Affinity distributions of antigen-specific IgG in patients with multiple sclerosis and in patients with viral encephalitis.

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.

Differential oligoclonal band patterns on polyvinyldifluoride membranes.

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.

Affinity of antigen-specific IgG distinguishes multiple sclerosis from encephalitis.

The characteristics of antigen-specific IgG in patients with multiple sclerosis and patients with encephalitis have been compared. Both groups of patients showed antigen-specific oligoclonal bands locally synthesised in the CSF. When the affinity distribution of the antigen-specific IgG was measured there was a marked difference between the two groups. Encephalitis patients had high affinity antibody against the causative antigen. This was consistent with the antibody undergoing affinity maturation as a result of the immune system fighting a primary infection. Multiple sclerosis patients lacked high affinity response. This lack of high affinity antibody was also seen in those encephalitis patients when antigens other than the causative antigen were studied.

Isoelectric focusing versus quantitative measurements in the detection of intrathecal local synthesis of IgG.

Specimens from 1007 patients with suspected neurological disturbances had quantitative and qualitative measurements made of cerebrospinal fluid and serum to investigate the presence of locally synthesised IgG. Qualitative measurement was recorded as the presence or absence of oligoclonal banding, and the quantitative measurement was derived by the use of the IgG index, the log index and the Reiber, Schuller and Tourtellotte formulae. The patients were divided into two categories, on the basis of banding: those with local synthesis and those without. The sensitivity, specificity and efficiency for each of the quantitative measurements were then calculated. Receiver-operator curves were also constructed for each of the quantitative measurements. 282 samples showed local synthesis of IgG by isoelectric focusing, whereas the best quantitative assay (log index) could only detect 198. Therefore, we conclude that oligoclonal banding should be adopted as the standard laboratory measurement of local synthesis of IgG in the diagnosis of neurological disorders, and that the diagnostic use of quantitative measurements should be abandoned for routine purposes. Furthermore, we suggest that quantitative analysis, at its current level, is misleading and has little value in the understanding of neurological disorders, but may be of use in serially monitoring individual patients as part of their therapeutic trials.

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.

A study of immunoglobulin G in the cerebrospinal fluid of 1007 patients with suspected neurological disease using isoelectric focusing and the Log IgG-Index. A comparison and diagnostic applications.

Cerebrospinal fluid and serum immunoglobulin G from 1007 patients with suspected neurological disease were analysed by 2 methods: isoelectric focusing for the detection of oligoclonal banding, and quantitative measurement of IgG and albumin for the formulation of a Log IgG-Index. A comparison of the 2 methods in the detection of local synthesis of IgG showed that isoelectric focusing not only gave a much higher yield overall, with 282 patients showing local synthesis versus 225 for the Log IgG-Index, but also a higher specificity, with a false positive rate of 0% versus 3.5%. In addition, of the 282 patients positive by isoelectric focusing only 163 (58%) were positive by the Log IgG-Index. Of the 1007 patients studied, 206 had multiple sclerosis (MS), and isoelectric focusing showed local synthesis in 95% of clinically definite cases, with a 90% detection rate overall. The Log IgG-Index was positive in only 67% of clinically definite cases, with an overall 59% detection rate. Thus with the exceptions noted above, local synthesis of IgG as defined by isoelectric focusing is confined to demyelinating, inflammatory, infectious and postinfectious disorders. Our results compare very favourably with the published sensitivities of magnetic resonance imaging in the detection of abnormalities in multiple sclerosis, and better than those for evoked potentials. Where both these investigations are readily available isoelectric focusing provides a useful adjunct. For the majority of physicians and neurologists who do not have ready access to magnetic resonance imaging, isoelectric focusing is an excellent alternative. We would also recommend that it become the standard for the measurement of IgG abnormalities in the cerebrospinal fluid and that the use of quantitative data be abandoned for routine purposes.

