Separation of cerebrospinal fluid specific proteins — A methodological study

Journal of the Neurological Sciences, 1980, 44:247-257 © Elsevier/North-Holland BiomedicalPress

247

SEPARATION OF CEREBROSPINAL FLUID SPECIFIC PROTEINS - - A M E T H O D O L O G I C A L STUDY PART I

C. WlKKELSO, C. BLOMSTRAND and L. RONNB.~CK Department of Neurology and Institute of Neurobiology, University of Gi~teborg, Gi~teborg (Sweden)

(Received 18 July 1979) (Accepted I1 September, 1979)

SUMMARY A method for preparative separation of cerebrospinal fluid specific proteins by affinity chromatography and isoelectric focusing is described. It has been tested on CSF from a group of human patients suffering from TIA. The advantage of the method is the production of a protein pattern easy to survey by eye. The sensitivity and reproducibility are adequate, as tested by model experiments described.

INTRODUCTION During recent years, neui obiologists have isolated and characterized nervous tissue -specific proteins (i.e. see Moore and McGregor, 1965; Hyd6n and R6nnb/ick 1979). In human patients the possibility of studying proteins in brain and spinal cord tissue is limited to material obtained from neurosurgical operations and postmortem material. Accordingly, a special interest has been focused on cerebrospinal fluid (CSF) proteins which reflect protein metabolic parameters in the central nervous system, including the cerebrovascular system and the choroidal plexus (for review, see Davson 1969; Lowenthal 1969). The existence of CSF proteins not found in serum has been proposed for many years. Using immunoelectrophoresis with antisera raised against CSF, Clausen (1961) found two proteins not present in serum. Laterre et al. (1964) and Bock (1973) using This work was supported by grants from the MedicalFaculty of G6teborg (Rederiaktiebolaget Transatlantics Fond), from the Multiple Sclerosis Foundation, The G6teborg Medical Society Foundation, and from Anna Ahrenbergs Foundation.

248 different immunological techniques, were able to show 9 precipitation lines in the cerebrospinal fluid, which were not present in serum. Two of these proteins, fl-trace and y-trace, have been further characterized by Link (1967) and Olsson et al. (1973). The largest portion of the CSF proteins is derived from blood plasma, the actual proportion depending on the functional state of the 'blood-CSF barrier' which is defective in several neurological diseases. This explains why small changes in CSF proteins which are not serum-derived, can easily be masked by changes in serum proteins or in 'barrier function'. In several degenerative brain disorders, characteristic protein changes have been demonstrated in the CSF (Laterre et al. 1964), but this is only found in a minority of the patients and therefore is of lesser importance in the diagnosis. A more specific study of the non-serum proteins in CSF would probably add new information concerning degenera*ive brain disorders. One way to do this is to study the content of separate brain-specific proteins. Murazio et al. (1977) were able to detect the nervous tissue-specific S 100 protein (Moore and McGregor 1965) in human CSF from some patients with multiple sclerosis. Myelin basic protein has been demonstrated in CSF and has shown correlations to episodes of multiple sclerosis (Roboz-Einstein 1969; Palfreyman et al. 1978). Glial fibrillary acidic protein was first extracted and purified by Eng et al. (1971) and quantitated in CSF by Mori et al. (1975). The fl-trace protein is a major constituent of the non-serum proteins, and comprises about 7 ~ of the total protein in CSF (Link 1967). The present work was aimed at developing another way of studying brainspecific protein. A method has been developed for preparative separation of the CSFspecific proteins through affinity chromatography (Ax6n and Porath 1966; Ax6n et al. 1967), separating CSF-specific proteins from proteins antigenically similar to serum proteins. The highly sensitive system of isoelectric focusing (Vesterberg 1972, 1973) was used for analytical estimation. MATERIAL AND METHODS Patients

CSF and serum samples were obtained from 7 consecutive patients (aged 45-70) who had suffered from transient ischemic attacks (TIA) according to the WHO classification. Thorough neurological and general somatic examinations lumbar puncture did not reveal any symptoms or signs of neurological disease. None of the patients had any history of previous neurological disease. Chest X-ray, electrocardiography, and measurements of blood sugar, lipids, uric acids, electrolytes, serum enzymes and other routine blood tests all proved to be normal. Furthermore, brain scan and electroencephalography were normal. CSF samples (15 ml) were obtained by lumbar puncture in the forenoon under standardized conditions. No blood contamination of the CSF was found. Routine CSF examinations, including total protein, cell count and pigment analysis, were carried out.

