Journal of
J. Neurol. 221,225-233 (1979)
Neurology © by Springer-Verlag1979
Isotachophoresis of CSF Proteins in Gel Tubes Especially Gammaglobulins An Analytical and Preparative Technique for High-Separation of CSF Proteins K. G. Kjellin and L. Hallander Department of Neurology, Karolinska Hospital, S-10401 Stockholm 60, Sweden
Summary. 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 ktl, and the separation time was 1.5-3.0h. 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.
Key words: Isotachophoresis - CSF proteins - Immunoglobulins. Zusammenfassung. Es wird eine isotachophoretische Methode beschrieben, welche Polyacrylamidgel (PAG-ITP) in einem einfachen selbstgebauten Scheibenelektrophoreseger~it mit Kunststoffr6hrchen benutzt. Die Methode ist besonders geeignet zum Studium von Gammaglobulin im Liquor und Serum, sowohl von Normalf'~illen wie auch bei Patienten mit neurologischen Krankheiten, im besonderen mit Multipler Sklerose. Es wird sowohl das Ger~tt selber beschrieben, wie auch das isotachophoretische (ITP) System, inklusive die Leiter und die Endelektrolyte sowie die Trennstoffe. Das ITP-System trennt die Gammaglobuline in reproduzierbarer Weise. Eine Ktihlung der Gelr6hrchen ist nicht notwendig. Das Volumen der untersuchten Proben schwankte 0340-5354/79/0221/0225/$1.80
226
K.G. Kjellin and L. Hallander zwischen 5 und 100 ~tl, die Trennzeit zwischen 1,5 und 3 Std. Im Liquor von gesicherten oder möglichen MS-Fällen zeigten sich charakteristische, langsam wandernde Gammaglobulinfraktionen. Mittels anderer ITP-Systeme oder anderer Trennstoffmischungen können auch die anodischen Proteine im Detail untersucht werden. PAG-ITP in Gelröhrchen stellt eine einfache und billige Untersuchungstechnik dar. Sie kann sowohl analytisch wie präparativ verwendet werden, für biologische Proben wie für Liquor, Serum und Extrakte aus Nervengewebe.
The cerebrospinal fluid (CSF) proteins have hitherto been analyzed mostly by agar or paper electrophoresis, while other methods, such as immunoelectrophoresis, have been used less frequently (Kjellin and Vesterberg, 1974). During recent years isoelectric focusing (IEF), a technique with separation capacities superior to those of conventional electrophoretic methods, has been adapted for analytical .studies of many proteins. Thin-layer IEF, in polyacrylamide gels (PAGIF), due to its extremely high resolving capacity, has been found to be very valuable for CSF protein examinations, and the method has given much new information about CSF proteins in many neurological diseases including degenerative (Kjellin and Stibler, 1974, 1975a, 1975b, 1976; Stibler et al., 1976, 1978), demyelinating and infectious (cf. Latner, 1973; Bollengier et al., 1976; Delmotte et al., 1977; Kjellin and Sidén, 1977a, b; Sidén and Kjellin, 1978) as weil as cerebrovascular disorders (Gudmundsson et al., 1977, 1978). Another high-separation method, isotachophoresis (ITP), was shown (Kjellin et al., 1975a, b, c, d; Kjellin, 1977; Hallander and Kjellin, 1977; Kjellin and Hallander, 1977) to be very suitable for CSF protein examinations in gel tubes and by using capillary tubes. Analytical ITP in capillary tubes has many advantages: very small CSF samples, a few lal of concentrated and 15-50 ~al of native CSF, can be quickly analyzed (30-60min) the results immediately obtained on a recorder; using unconcentrated CSF, losses due to concentration procedures are avoided; low molecular weight compounds as in CSF ultrafiltrates can also be examined; the technique gives high resolution, was found to be reproducible, and easy to perform for a given analytical system. The ITP findings using capillary tubes have been compared with those of PAGIF (Kjellin, Hallander, Moberg, 1975 a). A preliminary report on different analytical systems was given recently (Kjellin and Hallander, 1977; Hallander and Kjellin, 1977). With a micropreparative technique (Arlinger, L., 1977) delivering the ITP fractions on a cellulose acetate strip, the separated components could be further analyzed by crossed immunoelectrophoresis. According to our experience hitherto, the last mentioned technique will be very suitable for microanalytical identification tests, such as of immunoglobulins in MS (Kjellin and Sidén, 1978; Kjellin and Hallander, in publication). The main drawback of the apparatus for analytical ITP in capillary tubes, as well as the equipment for micropreparative examinations of the corresponding ITP fractions, is that they are rather expensive. The purpose of the present investigation was to study the usefulness of ITP in polyacrylamide gels (PAG-ITP) i.e. a rather inexpensive method. PAG-ITP in
Isotachophoresis of CSF Proteins in Gel Tubes
227
slabs o r p a r a l l e l t u b e s a l l o w s a n a l y s e s a n d c o m p a r i s o n b e t w e e n d i f f e r e n t s a m p l e s r u n at the s a m e t i m e a n d u n d e r i d e n t i c a l c o n d i t i o n s . V e r y s i m p l e a n d c h e a p l a b o r a t o r y d e v i c e s c a n be used, w h i c h are less critical in c o n s t r u c t i o n details t h a n m o r e c o m p l e x c o m m e r c i a l e q u i p m e n t as s u p p l i e d w i t h c o o l i n g systems. F u r t h e r m o r e , the s a m p l e v o l u m e s c a n be v a r i e d w i t h i n a b r o a d r a n g e e.g. ~tl : s to ml : s o f C S F by u s i n g P A G - I T P . T h e P A G - I T P m e t h o d u s e d in this s t u d y was e s p e c i a l l y d e s i g n e d f o r i n v e s t i g a t i n g the i m m u n o g l o b u l i n s , as in d e m y e l i n a t i n g d i s o r d e r s .
