Journal of
J. Neurol. 221,235-244 (1979)
Neurao © by Springer-Vedag 1979
Isotachophoresis in Capillary Tubes of CSF Proteins -Especially Gammaglobulins K. G. Kjellin and L. Hallander Department of Neurology, Karolinska Hospital, S-10401 Stockholm, Sweden
Summary. 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 lal 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, lal 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. Key words: Isotachophoresis - Capillary - CSF proteins - MS (= multiple sclerosis).
Zusammenfassung. Wie die Verfasser frtiher feststellen konnten, ist die Isotachophorese (ITP) in Polyakrylamid-R6hrchen (PAG-ITP) und in Teflonkapillaren (mit LKB-Tachophor®) eine gtinstige Methode for die Feintrennung, unter anderem der Proteine des Liquors (CSF) und des Serums. Sie sind bei der Diagnostik der Multiplen Sklerose verwendbar. Es wurde an anderer Stelle im Detail die analytische und prliparative PAG-ITP-Methode geschildert. Die vorliegende Publikation beschreibt kationische Systeme fiir ITP yon Gammaglobulinen aus Liquor und Serum in Teflonkapillaren, wobei Mikro-
0340-5354/79/0221/0235/$2.00
236
K.G. Kjellin and L. Hallander litermengen verwendet werden, so daß der Schwierigkeit der Injektionsverstopfung durch das Albumin begegnet wird und die Trennzeit verkürzt wird. Wenn eine Mikrodialyse zum Entsalzen der Liquorproben verwendet wird, können Mikrolitermengen unkonzentrierten Liquors innerhalb von 30min analysiert werden. In der Klinik und in der Forschung können die ITPFraktionen weiter untersucht werden, wenn das ITP-Gerät mit einem Mikrofraktionssammler (LKB-Tachofrac®) ausgerüstet wird. Hierbei werden die Fraktionen der Probe an einem Cellulose-Acetatstreifen angebracht. Die Fraktionen können im weiteren z. B. immunologisch analysiert werden. Die hier angewendeten Methoden sind besonders geeignet, um die Gammaglobuline im Liquor und im Serum zu untersuchen, und können unter anderem Verwendung finden, um demyelinisierende oder infektiöse Erkrankungen des Nervensystemes zu analysieren.
Introduetion
Isotachophoresis (ITP) has previously been shown [1--12], to be a very promising high-separating electromigratory method for the examination of CSF and serum proteins, e.g. in MS. The method has many advantages including fast and high separation capacity, where very small samples (1--10 pl of 10 x concentrated CSF and native serum; 15630~tl of original CSF) can be analyzed quickly (30--60 min), and the results obtained immediately on a recorder. Biological fluids with very low protein contents, such as CSF, are "self-concentrated" by the technique. Native CSF can be examined, avoiding losses by preceding concentration procedures, such as ultrafiltration. Furthermore, low-molecular weight compounds, e.g. in CSF ultrafiltrates, can be analyzed as well as high-molecular weight compounds such as protein compounds. The technique was easy to perform, and the results were reproducible for a given analytical ITP system. In order to identify the numerous peaks recorded, compounds with known mobility can be added to the sample [6, 7]. There are additional modifications which may increase the separation capacity of ITP, including a proper choice of spacers to split up a mobility area of interest, and the use of leading and terminating electrolytes to cut oft the relevant area from substances of no interest in the present analysis [6, 7]. Kjellin et al. [7] concluded that a combination of ITP of biological fluids with a low protein concentration such as CSF, followed by electrofocusing, might be very effective for analytical purposes. Our investigations regarding ITP of CSF from MS patients has recently been confirmed by Delmotte [2] who used the same ITP analytical system on a larger material of MS patients. The aim of the present report was to describe some analytical ITP systems elaborated especially for studying immunoglobulins in the CSF and serum of control subjects, and patients with known or probable abnormalities within the CSF gammaglobulins. The main object was to zoom in on this mobility range proteins in a physically proper way by carefully designed electrolyte systems in order to obtain smooth fast and reproducible analysis of the gammaglobulins.