249

Absorption chromatography Absorption chromatography was performed by insolubilisation of antibodies to human serum proteins on a carbohydrate matrix by means of cyanogen halides, essentially according to Ax6n et al. (1967). Pure rabbit anti-human antibody against more than 50 serum proteins (DAKO, Copenhagen, Denmark) was used (see below). This was compared with rabbit anti-human total serum (Svenska Hoechst, Stockholm, Sweden) and gave similar results*. The rabbit anti-human serum proteins were coupled to CNBr-activated Sepharose 4B (Pharmacia) and packed on columns K 15/30, volume 50 ml (Pharmacia), 10 mg to 1 ml Sepharose 4B according to Table 1. Then 2.0-2.5 mg of unconcentrated CSF protein was passed through the columns and eluted with a Tris-HCl buffer in 2.0 ml fractions. The eluate was read at 280 nm in a Zeiss spectrophotometer to detect proteins until the absorbance of the fractions was 0. These proteins were considered to be CSF-specific. Proteins adsorbed to the antibody-coated Sepharose matrix, considered as CSF non-specific, were eluted by lowering the p H to 2.8 using a glycine-HC1 buffer (Table 1). Thus, two fractions were obtained, one enriched in CSF-specific proteins (fraction I), the other containing proteins antigenically identical to serum proteins (fraction II). Protein determinations of total CSF and of the different fractions obtained by affinity chromatography (see below) were carried out according to Ponceau (Salo and H a n k a v a a r a 1974) and according to the Lowry protein determination technique TABLE 1 WASHING AND ELUTION SCHEME FOR CYANOGEN-BROMIDE ACTIVATED SEPHAROSE 4 B CNBr-activated Sepharose 4B swollen in 10-3 M HC! Anti serum proteins dissolved in 0.1 M NaHCO3 in 0.5 M NaC1 are coupled + Washing with coupling buffer (0.1 M NaHCO3 in 0.5 M NaCI) to remove unbound material 1 M ethanolamine (pH 8) to remove any remaining active groups 0.1 M acetate buffer in 1 M NaCI (pH 4) ~, 0.1 M borate buffer in 1 M NaCI (pH 8)

i ( to remove non-covalently adsorbed protein }

Cerebrospinal fluid Elute with 0.15 M Tris-/-IC! in 0.5 M NaCI (pH 8) until ODz8o = 0 Elute with 0.15 M glycine-HCl in 0.5 M NaCI (pH 2.8) The protein peak is pooled and quickly made pH 7 * The antisera were gifts from DAKO and Svenska Hoechst to whom the authors are grateful.

250 (Lowry et al. 1951), as modified by Lous et al. (1956). When using the Lowry technique, corrections for buffer interference was necessary. In some experiments, proteins were precipitated from the CSF using 10 ~ TCA. Equal results were found with the different techniques used, and the Lowry protein determination technique was preferred because of its high sensitivity. Concentration of protein fractions was measured using 47 mm stirred molecular filtration cells (Millipore) with Pelicon membranes. Two types were tested: PTCS 04710, which trap molecules with molecular weights above 10,000, and PSAC 04710, which trap molecules with molecular weights above 1,000-2,000 in solution. Differences were noticed in the isoelectric focusing pattern of fraction I concerning one protein band at a pI of 9.3 in relatively higher concentrations when the PSAC membrane was used (see below), the pressure used for filtration did not exceed 20 psi. Lyophilisation was tried but could not be used as a single concentration procedure, because the salt ion concentration became too high for isoelectric focusing.