Material Purified human gammaglobulins (16.5% w/v, AB Kabi), albumin (20% w/v, AB Kabi) and transferrin (research gift from AB Kabi) were used for test examinations. The samples of CSF and serum were collected from control subjects in which clinical data and CSF examinations, including IEF analyses, had shown no signs indicating aberrations of proteins within the CSF space, the final diagnoses being psychoneurosis or tension headache. The findings from the control cases were compared with those from patients with neurological disorders presumed to give abnormal CSF protein findings, especially MS patients.
Methods The CSF was obtained by lumbar puncture performed in a standardized way, and serum samples were collected at the same time. The cells in the CSF were examined by routine microscopy and counted per 3.2mm 3. The CSF protein concentration was determined by a modification of the method of Lowry, Rosebrough, Farr and Randall (1951). The samples were stored for no more than a few days at +4°C before electrophoresis, IEF and PAG-ITP were performed. The CSF was concentrated by vacuum ultrafiltration (Kjellin, 1967) prior to these examinations. Quantitative electrophoresis was performed and evaluated as previously described (Gärde and Kjellin, 1971). Thin-layer IEF was performed as described by Kjellin and Vesterberg (1974). The CSF samples applied to the IEF gels had a protein content of approximately 200Mg where the corresponding sample volumes and protein concentrations were respectively (20) 30-40 ~tl and 5-7.5 (10) g/1. The serum samples were diluted 10 times with distilled water and 30 MI was applied to the gel. The PA G-ITP Procedure. PAG-ITP was performed in a disc electrophoretic device, elaborated by the authors. The equipment was not cooled, and carbon rod electrodes were used. The gel tubes were made of plastic material (poly-methylmethacrylate), inner diameter 5 mm and length 120 mm. A gel retaining ridge was melted on the tubes by pressing one end against a heated coin. Six tubes were placed vertically in the upper buffer compartment by tightly fitting, vaseline lubricated, rubber grommets, before starting a run. Prior to the gel casting the gel tubes were positioned in modelling clay, freshly warmed by hand in order to obtain better adherence to the tubes. No leakage of the gel solution poured into the tubes was found. The gels were approximately 65 mm long, 5mm in diameter, and contained about 2.5 ml of the gel, made of T4C7 (T = acrylamide in % w/v; C = bisacrylamide cross-linker in % w/w of total amount of acrylamide); (BDH, laboratory grade). Polymerization was initiated by freshly dissolved ammonium persulphate (Merck, p.a.) 10mg/10ml gel solution, and TEMED (N,N,N',N'-tetramethyl-ethylene-diamine; E0stman Kodak Co.) 5.10 -4 v/v (5 gl in 10 ml gel solution) was used as accelerator. The gels were overlayered with distilled water. Gel meniscus was formed within 15min. No de-aeration was needed. PAG-ITP analysis was started 1 h after the initiation of the polymerization. Upper terminating electrolyte was 100 ml 10 mM EACA (epsilon-aminocapronic acid, Carl Roth, GFR puriss.) with Ba(OH)2 (Merck, p.a.) added to pH 9.5. Lower, "bulk leading" electrolyte was 100 mM phosphoric acid (Merck, puriss) with TRIS (Merck, puriss) added to pH 7.0.
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K . G . Kjellin and L. Hallander
I
1 2~ 4
8~
Fig. 1. a 1 2 3 4 5 6
Gammaglobulin Gammaglobulin Gammaglobulin Gammaglobulin Gammaglobulin Gammaglobulin
+ + + + + +
10 ~tl spacer 10 lal spacer 10 pl spacer 10 ~tl spacer 10 ~tl spacer 10 ~tl spacer
mlxture mixture mixture mlxture mixture mlxture
+ + + + +
50 pl 1% w / v glycine 50 pA 0.1 M valine 50 lal 1% w / v beta-alanine 501al 1% w / v G A B A 50 tll 0.1 M veronal
Isotachophoresis of CSF Proteins in Gel Tubes
229
The leading electrolyte in the gel consisted of TES (N-tris/hydroxymethyl/methyl-2-aminoethane sulfonic acid, Sigma Chem. Co., laboratory grade) 20 mM, and TRIS (Merck) 20 mM (pH 7.5 at +25°C before adding the TEMED and acrylamide). Samples and spacers were loaded and mixed in the tubes on top of the gels before they were assembled in the apparatus. 40 gl spacer mixture (Ampholines pI 7-9, 10 pl; pI 9-11, 20 gl; LKB-produkter, Bromma, Sweden; and GABA 1% w/v, 10 lal, Merck) as well as 100 gl 40% w/v sucrose solution with trace amounts of Bromophenol Blue as front tracking dye, were added to the sample volumes. 500 I~1plain (unconcentrated) CSF was applied to this mixture. In order to achieve similar separation conditions, 5001al physiological saline was added for investigating serum (5 gl per sample) and purified gammaglobulins (10 gl 1.65% w/v). PAG-ITP was performed with constant current: 1 mA/gel during the first 5 min in order to avoid heat convection in the sample above the gel. Thereafter 2 mA/gel was applied until the maximum voltage of 500V was obtained. PAG-ITP was continued at that constant voltage and decreasing current until the front of the bromophenol-blue colored albumin was observed 2 cm from the bottom of the gel. The total time for the PAG-ITP procedure illustrated in Figure la was 1.5 h, and for the results given in Figures 1b and 1c about 3 h. Immediately after the run was finished, the gels were blown out from the tubes by mouth into 12 ml 10% w/v of trichloracetic acid (TCA, Merck p.a.) into centrifuge tubes. After 30 min of fixation, the gels were stained ,wernight with 0.2% Coomassie Brilliant Blue R-250 (ICI) in acetic acid: ethanol : water (10 : 45 : 45 v/v/v). The samples were then destained in acetic acid : ethanol: water (10:25:65 v/v/v). The stained gels were finally photographed with a Polaroid (MP3) camera.
