Immunoglobulin Class and Light Chain Type of Oligoclonal Bands in CSF in Multiple Sclerosis Determined by Agarose Gel Electrophoresis and Immunofixation Hans Link, M D , and Maria Assunta Laurenzi, M D
Agarose gel electrophoresis and immunofixation of CSF and serum from 39 patients with multiple sclerosis (MS) revealed oligoclonal IgG in the CSF in all cases and oligoclonal IgA and IgM in 1 patient each. IgG kappa bands only were found in 10 patients, while no patient had IgG lambda bands alone. IgG kappa bands predominated in 20 patients and IgG lambda bands in 5, while 4 patients had the same number of IgG kappa and IgG lambda bands. Twenty-seven patients also displayed IgG bands with kappa and lambda present simultaneously. Bands of free lambda chains were found in 7 patients, while free kappa chain bands were not seen. One or 2 faint IgG bands in 4 patients constituted the only serum abnormality. In 4 additional MS patients selected on the basis of normal findings on agarose gel electrophoresis of the CSF, immunofixation did not reveal oligoclonal Ig, while isoelectric focusing showed bands in 1. Immunofixation is recommended for proving the presence of oligoclonal Ig in CSF and for characterizing oligoclonal Ig into classes and types of light chains. Link H, Laurenzi MA: Immunoglobulin class and light chain type of oligoclonal bands in CSF in multiple sclerosis determined by agarose gel electrophoresis and immunofixation. Ann Neurol 6: 107-1 10, 1979
The occurrence of oligoclonal immunoglobulins (Ig) in cerebrospinal fluid due to Ig synthesis within the central nervous system is a well-known phenomenon in multiple sclerosis (MS). Electrophoresis carried out on agar gel or agarose gel [ 8 ] and isoelectric focusing [ 51 are equally suitable for demonstrating oligoclonal Ig bands. At least 90% of MS patients display oligoclonal Ig when concentrated CSF is investigated with any of these methods [ 3 , 5 , 6, 9, 101. Due to its higher resolution power, isoelectric focusing also reveals oligoclonal Ig i n serum from about 40% of MS patients IS]. T h e oligoclonal Ig in MS CSF has been identified as IgG [6], and oligoclonal IgG is also present in MS brain [7].Additional evidence for the oligoclonal character of IgG in MS CSF has been obtained by demonstrating a predominance of kappa light chains [9, 12, 171 and y l heavy chains [ 151. The antibody character and function of the oligoclonal IgG in MS is still unknown, and further immunochemical characterization of the oligoclonal IgG is warranted. The aim of this investigation was to
characterize bands demonstrable in CSF and serum in MS patients with regard to Ig class, light chain type, and presence of free light chains. For this purpose, an immunofixation technique after agarose gel electrophoresis was used.
From the Department of Neurology, University Hospital, Linkiiping, Sweden.
Address reprint requests t o D r Link, Department of Neurology, University Hospital, S-58 1 85 Linkiiping, Sajeden.
Materials and Methods Cerebrospinal fluid and serum specimens were obtained simultaneously from 43 patients 2 3 to 59 years old (mean, 37 years) with clinically confirmed MS. Thirty-nine were consecutive patients visiting the department; the remaining 4 were selected on the basis of normal findings on agarose gel electrophoresis of CSF. CSF leukocytes were counted and differentiated by phase contrast microscopy. Albumin and IgG were determined simultaneously in previously unfrozen, unconcentrated CSF and in serum, mostly within 24 hours after lumbar puncture, by an automatic immunoprecipitation techn i q u e utilizing nephelometric analyses of antigen-antibody complexes in a flow system (Auto-analyzer 11, Technicon, New York, NY). T h e CSF IgG index [lo], equal t o (CSF IgGiserum IgG):(CSF albuminiserum albumin), was calculated; an index value above 0 . 7 0 , which constitutes the
Accepted for publication Feb 17, 1979.
