troca PlasmáticaRESUMO
OBJECTIVE: To determine the sensitivity and specificity of methods to detect anti-basal ganglia antibodies (ABGA) in Sydenham's chorea (SC). BACKGROUND: SC is a delayed manifestation of group Abeta hemolytic streptococcal infection typically associated with rheumatic fever (RHF). SC is characterized by chorea and motor and neuropsychiatric symptoms. Patients with SC produce antibodies that cross-react with streptococcal, caudate, and subthalamic nuclei antigens detected using an immunofluorescent (IF) method with inconsistent reports of positivity. METHODS: The authors developed ELISA and Western immunoblotting (WB) methods to detect ABGA and compared these assays to IF. They investigated samples from patients with acute SC (n = 20), persistent SC (n = 16), control samples from RHF (n = 16), and healthy pediatric volunteers (n = 11). RESULTS: ABGA ELISA had a sensitivity of 95% and specificity of 93% in acute SC. Both WB and IF had a sensitivity of 100% and specificity of 93%. In the persistent SC group, ABGA sensitivity dropped to 69% using WB and to 63% using IF. Three common basal ganglia antigens were identified by WB in both acute and persistent SC (40 kDa [n = 15], 45 kDa [n = 15], and 60 kDa [n = 13]). There was no antibody reactivity to cerebellum, cerebral cortex, or myelin antigen preparations in any group. CONCLUSIONS: These results support the hypothesis that Syndenham's chorea is an autoantibody-mediated disorder. Western immunoblotting and immunofluorescence are the best methods for detecting anti-basal ganglia antibodies, and reactivity to basal ganglia antigens of 40, 45, and 60 kDa were commonly seen in both acute and persistent cases of SC.
Assuntos
Autoanticorpos/análise , Gânglios da Base/imunologia , Coreia/imunologia , Doença Aguda , Autoanticorpos/sangue , Western Blotting , Estudos de Casos e Controles , Coreia/microbiologia , Doença Crônica , Ensaio de Imunoadsorção Enzimática , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Sensibilidade e Especificidade , Infecções Estreptocócicas/imunologia , Streptococcus pyogenes/imunologiaRESUMO
The authors report high intrathecal release of tumor necrosis factor alpha (TNF-alpha) and interleukin (IL)-1beta in five patients with sporadic or new-variant Creutzfeldt-Jakob disease (CJD) without activation of the humoral or lymphocytic immune responses. Increased release of TNF-alpha and IL-1beta was also detected in some patients with progressive dementias. CJD is associated with a local cerebral host response that involves the release of proinflammatory cytokines.
Assuntos
Síndrome de Creutzfeldt-Jakob/líquido cefalorraquidiano , Síndrome de Creutzfeldt-Jakob/imunologia , Citocinas/imunologia , Mediadores da Inflamação/líquido cefalorraquidiano , Humanos , Mediadores da Inflamação/imunologia , Interleucina-1/líquido cefalorraquidiano , Fator de Necrose Tumoral alfa/líquido cefalorraquidianoRESUMO
Using isoelectric focusing (IEF) and immunoperoxidase staining of proteins transferred to nitrocellulose membranes, we have examined the IgG band pattern in tears and matched serum and CSF specimens of 28 patients with MS, 4 patients with optic neuritis (ON), 30 individuals with systemic, inflammatory, or other neurologic diseases, and 5 patients with tension headache. We found no evidence of positive oligoclonal IgG in tears in any MS or ON patients, while 10 out of 16 cases with systemic immune disorders or infections of the CNS had positive tear oligoclonal bands. We are thus not able to support the hypothesis that tears from MS patients reveal abnormalities in their humoral immune response.