Isotachophoresis in Capillary Tubes of CSF Proteins
237
E~
m
m~
«
.«
Fig. 1. A. Block diagram of LKB Tachophor apparatus. B. Sample injection into LKB Tachophor apparatus. C. Comparative block diagrams of LKB Tachophor in normal and preparative mode. D. The LKB Tachofrac attachment
238
K.G. Kjellin and L. Hallander
Materials and Methods All amino acids were of reagent or pro analysis grade, obtained from Merck and Sigma. MES, 2 (N-morpholino)-ethanesulfonic acid) and TES, (N-tris/hydroxymetyl/methyl-2-aminoethane sulfonic acid) Bistris and ammediol (2-amino-2-methyl-l,3 propanediol) were obtained from Sigma, laboratory grade. EACA (epsilon-aminocaproic acid) was from Roth, Karlsruhe, GFR, laboratory grade. Ampholine was obtained from LKB Produkter Bromma, Sweden, and Ampholine in 0.5 pI intervals from Aminkemi AB, Stockholm, Sweden. Cacodylic acid was from Pfalz & Bauer Inc. Co., N.Y. USA, laboratory grade. Gammaglobulin and transferrin were from AB Kabi, Stockholm, Sweden. ITP in capillary tube was performed in a LKB 2127 Tachophor (Fig. 1), equipped with a 23 cm long teflon capillary, inner diameter 0.5 mm, UV, and thermal detector. The capillary was thermostated at 20°C and 10°C. CSF and serum samples from the same patient are taken in a standardized way (fasting, forenoon). After centrifugation to remove corpuscular elements samples of CSF and corresponding Serum were analyzed, and the results compared with those of electrophoresis and isoelectric focusing [7]. The centrifuged samples were stored not more than a few days at +4°C and were otherwise frozen at -23°C. The patient material is relatively limited, the largest groups including about 20 control cases and 30 patients with MS. Greater material, examined with the proper anionic and cationic isotachophoretic systems elaborated, is now being collected (Kjellin et al.,to be published).
Results T h e p r e v i o u s l y u s e d [6, 7] a n i o n i c s y s t e m M E S 5 m M , A m m e d i o l 10 m M p H 9.0, t e r m i n a t i n g E A C A w i t h B a ( O H ) 2 p H ~ 10 w i t h an a m i n o acid m i x t u r e , c o n s i s t i n g of: g a m m a - a m i n o b u t y r i c a c i d ( G A B A ) , b e t a - a l a n i n e , glycine, t a u r i n e , valine,
UV abs.
[#'.. 3.2
Fig. 2. ITP of 1 ~tl serum with amino acid spacers at 20°C. Leading electrolyte MES 5 mM, Ammediol pH 9.0
Isotachophoresis in Capillary Tubes of CSF Proteins
239
' UV abs. A
40' ,
~10~1CSF
lOx. MS patient
Fig. 3. ITP of CSF. Leading electrolyte: tris 5 mM, cacodylic acid pH 5.5
UV abs.
3
ll5' I 17'
Amf~--8. 6~t, 2%
L
Fig. 4. ITP of gammaglobulin. Leading electrolyte: tris 5 mM, cacodylic acid pH 5.5. Spacer trials. Versatility of modifying Ampholine mobility gradient to actual needs is shown (same amounts of gammaglobulin, 0.5 lal 0.8%, sufficiently diluted from KABI gammaglobulin 16.5%)
TES, serine, gluthamine and asparagine, all at 10 mM concentrations. A separation of 1 I~1 serum with 1 ~tl 2% Ampholine pI 3--9.5, and 2~tl amino acid mixture is shown in Figure 2. In all separations with the MES system at 20°C, clogging in the injection part of the capillary tube (presumably of albumin) was a serious problem, and all separations had to be started carefully at 501~A during the first 15 min. Separation of llal serum or 10~tl conc. 1 0 × C S F with a 2% mixture of Ampholine was possible, with separation times between 25--60 min, depending on the salt content of the sample.
240
K.G. Kjellin and L. Hallander
' U V abs.
U V abs. -4
Ampholines only. 15 I
16 I
MS-patient. 23 I
4
Case with very litt[e gamma-globulines.
