Isotachophoresis of CSF proteins in gel tubes especially gammaglobulins. An analytical and preparative technique for high-separation of CSF proteins.

An isotachophoretic method using polyacrylamide gel (PAG-ITP) in a simple disc electrophoretic equipment with plastic tubes containing the gels, was elaborated and especially designed for studying the gammaglobulins in CSF and serum from control subjects and patients with neurological disorders, especially known or probable MS. The device and the ITP system used, including leading and terminating electrolytes and spacer substances, dividing the gammaglobulins in a reproducible way, are described. No cooling of the gel tubes was needed. The sample volumes varied between 5--500 microliters, and the separation time was 1.5--3.0 h. CSF from patients with verified or probable MS revealed characteristic, increased low-mobility gammaglobulin fractions. Using other ITP systems, such as other spacer compositions, the anodic proteins can also be studied in more detail. PAG-ITP in gel tubes is a simple and inexpensive technique which can be used for both analytical and preparative procedures for biological material such as CSF, serum and extractions from nervous tissues.

Isotachophoresis in capillary tubes of CSF proteins -- especially gammaglobulins.

Isotachophoresis in polyacrylamide gel tubes (PAG-ITP) and in capillary tubes (Tachophor, LKB) have previously been found by the authors, to be very promising high-separation methods for CSF and serum proteins, especially regarding the diagnosis of MS. PAG-ITP methods for analytical and preparative use have been described by the authors elsewhere, while in this paper proper cationic systems for ITP in capillary tubes for studying gammaglobulins in microliter amounts of CSF and serum are described, i.e. the albumin injection-clog problem is avoided and the preparation time can be forced. By using microdialysis of the CSF samples for desalting, with a technique easy to perform and with high reproducibility, microliter amounts of native CSF can be performed in less than half an hour. The method seems to be even more applicable for clinical and scientific use if the capillary isotachophoretic apparatus is connected to a synchronized equipment (LKB Tachophrac) with a cellulosa acetate strip onto which the separated fractions are ejected for further analysis by immunological tests. The analytical systems used have been especially directed to gammaglobulins in CSF and serum regarding further studies on demyelinating and infectious disorders of the nervous system.

High-voltage isoelectric focusing in ultrathin gels and enzyme-amplified immunoassay: a new method for analysis of cerebrospinal fluid proteins.

A procedure using high-voltage isoelectric focusing (IF) in ultrathin (02. mm) gels and enzyme-amplified immuno-sandwich assay was elaborated to get optimal IF separation conditions, to avoid CSF concentration, e.g. by ultrafiltration preceding IF with the risk of unequal protein losses, to minimize the amounts of CSF and expensive reagents needed, especially antibodies and to shorten the analysis time, including the selective detection of proteins. The high voltage (2000-3000 V/10 cm) and efficient cooling during IF were obtained using ECPS 3000/150 and FBE 3000 (Pharmacia, Sweden). Ampholytes (Pharmalytes) of different pI intervals were used. The CSF and (diluted) serum samples were microdialysed in polyacrylamide gel before IF to minimize band curvature and to obtain optimal resolution. The IF separation was performed in about 1 h. Owing to the rapid fixation of ultrathin gels after IF, full use could be made of the high-voltage resolving capacity. The thin gels also made histochemical techniques applicable. Different immunological identification assays have been tested. An enzyme-amplified (alkaline phosphatase) immuno-sandwich method was found to be very sensitive and selective, and has so far given the best results. Many proteins in the same sample, applied as a line on the gel before IF, could be detected by overlaying antibody-soaked membrane strips. Furthermore, one specific protein could be examined in many samples simultaneously by overlaying or immersion of diluted antibody solutions. A few microlitres of unconcentrated CSF and diluted serum were used for the analysis performed within 1 day. The findings for albumin, transferrin and IgG in CSF and sera from patients with different neurological diseases, especially including cases with "normal" CSF, barrier damage, degenerative and demyelinating disorders, have been compared with the corresponding protein-stained (Coomassie R-250) patterns where the CSF had been concentrated by a special vacuum evaporation technique before IF.