Results Some typical results are collected in Figure l a, b,c. W h e n c o m p a r i n g sample b i (serum sample 5 ~tl) with sample b 2 ( g a m m a globulin 10 pl to which trace a m o u n t s of a l b u m i n was added: a l b u m i n / g l o b u l i n ratio 1 : 20 w / w ) it is evident that the spacer mixture used is m a i n l y spacing the g a m m a g l o b u l i n s ( m a r k e d "I" o n the serum sample). A l b u m i n , labelled "III", is on the limit to move electrophoretically ahead of the front. As in all I T P analyses the zones near the leading front are more c o n c e n t r a t e d than those following. Therefore the a l b u m i n fraction appears in a relatively short a n d condensed region (Fig. 1b, c). The proteins situated between the a l b u m i n - a n d the g a m m a g l o b u l i n regions, so called p o s t - a l b u m i n s (labelled "II" in the figures), were f o u n d to be crowded. They were f o u n d to include transferrins (in the middle of the range) a n d when present h e m o g l o b i n c o m p o u n d s in the rear part of the region. P r o m i n e n t p r o t e i n fractions a r o u n d G A B A ( g a m m a - a m i n o b u t y r i c acid) indicated with arrows in Figure 1 a, b were observed in patients with verified MS. The findings in C S F of a few patients with m y e l o p a t h y of u n k n o w n cause who Fig. 1.b 1 Serum 2 Gammaglobulin 3 Abducens-paresis N.U.D. 4 Encephalitis 5 Control case 6 MS? 7 MS
Fig. 1.c 1 Brain infarction 2 MS 3 MS? 4 Systemic disorder 5 Myelopathy N.U.D. 6 Control case 7 Control case 8 Control case
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were examined, should also be especially stressed (c5). Gammaglobulin samples (AB Kabi) were examined using discrete spacers of known mobility and concentration (Fig. 1a). It is evident that glycine (a2) is migrating ahead of the protein stack, but it is not overtaking albumin at the front. Veronal (aó) is not stacked at all. The run looks very much like sample al, but came to equilibrium 20 min:s later, where equilibrium means that the blurred front marker disc reappeared visually sharp. In sample a3 valine was found to stack in the "near front" gammaglobulins (not visualized on the photo). Beta-alanine and GABA (samples a4, a5) each divide the gammaglobulins in two easily observed groups. In samples b 3 - b 7 and c l - c 8 all CSF samples have prealbumin fractions (labelled "IV") which are moving ahead of the front. The study of this last mentioned fraction regarding different proteins is still in progress (Kjellin and Hallander, to be published).
Diseussion
The vertical disc electrophoresis equipment was chosen because it was easily constructed, and the proteins totally entered the gel. Earlier attempts to perform ITP in horizontal polyacrylamide flat beds with the samples applied directly onto the gel surface, resulted in long streaks of protein between the isotachophoretically concentrated ("stacked") zones. Such streaks could partially be washed away with physiological saline, indicating incomplete diffusion into the gel. It occurred on gels with the following compositions: T5C5; T5C3; T3.75,C7. This phenomenon has also been observed in our laboratory in electrofocusing gels with too small pore size. The rear part of gels bl and b2 (Fig. 1) were unfortunately unevenly destained. Some serum and plasma samples may have streaks in the rear part of the gel, depending on running conditions and cloudiness of the samples, the latter fact conceivably caused by lipoproteins. Acrylic gel tubes were preferred, as the gel did not adhere to the plastic tube, and could therefore be removed easily. In addition, the gels with their protein fractions were not deformed, which has been found by using glass tubes (Svendsen, 1973). Slow zone velocity with long "stacking" time to reach equilibrium (Routs, 1971) is beneficial for resolution (contrary to zone electrophoresis), and eliminates thermal convection before the proteins have entered the gel. Heat denaturation of proteins is also prevented by such a technique. However, with the low wattage conditions used in the present investigation no heat denaturation was observed. Thus no cooling arrangement was found necessary. The better heat conduction of glass tubes was not needed. However, ITP with maximum voltage 700-900 V was found to denaturate the slower gammaglobulins by heat, because of too high voltage gradient and resistance in the rear part of the sample under separation. The elaborated PAG-ITP method in plastic gel tubes was especially designed for studying the gammaglobulin regions in CSF and serum. The material from control subjects and patients with clinically verified MS, as well as from other neurological diseases with known possible abnormalities regarding gamma-
Isotachophoresis of CSF Proteins in Gel Tubes
231
Fig. 2. HDL fractions found by PAGIF. V3: relatively young patient with cerebral infarction; VT: control subject. To left applied on surgical lint, to right applied directly onto gel. Note the more distinct anodal fractions in the control case
globulins, are still limited. However, the increase of slower gammaglobulins observed in cases with verified or probable MS should be noticed as well as other aberrations found in such patients and in a case with myelopathy of unknown cause in which about 60% shows electrophoretic patterns in accordance with MS (Gärde and Kjellin, 1971). F r o m serum samples examined from patients with MS it seems that it might be possible to detect similar abnormalities as in the corresponding CSF (Kjellin, Moberg, Hallander, 1975 b; Kjellin, 1975). It should be pointed out that by using other ITP systems, such as other spacer compositions, the anodic proteins ("postalbumin", albumin, and prealbumin zones) can be studied in more detail, e.g. studies of high density lipoproteins ( H D L ) are in progress including comparison with I E F findings (Fig. 2). Furthermore, the P A G - I T P in gel tubes is a simple and inexpensive technique which can be used for both analytical and preparative investigations of biological material such as CSF and serum and extractions from nervous tissues. This work was supported by grants from the Karolinska Institute and the Swedish Society of Medical Sciences.