24 I
24 I
25 I
Control case. ùRepresentative" 23 I
24 I
1
-4 -4
1 t
pv Fig.5. ITP of CSF. Leading electrolyte: tris 5 mM, cacodylic acid pH 7.0. Terminating ion creatinine
In the following experiments, a new cationic system was tried: tris 5 mM, cacodylic acid 20mM pH c:a 5.5. Terminating ion was beta-atanine. Spacers were: 2% Ampholine mixture, 3)al, consisting of pI 6.5--7, 7--7.5, 7.5--8, in volume parts 2:1:3, and creatinine 25 mM, 1 lal. Temperature was thermostated at 20°C. The sample, 10pJ CSF, conc. 10X was acidified to 10mM of cacodylic acid 15 min before injection, to prevent gas bubbles (COfl), which can appear in the capillary tube and then block the current. Separation time was approximately l h at 50p.A. Typical results are shown in Figure 3, (cf. [12]). By doping experiments the last peaks in the diagram proved to be transferrins. The difference in the gammaglobulin region between the two patients is obvious, and in accordance with earlier results with thin layer electrofocusing (PAGIF). The system was optimized on 0.5 gl 0.8% gammaglobulin (Fig. 4). Ampholine pI 6--8 worked efficiently, but the special mixture pI 6.5--8 gave a more even peak distribution, not resolving the more acidic proteins, which would have eonfused interpretation. In Figure 5 a more restrictive system is tried, useful for separation ofgammaglobulins only: tris 5 mM, cacodylic acid 5 mM pH 7.0, terminating ion creatinine. Spacers were 1 lal 2% Ampholine pI 7--8. Sample was 9 lal CSF 10X, not dialyzed. The relatively high leading pH was found to eliminate the gas bubble problem. The last system used was: tris 5 mM, cacodylic acid 7.5 mM pH 6.3. Terminating ion was beta-alanine. Spacers were 1 lal 1% Ampholine pI 6.5--7, 1 lal 1% Ampholine 7--7.5 and 1 p,l 10 mM bistris. Samples were 20)al unconcentrated CSF, microdialyzed for 1 h. Separation time was less than 30 min. This system is illustrated in Figure 6.
Isotachophoresis in Capillary Tubes of CSF Proteins
241
' U V abs.
? ;' "8 /t
U V abs.
Control case 18
19
20
!
I
!
Fig. 6. ITP of CSF. Leading electrolyte: tris 5 mM, cacodylic acid pH 6.3.20 ~tl unconcentrated CSF, microdialyzed for I h, with similar total protein concentrations (0.5 g/l in control case and 0.6g/l in MS case)
Discussion A simple vertical disc-electrophoresis equipment has previously been described by the authors [3,4,9] for studying CSF proteins by isotachoporesis in polyacrylamide gel (PAG-ITP). Earlier attempts to perform ITP in horizontal polyacrylamide flatbeds with the sample applied directly onto the gel surface, resulted in long streaks of protein between the ITP concentrated ("stacked") zones, which streaks could partially be washed away with physiological saline indicating incomplete diffusion into the gel. Acrylic gel tubes were preferred, as the gel did not adhere to the plastic material and could be removed easily, e.g. by blowing by mouth. With the low wattage conditions used no heat denaturation was observed, and thus no cooling system was needed. The elaborated PAG-ITP method in
242
K.G. Kjellin and L. Hallander
plastic gel tubes was especially designed for studying the gammaglobulins in CSF and serum. The increase of slow gammaglobulins found in cases of known or probable MS should be especially mentioned as well as other aberrations observed in the gammaglobulin range of those patients and in myelopathy without any known cause (Kjellin and Hallander, in press). By using other PAG-ITP systems with other spacer compositions, the anodic proteins can be studied in more detail. Furthermore, the PAG-ITP in gel tubes is a simple and inexpensive technique which can be used for analytical and preparative investigations of biological material such as CSF and serum and extractions from nervous tissues. However, for studying very small samples of CSF and serum (lal amounts) quickly (within 30--60 min) with the results immediately obtained on a recorder, a more sophisticated apparatus, ITP in capillary tubes, is to be used [6--9]. Special ITP systems, including leading and terminating electrolytes and proper spacer substances, are to be elaborated for studying gammaglobulins in CSF and serum. These systems are described in the present paper. It was found that for studying gammaglobulins in CSF and serum, cationic systems obviously are the method of choice, which means that the problem of albumin injection clog is avoided, and thus the separation time can be forced. When using the anionic MES-pH 9.0 system at 20°C, the CSF protein separations were found difficult to reproduce. However, no dialysis of the CSF samples was performed. The terminating creatinine in the pH 7.0 system (moving faster than betaalanine) makes it possible to use longer capillary tubes, the disadvantage being longer separation times. This modification means more detailed information about the gammaglobulins. In the present state however, the system can be used for rapid diagnoses of the gammaglobulin profile. The pH 6.3 system can obviously be of value, but the Ampholine blank peaks were in the same order of magnitude as the gammaglobulins ofcontrol cases. It is not possible to inject more than 15--201al into the 25cm capillary tube, and at least 100B1 would probably be needed. Counterflow and continuous injection [14] seems appropriate, and will be tried in this laboratory. When working with such small CSF volumes (lals) as in ITP in capillary tubes, it is necessary to use a microdialysis procedure for desalting, easy to perform and with high reproducibility. Such a microdialysis technique has been elaborated at our laboratory for (5)-15-25-1501al of CSF and will be described in a separate paper (Hallander and Kjellin, to be published). Another technique has also been described by Delmotte [1]. However, no autoanalyser-based dialysis system was available to the present authors, so Delmotte's technique was not practicable. Our need was a dialysis procedure with no charge-dependent absorption, easily changed pore exclusion limits, and ability to recover more than 90% of CSF proteins from the sample. It is of special interest that submicrogram amounts of a certain protein can be detected by connecting the equipment for capillary ITP (Tachophor, AB LKBprodukter) to equipment with a synchronized cellulosa acetate strip where the fractions are collected and then can be analyzed by microimmunological techniques (LKB-Tachofrac, Fig. 1). This method seems very promising for rapid
UV abs
Mixed monovalent anti ALB TR - IgG - antibodies
Immunopreci pitate
CAlbumin C%
~v_
Transferin
~
Transferin 4
_____o, Immunoglobulin - G
i
-h UV abs.
