Journal of
J. Neurol. 217, 103--109 (1977)
Neurology © by Springer-Verlag 1977
Crossed Immunoeleetrofocusing of Cerebrospinal Fluid Immunoglobulins A. Sid6n Department of Neurology, Karolinska Hospital, S-10401 Stockholm,Sweden
Summary. The combination of the high separation capacity of isoelectric focusing and the specificity of immunoelectrophoresis gives a valuable tool for protein studies. This technic can be applied to immunoglobulin examinations by using KOCN-treated antibodies isoelectric at pH 5 and performing the immunoelectrophoretic procedure at this pH. CSF and serum samples of patients exhibiting CSF immunoglobulin abnormalities and blood-CSF barrier damages were examined by this method. Key words: Crossed immunoelectrofocusing - Carbamylated antibodies Immunoglobulins. Zusammeufassung. Die hohe Separationskapazit~it isoelektrischer Fokusierung in Verbindung mit immunoelektrophoretischer Spezifizit~it macht sie zu einem wertvollen Instrument fiir das Studium der Proteine. Diese Technik kann fiir die Untersuchung der Immunoglobuline angewandt werden, indem das immunoelektrophoretische Verfahren mit KOCN-behandelten, bei pH 5 isoelektrischer Antik6rper bei dem genannten pH durchgefiihrt wird. Liquorund Serumproben von Patienten mit abnormalen Liquor-Immunoglobulinen und Sch~iden der Blut-Liquor-Schranke wurden durch diese Methode untersucht. Introduction Isoelectric focusing has been adapted for analytical studies of many proteins because of its excellent resolving capacity. One method for the examination of cerebrospinal fluid (CSF) proteins was reported by Delmotte (1971, 1972) and another by Kjellin and Vesterberg (1972) and the technic has been found very valuable for studies of CSF proteins in several neurological disorders, e.g. degenerative, demyelinating and infectious diseases (Delmotte and Gonsette, 1977; Kjellin and Versterberg, 1974; Kjellin ans Stibler, 1976; Kjellin and Sid6n,
104
A. Sid6n
Fig. 1, Isoelectric focusing of CSF. The anode was at the top and the cathode at the bottom. A: normal CSF. Serum sample to the left. The bracket shows the approximate range where the majority of native gammaglobulins (normal and pathological) are focused; the normal gammaglobulin region extends from the sample application (arrow) to the cathodat end of serum. Note the weakly stained CSF gammaglobulin range. B: CSF findings in multiple sclerosis. Serum sample to the right. Increased concentration of CSF gammaglobulins, especially those with alkaline pI values. C: blood-CSF barrier damage. Serum sample to the right. Note the uniform and serum-like increase of CSF gammaglobulins
1977). Some examples o f C S F protein findings on isoelectric focusing are shown in Figure 1. Crossed immunoelectrofocusing, since its description (Rotbol, 1970), has been developed further into a reproducible and relatively simple m e t h o d (SOderholm and Smyth, 1975; S6derholm, Smyth and Wadstr6m, 1975) and has been f o u n d applicable to examinations o f n o n - i m m u n o g l o b u l i n C S F proteins with acidic isoelectric points (pI) (Stibler, 1977). Problems are, however, encountered when i m m u n o g l o b u l i n s are to be studied by crossed immunoelectrofocusing. This is primarily due to the similarities o f electrophoretic mobility between the antigens (the i m m u n o g l o b u l i n s to be examined) and the antibodies in the antisera to immunoglobulins. A crossed immunoelectrofocusing technic which was f o u n d suitable for examinations o f immunoglobulins, e.g. the C S F immunoglobulin abnormalities f o u n d with demyelinating and infectious neurological disorders, is presented in this paper.