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References Arlinger, L.: The principle of preparative capillary isotachophoresis. In: Electrofocusing and isotachophoresis (eds. Radola, B. J., and Graesslin, D.), pp. 505--514. Berlin, New York: Walter de Gruyter 1977 Bollengier, F., Delmotte, P., Lowenthal, A.: Biochemical findings in multiple sclerosis, Part 3 (Immunoglobulins of restricted heterogeneity and light chain distribution in cerebrospinal fluid of patients with multiple sclerosis). J. Neurol. 212, 151--158 (1976) Delmotte, P., Gonsette, R.: Biochemical findings in multiple sclerosis, Part 4 (Isoelectric focusing of the CSF gammaglobulins in rnultiple sclerosis (262 cases) and other neurological diseases (272 cases)). J. Neurol. 215, 27--37 (1977) Gärde, A., Kjellin, K. G.: Diagnostic significance of cerebrospinal fluid examinations in myelo= pathy. Acta Neurol. Scand. 47, 555--568 (1971) Gudmundsson, G., Kjellin, K. G., Mettinger, K. L., Sidén, Ä., Söderström, C. E.: CSF protein abnormalities found by isoelectric focusing in ischaemic cerebrovascular diseases. Abstracts of 1Ith World Congress ofNeurology, p. 136. Amsterdam, 11-16 Sept. 1977 (International Congress Series No. 427). Amsterdam: Excerpta Medica 1977 Gudmundsson, G., Kjellin, K. G., Mettinger, K. L., Sidén, •., Söderström, C. E.: In: International Symposium "Cerebrovascular disorders and stroke", Florence, April 5-7, 1978. (To be published by Raven Press) Hallander, L. B., Kjellin, K. G.: Isotachophoresis for investigation of CSF proteins. Sixth Internat. Meeting of the Internat. Soc. for Neurochem. Aug. 1977, p. 339. Copenhagen, Denmark 1977 Kjellin, K. G.: Trace elements in the cerebrospinal fluid. In: Nuclear activation techniques in the life sciences. Vienna: International Atomic Energy Agency 1967 Kjellin, K. G.: Isoelectric focusing and isotachophoresis: Two new methods applied to cerebrospinal fluid proteins. Fifth Internat. Meeting of the Internat. Soc. for Neurochemistry 1975, Barcelona, Spain, 1975c Kjellin, K. G.: High separation methods, isoelectric focusing and isotachophoresis for investigation of CSF and serum proteins in neurological diseases. Sixth Internat. Meeting of the Internat. Soc. for Neurochemistry, p. 340. Copenhagen, Denmark, 1977 Kjellin, K. G., Hallander, L. B.: Proceedings of the Annual Meeting of the Swedish Soc. of Medical Sciences, p. 10. Stockholm, 1975d Kjellin, K. G., Hallander, L. B.: 1lth World Congress of Neurology, Amsterdam, The Netherlands, 1977. Excerpta Medica 427, 96, 348--349 (1977) Kjellin, K. G., Sidén, A.: Electrofocusing and electrophoresis of cerebrospinal fluid proteins in CNS disorders of known or probable infectious etiology. Europ. Neurol. 16, 79--89 (1977a) Kjellin, K. G., Sidén, A.: Aberrant CSF protein fractions found by electrofocusing in multiple sclerosis. Europ. Neurol. 15, 40--50 (1977b) Kjellin, K. G., Sidén,/~.: Isoelectric focusing and isotachophoresis for investigation of CSF and serum proteins in demyelinating and infectious neurological diseases. In: Myelination and demyelination (ed. Jorma Palo). Plenum 1978 Kjellin, K. G., Stibler, H.: CSF protein patterns in extrapyramidal diseases. Europ. Neurol. 12, 186--194 (1974) Kjellin, K. G., Stibler, H.: Protein patterns of cerebrospinal fluid in hereditary ataxias and hereditary spastic paraplegia. J. Neurol. Sci. 25, 65--74 (1975 a) Kjellin, K. G., Stibler, H.: Cerebrospinal fluid protein patterns in spasmodic torticollis. Europ. Neurol. 13,461--475 (1975b) Kjellin, K. G., Stibler, H.: Isoelectric focusing and electrophoresis ofcerebrospinal fluid proteins in muscular dystrophies and spinal muscular atrophies. J. Neurol. Sci. 27, 45--57 (1976) Kjellin, K. G., Vesterberg, O.: Isoelectric focusing of CSF-proteins in neurological diseases. J. Neurol. Sci. 23, 199--213 (1974) Kjellin, K. G., Hallander, L. B., Moberg, U.: Analytical isotachophoresis: a new method for analysis of cerebrospinal fluid proteins, J. Neurol. Sci. 26, 617--622 (1975a) Kjellin, K. G., Moberg, U., Hallander, L. B.: Analytical isotachophoresis of cerebrospinal fluid proteins. A preliminary report. Sci. Tools 22, 3--7 (1975b)
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Latner, A.: Some clinical biochemical aspects of isoelectric focusing. Ann. N.Y. Acad. Sci. 209, 281--298 (1973) Lowry, O. H., Rosebrough, Farr, Randall: Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193, 265--275 (1951) Routs, R. J.: Electrolyte systems in isotachophoresis and their application to some protein separations. Thesis, 1971. University of Technology, Eindhoven, The Netherlands Sidén, /~., Kjellin, K. G.: CSF protein examinations with thin-layer isoelectric focusing in multiple sclerosis. J. Neurol. Sci. 39, 131--146 (1978) Stibler, H., Allgulander, S., Borg, S., Kjellin, K. G.: Abnormal microheterogeneity of transferrin in serum and cerebrospinal fluid in alcoholism. Acta Med. Scand. 204, 49--56 (1978) Stibler, H., Kjellin, K. G.: Isoelectric focusing and electrophoresis of the CSF proteins in tremor of different origins. J. Neurol. Sci. 30, 269--285 (1976) Svendsen, J. P.: On the procedure of preparative isotachophoresis. Sci. Tools 20, 1--4 (1973) Received January 23, 1979
J. Neurol. 221,225-233 (1979)
Neurology © by Springer-Verlag1979
Isotachophoresis of CSF Proteins in Gel Tubes Especially Gammaglobulins An Analytical and Preparative Technique for High-Separation of CSF Proteins K. G. Kjellin and L. Hallander Department of Neurology, Karolinska Hospital, S-10401 Stockholm 60, Sweden
Summary. 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 ktl, and the separation time was 1.5-3.0h. 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.