0364-5134/79/080107-04%01.25 @ 1078 by Hans Link
107
u p p e r limit of normal in our laboratory, is considerecl to indicate I g G synthesis within t h e C N S . K a p p a and lambda light chains were quantitated in previously frozen, unconcentrated (:SF a n d s e r u m by single radial immunodiffusion, and the k a p p d l a m b d a ratio 112, 171 was determined. T h e 95% confidence limits in our laboratory a r e 0.7 t o 1.7 f o r CSF and 0 . 7 to I .3 for serum. Agarose gel electrophoresis was performed o n CSF concentrated by ultrafiltration at 4°C in collodion bags (Sartorius Membranfilter, Giittingen, W e s t Germany). Four microliters of concentrated CSF with an IgG level o f 2 t o 4 gm p e r liter ~ v a srun i n parallel with t h e corresponding serum, diluted \vith physiological saline to a similar IgG level. J’reformvd agarose gels (Panagel slide, Millipore Biomedica, Acton, M A ) and a Panagel electrophoresis apparatus (Millipore Biomedica) w e r e used as previously d e scribed I 1 , 2 I. Twenty-six previously unfrozen samples of CSF and associated s e r u m w e r e investigated within o n e k after lumbar puncture, while 17 CSF and serum samples had b e e n previously frozen and stored a t -20°C for u p t o five months. T h e occurrence of 1 o r m o r e homogeneous bands in the g a m m a globulin region in addition t o those normally seen mas considered abnormal. For immunofixation, five aliquots from t h e same C S F specimen and five of s e r u m f r o m each patient were subjected simultaneously t o agarose gel electrophoresis. For demonstration of IgG, C S F and s e r u m with an IgG concentration of 1.5 t o 2.0 g m p e r liter w e r e used; CSF and serum Tvith an 1gG conc.entration of 3 to 4 g m per liter was utilized for kappa and lambda light chains. For demonstration of IgA and IgM, CSF was concentrated 100 to 200 times, and undiluted s e r u m was used. Electrophoresis was carriecl o u t as described in the preceding paragraph. For subsequent characterization of t h e Ig class and light chain type of oligoclonal Ig, cellulose acetate strips (Sepraphore 111, G e l m a n Instrunient Company, Ann Arbor, MI) w e r e clipped in the corresponding antiserum and applieci t o the gel surface immediately after electrophoresis I I , 1.211. D u r i n g incubation in a moist chamber for o n e hour, specific i m m u n e complexes a r e formed as insoluble precipitates. Proteiiis not precipitated w e r e then removed by washing in 1 t o 2 liters of physiological saline during o v e r night stirring. This Lvas followed by absorption o n filter paper moistened in physiological saline, whereupon t h e plate was dried, stained with a m i d o black diluted in 5‘’f acetic acid, destained in 57f’ acetic acid, and clried. T h e band pattern o n the gel was compared with that obtained o n agarose gel electrophoresis without immunofixation. Isoelec rric focusing was carried o u t as previously described 141. All antisera were purchased from Dakopatts (Copenhag e n , D e n m a r k ) . The antisera against IgG, IgA, and I g M were specific for t h e corresponding heavy chains, while t h e antisera against kappa and lambda light chains w e r e monospecific against human Bence J o n e s protein and directed against surface as well as hidden determinants.
Results Oligoclonal bands were found by agarose gel electrophoresis i n CSF from 39 MS patients. In 4 additional MS patients, no abnormality was recognized by 108 Annals of Neurology
Vol 6 No 2
August
197‘)
1
2
3
4
5
6
Agarose gel e1rr.trophorr.ti.rpattern of CSF fvoni I hIS patient ( 1) and patterns obtained after immunofixationof this CSF usinguntisera against IgG (2). kappa ( 3 ) ,lambda (4).IgA ( 5 ) . and 1gM (6). The horizontal line m m p n n d s to areas id .raniph applitztion; thr anode is in the ripper part. the cathode in the lort,rr. H e a y arroii8.r denote the quantitatioely most iinportant oligoi.lowall g and the slender arroiis additional, i ~ a k oligoclonal bands. This patient had one strong and one weak band in the cathodir-area cansisting of I g G kappa and IgG lanibda simultaneous[y: one strong cathodic band coitsisting of IgG kappa; one uvuk anodiir-band 1-onsistiwi:of IgG lambda; and. jinally, one iireak anodic band ionsistiizg offret lanibda light chains not clearlj~disceriiible in the pattern of total CSF.
agarose gel electrophoresis; this group is discussed separately later. Immunofixation (Figure) revealed IgG bands in all 39 CSF specimens and in 4 of the serum specimens. The oligoclonal CSF IgG formed 1 to 10 bands (mean, 5 bands), while thc oligoclonal serum IgG formed 1 or 2 bands with the same mobility as bands in the corresponding CSF. In addition, 1 IgA band was found in 1 CSF sample and 1 IgM band in another. Cathodic oligoclonal bands-i.e., bands migrating on the cathodic side of the application slitwere found i n 39 CSF and 3 serum specimens. CSF specimens from I6 patients also displayed 1 or 2 bands migrating on the anodic side of the slit, while the remaining serum had only 1 anodic band. Oligoclonal IgA and IgM migrated on the anodic side. IgG bands of kappa type only were found in 10 CSF and 3 serum specimens, while no specimen displayed IgG bands of lambda type only (Table). In the remaining 1 serum and 29 CSF specimens with oligoclonal bands, IgG bands of both kappa and lambda type were found. A predominance of IgG bands of the kappa type was observed in 20 CSF