Electrophoretic systems (1) The isoelectric focusing procedure was carried out in flat beds of polyacrylamide gel of dimensions 11 x 25 x 0.1 cm, according to Vesterberg (1972, 1973), the pH range in the gel being 3.5-9.5. The gels contain 5 ~ polyacrylamide total concentration, a cross-linkage of 3 ~o with a final ampholine (LKB-products, Bromma, Sweden) concentration of 2 ~o and the following composition: pH 4-6 (0.2 ml), pH 5-7 (0.2 ml), pH 9-11 (0.4 ml) and pH 3.5-10 (2.8 ml). The sample solutions were soaked in pieces of special filter paper (LKB, Beckman) to a final amount of 125-130/~g. The isoelectric focusing was started at 300 V with 50 mA and the separation was performed for 120 min at 20 °C with a final voltage of 1150 V and a final current of 18 mA. Close to each electrode, 5 #1 of hemoglobin solution containing 5 #g hemoglobin//A was applied. The joining of the hemoglobin bands migrating from both electrodes took place within 1.5 h. Usually 16 samples were focused on each gel slab. pH was measured using a surface electrode Type LOT 403-30 M8, Ingold, Ziirich, Switzerland. After the separation, the gel was fixed in methanol, sulfosalicylic acid and trichloroacetic acid, stained in Coomassie Brilliant Blue and destained in ethanol and acetic acid. The gels were preserved in glycerol. (2) Agar gel electrophoresis was performed according to Wieme (1959) on microscopic slides in a 0.9 ~ agar solution and a continuous buffer system of 0.04 M sodium diethyl-barbiturate, pH 8.4 (ionic strength 0.05). Samples were run (with human serum, albumin, transferrin and IgG as references) in a cooled chamber for 25 min at 200 V (approximately 27 V/era). The slides were fixed in 5 ~o acetic acid, stained in 0.5 ~ Amido black and dried.

Immunological test systems Immunological analyses were carried out by double diffusion according to Ouchterlony (1948) in 1 ~ agarose gel containing barbital buffer (pH 8.6, ionic

251 TABLE 2 ROUTINE TESTS ON SERUM AND CSF Normal values ± SD are given.

Total protein Uric acid LD-activity

Serum

CSF

74 4- 7 g/1 (60-80) 5.3 ~: 6 mg/l (26-80) 273 4- 58 units/ml (10{)-400)

450 4- 88 mg/l (300--600) 4.4 4- 2.4 mg/l~o(2-9) 26 4- 7.7 units/ml (8--40)

strength 0.02). Immunoelectrophoresis was performed according to Laurell (1966) in 1 ~o Litex agarose (Glostrup, Denmark) containing 1 ~ polyethyleneglycol (m.w. 6,000) in barbital buffer (pH 8.6, ionic strength 0.02). The gel was run for 6 h with a current of 1 mA/cm at 15 °C. RESULTS

Routine tests As shown in Table 2, routine tests on CSF and serum were all normal. No xanthochromia was seen. The cell count did not exceed 2 x 106 cells/l in any sample.

Amount of protein in different fractions In fraction I approximately 10 ~o of total protein was obtained, while 70-80 ~o of the applied CSF protein (100 ~o) was found in fraction II. During the concentration procedure 20-30 ~o of fraction I or II total protein was lost.

Isoelectric focusing Approximately 50 individual bands were obtained in total CSF and serum by isoelectric focusing, and the pattern was similar to that found in control material (Stibler 1978). The CSF-specific fraction I was characterized by 12-14 bands not seen in fraction II (Figs. 1 and 2). the most prominent bands were found at pI values of 4.6, 4.7 and 5.8. A band which was less distinct than the others was consistently found at pI 4.5, and bands at 5.3 and 7.4 were always visible. The bands at pI 4.2, 6.9, 7.2 and 8.0 showed more variation than those previously mentioned, but were always visible. On the contrary, bands at pI 3.6 and 6.4 were not found in all samples. A band at pI 9.3 varied greatly in intensity. The bands at pI 4.6, 4.7 and 5.8 were constantly seen in total CSF but were often very weak. The other characteristic bands were not visible except for those at pI 4.2, 7.4 and 9.3 which could be seen in a few of the samples of total CSF run in parallel.