Key words: Isotachophoresis - CSF proteins - Immunoglobulins. Zusammenfassung. Es wird eine isotachophoretische Methode beschrieben, welche Polyacrylamidgel (PAG-ITP) in einem einfachen selbstgebauten Scheibenelektrophoreseger~it mit Kunststoffr6hrchen benutzt. Die Methode ist besonders geeignet zum Studium von Gammaglobulin im Liquor und Serum, sowohl von Normalf'~illen wie auch bei Patienten mit neurologischen Krankheiten, im besonderen mit Multipler Sklerose. Es wird sowohl das Ger~tt selber beschrieben, wie auch das isotachophoretische (ITP) System, inklusive die Leiter und die Endelektrolyte sowie die Trennstoffe. Das ITP-System trennt die Gammaglobuline in reproduzierbarer Weise. Eine Ktihlung der Gelr6hrchen ist nicht notwendig. Das Volumen der untersuchten Proben schwankte 0340-5354/79/0221/0225/$1.80
226
K.G. Kjellin and L. Hallander zwischen 5 und 100 ~tl, die Trennzeit zwischen 1,5 und 3 Std. Im Liquor von gesicherten oder möglichen MS-Fällen zeigten sich charakteristische, langsam wandernde Gammaglobulinfraktionen. Mittels anderer ITP-Systeme oder anderer Trennstoffmischungen können auch die anodischen Proteine im Detail untersucht werden. PAG-ITP in Gelröhrchen stellt eine einfache und billige Untersuchungstechnik dar. Sie kann sowohl analytisch wie präparativ verwendet werden, für biologische Proben wie für Liquor, Serum und Extrakte aus Nervengewebe.
The cerebrospinal fluid (CSF) proteins have hitherto been analyzed mostly by agar or paper electrophoresis, while other methods, such as immunoelectrophoresis, have been used less frequently (Kjellin and Vesterberg, 1974). During recent years isoelectric focusing (IEF), a technique with separation capacities superior to those of conventional electrophoretic methods, has been adapted for analytical .studies of many proteins. Thin-layer IEF, in polyacrylamide gels (PAGIF), due to its extremely high resolving capacity, has been found to be very valuable for CSF protein examinations, and the method has given much new information about CSF proteins in many neurological diseases including degenerative (Kjellin and Stibler, 1974, 1975a, 1975b, 1976; Stibler et al., 1976, 1978), demyelinating and infectious (cf. Latner, 1973; Bollengier et al., 1976; Delmotte et al., 1977; Kjellin and Sidén, 1977a, b; Sidén and Kjellin, 1978) as weil as cerebrovascular disorders (Gudmundsson et al., 1977, 1978). Another high-separation method, isotachophoresis (ITP), was shown (Kjellin et al., 1975a, b, c, d; Kjellin, 1977; Hallander and Kjellin, 1977; Kjellin and Hallander, 1977) to be very suitable for CSF protein examinations in gel tubes and by using capillary tubes. Analytical ITP in capillary tubes has many advantages: very small CSF samples, a few lal of concentrated and 15-50 ~al of native CSF, can be quickly analyzed (30-60min) the results immediately obtained on a recorder; using unconcentrated CSF, losses due to concentration procedures are avoided; low molecular weight compounds as in CSF ultrafiltrates can also be examined; the technique gives high resolution, was found to be reproducible, and easy to perform for a given analytical system. The ITP findings using capillary tubes have been compared with those of PAGIF (Kjellin, Hallander, Moberg, 1975 a). A preliminary report on different analytical systems was given recently (Kjellin and Hallander, 1977; Hallander and Kjellin, 1977). With a micropreparative technique (Arlinger, L., 1977) delivering the ITP fractions on a cellulose acetate strip, the separated components could be further analyzed by crossed immunoelectrophoresis. According to our experience hitherto, the last mentioned technique will be very suitable for microanalytical identification tests, such as of immunoglobulins in MS (Kjellin and Sidén, 1978; Kjellin and Hallander, in publication). The main drawback of the apparatus for analytical ITP in capillary tubes, as well as the equipment for micropreparative examinations of the corresponding ITP fractions, is that they are rather expensive. The purpose of the present investigation was to study the usefulness of ITP in polyacrylamide gels (PAG-ITP) i.e. a rather inexpensive method. PAG-ITP in
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slabs o r p a r a l l e l t u b e s a l l o w s a n a l y s e s a n d c o m p a r i s o n b e t w e e n d i f f e r e n t s a m p l e s r u n at the s a m e t i m e a n d u n d e r i d e n t i c a l c o n d i t i o n s . V e r y s i m p l e a n d c h e a p l a b o r a t o r y d e v i c e s c a n be used, w h i c h are less critical in c o n s t r u c t i o n details t h a n m o r e c o m p l e x c o m m e r c i a l e q u i p m e n t as s u p p l i e d w i t h c o o l i n g systems. F u r t h e r m o r e , the s a m p l e v o l u m e s c a n be v a r i e d w i t h i n a b r o a d r a n g e e.g. ~tl : s to ml : s o f C S F by u s i n g P A G - I T P . T h e P A G - I T P m e t h o d u s e d in this s t u d y was e s p e c i a l l y d e s i g n e d f o r i n v e s t i g a t i n g the i m m u n o g l o b u l i n s , as in d e m y e l i n a t i n g d i s o r d e r s .