Material and Methods The CSF was obtained by lumbar puncture and a serum sample was collected at the same time. The CSF and serum samples were collected from five patients exhibiting abnormal bands in the CSF gammaglobulin region and five subjects with CSF protein findings compatible with a
Crossed Immunoelectrofocusing
105
blood-CSF barrier damage. The CSF samples were ultrafiltered (Kjellin, 1967) and concentrated to a protein content of 5 g/l; the serum samples were diluted 10 times with 0.15 M NaC1. Isoelectric focusing was performed in thin layers of polyacrylamide gel (Kjellin and Vesterberg, 1974). The gel was prepared from 6ml of an aqueous solution of 30.5% acrylamide (British Drug House) and 1% bisacrylamide, 22 ml aqua dest and 3.75 g sucrose. The following Ampholine solutions (LKB) were used: pH 3.5--10 (1.4ml), pH 4--6(0.1 ml),pH 5--7 (0.1 ml) and pH 9--11 (0.4 ml). The polymerization was initiated by riboflavin and then performed by using a daylight fluorescent light tube. The volume of each CSF and serum sample applied to the gel had a protein content of 150--200 and 200--250 ~tg respectively. The isoelectric focusing was startet at 550V (94mA) and performed for 60min with a final voltage of 1000V (final current 15 mA). The antibodies of the antisera to human immunoglobulins were carbamylated essentially as described by Bjerrum, Ingild, Lowenstein and Weeke (1973). One ml of rabbit antiserum (Dakopatts) was mixed in a test-tube with 1 ml 0.1 M NaC1 and 8 ml borate buffer (2.13 g NaCI + 5.18 g Na2BnO7 • 10 H20 + 9.03 g H3BO3 to one liter, pH 8.0). The reaction was started by the addition of 160mg KOCN. After incubating at + 47°C for 120min, 10ml 0.01 M phosphate buffer (pH 6.0) at + 4°C was added and the mixture cooled to this temperature. The solution was then ultrafiltered to a volume of 1 ml and 15 mi phosphate buffer at + 4°C was added and the ultrafiltration procedure was continued to a final volume of 1 ml. Electrophoresis in 1% agarose (10 V/cm, 60 rain) at pH 5.0 was used to check that the carbamylated antibodies were essentially immobile at this pH. The gels for the second dimension of the crossed immunoelectrofocusing procedure were prepared on glass plates (100 x 100 x 1 mm). The agarose (Litex) was dissolved in aqua dest followed by the addition of 1.0 M acetate buffer (pH 5.0) giving a 1% (w/v) agarose solution in 0.07 M acetate buffer. An antibody-free agarose strip of 25 mm breadth was cast along one side of the glass plate and then the antibody-containing gel was cast on the remaining part; the thickness of the gel was approximately 1.5 mm. After completion of the isoelectric focusing a 4 m m broad strip was cut from the protein track in the polyacrylamide gel. This strip was transferred to the antibody-free agarose gel by the laying-on method (S/Sderholm and Smyth, 1975) and placed parallel and close to the junction to the antibody-containing gel. The electrophoresis was run in 0.07 M acetate buffer (pH 5.0) for 4 or 9 h at 7 and 3 V/cm respectively. An electrophoresis apparatus (Electrophoresis Cell Type C, AB Analysteknik) with effective cooling and circulating buffer was used. The anode was connected to the antibody-free agarose strip and the immunoglobulins in the polyacrylamide gel migrated towards the cathode. After pressing, washing and drying of the gel the immunoprecipitates were stained with Coomassie Brilliant Blue.