I mmunoprecipitate
S !
~
ù~L', '',~
1 minute
-'v~~':~ Anti-human-antibodies
I~~~-~
~./-~--~ 9
~
,~ '
Immunoglobulin - G
UV abs.
Immunoprecipitate
o
Immunoglobulin - G
Fig. 7a--c. Composite diagrams of Tachofrac separations and consecutive immunologic crosselectrophoretic detection, method according to Moberg et al. [13]. a CSF, MS patient, b Serum, control subject, c CSF, control subject
244
K.G. Kjellin and L. Hallander
diagnosis of p r o b a b l e MS or different infectious neurological diseases. Some p r e l i m i n a r y findings are presented in Figure 7. The cellulosa acetate strip was simply laid o n a tris-barbital (pH 8.6) antib o d y l o a d e d (approx. 2001al/20 ml) 1% agarose gel. I m m u n o e l e c t r o p h o r e s i s was performed over-night ( 1 6 - - 1 8 h) at 2.5 V / c m . F o r further details, see Moberg et al.,[13].
Acknowledgements. This work was supported by grants from the Karolinska Institute and the Swedish Society of Medical Sciences. The skilful technical assistance of Mrs. Gunborg Strand and Vera Snikvalds is gratefully acknowledged.
References 1. Delmotte, .: Isotachophoresis of the cerebrospinal fluid proteins: Advantage of prior dialysis and its practical realization. In: Electrofocusing and isotachophoresis, Radola, B. J., Graesslin, D. (eds.), pp. 559--564. Berlin, New York: Gruyter 1977 2. Delmotte, .: Analysis of complex protein mixtures by capillary isotachophoresis - - some qualitative and quantitative aspects. Sci. Tools 24, 33--41 (1977) 3. Hallander, L. B., Kjellin, K. G.: A technical report on high-separation methods, isoelectric focusing and isotachophoresis, for investigation of CSF and serum proteins. Sixth Internat. Meeting of the Internat. Soc. for Neurochem., p. 321. August 1977, Copenhagen, Denmark 4. Hallander, L. B., Kjellin, K. G.: Isotachophoresis for investigation of CSF proteins. Sixth. Internat. Meeting of the Internat. Soc. for Neurochem., p. 339. August 1977, Copenhagen, Denmark 5. Hallander, L. B., Kjellin, K. G.: High-separation methods, isoelectric focusing (IEF) and isotachophoresis (ITP), for investigation of CSF- and serum proteins in neurological diseases. 1lth World Congress of Neurology 1977, Amsterdam, The Netherlands. Excerpta Medica 427, 96 (1977) 6. Kjellin, K. G., Moberg, U., Hallander, L. B.: Analytical isotachophoresis of cerebrospinal fluid proteins, a preliminary report. Sci. Tools 22, 3--7 (1975) 7. 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 (1975) 8. 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 9. Kjellin, K. G., et al.: Proceedings of the Annual Meeting of the Swedish Soc. of Medical Sciences, p. 10. Stockholm 1975 I0. 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. August 1977, Copenhagen, Denmark 11. Kjellin, K. G.: High separation techniques (isoelectric focusing and isotachophoresis) for investigation of CSF and serum proteins in neurological diseases. 1lth World Congress of Neurology, 1977. Amsterdam. The Netherlands. Excerpta Medica 427, 348--349 12. 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. New York: Plenum 1978 13. Moberg, U., Hjalmarsson, S.-G., Arlinger, L., Lundin, H.: Preparative capillary isotachophoresis. Separati0n of microgram amounts of some human serum proteins. In: Electrofocusing and isotachophoresis, Radola, B. J., Graesslin, D. (eds.), pp. 515--526. Berlin, New York: Gruyter 1977 14. Ryslavy, Z., Bocek, P., Deml, M., Janak, J.: Capillary isotachophoresis: Continuous sampling technique in the analyses of low concentration samples. J. Chromatogr. 147, 369--373 (1978) Received January 19, 1979
J. Neurol. 221,235-244 (1979)
Neurao © by Springer-Vedag 1979
Isotachophoresis in Capillary Tubes of CSF Proteins -Especially Gammaglobulins K. G. Kjellin and L. Hallander Department of Neurology, Karolinska Hospital, S-10401 Stockholm, Sweden
Summary. 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 lal 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, lal 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. Key words: Isotachophoresis - Capillary - CSF proteins - MS (= multiple sclerosis).
