sepharose and Multiple-Sclerosis

We combined a vector decomposition technique with Gaussian probability thresholding in feature space to segment normal brain tissues, tumors, or other abnormalities on dual-echo MR images. The vector decomposition technique assigns to each voxel a fractional volume for each of two tissues. A probability threshold, based on an assumed Gaussian probability density function describing random noise, isolates a region in feature space for fractional volume calculation that minimizes contamination from other tissues. The calculated fractional volumes are unbiased estimates of the true fractional volumes. The contrast-to-noise ratio (CNR) between tissues on the segmented images is the same as the Euclidean norm of CNRs in the original images. The method is capable of segmenting more than two tissues from a set of dual-echo images by sequentially analyzing different pairs of tissues. The model is analyzed mathematically and in experiments with a phantom. Two clinical examples are presented.

Topics: Adenocarcinoma; Algorithms; Artifacts; Brain; Brain Neoplasms; Cerebrospinal Fluid; Copper; Copper Sulfate; Cysts; Gels; Humans; Image Enhancement; Image Processing, Computer-Assisted; Magnetic Resonance Imaging; Models, Biological; Models, Structural; Models, Theoretical; Multiple Sclerosis; Probability; Sepharose

Seven different formulae and agarose isoelectrofocusing (AIF) using immunolabelling for IgG were compared for their ability to discriminate between intrathecally produced IgG and transudated IgG in cerebrospinal fluid. All reference limits were set to a specificity of 97.5% (reference group, n = 211). The probability of a positive test (p+) was evaluated for 112 patients with multiple sclerosis (MS), 42 with meningitis, 114 with noninflammatory diseases affecting the central nervous system (CNS), 23 with Guillain-Barré syndrome, and 56 with various diseases not affecting the CNS. Agarose isoelectrofocusing had the best diagnostic sensitivity (93%) for MS, combined with a low p+ (0-19%) for other diseases. Among the formulae, the IgG extended index and Reiber's hyperbolic formula were equivalent, giving high (75-79%) diagnostic sensitivity for MS combined with low p+ (4-22%) for other diseases. All other formulae, although sensitive for MS, had a higher rate of false positive results.

Topics: Algorithms; Humans; Immunoglobulin G; Isoelectric Focusing; Meningitis; Multiple Sclerosis; Nervous System Diseases; Polyradiculoneuropathy; Reference Values; Sensitivity and Specificity; Sepharose

To detect immunoglobulin G (IgG) oligoclonal bands in unconcentrated cerebrospinal fluid (CSF) we used a recently developed method combining agarose isoelectric focusing (IEF) and double immunofixation peroxidase staining with Avidin-Biotin amplification. We studied 65 CSF and serum paired specimens from normals, multiple sclerosis (MS), other neurological diseases (OND) and benign monoclonal gammopathies (BMG). We found that the oligoclonal IgG pattern can be demonstrated after IEF of 15 microliter of CSF specimens with an IgG concentration of 15 mg/L. In 98% of CSF from patients with clinically definite MS a sharp oligoclonal band pattern was detected. The reliability and the sensitivity of this powerful technique is compared to agarose IEF of concentrated CSF, followed by Coomassie Brilliant Blue staining. This method constitutes a real improvement in the detection of CSF IgG oligoclonal bands because it avoids CSF concentration and allows the detection of IgG bands only.

Topics: Avidin; Biotin; Humans; Immunoenzyme Techniques; Immunoglobulins; Isoelectric Focusing; Monoclonal Gammopathy of Undetermined Significance; Multiple Sclerosis; Nervous System Diseases; Oligoclonal Bands; Ovalbumin; Sepharose; Staining and Labeling

CSF oligoclonal IgG bands are often found in MS or cerebral diseases in which there is chronic antigenic stimulation. Using agarose isoelectric focusing followed by immunoblotting, we found oligoclonal IgA bands in CSF from 16 of 20 randomly selected patients with MS, 7 of 7 with subacute sclerosing panencephalitis (SSPE), and 0 of 10 with noninflammatory neurologic or psychiatric disease. IgA bands did not correlate with the course or stage of MS. Serial samples from two patients with MS and one with SSPE demonstrated only minor changes in IgA banding pattern. One MS patient without oligoclonal IgG bands had oligoclonal IgA bands, indicating that the latter test may be helpful in the diagnosis of MS.

Topics: Adolescent; Adult; Child; Humans; Immunoenzyme Techniques; Immunoglobulin A; Isoelectric Focusing; Middle Aged; Multiple Sclerosis; Sepharose; Subacute Sclerosing Panencephalitis

Specificity of antibodies to Victoria strain of Newcastle disease virus (NDV) found in infectious mononucleosis (IM) and other pathologic sera was investigated by agglutination of NDV-modified human O red blood cells, as well as by immunodiffusion and enzyme immunoassay with various preparations of the virus. These studies clearly demonstrated that the NDV antibodies are distinct from P-B or H-D antibodies. The unexpected observation that guinea pig kidney (GPK) tissues absorbed NDV antibodies allowed their classification into a group of 'GPK-positive' heterophile antibodies. The simultaneous occurrence of the NDV antibodies and H-D antibodies in IM and other diseases suggests the possibility that multiple new antigenic determinants, especially those of carbohydrate nature, may appear due to the alteration of self-antigens as a result of various pathologic processes.