Results No electroendosmotic or other significant technical problems were encountered. T h e q u a l i t a t i v e r e p r o d u c i b i l i t y o f t h e t e c h n i c was g o o d . It is p r e s e n t l y , h o w e v e r , n o t s u i t a b l e f o r q u a n t i t a t i v e p u r p o s e s m a i n l y b e c a u s e o f the difficulties i n v o l v e d in t r a n s f e r r i n g a s t a n d a r d i z e d a m o u n t o f s a m p l e f r o m the p o l y a c r y l a m i d e gel a n d t h e p o s s i b i l i t y o f r e t e n t i o n o f s o m e p r o t e i n in the p o l y a c r y l a m i d e gel strip. A slight t e n d e n c y to m o r e d i s t i n c t i m m u n o p r e c i p i t a t e s w a s s o m e t i m e s o b s e r v e d w h e n the i m m u n o e l e c t r o p h o r e t i c step was p e r f o r m e d at 3 V / c m f o r 9 h c o m p a r e d to r u n s at 7 V / c m f o r 4 h. G e n e r a l l y , h o w e v e r , the t e c h n i c a l q u a l i t y s h o w e d n o d i f f e r e n c e s , a n d t h e c h o i c e b e t w e e n the t w o a l t e r n a t i v e s m a y be d i c t a t e d p r i m a r i l y b y t h e t i m e t h a t is f o u n d m o s t p r a c t i c a l . A h i g h e r field s t r e n g t h (9 V / c m f o r 3 h) was u s e d in s o m e e x p e r i m e n t s b u t s e e m e d to give slightly i n f e r i o r results, a n d it was c o n c l u d e d t h a t f i e l d s t r e n g t h s o f 3 - - 7 V / c m w e r e o p t i m a l f o r this s y s t e m .
106
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Fig. 2. Crossed immunoelectrofocusing of CSF and serum from two patients with a blood-CSF barrier damage. The agarose gel contained 1pl/cm: of rabbit antiserum to IgG. The cathodal end of the polyacrylamide gel strip was to the left and the sample application was at the arrow. The immunoelectrophoresis was performed at 7 V/cm for 4 h in A and 3 V/cm for 9 h in B. The corresponding isoelectric focusing findings are shown below
Some examples of findings on crossed i m m u n o e l e c t r o f o c u s i n g of C S F a n d serum are s h o w n in Figure 2 a n d 3. The i m m u n o p r e c i p i t a t e s were stained inside a n d h a d distinct outlines. W h e n the p r o t e i n samples subjected to isoelectric focusing were r u n into a gel c o n t a i n i n g 1 l~l/cm 2 o f rabbit a n t i s e r u m to h u m a n
Crossed Immunoelectrofocusing
107
Fig, 3. Crossed immunoelectrofocusing of CSF from two patients exhibiting abnormal bands in the CSF gammaglobulin range. The corresponding 2serum samples showed pictures similar to those in Figure 2. The agarose gel contained 1txl/cm of rabbit antiserum to IgG. The cathodal end of the polyacrylamide gel strip was to the left and the sample application was at the arrow. The immunoelectrophoresis was performed at 7V/cm for 4h in A and 3V/cm for 9h in B. The corresponding isoelectric focusing findings are shown to the right
IgG, the immunoprecipitates exhibited maximal heights o f 15 to 40 mm, Sera appearing normal on isoelectric focusing showed a relatively typical picture (Fig. 2)." a rather smoothly outlined immunoprecipitate with the maximal peaks in the pI range of a b o u t 7.7--8.4 p H units. The CSF of patients with a blood-CSF barrier damage gave a profile very similar to that of the corresponding serum sample (Fig. 2). When CSF samples exhibiting abnormal bands in the g a m m a globulin range on isoelectric focusing were examined by crossed immunoelectrofocusing the results were quite different (Fig. 3). The immunoprecipitates had an irregular outline with distinct peaks corresponding to the abnormal bands. In all cases these bands were found to be of I g G identity.