Material Purified human gammaglobulins (16.5% w/v, AB Kabi), albumin (20% w/v, AB Kabi) and transferrin (research gift from AB Kabi) were used for test examinations. The samples of CSF and serum were collected from control subjects in which clinical data and CSF examinations, including IEF analyses, had shown no signs indicating aberrations of proteins within the CSF space, the final diagnoses being psychoneurosis or tension headache. The findings from the control cases were compared with those from patients with neurological disorders presumed to give abnormal CSF protein findings, especially MS patients.
Methods The CSF was obtained by lumbar puncture performed in a standardized way, and serum samples were collected at the same time. The cells in the CSF were examined by routine microscopy and counted per 3.2mm 3. The CSF protein concentration was determined by a modification of the method of Lowry, Rosebrough, Farr and Randall (1951). The samples were stored for no more than a few days at +4°C before electrophoresis, IEF and PAG-ITP were performed. The CSF was concentrated by vacuum ultrafiltration (Kjellin, 1967) prior to these examinations. Quantitative electrophoresis was performed and evaluated as previously described (Gärde and Kjellin, 1971). Thin-layer IEF was performed as described by Kjellin and Vesterberg (1974). The CSF samples applied to the IEF gels had a protein content of approximately 200Mg where the corresponding sample volumes and protein concentrations were respectively (20) 30-40 ~tl and 5-7.5 (10) g/1. The serum samples were diluted 10 times with distilled water and 30 MI was applied to the gel. The PA G-ITP Procedure. PAG-ITP was performed in a disc electrophoretic device, elaborated by the authors. The equipment was not cooled, and carbon rod electrodes were used. The gel tubes were made of plastic material (poly-methylmethacrylate), inner diameter 5 mm and length 120 mm. A gel retaining ridge was melted on the tubes by pressing one end against a heated coin. Six tubes were placed vertically in the upper buffer compartment by tightly fitting, vaseline lubricated, rubber grommets, before starting a run. Prior to the gel casting the gel tubes were positioned in modelling clay, freshly warmed by hand in order to obtain better adherence to the tubes. No leakage of the gel solution poured into the tubes was found. The gels were approximately 65 mm long, 5mm in diameter, and contained about 2.5 ml of the gel, made of T4C7 (T = acrylamide in % w/v; C = bisacrylamide cross-linker in % w/w of total amount of acrylamide); (BDH, laboratory grade). Polymerization was initiated by freshly dissolved ammonium persulphate (Merck, p.a.) 10mg/10ml gel solution, and TEMED (N,N,N',N'-tetramethyl-ethylene-diamine; E0stman Kodak Co.) 5.10 -4 v/v (5 gl in 10 ml gel solution) was used as accelerator. The gels were overlayered with distilled water. Gel meniscus was formed within 15min. No de-aeration was needed. PAG-ITP analysis was started 1 h after the initiation of the polymerization. Upper terminating electrolyte was 100 ml 10 mM EACA (epsilon-aminocapronic acid, Carl Roth, GFR puriss.) with Ba(OH)2 (Merck, p.a.) added to pH 9.5. Lower, "bulk leading" electrolyte was 100 mM phosphoric acid (Merck, puriss) with TRIS (Merck, puriss) added to pH 7.0.
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I
1 2~ 4
8~
Fig. 1. a 1 2 3 4 5 6
Gammaglobulin Gammaglobulin Gammaglobulin Gammaglobulin Gammaglobulin Gammaglobulin
+ + + + + +
10 ~tl spacer 10 lal spacer 10 pl spacer 10 ~tl spacer 10 ~tl spacer 10 ~tl spacer
mlxture mixture mixture mlxture mixture mlxture
+ + + + +
50 pl 1% w / v glycine 50 pA 0.1 M valine 50 lal 1% w / v beta-alanine 501al 1% w / v G A B A 50 tll 0.1 M veronal
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The leading electrolyte in the gel consisted of TES (N-tris/hydroxymethyl/methyl-2-aminoethane sulfonic acid, Sigma Chem. Co., laboratory grade) 20 mM, and TRIS (Merck) 20 mM (pH 7.5 at +25°C before adding the TEMED and acrylamide). Samples and spacers were loaded and mixed in the tubes on top of the gels before they were assembled in the apparatus. 40 gl spacer mixture (Ampholines pI 7-9, 10 pl; pI 9-11, 20 gl; LKB-produkter, Bromma, Sweden; and GABA 1% w/v, 10 lal, Merck) as well as 100 gl 40% w/v sucrose solution with trace amounts of Bromophenol Blue as front tracking dye, were added to the sample volumes. 500 I~1plain (unconcentrated) CSF was applied to this mixture. In order to achieve similar separation conditions, 5001al physiological saline was added for investigating serum (5 gl per sample) and purified gammaglobulins (10 gl 1.65% w/v). PAG-ITP was performed with constant current: 1 mA/gel during the first 5 min in order to avoid heat convection in the sample above the gel. Thereafter 2 mA/gel was applied until the maximum voltage of 500V was obtained. PAG-ITP was continued at that constant voltage and decreasing current until the front of the bromophenol-blue colored albumin was observed 2 cm from the bottom of the gel. The total time for the PAG-ITP procedure illustrated in Figure la was 1.5 h, and for the results given in Figures 1b and 1c about 3 h. Immediately after the run was finished, the gels were blown out from the tubes by mouth into 12 ml 10% w/v of trichloracetic acid (TCA, Merck p.a.) into centrifuge tubes. After 30 min of fixation, the gels were stained ,wernight with 0.2% Coomassie Brilliant Blue R-250 (ICI) in acetic acid: ethanol : water (10 : 45 : 45 v/v/v). The samples were then destained in acetic acid : ethanol: water (10:25:65 v/v/v). The stained gels were finally photographed with a Polaroid (MP3) camera.