Agar gel electrophoresis The normal protein pattern of total CSF is shown in Fig. 3. In fraction I three prominent bands could be seen (Fig. 3). One band had the mobility ofprealbumin, one

252

4

--

q

al, i l l

1

5

--

6

--

7

9

p~t

1

2

3

1

_

1

--

--

--

position

of bands

fraction

I

4

Fig. 1. Typical patterns produced by isoelectric focusing of (from left to right) whole CSF proteins, fraction I, fraction I1 and serum proteins. Fig. 2. Schematic drawing of the protein pattern of fraction I. correlated to a pH scale.

Fig. 3. Normal agar gel patterns of whole CSF (right) and fraction I (left).

253 with a mobility of fl-trace protein, and one with a mobility in the cathodal part of the ),-globulin. A few weak bands with mobilities between post-), and fl could also be seen.

Control experiments To test the binding capacity of the columns, human serum proteins were loaded onto the columns until no more protein was bound. It was found that 4 mg protein could be adsorbed. In the experiments performed, less than 2.5 mg CSF protein was fractionated. The specificity of the affinity chromatographic method was tested by rocket immunoelectrophoresis and immunodiffusion in agar (Ouchterlony 1948). Unconcentrated or concentrated fraction I showed no reaction with transferrin, albumin or IgG. In unconcentrated fraction II, 75-80 9/00of normal CSF transferrin, albumin and IgG could be obtained; if fraction II was concentrated (as described above) a further 10-15 ~o loss of protein resulted. Densitometric tracings, i.e. registration of dye uptake of different protein bands, of the isoelectric focusing gels were performed on a Vitatron-Linear log recorder. No albumin, transferrin or IgG of fraction I were revealed while in fraction II the relative amounts of these 3 protein bands as measured by dye uptake was 60--80 ~o of those observed in total CSF. In a model experiment using rat albumin and purified IgG from sheep anti-ratalbumin, the latter coupled to the Sepharose, a 15-20 ~o loss of immunological activity of albumin adsorbed to the antibody-coated gel matrix (fraction II) was found after the antigen-antibody reaction. No albumin could be found in fraction I, if the column was not overloaded. This figure agrees well with the total protein amount in fractions I and II, respectively, suggesting that most of the specific proteins in fraction I could be eluted immunologically well preserved. Thus, it was concluded that the specificity of the affinity chromatographic method was adequate in our experimental procedure.

Concentration procedure The excluding capacity of molecules by the Millipore filter was checked by lyophilisation of the ultrafiltrates from fractions I and II. Protein determination according to the Ponceau technique (see above), and isoelectric focusing were performed on the lyophisate. No proteins or peptides were detected. Proteinase inhibitor (Trasylol®, Bayer) was, in some experiments, added to half the CSF sample (1000 KIE/ml CSF) and examined in a similar way as above. No differences were noticed in the isoelectric protein pattern. Neither storing at + 4 °C for one week, nor freezing to --20 °C for 2 month had any effect, except in the latter case, one band at pI 9.3 diminished in dye uptake on isoelectric focusing. Instead, a band of pI 8.0 was seen (Stibler 1978). DISCUSSION To identify CSF specific proteins we have developed a separation method by