Discussion Crossed immunoelectrofocusing is a valuable method for protein studies since it makes use of the excellent separation capacity o f isoelectric focusing and the specificity o f immunoelectrophoresis. Immunoglobulin studies with this technic, however, offer difficulties at the immunoelectrophoretic step in the second
108
A. Sid6n
dimension. One convenient solution to the problem is to change the pI of the antibodies in the antiserum. By such a procedure the antibodies can be made essentially immobile at a p H where the native immunoglobulins migrate in the electrical field. Proteins can be modified chemically in several ways (Stark, 1970) and methods for either elevating or lowering the pI of immunoglobulins have been described. The antibody population of the antiserum can be treated with methyla m m o n i u m chloride and carbodiimide hydrochloride resulting in an elevation of the pI (Grubb, 1974). Antibodies made isoelectric at p H 10.3 were used in some experiments where the immunoelectrophoretic step of crossed immunoelectrofocusing was performed at this pH. The results were good but one problem was encountered: it proved difficult to achieve a satisfying migration of immunoglobulins with high pI values. The CSF immunoglobulins with neurological diseases (primarily demyelinating and infectious disorders) with distinct abnormalities in these proteins are generally found to have high pI values (approximately > 8 p H units). For this reason it was considered more appropriate to run the immunoelectrophoresis in an acidic buffer and to use chemically modified antibodies isoelectric at a low pH. The pI of antibodies can be lowered by carbamylation (Bjerrum et al., 1973), treatment with fl-propiolacton (Stephan and Frahm, 1971) or formaldehyde (Scherer and Ruhenstroth-Bauer, 1976). The carbamylation procedure was chosen because of its relative simplicity and good reproducibility. Studies of CSF proteins are of great importance in many neurological disorders, not only for diagnostic purposes but also to collect information of etiological and pathophysiological interest. Crossed immunoelectrofocusing can be used for immunoglobulin examinations if the technic is modified, e.g. by performing the immunoelectrophoretic procedure at p H 5.0 and using carbamylated antibodies isoelectric at this pH. A still greater separation and identification capacity can probably be achieved if a more shallow alkaline p H gradient is used in the isoelectric focusing procedure. Further studies of CSF immunoglobulins in neurological diseases by crossed immunoelectrofocusing should give interesting results.
Acknowledgements. This work was supported by grants from the Swedish Multiple Sclerosis Society. An introduction into the basic principles of crossed immunoelectrofocusing was kindly given by O. Vesterberg (Occupational Health Department, Chemical Division, Stockholm) and valuable advice was given by K. G. Kjellin (Department of Neurology, Karolinska Hospital, Stockholm).
References Bjerrum, O. J., Ingild, A., Lowenstein, H., Weeke, B.: Carbamylated antibodies used for quantitation of human IgG. A routine method. In: A manual of quantitative immunoelectrophoresis (N. H. Axelsen, J. Kroll, B. Weeke, eds.). Scand. J. Immunol. 2, Suppl. 1, 145--148 (1973) Delmotte, P.: Gel isoelectric focusing of cerebrospinal fluid proteins: a potential diagnostic tool. Z. klin. Chem. u. klin. Biochem. 9, 334--336 (1971) Delmotte, P.: Comparative results of agar gel electrophoresis and isoelectric focusing examina-
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109