Topics: Antibodies, Viral; Antibody Specificity; Gels; Humans; Immunodiffusion; Infectious Mononucleosis; Leprosy; Lupus Erythematosus, Systemic; Multiple Sclerosis; Neoplasms; Newcastle disease virus; Sepharose; Syphilis

To demonstrate oligoclonal IgG bands (I) in unconcentrated cerebrospinal fluid, we used isoelectric focusing in agarose followed by protein transfer to cellulose nitrate membrane, double-antibody peroxidase labeling, and avidin-biotin amplification. I can be reliably seen after isoelectric focusing of 5-microL specimens containing 125 ng of IgG (25 mg/L). Thus the technique is more sensitive than others (e.g., silver staining) and more reliable than radioimmunofixation. When we used this technique with fluids from 62 patients with multiple sclerosis and infectious disease of the central nervous system, 84% displayed I, a percentage not increased when the same specimens were concentrated to 3.5 g of IgG per liter, examined by agarose isoelectric focusing, and stained with Coomassie Blue. Results for 53 patients with tension headache and psychoneurosis were all negative. By obviating the need to concentrate samples of cerebrospinal fluid the present method is a useful, sensitive alternative for demonstrating I.

Topics: Avidin; Biotin; Central Nervous System Diseases; Collodion; Fluorescent Antibody Technique; Humans; Immunoenzyme Techniques; Immunoglobulin G; Indicators and Reagents; Infections; Isoelectric Focusing; Multiple Sclerosis; Rosaniline Dyes; Sepharose

Agarose isoelectric focusing (AIEF) of concentrated CSF was compared with AIEF of unconcentrated CSF and subsequent immunofixation with radiolabeled antihuman IgG Fc Fragment antiserum and autoradiography for the demonstration of oligoclonal bands in CSF from 287 neurological patients. Oligoclonal bands were demonstrated by AIEF in 98% of 43 patients with multiple sclerosis, 72% of 18 patients with infectious CNS diseases, and 23% of 226 patients with other neurological diseases. The corresponding figures obtained with AIEF of unconcentrated CSF and radioimmunofixation were 98%, 67% and 21%, respectively. In 15 of the patients, oligoclonal bands were demonstrated in CSF and serum by both techniques. They are both useful alternatives for the demonstration of oligoclonal bands in CSF, and the method for unconcentrated CSF can be safely applied when only small CSF volumes are available. The oligoclonal IgG pattern obtained by AIEF was not influenced by concentration of CSF by ultrafiltration and subsequent dilution to the original IgG concentration, nor by storage for 6 months.

Topics: Adult; Aged; Female; Humans; Immunoglobulins; Immunologic Techniques; Iodine Radioisotopes; Isoelectric Focusing; Male; Meningitis; Meningoencephalitis; Middle Aged; Multiple Sclerosis; Nervous System Diseases; Oligoclonal Bands; Polysaccharides; Preservation, Biological; Sepharose; Specimen Handling

A newly developed peroxidase-linked immunoassay is described which is sensitive enough to quantify herpes simplex antibodies in serum and cerebrospinal fluid diluted to an IgG level of 1 mg/dl. Thus, a comparison of photometric signals allows the direct detection of specific antibodies which have been secreted by activated tissue B lymphocytes into the CSF compartment during the humoral immune phase of herpes simplex encephalitis. The technique utilizes urea-Triton-dissolved virus antigens covalently bound to Sepharose 4B pearls. A highly specific sandwich antibody was purified by immune absorption column chromatography and labelled in its protected state. In the majority of cases the antibody level increased around the 10th day, to reach its maximum a few days after. In some cases however the serum levels gradually rose over a period of several weeks. The antibody levels in the CSF increase uniformly at the same time, irrespective of the general immune response and soared up to higher than serum levels within a few days. Local antibody production may persist for years so that late diagnosis of herpes encephalitis becomes possible with a single side by side test of serum and CSF from the patient.

Topics: Acute Disease; Animals; Antibodies, Viral; Brain; Chronic Disease; Complement Fixation Tests; Encephalitis, Arbovirus; Enzyme-Linked Immunosorbent Assay; Herpes Simplex; Humans; Immunoglobulin G; Meningoencephalitis; Multiple Sclerosis; Rabbits; Sepharose

Demonstration of oligoclonal bands by electrophoresis of cerebrospinal fluid is an important aid in establishing the diagnosis of multiple sclerosis. Electrophoretic systems vary in their effectiveness in doing so. We compared two systems in this respect. For a thin-layer agarose system, sensitivity was less (47%) than for a high-resolution agarose system (87%). Each system had good specificity (92 and 85%, respectively). Interpretation of electrophoretic patterns for cerebrospinal fluid should be available in clinical laboratories. Further, the best available system should be used for demonstration of oligoclonal bands.