254 means of affinity chromatography and combined this with the high resolving capacity of isoelectric focusing. Antibodies to human serum proteins were insolubilized on a carbohydrate matrix by means of cyanogen halides. This reaction involves the formation of a reactive intermediate by treating the polysaccharide for a short period with aqueous cyanogen halide solution under alkaline conditions, and coupling the intermediate with the antibodies in neutral or slightly alkaline aqueous medium. By passing a solution containing the respective antigens through the column, an immunoreaction takes place. The antigen-antibody complexes can be disaggregated by lowering the pH value to < 3.0. The reaction between cyanogen halides and carbohydrates seems to be rather complex. Carbohydrate derivatives containing primary amino groups presumably form cyanamide intermediates, capable of reacting with proteins or peptides, with the formation of guanidino derivatives. This kind of coupling is of considerable interest because the charge of the fixed protein is essentially unchanged, a fact that might be important for satisfactory retention of biological activity (Ax6n et al. 1967). Affinity chromatography offers unique possibilities for achieving separations which are difficult or even impossible, using less specific techniques. In principle, affinity chromatography can be used to isolate either of the components of a reversibly reacting system. The basic requirement is that it must be possible to couple one component of the system to an insoluble matrix and at the same time retain its specific activity. As model systems for the method, insoluble enzymes have been packed and used as a bed 'reactor'. By passing a solution of a substrate through the enzyme bed, large quantities were converted into products (Bar-Eli and Katchalclin 1963). The reaction stopped automatically when the solution left the bed, and the solid enzyme could be used continuously for very long periods of time. In the present study, fractions highly enriched in CSF-specific proteins and fractions of serum-derived proteins from a CSF sample were prepared, the method is reproducible and gives a new approach to the study of CSF proteins in neurological disease. Thus, we have been able to separate CSF proteins into one 'specific group' (fraction I) and one group, immunologically similar to serum proteins (fraction 1I). Fraction I revealed 10 ~ of total CSF protein while 70-80 ~ was obtained in fraction I1. A 10-20 ~ loss of immunological activity was found in proteins adsorbed to serum antibody-containing gel matrix. This decrease in activity could be due to conformational changes of the proteins, resulting in exposure of different antigenic sites. There could also be some degeneration of the proteins put on the columns. No albumin was found in fraction I, indicating a capacity of the Sepharose column to separate CSF proteins from those antigenically similar to serum proteins. To eliminate the risk of artifactual protein species, many control experiments, including specificity tests, loading of columns, tests for concentration artifacts and protein determination, were made. All these tests showed that we were within the limit of safety in our experimental set-up. Progress in the analytical field of CSF proteins was made by the introduction of

255 isoelectric focusing (Vesterberg and Svensson 1966), whereby proteins were separated according to their isoelectric points in a pH-gradient made by ampholytes. When whole CSF preparations are fractiortated by a high resolving system like isoelectric focusing (Kjellin and Vesterberg 1974; Stibler 1978), many protein bands can be demonstrated. The electrophoretic technique used in the present study is extremely sensitive, separating proteins with differences in isoelectric points of 0.01-0.02 pH-units. The technique is even more sensitive than disc-electrophoresis (Davis 1964; Ornstein 1964). We used the fiat-gel method, i.e. a thin layer of polyacrylamide was poured onto a glass-plate. The advantage is that 15 samples with a low protein content could be run in parallel on the same plate, thus facilitating comparison between the different samples. The great advantage of our technical approach is that one obtains a protein pattern, which one can readily survey in both CSF fractions. In fraction I, 12-14 specific bands were noted. The present results on patterns from whole CSF correspond to those described earlier by Stibler (1978). In the latter study, several of the protein bands could be identified by comparison with purified serum proteins and by crossed immunoelectrofocusing. However, the only CSF-characteristic proteins studied were the/~-trace and the ),-trace proteins (Link 1967; Stibler 1978). Thus the overwhelmingly large portion studied by Kjellin and Stibler (1976) were the serum-derived proteins. With regard to transferrin, a desialized form, the tau fraction, was characteristically found in CSF, probably due to intrathecal desialization of transferrin (Pette and Stupp 1960; Stibler 1978). The desialized transferrin reacts in art antigenically similar fashion to the main transferrin, which is in accordance with the results from the present study where no tau fraction could be demonstrated in the 'CSF-specific' fraction I. The relative amount of serum proteins is determined by the state of the blood-CSF barrier, impairment of which commonly accompanies diseases of the CNS. Furthermore, the concentration ratios of several proteins between serum and CSF can be correlated to the hydrodynamic volumes of the protein molecules (Felgenhauer 1974) which means that a barrier damage may change the relative distribution of the different serum-derived proteins in the CSF. These facts have made it difficult to distinguish specific protein patterns of 'degenerative type' as that earlier described by Laterre et al. (1964). The present method increases the possibility of detecting changes in the CSFcharacteristic proteins which are probably of greatest interest when degenerative diseases are studied. In this methodological work, patients with transient ischemic attacks (TIA) were chosen, since they represent a relatively homogenous group of patients without signs of brain damage. Furthermore, a relatively comprehensive clinical investigation had been carried out, including aortocervical and cerebral angiography. No brain damage or severe cerebral vessel disease could be demonstrated. The results have also been compared to other groups of 'controls' without signs of neurological disease, such as patients with functional neurotic disturbances, headache etc., and results obtained were identical to those in the TIA-group.