tion of the gamma globulins of the cerebrospinal fluid. Acta neurol, belg. 72, 226---234 (1972) Delmotte, P., Gonsette, R.: Isoelectric focusing of the CSF gamma giobulins in multiple sclerosis (262 cases) and otrher neurological diseases (272 cases). J. Neurol. 215, 27--37 (1977) Grubb, A. O.: Crossed immunoelectrophoresis and electroimmunoassay of IgM. J. Immunol. 112, 1420--1423 (1974) Grubb, A. O.: Crossed immunoelectrophoresis and electroimmunoassay of IgG. J. Immunol. 113, 343w347 (1974) Kjellin, K. G.: Trace elements in the eerebrospinal fluid. "Nuclear activation techniques in the life sciences". Vienna: International Atomic Energy Agency 1967 Kjellin, K. G., Vesterberg, O.: Thin-layer isoelectric focusing of cerebrospinal fluid proteins. A preliminary report with special reference to the diagnostic significance in multiple sclerosis. In: Proceedings of the 20th Congress of Scandinavian Neurologists, pp. 379w380. Oslo: Universitetsforlaget 1972 Kjellin, K. G., Vesterberg, O.: Isoelectric focusing of CSF in neurological diseases. J. neurol. Sci. 23, 199m213 (1974) 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., Sidtn, ]k.: Aberrant CSF protein fractions found by electrofocusing in multiple sclerosis. Eur. Neurol. 15, 40~50 (1977) Rotbol, L.: Isoelectric focusing of human proteins in polyacrylamide gel. Clin. Chim. Acta 29, 101--105 (1970) Scherer, R., Ruhenstroth-Bauer, G.: Plasma protein profiling: the diagnostic evaluation of disorders in plasma protein composition by a new immunoelectrophoretic method. Clin. Chim. Acta 66, 417---433 (1976) Stark, G. R.: Recent developments in chemical modification and sequential degradation of proteins. Advances Protein Chem. 24, 261~308 (1970) Stephan, W., Frahm, U.: Quantitative Simultan-Immunelektrophorese. Z. klin. Chem. u. kiln. Biochem. 9, 224--228 (1971) Stibler, H.: Crossed immunoelectrofocusing for identification of normal and abnormal cerebrospinal fluid proteins. J. neurol. Sci. 32, 331--336 (1977) Stderholm, J., Smyth, C. J.: Crossed immunoelectrofocusing for studies on protein microheterogeneity. In: Progress in isoelectric focusing and isotachophoresis (P. G. Righetti, ed.), pp. 99--114. Amsterdam: North-Holland 1975 Stderholm, J. Smyth, C. J., Wadstrtm, T.: A simple and reproducible method for crossed immunoelectrofocusing. Scand. ~1. Immunol. 4, Suppl. 2, 107w113 (1975) Received July 18, 1977
J. Neurol. 217, 103--109 (1977)
Neurology © by Springer-Verlag 1977
Crossed Immunoeleetrofocusing of Cerebrospinal Fluid Immunoglobulins A. Sid6n Department of Neurology, Karolinska Hospital, S-10401 Stockholm,Sweden
Summary. The combination of the high separation capacity of isoelectric focusing and the specificity of immunoelectrophoresis gives a valuable tool for protein studies. This technic can be applied to immunoglobulin examinations by using KOCN-treated antibodies isoelectric at pH 5 and performing the immunoelectrophoretic procedure at this pH. CSF and serum samples of patients exhibiting CSF immunoglobulin abnormalities and blood-CSF barrier damages were examined by this method. Key words: Crossed immunoelectrofocusing - Carbamylated antibodies Immunoglobulins. Zusammeufassung. Die hohe Separationskapazit~it isoelektrischer Fokusierung in Verbindung mit immunoelektrophoretischer Spezifizit~it macht sie zu einem wertvollen Instrument fiir das Studium der Proteine. Diese Technik kann fiir die Untersuchung der Immunoglobuline angewandt werden, indem das immunoelektrophoretische Verfahren mit KOCN-behandelten, bei pH 5 isoelektrischer Antik6rper bei dem genannten pH durchgefiihrt wird. Liquorund Serumproben von Patienten mit abnormalen Liquor-Immunoglobulinen