Topics: Electrophoresis; Electrophoresis, Agar Gel; Humans; Immunoglobulins; Multiple Sclerosis; Sepharose; Time Factors

A method is described for preparative isoelectric focusing in agarose using low electroendosmotic agarose. Resolution comparable to that seen on analytical polyacrylamide gels is attainable as demonstrated by the isolation of bands with identical idiotypes from the serum of a patient with a monoclonal gammopathy.

Topics: Electrophoresis, Agar Gel; Humans; Hypergammaglobulinemia; Immunoglobulin Idiotypes; Isoelectric Focusing; Multiple Sclerosis; Polysaccharides; Sepharose

Using a leucocyte migration test (Clausen's direct agarose gel migration method) hypersensitivity to human encephalitogenic protein has been examined in 50 multiple sclerosis patients (group 1), 50 healthy persons (group 2) and 25 patients with other neurological diseases (group 3). In group 1, 30 MS patients (60%) show an abnormal migration index, manifested either as inhibition or stimulation of migration; 29 controls in group 2 (58%), 11 O.N.D. patients in group 3 (44%) show an abnormal migration index. These results mean that lymphocyte hypersensitivity to myelin basic protein appears neither to be constant nor specific to multiple sclerosis. Three migration index curve types at different antigen concentration are obtained: monophasic curves within the normal index zones; monophasic curves staying in the inhibition or stimulation zone and biphasic curves with dose-effect relationship. Whatever the antigen used, this dose-effect relationship implies that the test must be carried out at different concentrations. The meaning of spontaneous sensitisation in healthy controls is discussed.

Topics: Adolescent; Adult; Age Factors; Cell Migration Inhibition; Cell Movement; Dose-Response Relationship, Immunologic; Female; Humans; Hypersensitivity, Delayed; Lymphocytes; Male; Middle Aged; Multiple Sclerosis; Myelin Basic Protein; Sepharose; Sex Factors

Ten patients with MS were studied with leukocyte migration in agarose technique to detect changes in reactivity to encephalitogenic protein in connexion with a relapse. Six showed significant reactivity within a few days after the relapse. It decreased or disappeared during the 2 weeks after the relapse, but sometimes reappeared and was found in tests performed 2-3 months later. Five patients with cerebral infarction were studied in a similar way--in three, marked reactivity was noted within a few days after the stroke; in these, reactivity decreased or disappeared in later tests. In the two other patients, reactivity appeared in the second and/or third test. The possibility of a reactivity as in epiphenomenon due to CNS tissue destruction is discussed, and the need for antigens with a more restricted specificity for such an analysis is stressed.

Topics: Cell Migration Inhibition; Cells, Cultured; Humans; Intracranial Embolism and Thrombosis; Leukocytes; Longitudinal Studies; Lymphocytes; Multiple Sclerosis; Myelin Basic Protein; Recurrence; Sepharose

Ten patients with MS were studied with leukocyte migration in agarose technique to detect changes in reactivity ot encephalitogenic protein in connexion with a relapse. Six showed significant reactivity within a few days after the relapse. It decreased or disappeared during the 2 weeks after the relapse, but sometimes reappeared and was found in tests performed 2-3 months later. Five patients with cerebral infarction were studied in a similar way--in three, marked reactivity was noted within a few days after the stroke; in these, reactivity decreased or disappeared in later tests. In the two other patients, reactivity appeared in the second and/or third test. The possbility of a reactivity as an epiphenomenon due to CNS tissue destruction is discussed, and the need for antigens with a more restricted specificity for such an analysis is stressed.

Topics: Cell Movement; Humans; Intracranial Embolism and Thrombosis; Leukocytes; Multiple Sclerosis; Myelin Basic Protein; Recurrence; Sepharose; Time Factors

The literature on in vitro test of sensitivity to brain antigen in multiple sclerosis is summarized in the tables. A study is presented using leukocyte migration in agarose as test system and a wide range of concentration of bovine encephalitogenic protein as antigen. Definite reactivity was seen in 12 out of 28 patients with MS. Some occurred in patients who had had no signs of disease activity for at least 5 years. Reactivity occurred more often in patients with active disease studied within 5 days after a relapse. The significance of the in vitro reactivity is briefly discussed.

Topics: Antigens; Cell Migration Inhibition; Cell Movement; Cells, Cultured; Dose-Response Relationship, Immunologic; Epitopes; Humans; Leukocytes; Lymphocytes; Multiple Sclerosis; Myelin Basic Protein; Sepharose