256 Studies are now in progress to investigate different degenerative disorders a n d to characterize further some o f the p r o t e i n b a n d s h i t h e r t o n o t described. I n an investigation o f patients with senile dementia, highly characteristic changes have been shown in c o n t r a s t to the findings in patients with m u l t i i n f a r c t i o n d e m e n t i a ( W i k k e l s o et al., to be published). P r e l i m i n a r y experiments for identification o f the /~-trace p r o t e i n have been carried o u t using b o t h crossed i m m u n o e l e c t r o p h o r e s i s a n d the i m m u n o f i x a t i o n technique a n d have shown t h a t the b a n d s at p H 4.6, 4.7, 5.3 a n d 5.8 p r o b a b l y c o r r e s p o n d t o / % t r a c e protein. ACKNOWLEDGEMENT R u t h A n d e r s s o n , h e a d nurse at the C e r e b r o s p i n a l F l u i d L a b o r a t o r y , Sahlgren's Hospital, is gratefully a c k n o w l e d g e d for her valuable help d u r i n g this work.

REFERENCES Ax6n, R. and J. Porath (1966) Chemical coupling of enzymes to cross-linked dextran (Sephadex), Nature (Lond.), 210: 367-369. Ax6n, R., J. Porath and S. Ernback (1967) Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides, Nature (Lond.), 214:1302-1304. Bar-Edi, A. and E. Katchalclin (1963) Preparation and properties of water-insoluble derivatives of trypsin, at. Biol. Chem., 238: 1690. Bock, E. (1973) Non-plasma proteins in cerebrospinal fluid. In: N. H. Axelen, J. Kr611 and B. Weeke (Eds.), A Manual of Quantitative Immunoelectrophoresis, Universitetsf6rlaget, Oslo, pp. 119-124. Clausen, J. (1961) Proteins in normal cerebrospinal fluid not found in serum, Proc. Soc. exp. BioL (N. Y.), 107: 170--172. Davis, B. J. (1964) Disc electrophoresis, Part 2 (Method and application to human serum proteins), Ann. N.Y. Acad. Sci., 121: 404-427. Davson, H. (1969) The cerebrospinal fluid. In: E. Lajtha (Ed.), Handbook ofNeurochemistry, Vol. II, pp. 23-48. Eng, L. F., J. J. Vanderhaeghen, A. Bignami and B. Gerstl (1971) An acidic protein isolated from fibrous astrocytes, Brain Res., 28: 351-354. Felgenhauer, K. (1974) Protein size and cerebrospinal fluid composition, Klin. Wschr., 52: 1158-1164. Hyd~n, H. and L. R/Snnb~ck (1979) Proteins S-100 and 14-3-2 in nerve cells of rats raised in enriched and impoverished environments. Distribution, quantification and cell separation by surface antigens, Behav. Neural. Biol., 25: 371-379. Kjellin, K. G. and H. Stibler (1976) Isoelectric focusing and electrophoresis of cerebrospinal fluid proteins in muscular dystrophies and spinal muscular atrophies, J. neurol. Sci., 27 : 45-57. Kjellin, K. G. and O. Vesterberg (1974) Isoelectric focusing of CSF protein in neurological diseases, J. neurol. Sci., 23: 199-213. Laterre, C., J. I-Ieremans and A. Carbonara (1964) Immunological comparison of some proteins found in cerebrospinal fluid, urine and extracts from brain and kidney, Clin. chim. Aeta, 10: 197-209. Laurell, C. B. (1966) Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies, Analyt Biochem., 15 : 45-52. Link, H. (1967) Immunoglobulin G and low molecular weight proteins in human cerebrospinal fluid, Acta neurol, scand., Suppl. 28: 43: 1-136. Lous, P., C. M. Plum and M. Schou (1956) Kolorimetrisk bestemmelse af lokal protein og globulin i spinalvaeske, Nord. Med., 55: 693-695. Lowenthal, A. (1969) Chemical physiopathology of the cerebrospinal fluid, In: A. Lajtha (Ed.), Handbook of Neurochemistry, Vol. VII, pp. 429-464. Lowry, O., N. Rosebrough, A. Farr and R. Randall (1951) Protein measurements with the Folin phenol reagent J. biol. Chem., 193: 265-275.