und Sch~iden der Blut-Liquor-Schranke wurden durch diese Methode untersucht. Introduction Isoelectric focusing has been adapted for analytical studies of many proteins because of its excellent resolving capacity. One method for the examination of cerebrospinal fluid (CSF) proteins was reported by Delmotte (1971, 1972) and another by Kjellin and Vesterberg (1972) and the technic has been found very valuable for studies of CSF proteins in several neurological disorders, e.g. degenerative, demyelinating and infectious diseases (Delmotte and Gonsette, 1977; Kjellin and Versterberg, 1974; Kjellin ans Stibler, 1976; Kjellin and Sid6n,
104
A. Sid6n
Fig. 1, Isoelectric focusing of CSF. The anode was at the top and the cathode at the bottom. A: normal CSF. Serum sample to the left. The bracket shows the approximate range where the majority of native gammaglobulins (normal and pathological) are focused; the normal gammaglobulin region extends from the sample application (arrow) to the cathodat end of serum. Note the weakly stained CSF gammaglobulin range. B: CSF findings in multiple sclerosis. Serum sample to the right. Increased concentration of CSF gammaglobulins, especially those with alkaline pI values. C: blood-CSF barrier damage. Serum sample to the right. Note the uniform and serum-like increase of CSF gammaglobulins
1977). Some examples o f C S F protein findings on isoelectric focusing are shown in Figure 1. Crossed immunoelectrofocusing, since its description (Rotbol, 1970), has been developed further into a reproducible and relatively simple m e t h o d (SOderholm and Smyth, 1975; S6derholm, Smyth and Wadstr6m, 1975) and has been f o u n d applicable to examinations o f n o n - i m m u n o g l o b u l i n C S F proteins with acidic isoelectric points (pI) (Stibler, 1977). Problems are, however, encountered when i m m u n o g l o b u l i n s are to be studied by crossed immunoelectrofocusing. This is primarily due to the similarities o f electrophoretic mobility between the antigens (the i m m u n o g l o b u l i n s to be examined) and the antibodies in the antisera to immunoglobulins. A crossed immunoelectrofocusing technic which was f o u n d suitable for examinations o f immunoglobulins, e.g. the C S F immunoglobulin abnormalities f o u n d with demyelinating and infectious neurological disorders, is presented in this paper.
Material and Methods The CSF was obtained by lumbar puncture and a serum sample was collected at the same time. The CSF and serum samples were collected from five patients exhibiting abnormal bands in the CSF gammaglobulin region and five subjects with CSF protein findings compatible with a
Crossed Immunoelectrofocusing
105
blood-CSF barrier damage. The CSF samples were ultrafiltered (Kjellin, 1967) and concentrated to a protein content of 5 g/l; the serum samples were diluted 10 times with 0.15 M NaC1. Isoelectric focusing was performed in thin layers of polyacrylamide gel (Kjellin and Vesterberg, 1974). The gel was prepared from 6ml of an aqueous solution of 30.5% acrylamide (British Drug House) and 1% bisacrylamide, 22 ml aqua dest and 3.75 g sucrose. The following Ampholine solutions (LKB) were used: pH 3.5--10 (1.4ml), pH 4--6(0.1 ml),pH 5--7 (0.1 ml) and pH 9--11 (0.4 ml). The polymerization was initiated by riboflavin and then performed by using a daylight fluorescent light tube. The volume of each CSF and serum sample applied to the gel had a protein content of 150--200 and 200--250 ~tg respectively. The isoelectric focusing was startet at 550V (94mA) and performed for 60min with a final voltage of 1000V (final current 15 mA). The