Determination of CSF proteins by a simple and rapid immunonephelometric method.

A simple, fast and reliable immunonephelometric (I-NEPH) method has been worked out for the determination of CSF proteins. A good quality and low price I-NEPH apparatus was used. The results were obtained from regression lines, constructed from various parts of the calibration curve, calculated by using a simple pocket calculator. Ultrasound was found to be a simple and effective cleaning technique for nephelometry. The method was used for determining concentrations of albumin, IgG, IgA, IgM and the ratio of the light kappa and lambda chains in CSF and serum from 15 control cases, 11 MS patients, and three patients with plasma cell dyscrasias. The I-NEPH method was found to be a valuable complement to high separation techniques including especially isoelectric focusing used for CSF examinations, e.g., for evaluating influence of serum protein composition and degree of barrier damage. An increased kappa-lambda ratio was observed in some of the patients with MS in accordance with previous investigations but was normal in four of the nine MS cases where the ratio was examined.

Analytical isotachophoresis: a new method for analysis of cerebrospinal fluid proteins. A preliminary report.

Isotachophoresis of CSF proteins seems to be a very promising method. Very small CSF samples, a few mul of concentrated and 15-30 mul of unconcentrated CSF, can be quickly analysed (30-60 min), and the results immediately obtained on a recorder. Using unconcentrated CSF, losses due to concentration procedures, are avoided. Low-molecular weight compounds, e.g. in CSF ultrafiltrates, can also be examined. The method gives high resolution, is reproducible, and is easy to perform. The isotachophoretic findings have been compared with those of electrofocusing.

Microdialysis of cerebrospinal fluid in polyacrylamide gels: a suitable procedure prior to electrophoretic analysis.

Most electrophoresis methods for separation of CSF proteins are generally preceded by some procedure of concentration and desalting of the specimen. Generally ultrafiltration techniques are used. The risk of losses, which may be unequal for different CSF proteins during such procedures, is to be stressed. On the other hand, desalting prior to isoelectric focusing (IEF) will minimize the curvature of the protein bands, and in isotachophoresis (ITP) faster separation and increased capacity with repeated sample application are made possible. Since some years microdialysis of samples has been performed by the authors and found to be a valuable procedure both prior to IEF and ITP. With respect to microheterogeneity and recovery, tested by IEF, immunonephelometry and radioiodinated proteins no losses were observed. Ion exchange of acrylamide in a mixed resin, and recrystallization of bisacrylamide, were found necessary to avoid absorption from very dilute protein solutions as CSF. Gel structure and performance were very dependent on polymerization conditions (time, temperature, initiator and accelerator concentrations).

Glucose determination in samples taken by microdialysis by peroxidase-catalyzed luminol chemiluminescence.

An automatic, luminometric assay of glucose in samples of the extracellular water space obtained by microdialysis is described. The assay involves oxidation by glucose oxidase (EC 1.1.3.4) and mutarotation of glucose by aldose mutarotase (EC 5.1.3.3.). The H2O2 formed is subsequently determined in a reaction catalyzed by horseradish peroxidase (EC 1.11.1.7) using luminol as electron donor. The assay is linear between 0.01 and 1 nmol in the cuvette. The detection limit, defined as 3 standard deviations of the reagent blank, was 0.008 mumol/liter in the cuvette. A complete oxidation of glucose is obtained within 4 min and 25 samples are automatically assayed within 75 min. Addition of microdialysate sample obtained from human adipose tissue in vivo did not interfere with the standard curves. Glucose added to microdialysate resulted in a complete recovery compared to a H2O2 standard. Analytical interference from different factors was investigated. No interference was observed up to the following concentrations: 5 mumol/liter epinephrine, 1 mumol/liter norepinephrine, 100 mumol/liter insulin, 500 mumol/liter pyruvate, 50 mmol/liter lactate, and 1 mumol/liter ascorbate. The glucose values with the present method correlated strongly (r = 0.984) with values obtained using a routine method involving glucose oxidase and peroxidase.