Zusammenfassung. Wie die Verfasser frtiher feststellen konnten, ist die Isotachophorese (ITP) in Polyakrylamid-R6hrchen (PAG-ITP) und in Teflonkapillaren (mit LKB-Tachophor®) eine gtinstige Methode for die Feintrennung, unter anderem der Proteine des Liquors (CSF) und des Serums. Sie sind bei der Diagnostik der Multiplen Sklerose verwendbar. Es wurde an anderer Stelle im Detail die analytische und prliparative PAG-ITP-Methode geschildert. Die vorliegende Publikation beschreibt kationische Systeme fiir ITP yon Gammaglobulinen aus Liquor und Serum in Teflonkapillaren, wobei Mikro-
0340-5354/79/0221/0235/$2.00
236
K.G. Kjellin and L. Hallander litermengen verwendet werden, so daß der Schwierigkeit der Injektionsverstopfung durch das Albumin begegnet wird und die Trennzeit verkürzt wird. Wenn eine Mikrodialyse zum Entsalzen der Liquorproben verwendet wird, können Mikrolitermengen unkonzentrierten Liquors innerhalb von 30min analysiert werden. In der Klinik und in der Forschung können die ITPFraktionen weiter untersucht werden, wenn das ITP-Gerät mit einem Mikrofraktionssammler (LKB-Tachofrac®) ausgerüstet wird. Hierbei werden die Fraktionen der Probe an einem Cellulose-Acetatstreifen angebracht. Die Fraktionen können im weiteren z. B. immunologisch analysiert werden. Die hier angewendeten Methoden sind besonders geeignet, um die Gammaglobuline im Liquor und im Serum zu untersuchen, und können unter anderem Verwendung finden, um demyelinisierende oder infektiöse Erkrankungen des Nervensystemes zu analysieren.
Introduetion
Isotachophoresis (ITP) has previously been shown [1--12], to be a very promising high-separating electromigratory method for the examination of CSF and serum proteins, e.g. in MS. The method has many advantages including fast and high separation capacity, where very small samples (1--10 pl of 10 x concentrated CSF and native serum; 15630~tl of original CSF) can be analyzed quickly (30--60 min), and the results obtained immediately on a recorder. Biological fluids with very low protein contents, such as CSF, are "self-concentrated" by the technique. Native CSF can be examined, avoiding losses by preceding concentration procedures, such as ultrafiltration. Furthermore, low-molecular weight compounds, e.g. in CSF ultrafiltrates, can be analyzed as well as high-molecular weight compounds such as protein compounds. The technique was easy to perform, and the results were reproducible for a given analytical ITP system. In order to identify the numerous peaks recorded, compounds with known mobility can be added to the sample [6, 7]. There are additional modifications which may increase the separation capacity of ITP, including a proper choice of spacers to split up a mobility area of interest, and the use of leading and terminating electrolytes to cut oft the relevant area from substances of no interest in the present analysis [6, 7]. Kjellin et al. [7] concluded that a combination of ITP of biological fluids with a low protein concentration such as CSF, followed by electrofocusing, might be very effective for analytical purposes. Our investigations regarding ITP of CSF from MS patients has recently been confirmed by Delmotte [2] who used the same ITP analytical system on a larger material of MS patients. The aim of the present report was to describe some analytical ITP systems elaborated especially for studying immunoglobulins in the CSF and serum of control subjects, and patients with known or probable abnormalities within the CSF gammaglobulins. The main object was to zoom in on this mobility range proteins in a physically proper way by carefully designed electrolyte systems in order to obtain smooth fast and reproducible analysis of the gammaglobulins.