antibodies of the antisera to human immunoglobulins were carbamylated essentially as described by Bjerrum, Ingild, Lowenstein and Weeke (1973). One ml of rabbit antiserum (Dakopatts) was mixed in a test-tube with 1 ml 0.1 M NaC1 and 8 ml borate buffer (2.13 g NaCI + 5.18 g Na2BnO7 • 10 H20 + 9.03 g H3BO3 to one liter, pH 8.0). The reaction was started by the addition of 160mg KOCN. After incubating at + 47°C for 120min, 10ml 0.01 M phosphate buffer (pH 6.0) at + 4°C was added and the mixture cooled to this temperature. The solution was then ultrafiltered to a volume of 1 ml and 15 mi phosphate buffer at + 4°C was added and the ultrafiltration procedure was continued to a final volume of 1 ml. Electrophoresis in 1% agarose (10 V/cm, 60 rain) at pH 5.0 was used to check that the carbamylated antibodies were essentially immobile at this pH. The gels for the second dimension of the crossed immunoelectrofocusing procedure were prepared on glass plates (100 x 100 x 1 mm). The agarose (Litex) was dissolved in aqua dest followed by the addition of 1.0 M acetate buffer (pH 5.0) giving a 1% (w/v) agarose solution in 0.07 M acetate buffer. An antibody-free agarose strip of 25 mm breadth was cast along one side of the glass plate and then the antibody-containing gel was cast on the remaining part; the thickness of the gel was approximately 1.5 mm. After completion of the isoelectric focusing a 4 m m broad strip was cut from the protein track in the polyacrylamide gel. This strip was transferred to the antibody-free agarose gel by the laying-on method (S/Sderholm and Smyth, 1975) and placed parallel and close to the junction to the antibody-containing gel. The electrophoresis was run in 0.07 M acetate buffer (pH 5.0) for 4 or 9 h at 7 and 3 V/cm respectively. An electrophoresis apparatus (Electrophoresis Cell Type C, AB Analysteknik) with effective cooling and circulating buffer was used. The anode was connected to the antibody-free agarose strip and the immunoglobulins in the polyacrylamide gel migrated towards the cathode. After pressing, washing and drying of the gel the immunoprecipitates were stained with Coomassie Brilliant Blue.
Results No electroendosmotic or other significant technical problems were encountered. T h e q u a l i t a t i v e r e p r o d u c i b i l i t y o f t h e t e c h n i c was g o o d . It is p r e s e n t l y , h o w e v e r , n o t s u i t a b l e f o r q u a n t i t a t i v e p u r p o s e s m a i n l y b e c a u s e o f the difficulties i n v o l v e d in t r a n s f e r r i n g a s t a n d a r d i z e d a m o u n t o f s a m p l e f r o m the p o l y a c r y l a m i d e gel a n d t h e p o s s i b i l i t y o f r e t e n t i o n o f s o m e p r o t e i n in the p o l y a c r y l a m i d e gel strip. A slight t e n d e n c y to m o r e d i s t i n c t i m m u n o p r e c i p i t a t e s w a s s o m e t i m e s o b s e r v e d w h e n the i m m u n o e l e c t r o p h o r e t i c step was p e r f o r m e d at 3 V / c m f o r 9 h c o m p a r e d to r u n s at 7 V / c m f o r 4 h. G e n e r a l l y , h o w e v e r , the t e c h n i c a l q u a l i t y s h o w e d n o d i f f e r e n c e s , a n d t h e c h o i c e b e t w e e n the t w o a l t e r n a t i v e s m a y be d i c t a t e d p r i m a r i l y b y t h e t i m e t h a t is f o u n d m o s t p r a c t i c a l . A h i g h e r field s t r e n g t h (9 V / c m f o r 3 h) was u s e d in s o m e e x p e r i m e n t s b u t s e e m e d to give slightly i n f e r i o r results, a n d it was c o n c l u d e d t h a t f i e l d s t r e n g t h s o f 3 - - 7 V / c m w e r e o p t i m a l f o r this s y s t e m .