Assuntos
Imunoglobulina G/análise , Esclerose Múltipla/imunologia , Lágrimas/imunologia , Adulto , Feminino , Humanos , Doenças do Sistema Imunitário/imunologia , Técnicas Imunoenzimáticas , Fragmentos Fc das Imunoglobulinas/análise , Imunoglobulina G/líquido cefalorraquidiano , Focalização Isoelétrica , Masculino , Esclerose Múltipla/líquido cefalorraquidiano , Doenças do Sistema Nervoso/imunologia , Valores de ReferênciaRESUMO
OBJECTIVE: To assess whether serial serum levels of soluble forms of intercellular adhesion molecule-1 (sICAM-1) and vascular cell adhesion molecule-1 (sVCAM-1) are useful as surrogate markers of disease activity in multiple sclerosis (MS). BACKGROUND: Increased levels of sICAM-1 and sVCAM-1 have been described in cross-sectional, but not longitudinal, studies of patients with MS. Although they appear to correlate with clinical and MRI markers of disease activity, their role as potential surrogate markers remains undefined. METHODS: Serial serum levels of sICAM-1 and sVCAM-1 were measured in patients with MS undergoing monthly gadolinium-enhanced MRI studies of the brain (462 gadolinium-enhanced MRI in 57 patients) and in 12 normal control subjects. Ten patients had primary progressive (PP), 22 relapsing remitting (RR), and 25 secondary progressive (SP) disease. RESULTS: Levels of sICAM-1 and sVCAM-1 were increased intermittently in patients with all subtypes of MS. Median levels of sICAM-1 were elevated in patients with MS compared with normal controls (normal controls median [interquartile range] = 176[119-209] compared with PP = 502[194-1768], RR = 419[158-481], and SP = 352[196-469] ng/mL; p = 0.04). After excluding patients with PP MS, patients with high sICAM-1 levels had a greater number of gadolinium-enhancing lesions per study (1.9[0.9-4.3]) than patients with normal levels (0.4[0-2.7], p = 0.03), and patients with MRI studies with no gadolinium-enhancing lesions had lower associated sICAM-1 levels (200 ng/mL[85-561]) than patients with only persistent (349 ng/mL[82-615]) or new enhancing lesions (497 ng/mL[108-667], p = 0.03). Patients with RR or SP disease that progressed clinically during the study had a greater number of gadolinium-enhancing lesions per MRI study (3.5[0.4-5.5]) than did patients in whom disease did not progress (1.2 [0.3-2.7], p = 0.03). The patients with progressive disease tended to have higher sICAM-1 levels (469 ng/mL [196-1019]) than patients in whom disease did not progress (353 ng/mL [171-469], p = 0.07). Although MS patients tended to have higher sVCAM-1 levels than did normal controls, this finding was not significant. No correlation could be found between levels of sVCAM-1 and gadolinium enhancement on MRI. CONCLUSIONS: Patients with MS have elevated levels of sICAM-1, which correlate with gadolinium enhancement on MRI and possibly short-term disease progression. Soluble ICAM-1, and not sVCAM-1, may therefore be suitable as a long-term surrogate marker of disease activity in MS.
Assuntos
Molécula 1 de Adesão Intercelular/sangue , Esclerose Múltipla/sangue , Molécula 1 de Adesão de Célula Vascular/sangue , Adulto , Biomarcadores , Progressão da Doença , Feminino , Gadolínio , Humanos , Estudos Longitudinais , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/diagnóstico , Fatores Sexuais , SolubilidadeRESUMO
Pervasive retrograde amnesia without anterograde memory impairment has rarely been described as a consequence of circumscribed brain damage. We report this phenomenon in a 33 yr-old, right-handed man (JG) in association with the extension in the right thalamus of a previously small, bilateral thalamic lesion. JG presented with a dense amnesia for autobiographical material more than a few years old, with some sparing of recent memories. Furthermore, he was completely unable to recognise famous people or world events. Many other aspects of semantic knowledge were intact and there was no evidence of general intellectual impairment, executive dysfunction or loss of visual imagery. Magnetic resonance imaging revealed an acute lesion in the right thalamus and two small, symmetrical, bilateral non-acute thalamic lesions. Follow-up neuropsychological assessment indicated a stable pattern of impaired retrograde and spared anterograde memory over 18 months and psychiatric assessments yielded no evidence of confabulation, malingering or other symptoms to suggest psychogenic amnesia. JG's profile indicates that the division of declarative memory into just two categories - episodic and semantic - is inadequate. Rather, his case adds to the growing body evidence to suggest that world knowledge pertaining to people and events is stored or accessed similarly to autobiographical information and differently from other types of more general factual knowledge. We hypothesize that the right mediodorsal thalamic nucleus and immediately surrounding regions comprise the central processing mechanism referred to by McClelland (Revue Neurologique, 150 (1994) 570) and Markowitsch (Brain Research Review, 21 (1995) 117) as responsible for inducing and co-ordinating the recall of these sorts of cortically stored memory engrams.
Assuntos
Amnésia Retrógrada/diagnóstico , Núcleo Mediodorsal do Tálamo , Doenças Talâmicas/diagnóstico , Adulto , Amnésia Retrógrada/fisiopatologia , Infarto Cerebral/diagnóstico , Infarto Cerebral/fisiopatologia , Diagnóstico Diferencial , Dominância Cerebral/fisiologia , Encefalomielite/diagnóstico , Encefalomielite/fisiopatologia , Seguimentos , Humanos , Imageamento por Ressonância Magnética , Masculino , Núcleo Mediodorsal do Tálamo/patologia , Núcleo Mediodorsal do Tálamo/fisiopatologia , Testes Neuropsicológicos , Doenças Talâmicas/fisiopatologia , Tomografia Computadorizada por Raios XRESUMO
We report a method for displaying the affinity distribution of a polyclonal antibody population using sodium thiocyanate elution followed by an ELISA detection technique. We have used this method to study the affinity distribution of antibodies in samples of CSF and serum from patients with MS, and compared the results to those obtained from patients with viral encephalitis. Patients with MS had predominantly low affinity antibody against a particular antigen whilst patients with a primary viral infection had predominantly high affinity antibody against the causative organism.