Isotachophoresis in Capillary Tubes of CSF Proteins
237
E~
m
m~
«
.«
Fig. 1. A. Block diagram of LKB Tachophor apparatus. B. Sample injection into LKB Tachophor apparatus. C. Comparative block diagrams of LKB Tachophor in normal and preparative mode. D. The LKB Tachofrac attachment
238
K.G. Kjellin and L. Hallander
Materials and Methods All amino acids were of reagent or pro analysis grade, obtained from Merck and Sigma. MES, 2 (N-morpholino)-ethanesulfonic acid) and TES, (N-tris/hydroxymetyl/methyl-2-aminoethane sulfonic acid) Bistris and ammediol (2-amino-2-methyl-l,3 propanediol) were obtained from Sigma, laboratory grade. EACA (epsilon-aminocaproic acid) was from Roth, Karlsruhe, GFR, laboratory grade. Ampholine was obtained from LKB Produkter Bromma, Sweden, and Ampholine in 0.5 pI intervals from Aminkemi AB, Stockholm, Sweden. Cacodylic acid was from Pfalz & Bauer Inc. Co., N.Y. USA, laboratory grade. Gammaglobulin and transferrin were from AB Kabi, Stockholm, Sweden. ITP in capillary tube was performed in a LKB 2127 Tachophor (Fig. 1), equipped with a 23 cm long teflon capillary, inner diameter 0.5 mm, UV, and thermal detector. The capillary was thermostated at 20°C and 10°C. CSF and serum samples from the same patient are taken in a standardized way (fasting, forenoon). After centrifugation to remove corpuscular elements samples of CSF and corresponding Serum were analyzed, and the results compared with those of electrophoresis and isoelectric focusing [7]. The centrifuged samples were stored not more than a few days at +4°C and were otherwise frozen at -23°C. The patient material is relatively limited, the largest groups including about 20 control cases and 30 patients with MS. Greater material, examined with the proper anionic and cationic isotachophoretic systems elaborated, is now being collected (Kjellin et al.,to be published).
Results T h e p r e v i o u s l y u s e d [6, 7] a n i o n i c s y s t e m M E S 5 m M , A m m e d i o l 10 m M p H 9.0, t e r m i n a t i n g E A C A w i t h B a ( O H ) 2 p H ~ 10 w i t h an a m i n o acid m i x t u r e , c o n s i s t i n g of: g a m m a - a m i n o b u t y r i c a c i d ( G A B A ) , b e t a - a l a n i n e , glycine, t a u r i n e , valine,
UV abs.
[#'.. 3.2
Fig. 2. ITP of 1 ~tl serum with amino acid spacers at 20°C. Leading electrolyte MES 5 mM, Ammediol pH 9.0
Isotachophoresis in Capillary Tubes of CSF Proteins
239
' UV abs. A
40' ,
~10~1CSF
lOx. MS patient
Fig. 3. ITP of CSF. Leading electrolyte: tris 5 mM, cacodylic acid pH 5.5
UV abs.
3
ll5' I 17'
Amf~--8. 6~t, 2%
L
Fig. 4. ITP of gammaglobulin. Leading electrolyte: tris 5 mM, cacodylic acid pH 5.5. Spacer trials. Versatility of modifying Ampholine mobility gradient to actual needs is shown (same amounts of gammaglobulin, 0.5 lal 0.8%, sufficiently diluted from KABI gammaglobulin 16.5%)
TES, serine, gluthamine and asparagine, all at 10 mM concentrations. A separation of 1 I~1 serum with 1 ~tl 2% Ampholine pI 3--9.5, and 2~tl amino acid mixture is shown in Figure 2. In all separations with the MES system at 20°C, clogging in the injection part of the capillary tube (presumably of albumin) was a serious problem, and all separations had to be started carefully at 501~A during the first 15 min. Separation of llal serum or 10~tl conc. 1 0 × C S F with a 2% mixture of Ampholine was possible, with separation times between 25--60 min, depending on the salt content of the sample.
240
K.G. Kjellin and L. Hallander
' U V abs.
U V abs. -4
Ampholines only. 15 I
16 I
MS-patient. 23 I
4
Case with very litt[e gamma-globulines.