106
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Fig. 2. Crossed immunoelectrofocusing of CSF and serum from two patients with a blood-CSF barrier damage. The agarose gel contained 1pl/cm: of rabbit antiserum to IgG. The cathodal end of the polyacrylamide gel strip was to the left and the sample application was at the arrow. The immunoelectrophoresis was performed at 7 V/cm for 4 h in A and 3 V/cm for 9 h in B. The corresponding isoelectric focusing findings are shown below
Some examples of findings on crossed i m m u n o e l e c t r o f o c u s i n g of C S F a n d serum are s h o w n in Figure 2 a n d 3. The i m m u n o p r e c i p i t a t e s were stained inside a n d h a d distinct outlines. W h e n the p r o t e i n samples subjected to isoelectric focusing were r u n into a gel c o n t a i n i n g 1 l~l/cm 2 o f rabbit a n t i s e r u m to h u m a n
Crossed Immunoelectrofocusing
107
Fig, 3. Crossed immunoelectrofocusing of CSF from two patients exhibiting abnormal bands in the CSF gammaglobulin range. The corresponding 2serum samples showed pictures similar to those in Figure 2. The agarose gel contained 1txl/cm of rabbit antiserum to IgG. The cathodal end of the polyacrylamide gel strip was to the left and the sample application was at the arrow. The immunoelectrophoresis was performed at 7V/cm for 4h in A and 3V/cm for 9h in B. The corresponding isoelectric focusing findings are shown to the right
IgG, the immunoprecipitates exhibited maximal heights o f 15 to 40 mm, Sera appearing normal on isoelectric focusing showed a relatively typical picture (Fig. 2)." a rather smoothly outlined immunoprecipitate with the maximal peaks in the pI range of a b o u t 7.7--8.4 p H units. The CSF of patients with a blood-CSF barrier damage gave a profile very similar to that of the corresponding serum sample (Fig. 2). When CSF samples exhibiting abnormal bands in the g a m m a globulin range on isoelectric focusing were examined by crossed immunoelectrofocusing the results were quite different (Fig. 3). The immunoprecipitates had an irregular outline with distinct peaks corresponding to the abnormal bands. In all cases these bands were found to be of I g G identity.
Discussion Crossed immunoelectrofocusing is a valuable method for protein studies since it makes use of the excellent separation capacity o f isoelectric focusing and the specificity o f immunoelectrophoresis. Immunoglobulin studies with this technic, however, offer difficulties at the immunoelectrophoretic step in the second
108
A. Sid6n
dimension. One convenient solution to the problem is to change the pI of the antibodies in the antiserum. By such a procedure the antibodies can be made essentially immobile at a p H where the native immunoglobulins migrate in the electrical field. Proteins can be modified chemically in several ways (Stark, 1970) and methods for either elevating or lowering the pI of immunoglobulins have been described. The antibody population of the antiserum can be treated with methyla m m o n i u m chloride and carbodiimide hydrochloride resulting in an elevation of the pI (Grubb, 1974). Antibodies made isoelectric at p H 10.3 were used in some experiments where the immunoelectrophoretic step of crossed immunoelectrofocusing was performed at this pH. The results were good but one problem was encountered: it proved difficult to achieve a satisfying migration of immunoglobulins with high pI values. The CSF immunoglobulins with neurological diseases (primarily demyelinating and infectious disorders) with distinct abnormalities in these proteins are generally found to have high pI values (approximately > 8 p H units). For this reason it was considered more appropriate to run the immunoelectrophoresis in an acidic buffer and to use chemically modified antibodies isoelectric at a low pH. The pI of antibodies can be lowered by carbamylation (Bjerrum et al., 1973), treatment with fl-propiolacton (Stephan and Frahm, 1971) or formaldehyde (Scherer and Ruhenstroth-Bauer, 1976). The carbamylation procedure was chosen because of its relative simplicity and good reproducibility. Studies of CSF proteins are of great importance in many neurological disorders, not only for diagnostic purposes but also to collect information of etiological and pathophysiological interest. Crossed immunoelectrofocusing can be used for immunoglobulin examinations if the technic is modified, e.g. by performing the immunoelectrophoretic procedure at p H 5.0 and using carbamylated antibodies isoelectric at this pH. A still greater separation and identification capacity can probably be achieved if a more shallow alkaline p H gradient is used in the isoelectric focusing procedure. Further studies of CSF immunoglobulins in neurological diseases by crossed immunoelectrofocusing should give interesting results.
Acknowledgements. This work was supported by grants from the Swedish Multiple Sclerosis Society. An introduction into the basic principles of crossed immunoelectrofocusing was kindly given by O. Vesterberg (Occupational Health Department, Chemical Division, Stockholm) and valuable advice was given by K. G. Kjellin (Department of Neurology, Karolinska Hospital, Stockholm).
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