Assuntos
Afinidade de Anticorpos , Encefalite/imunologia , Imunoglobulina G/imunologia , Esclerose Múltipla/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Viroses/imunologiaRESUMO
The intrathecal production of IgM antibodies to different viral antigens was measured by a modification of ELISA that was both sensitive and specific. Suitably diluted CSF and homologous serum samples containing similar amount of IgM were examined, and a comparison of the photometric signals permitted the detection of specific antibodies secreted from activated lymphocytes into the CSF compartment during the course of viral infections of the central nervous system. Polyvinyl chloride (PVC) microtitre plates were activated by glutaraldehyde and then coated with different viral antigens. Test samples were incubated on these solid-phase antigens and virus-specific IgM antibodies were detected using a peroxidase-conjugated F (ab')2 fragment of anti-human IgM antibody to avoid interference from rheumatoid factors.
Assuntos
Anticorpos Antivirais/líquido cefalorraquidiano , Encefalite/líquido cefalorraquidiano , Imunoglobulina M/líquido cefalorraquidiano , Citomegalovirus/imunologia , Encefalite/imunologia , Glutaral/farmacologia , Herpes Simples/líquido cefalorraquidiano , Herpes Simples/imunologia , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Vírus do Sarampo/imunologia , Vírus da Caxumba/imunologia , Neurossífilis/líquido cefalorraquidiano , Neurossífilis/imunologia , Plásticos , Simplexvirus/imunologiaRESUMO
We report a novel observation which has been reproducibly noted whilst studying immunoglobulin G binding to antigen immobilized on polyvinyldifluoride membranes. In some samples we have observed a difference between the pattern of oligoclonal bands on the front surface when compared to the reverse side of the membrane. We postulate that this observation results from differing affinities of the specific antibody binding to the antigen immobilised on the membrane. High affinity antibody will bind to antigen on the surface of the membrane next to the gel, while lower affinity antibody appears to diffuse through to the reverse side of the membrane.
Assuntos
Immunoblotting/métodos , Imunoglobulina G/análise , Afinidade de Anticorpos , Colódio , Humanos , PolivinilRESUMO
A sensitive technique was developed for the quantitative detection of intrathecal production of interleukin-2 (IL-2). Concentrations of IL-2 in paired cerebrospinal fluid (CSF) and serum samples were measured by an enzyme-linked immunosorbent assay using a monoclonal antibody and an affinity purified polyclonal antibody. The assay produced a linear response with respect to IL-2 concentration, and could readily detect levels of IL-2 as low as 1.5 international units/ml. Concentrations of IL-2 in CSF and serum samples were standardised by calculating their ratio to albumin concentration in order to correct for passive transudation of IL-2 across blood-CSF barriers. CSF IL-2/albumin ratios higher than concomitant serum ratios were considered indicative of intrathecal IL-2 production. The technique provides a sensitive, specific, and reproducible method for the determination of in vivo synthesis of IL-2 within the central nervous system.
Assuntos
Interleucina-2/líquido cefalorraquidiano , Ensaio de Imunoadsorção Enzimática , Humanos , Interleucina-2/biossíntese , Interleucina-2/imunologiaRESUMO
A sensitive, simple and specific sandwich ELISA for S-100b is described. This method involves the binding of a monoclonal anti-S-100b antibody to the wall of a microtitre plate. This capture antibody is subsequently incubated with S-100b standard, control or patient sample in the form of cerebrospinal fluid (CSF). After incubation, the microtitre plate is washed and horseradish peroxidase-labelled polyclonal anti-S-100b is added (detector antibody). The amount of detector antibody bound to the microtitre plate is proportional to the amount of S-100b in the sample. The assay has a lower limit of detection of 0.04 ng/ml and shows < 0.006% reactivity with the closely related polypeptide S-100a. The assay has a mean within-batch precision of 9.3 and 5.6% at S-100b concentrations of 0.38 and 0.8 ng/ml, respectively. The between batch precision is 8.9 and 8.1% at S-100b concentrations of 0.12 and 0.34 ng/ml, respectively. The recovery of S-100b from CSF spiked with 0.5 ng/ml was 94% with a CV of 8.5%. The assay may be completed in less than 5 h using precoated microtitre plates, thus lending itself to routine use in clinical laboratories. Using this ELISA, 154 CSF samples were analysed and 19% of samples were found to have elevated levels. The highest levels were found in patients with cerebral haemorrhage or central nervous system malignancy. S-100b concentrations from individuals without evidence of neurological disease were found to be less than 0.4 ng/ml. Only 5% of patients with multiple sclerosis were found to have elevated CSF S-100b concentrations. Serial CSF samples taken from a patient with an infected in-dwelling shunt showed a dramatic decline, suggesting that S-100b is rapidly cleared.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Proteínas S100/líquido cefalorraquidiano , Humanos , Fatores de Crescimento Neural , Subunidade beta da Proteína Ligante de Cálcio S100 , Sensibilidade e EspecificidadeRESUMO
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.