24 I
24 I
25 I
Control case. ùRepresentative" 23 I
24 I
1
-4 -4
1 t
pv Fig.5. ITP of CSF. Leading electrolyte: tris 5 mM, cacodylic acid pH 7.0. Terminating ion creatinine
In the following experiments, a new cationic system was tried: tris 5 mM, cacodylic acid 20mM pH c:a 5.5. Terminating ion was beta-atanine. Spacers were: 2% Ampholine mixture, 3)al, consisting of pI 6.5--7, 7--7.5, 7.5--8, in volume parts 2:1:3, and creatinine 25 mM, 1 lal. Temperature was thermostated at 20°C. The sample, 10pJ CSF, conc. 10X was acidified to 10mM of cacodylic acid 15 min before injection, to prevent gas bubbles (COfl), which can appear in the capillary tube and then block the current. Separation time was approximately l h at 50p.A. Typical results are shown in Figure 3, (cf. [12]). By doping experiments the last peaks in the diagram proved to be transferrins. The difference in the gammaglobulin region between the two patients is obvious, and in accordance with earlier results with thin layer electrofocusing (PAGIF). The system was optimized on 0.5 gl 0.8% gammaglobulin (Fig. 4). Ampholine pI 6--8 worked efficiently, but the special mixture pI 6.5--8 gave a more even peak distribution, not resolving the more acidic proteins, which would have eonfused interpretation. In Figure 5 a more restrictive system is tried, useful for separation ofgammaglobulins only: tris 5 mM, cacodylic acid 5 mM pH 7.0, terminating ion creatinine. Spacers were 1 lal 2% Ampholine pI 7--8. Sample was 9 lal CSF 10X, not dialyzed. The relatively high leading pH was found to eliminate the gas bubble problem. The last system used was: tris 5 mM, cacodylic acid 7.5 mM pH 6.3. Terminating ion was beta-alanine. Spacers were 1 lal 1% Ampholine pI 6.5--7, 1 lal 1% Ampholine 7--7.5 and 1 p,l 10 mM bistris. Samples were 20)al unconcentrated CSF, microdialyzed for 1 h. Separation time was less than 30 min. This system is illustrated in Figure 6.
Isotachophoresis in Capillary Tubes of CSF Proteins
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Discussion A simple vertical disc-electrophoresis equipment has previously been described by the authors [3,4,9] for studying CSF proteins by isotachoporesis in polyacrylamide gel (PAG-ITP). Earlier attempts to perform ITP in horizontal polyacrylamide flatbeds with the sample applied directly onto the gel surface, resulted in long streaks of protein between the ITP concentrated ("stacked") zones, which streaks could partially be washed away with physiological saline indicating incomplete diffusion into the gel. Acrylic gel tubes were preferred, as the gel did not adhere to the plastic material and could be removed easily, e.g. by blowing by mouth. With the low wattage conditions used no heat denaturation was observed, and thus no cooling system was needed. The elaborated PAG-ITP method in
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plastic gel tubes was especially designed for studying the gammaglobulins in CSF and serum. The increase of slow gammaglobulins found in cases of known or probable MS should be especially mentioned as well as other aberrations observed in the gammaglobulin range of those patients and in myelopathy without any known cause (Kjellin and Hallander, in press). By using other PAG-ITP systems with other spacer compositions, the anodic proteins can be studied in more detail. Furthermore, the PAG-ITP in gel tubes is a simple and inexpensive technique which can be used for analytical and preparative investigations of biological material such as CSF and serum and extractions from nervous tissues. However, for studying very small samples of CSF and serum (lal amounts) quickly (within 30--60 min) with the results immediately obtained on a recorder, a more sophisticated apparatus, ITP in capillary tubes, is to be used [6--9]. Special ITP systems, including leading and terminating electrolytes and proper spacer substances, are to be elaborated for studying gammaglobulins in CSF and serum. These systems are described in the present paper. It was found that for studying gammaglobulins in CSF and serum, cationic systems obviously are the method of choice, which means that the problem of albumin injection clog is avoided, and thus the separation time can be forced. When using the anionic MES-pH 9.0 system at 20°C, the CSF protein separations were found difficult to reproduce. However, no dialysis of the CSF samples was performed. The terminating creatinine in the pH 7.0 system (moving faster than betaalanine) makes it possible to use longer capillary tubes, the disadvantage being longer separation times. This modification means more detailed information about the gammaglobulins. In the present state however, the system can be used for rapid diagnoses of the gammaglobulin