257 Moore, B. W. and D. McGregor (1965) Chromatographic and electrophoretic fractionation of soluble proteins of brain and liver, J. biol. Chem., 240: 1647-1653. Mori, T., K. Morimolo, Y. Ushio, T. Hayakawa and H. Mogami (1975) Radioimmunoassay of astroprotein (an astrocyte-specific cerebroprotein) in cerebrospinal fluid from patients with glioma - - A preliminary study, Neurol. Med. Chit. (Tokyo), 15: 23-35. Murazio, M., A. Massaro and F. Michetti (1977) A brain-specifiic protein (S-100 protein)in cerebrospinal fluid of multiple sclerosis patients, 6th Int. Meet. Int. Soc. Neurochem., p. 324 (Abstr.). Olsson, J.-E., H. Link and B. Nosslin (1973) Metabolic studies on j125 beta-trace protein, with special reference to synthesis within the central nervous system, J. Neurochem., 21 : 1153-1159. Ornstein, L. (1964) Disc electrophoresis, Part 1 (Background and theory), Ann. N. Y. Acad. Sci., 121 : 321-349. Ouchterlony, O. (1948) In vitro method for testing toxin-producing capacity of diphtheria bacteria, Acta path. microbiol, scand., 25 : 186-191. Palfreyman, J. W., D. G. T. Thomas and J. G. Ratcliffe (1978) Radioimmunoassay of human myelin basic protein in tissue extract, cerebrospinal fluid and serum and its clinical application to patients with head injury, Clin. chim. acta, 82: 259-270. Pette, D. and I. Stupp (1960) Die Tau-Fraktion im Liquor cerebrospinalis, Klin. Wschr., 28 : 109-110. Roboz-Einstein, E. (1968) Basic protein of myelin and its role in experimental allergic encephalomyelitis and multiple sclerosis, In A. Lajtha (Ed.), Handbook of Neurochemistry, Vol. VII, pp. 107-129. Salo, R. J. and E. I. Hankavaara (1974) A linear single reagent method for determination of protein in cerebrospinal fluid, Scand. J. din. Lab. Invest., 34: 283-288. Stibler, H. (1978) The normal cerebrospinal fluid proteins identified by means of thin-layer isoelectric focusing and crossed immunoelectrofocusing, J. neurol. Sci., 36: 273-288. Vesterberg, O. (1972) Isoelectric focusing of proteins in polyacrylamide gels, Biochim. biophys. Acta, 257: 11-19. Vesterberg, O. (1973) Isoelectrie focusing of proteins in thin-layers of polyacrylamide gel, Sci. Tools, 20: 22-29. Vesterberg, O. and H. Svensson (1966) Isoelectric fractionation analysis and characterization of ampholytes in natural pI-I-gradient, Part 4 (Further studies on the resolving power in connection with separation of myoglobins), Acta chem. scand., 20: 820-934. Wieme, R. J. (1959) Studies on Agar Gel Electrophoresis - - Techniques, Applications Thesis, Arscia, Brussels.