Results Some typical results are collected in Figure l a, b,c. W h e n c o m p a r i n g sample b i (serum sample 5 ~tl) with sample b 2 ( g a m m a globulin 10 pl to which trace a m o u n t s of a l b u m i n was added: a l b u m i n / g l o b u l i n ratio 1 : 20 w / w ) it is evident that the spacer mixture used is m a i n l y spacing the g a m m a g l o b u l i n s ( m a r k e d "I" o n the serum sample). A l b u m i n , labelled "III", is on the limit to move electrophoretically ahead of the front. As in all I T P analyses the zones near the leading front are more c o n c e n t r a t e d than those following. Therefore the a l b u m i n fraction appears in a relatively short a n d condensed region (Fig. 1b, c). The proteins situated between the a l b u m i n - a n d the g a m m a g l o b u l i n regions, so called p o s t - a l b u m i n s (labelled "II" in the figures), were f o u n d to be crowded. They were f o u n d to include transferrins (in the middle of the range) a n d when present h e m o g l o b i n c o m p o u n d s in the rear part of the region. P r o m i n e n t p r o t e i n fractions a r o u n d G A B A ( g a m m a - a m i n o b u t y r i c acid) indicated with arrows in Figure 1 a, b were observed in patients with verified MS. The findings in C S F of a few patients with m y e l o p a t h y of u n k n o w n cause who Fig. 1.b 1 Serum 2 Gammaglobulin 3 Abducens-paresis N.U.D. 4 Encephalitis 5 Control case 6 MS? 7 MS
Fig. 1.c 1 Brain infarction 2 MS 3 MS? 4 Systemic disorder 5 Myelopathy N.U.D. 6 Control case 7 Control case 8 Control case
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were examined, should also be especially stressed (c5). Gammaglobulin samples (AB Kabi) were examined using discrete spacers of known mobility and concentration (Fig. 1a). It is evident that glycine (a2) is migrating ahead of the protein stack, but it is not overtaking albumin at the front. Veronal (aó) is not stacked at all. The run looks very much like sample al, but came to equilibrium 20 min:s later, where equilibrium means that the blurred front marker disc reappeared visually sharp. In sample a3 valine was found to stack in the "near front" gammaglobulins (not visualized on the photo). Beta-alanine and GABA (samples a4, a5) each divide the gammaglobulins in two easily observed groups. In samples b 3 - b 7 and c l - c 8 all CSF samples have prealbumin fractions (labelled "IV") which are moving ahead of the front. The study of this last mentioned fraction regarding different proteins is still in progress (Kjellin and Hallander, to be published).
Diseussion
The vertical disc electrophoresis equipment was chosen because it was easily constructed, and the proteins totally entered the gel. Earlier attempts to perform ITP in horizontal polyacrylamide flat beds with the samples applied directly onto the gel surface, resulted in long streaks of protein between the isotachophoretically concentrated ("stacked") zones. Such streaks could partially be washed away with physiological saline, indicating incomplete diffusion into the gel. It occurred on gels with the following compositions: T5C5; T5C3; T3.75,C7. This phenomenon has also been observed in our laboratory in electrofocusing gels with too small pore size. The rear part of gels bl and b2 (Fig. 1) were unfortunately unevenly destained. Some serum and plasma samples may have streaks in the rear part of the gel, depending on running conditions and cloudiness of the samples, the latter fact conceivably caused by lipoproteins. Acrylic gel tubes were preferred, as the gel did not adhere to the plastic tube, and could therefore be removed easily. In addition, the gels with their protein fractions were not deformed, which has been found by using glass tubes (Svendsen, 1973). Slow zone velocity with long "stacking" time to reach equilibrium (Routs, 1971) is beneficial for resolution (contrary to zone electrophoresis), and eliminates thermal convection before the proteins have entered the gel. Heat denaturation of proteins is also prevented by such a technique. However, with the low wattage conditions used in the present investigation no heat denaturation was observed. Thus no cooling arrangement was found necessary. The better heat conduction of glass tubes was not needed. However, ITP with maximum voltage 700-900 V was found to denaturate the slower gammaglobulins by heat, because of too high voltage gradient and resistance in the rear part of the sample under separation. The elaborated PAG-ITP method in plastic gel tubes was especially designed for studying the gammaglobulin regions in CSF and serum. The material from control subjects and patients with clinically verified MS, as well as from other neurological diseases with known possible abnormalities regarding gamma-
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Fig. 2. HDL fractions found by PAGIF. V3: relatively young patient with cerebral infarction; VT: control subject. To left applied on surgical lint, to right applied directly onto gel. Note the more distinct anodal fractions in the control case
globulins, are still limited. However, the increase of slower gammaglobulins observed in cases with verified or probable MS should be noticed as well as other aberrations found in such patients and in a case with myelopathy of unknown cause in which about 60% shows electrophoretic patterns in accordance with MS (Gärde and Kjellin, 1971). F r o m serum samples examined from patients with MS it seems that it might be possible to detect similar abnormalities as in the corresponding CSF (Kjellin, Moberg, Hallander, 1975 b; Kjellin, 1975). It should be pointed out that by using other ITP systems, such as other spacer compositions, the anodic proteins ("postalbumin", albumin, and prealbumin zones) can be studied in more detail, e.g. studies of high density lipoproteins ( H D L ) are in progress including comparison with I E F findings (Fig. 2). Furthermore, the P A G - I T P in gel tubes is a simple and inexpensive technique which can be used for both analytical and preparative investigations of biological material such as CSF and serum and extractions from nervous tissues. This work was supported by grants from the Karolinska Institute and the Swedish Society of Medical Sciences.