profile. The pH 6.3 system can obviously be of value, but the Ampholine blank peaks were in the same order of magnitude as the gammaglobulins ofcontrol cases. It is not possible to inject more than 15--201al into the 25cm capillary tube, and at least 100B1 would probably be needed. Counterflow and continuous injection [14] seems appropriate, and will be tried in this laboratory. When working with such small CSF volumes (lals) as in ITP in capillary tubes, it is necessary to use a microdialysis procedure for desalting, easy to perform and with high reproducibility. Such a microdialysis technique has been elaborated at our laboratory for (5)-15-25-1501al of CSF and will be described in a separate paper (Hallander and Kjellin, to be published). Another technique has also been described by Delmotte [1]. However, no autoanalyser-based dialysis system was available to the present authors, so Delmotte's technique was not practicable. Our need was a dialysis procedure with no charge-dependent absorption, easily changed pore exclusion limits, and ability to recover more than 90% of CSF proteins from the sample. It is of special interest that submicrogram amounts of a certain protein can be detected by connecting the equipment for capillary ITP (Tachophor, AB LKBprodukter) to equipment with a synchronized cellulosa acetate strip where the fractions are collected and then can be analyzed by microimmunological techniques (LKB-Tachofrac, Fig. 1). This method seems very promising for rapid
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244
K.G. Kjellin and L. Hallander
diagnosis of p r o b a b l e MS or different infectious neurological diseases. Some p r e l i m i n a r y findings are presented in Figure 7. The cellulosa acetate strip was simply laid o n a tris-barbital (pH 8.6) antib o d y l o a d e d (approx. 2001al/20 ml) 1% agarose gel. I m m u n o e l e c t r o p h o r e s i s was performed over-night ( 1 6 - - 1 8 h) at 2.5 V / c m . F o r further details, see Moberg et al.,[13].
Acknowledgements. This work was supported by grants from the Karolinska Institute and the Swedish Society of Medical Sciences. The skilful technical assistance of Mrs. Gunborg Strand and Vera Snikvalds is gratefully acknowledged.
References 1. Delmotte, .: Isotachophoresis of the cerebrospinal fluid proteins: Advantage of prior dialysis and its practical realization. In: Electrofocusing and isotachophoresis, Radola, B. J., Graesslin, D. (eds.), pp. 559--564. Berlin, New York: Gruyter 1977 2. Delmotte, .: Analysis of complex protein mixtures by capillary isotachophoresis - - some qualitative and quantitative aspects. Sci. Tools 24, 33--41 (1977) 3. Hallander, L. B., Kjellin, K. G.: A technical report on high-separation methods, isoelectric focusing and isotachophoresis, for investigation of CSF and serum proteins. Sixth Internat. Meeting of the Internat. Soc. for Neurochem., p. 321. August 1977, Copenhagen, Denmark 4. Hallander, L. B., Kjellin, K. G.: Isotachophoresis for investigation of CSF proteins. Sixth. Internat. Meeting of the Internat. Soc. for Neurochem., p. 339. August 1977, Copenhagen, Denmark 5. Hallander, L. B., Kjellin, K. G.: High-separation methods, isoelectric focusing (IEF) and isotachophoresis (ITP), for investigation of CSF- and serum proteins in neurological diseases. 1lth World Congress of Neurology 1977, Amsterdam, The Netherlands. Excerpta Medica 427, 96 (1977) 6. Kjellin, K. G., Moberg, U., Hallander, L. B.: Analytical isotachophoresis of cerebrospinal fluid proteins, a preliminary report. Sci. Tools 22, 3--7 (1975) 7. 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 (1975) 8. 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 9. Kjellin, K. G., et al.: Proceedings of the Annual Meeting of the Swedish Soc. of Medical Sciences, p. 10. Stockholm 1975 I0. 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. August 1977, Copenhagen, Denmark 11. Kjellin, K. G.: High separation techniques (isoelectric focusing and isotachophoresis) for investigation of CSF and serum proteins in neurological diseases. 1lth World Congress of Neurology, 1977. Amsterdam. The Netherlands. Excerpta Medica 427, 348--349 12. 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. New York: Plenum 1978 13. Moberg, U., Hjalmarsson, S.-G., Arlinger, L., Lundin, H.: Preparative capillary isotachophoresis. Separati0n of microgram amounts of some human serum proteins. In: Electrofocusing and isotachophoresis, Radola, B. J., Graesslin, D. (eds.), pp. 515--526. Berlin, New York: Gruyter 1977 14. Ryslavy, Z., Bocek, P., Deml, M., Janak, J.: Capillary isotachophoresis: Continuous sampling technique in the analyses of low concentration samples. J. Chromatogr. 147, 369--373 (1978) Received January 19, 1979