Towards the development of molecularly imprinted polymer based screen-printed sensors for metabolites of PAHs.

This paper describes the fabrication of a sensor for 1-hydroxypyrene (1-OHP) based on a screen-printed carbon electrode (SPCE) modified with a molecularly imprinted polymer (MIP); 1-OHP was chosen as a model metabolite of polyaromatic hydrocarbons (PAHs). It was shown that 1-OHP could be readily oxidised at a plain SPCE and the electrochemical mechanism was found to involve an ECE (electron transfer-chemical reaction-electron transfer) process. The MIP for 1-OHP was prepared using only divinylbenzene (DVB) and styrene as monomers and the binding was only based on hydrophobic interactions. Batch binding studies revealed that optimum uptake of 1-OHP by the MIP occurred from solutions containing 35% water in methanol. Selectivity of the binding sites in the MIP was examined by performing uptake studies in the same solution containing either phenol or 1-naphthol; the specific binding of 1-OHP was twenty times greater than the former and five times greater than the latter. Preliminary calibration studies were performed with the MIP-SPCE using a two-step approach; accumulation was carried out in 35% water in methanol followed by measurement in 50% methanol-0.025 mol dm(-3) phosphate buffer pH 12. This two-step non-competitive affinity assay gave encouraging results and indicated potential for use in pollution studies.

A micro-method for measuring total protein in cerebrospinal fluid by using benzethonium chloride in microtiter plate wells.

By using a benzethonium chloride concentration 12-fold that described originally (Clin Chem 1979;25:1317-9), we developed a reliable method suitable for routine measurement of total protein in cerebrospinal fluid. Only 10 microL of sample is required. Reactivity to immunoglobulin G (IgG) and to albumin (Alb) is similar, as is necessary for specimens that can have very varied IgG/Alb ratios. The assay, performed in a microtiter plate for ease of use, has a between-batch coefficient of variation of 3.4% for a protein concentration of 450 mg/L. This contrasts with a dye-binding technique with Ponceau S, for which the CV was 9% at the same concentration.

The JC virus antibody response in serum and cerebrospinal fluid in progressive multifocal leucoencephalopathy.

BACKGROUND: A clinical diagnosis of progressive multifocal leucoencephalopathy (PML) can be confirmed by histological or virological examination of brain material. Whilst a less invasive method is provided by the detection of JC DNA in cerebrospinal fluid (CSF), very few studies have been done to assess the value of JC virus (JCV) serology in PML diagnosis. OBJECTIVES: To study the JCV antibody response in the serum and CSF of PML patients. STUDY DESIGN: A retrospective study was done using haemagglutination inhibition (HI), M-antibody capture radioimmunoassay (MACRIA) and JC-specific oligoclonal IgG banding on one or more sera and/or CSFs from 28 confirmed PML patients. Seventy-one serum and CSF samples were tested from patients with memory loss or dementia as a control group. RESULTS: Twenty-seven PML patients (96%) had detectable JCV HI antibody in the serum, with titres ranging from 1 : 10 to > 1 : 20480, compared to 48 (68%) of the controls (P = <0.005). JCV IgM antibody was detected in the serum of 12/22 (55%) PML patients. JCV HI antibody was detected in the CSF in 12 of 18 (67%) PML patients, antibody index measurements being used to control for a possible breakdown of the blood-brain barrier. Intrathecal JCV antibody was not found in any control patient. Locally produced JCV-specific IgG bands were detected in the CSF of 7 PML patients tested, confirming the intrathecal origin and specificity of the HI antibody. CONCLUSIONS: The presence of intrathecal JCV antibody indicates active central nervous system infection with JC virus, and provides a useful diagnostic test for PML, with a sensitivity of 67% and a specificity of 100%. The absence of serum JCV antibody nearly always excludes a diagnosis of PML, but the titre of antibody, IgG or IgM, correlates with the underlying condition rather than the development of neurological symptoms.