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References Arlinger, L.: The principle of preparative capillary isotachophoresis. In: Electrofocusing and isotachophoresis (eds. Radola, B. J., and Graesslin, D.), pp. 505--514. Berlin, New York: Walter de Gruyter 1977 Bollengier, F., Delmotte, P., Lowenthal, A.: Biochemical findings in multiple sclerosis, Part 3 (Immunoglobulins of restricted heterogeneity and light chain distribution in cerebrospinal fluid of patients with multiple sclerosis). J. Neurol. 212, 151--158 (1976) Delmotte, P., Gonsette, R.: Biochemical findings in multiple sclerosis, Part 4 (Isoelectric focusing of the CSF gammaglobulins in rnultiple sclerosis (262 cases) and other neurological diseases (272 cases)). J. Neurol. 215, 27--37 (1977) Gärde, A., Kjellin, K. G.: Diagnostic significance of cerebrospinal fluid examinations in myelo= pathy. Acta Neurol. Scand. 47, 555--568 (1971) Gudmundsson, G., Kjellin, K. G., Mettinger, K. L., Sidén, Ä., Söderström, C. E.: CSF protein abnormalities found by isoelectric focusing in ischaemic cerebrovascular diseases. Abstracts of 1Ith World Congress ofNeurology, p. 136. Amsterdam, 11-16 Sept. 1977 (International Congress Series No. 427). Amsterdam: Excerpta Medica 1977 Gudmundsson, G., Kjellin, K. G., Mettinger, K. L., Sidén, •., Söderström, C. E.: In: International Symposium "Cerebrovascular disorders and stroke", Florence, April 5-7, 1978. (To be published by Raven Press) Hallander, L. B., Kjellin, K. G.: Isotachophoresis for investigation of CSF proteins. Sixth Internat. Meeting of the Internat. Soc. for Neurochem. Aug. 1977, p. 339. Copenhagen, Denmark 1977 Kjellin, K. G.: Trace elements in the cerebrospinal fluid. In: Nuclear activation techniques in the life sciences. Vienna: International Atomic Energy Agency 1967 Kjellin, K. G.: Isoelectric focusing and isotachophoresis: Two new methods applied to cerebrospinal fluid proteins. Fifth Internat. Meeting of the Internat. Soc. for Neurochemistry 1975, Barcelona, Spain, 1975c Kjellin, K. G.: High separation methods, isoelectric focusing and isotachophoresis for investigation of CSF and serum proteins in neurological diseases. Sixth Internat. Meeting of the Internat. Soc. for Neurochemistry, p. 340. Copenhagen, Denmark, 1977 Kjellin, K. G., Hallander, L. B.: Proceedings of the Annual Meeting of the Swedish Soc. of Medical Sciences, p. 10. Stockholm, 1975d Kjellin, K. G., Hallander, L. B.: 1lth World Congress of Neurology, Amsterdam, The Netherlands, 1977. Excerpta Medica 427, 96, 348--349 (1977) Kjellin, K. G., Sidén, A.: Electrofocusing and electrophoresis of cerebrospinal fluid proteins in CNS disorders of known or probable infectious etiology. Europ. Neurol. 16, 79--89 (1977a) Kjellin, K. G., Sidén, A.: Aberrant CSF protein fractions found by electrofocusing in multiple sclerosis. Europ. Neurol. 15, 40--50 (1977b) Kjellin, K. G., Sidén,/~.: Isoelectric focusing and isotachophoresis for investigation of CSF and serum proteins in demyelinating and infectious neurological diseases. In: Myelination and demyelination (ed. Jorma Palo). Plenum 1978 Kjellin, K. G., Stibler, H.: CSF protein patterns in extrapyramidal diseases. Europ. Neurol. 12, 186--194 (1974) Kjellin, K. G., Stibler, H.: Protein patterns of cerebrospinal fluid in hereditary ataxias and hereditary spastic paraplegia. J. Neurol. Sci. 25, 65--74 (1975 a) Kjellin, K. G., Stibler, H.: Cerebrospinal fluid protein patterns in spasmodic torticollis. Europ. Neurol. 13,461--475 (1975b) Kjellin, K. G., Stibler, H.: Isoelectric focusing and electrophoresis ofcerebrospinal fluid proteins in muscular dystrophies and spinal muscular atrophies. J. Neurol. Sci. 27, 45--57 (1976) Kjellin, K. G., Vesterberg, O.: Isoelectric focusing of CSF-proteins in neurological diseases. J. Neurol. Sci. 23, 199--213 (1974) Kjellin, K. G., Hallander, L. B., Moberg, U.: Analytical isotachophoresis: a new method for analysis of cerebrospinal fluid proteins, J. Neurol. Sci. 26, 617--622 (1975a) Kjellin, K. G., Moberg, U., Hallander, L. B.: Analytical isotachophoresis of cerebrospinal fluid proteins. A preliminary report. Sci. Tools 22, 3--7 (1975b)
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Latner, A.: Some clinical biochemical aspects of isoelectric focusing. Ann. N.Y. Acad. Sci. 209, 281--298 (1973) Lowry, O. H., Rosebrough, Farr, Randall: Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193, 265--275 (1951) Routs, R. J.: Electrolyte systems in isotachophoresis and their application to some protein separations. Thesis, 1971. University of Technology, Eindhoven, The Netherlands Sidén, /~., Kjellin, K. G.: CSF protein examinations with thin-layer isoelectric focusing in multiple sclerosis. J. Neurol. Sci. 39, 131--146 (1978) Stibler, H., Allgulander, S., Borg, S., Kjellin, K. G.: Abnormal microheterogeneity of transferrin in serum and cerebrospinal fluid in alcoholism. Acta Med. Scand. 204, 49--56 (1978) Stibler, H., Kjellin, K. G.: Isoelectric focusing and electrophoresis of the CSF proteins in tremor of different origins. J. Neurol. Sci. 30, 269--285 (1976) Svendsen, J. P.: On the procedure of preparative isotachophoresis. Sci. Tools 20, 1--4 (1973) Received January 23, 1979