Relationship between Light Chain Type of Olrgoclonal CSF IgG Bands, CSF KabbdLamb‘ta Ratio, and CSF IgG Index in 39 MS Patients CSF KamdLambda Ratio Oligoclonal IgG Kappa only (N = 10) Predominance of IgG kappa bands (N = 20) Predominance of IgG lambda bands ( N = 5 ) Same number of IgG kappa and IgG lambda bands ( N = 4 )
>1.7 ( N = 14)
CSF IgG Index
0.7-1.7‘‘
I; this was also the case in 1 of our 4 MS patients with normal findings on agarose gel electrophoresis of the CSF. This increase i n positive findings is unimportant from a clinical standpoint and does not diminish the importance of agar gel or agarose gel electrophoresis as a routine method for CSF investigation. Oligoclonal IgG bands in serum were found in only a few o f the MS serum specimens studied by agarose gel electrophoresis and immunofixation. Using isoelectric focusing, we previously found that no fewer than 4 1‘;i of MS serum specimens revealed oligoclonal Ig bands 151. Free light chains are regularly present in normal CSF as well as in the CSF in MS [6]. Using crossed immunoelectrophoresis, Vandvik 141 described free light chains migrating as bands in CSF in 4 of his 2 0 MS patients. These bands were of kappa type in 1 patient, lambda type in 2, and both kappa and lambda type in 1. Bands of free light chains have also been demonstrated in CSF in acute aseptic meningitis [ 11, subacute sclerosing panencephalitis [ 141, varicella zoster virus myelitis [ 141, and progressive rubella virus panencephalitis [ 161. O u r findings of bands consisting of free lambda light chains only in 7 of 39 MS patients is remarkable in view of the predominance of oligoclonal IgG of kappa type synthesized within the CNS in this disorder. The relevance of the occurrence of electrophoretically restricted populations of free light chains is not clear, but this may reflect a by-product during antibody production under intense immunogenic stimulation. Commercially available agarose gel plates and electrophoresis equipment, as recommended by Johnson et a1 [2], were used in the present investigation. T h e choice makes electrophoresis an easy, quick, and reliable procedure that is extremely useful for routine work. Subsequent immunofixation did not detect oligoclonal Ig in 4 MS patients with a normal agarose gel electrophoresis pattern. Experience with other patients (Link H: unpublished observations) has indicated, however, that in individuals with indeterminate oligoclonal bands, subsequent immunofixation, as carried out in the present investigation, may prove the presence of oligoclonal Ig. Another advantage of immunofixation is that it allows a thorough characterization of oligoclonal Ig in CSF regarding class and light chain type as well as, probably, subclass. T h e method can be recommended for further characterization of the humoral immune response in other neurological disorders involving an inflammatory reaction within the nervous system.
110 Annals of Neurology
Vol 6
No 2
August 1979
Supported by the Swedish Medical Research Council (Project N o . 3381) and by the Swedish Institute through a scholarship to D r Laurenzi.
References 1. Frydi-n A, Link H: Predominance of oligoclonal IgG type lambda in CSF in aseptic meningitis. Arch Neurol (in press) 2. Johnson KP, Arrigo SC, Nelson BJ, et al: Agarose electrophoresis of cerebrospinal fluid in multiple sclerosis. Neurology (Minneap) 27:273-277,1977 3. Iaterre EC, Callewaert A , Heremans JF: Electrophoretic morphology of gamma globulins in cerebrospinal fluid of multiple sclerosis and other diseases of the nervous system. Neurology (Minneap) 20:982-990, 1970 4 Laurenzi M A , Link H: Localization of the immunoglobulins G , A and M, beta trace protein and gamma trace protein o n isoelectric focusing of serum and cerebrospinal fluid. Acta Neurol Scand 58:141-147, 1978 5 Laurenzi M A , Link H: Comparison of agarose gel electrophoresis and isoelectric focusing in the demonstration of oligoclonal immunoglobulins in CSF and serum. Acta Neurol Scand 58:148-156. 1978 6 Link H: Immunoglobulin G and low molecular weight proteins in human cerebrospinal fluid: chemical and immunological characterization with special reference t o multiple sclerosis. Acta Neurol Scand 43:suppl 28: 1-136, 1967 7 Link H: Oligoclonal immunoglobulin G in multiple sclerosis brains. J Neurol Sci 16:103-114, 1972 8 Link H: Comparison of electrophoresis on agar gel and agarose gel in the evaluation of gamma globulin abnormalities in cerebrospinal fluid and serum in multiple sclerosis. Clin Chim Acta 46:383-389, 1973 9 Link H , Muller R: Immunoglobulins in multiple sclerosis and infections of the nervous system. Arch Neurol 25:326-344, 197 I 1 0 Link H, Tibbling G: Principles of albumin and IgG analyses in neurological disorders: 111. Evaluation of IgG synthesis within the central nervous system in multiple sclerosis. Stand J Clin Lab Invest 37:397-401, 1977 11 Link H, Wahren B, Norrby E: Prolonged pleocytosis and immunoglobulin changes in the CSF of patients with GuillainBar& syndrome and acute and reactivated C M V and EBV infections. J Clin Microbiol (in press) 12 Link H, Zettervall 0: Multiple sclerosis: disturbeci kappa/ lambda chain ratio of immunoglobulin G in cerebrospinal fluid. Clin Exp Immunol 6:435-438, 1970 13 Rirchie RF, Smith R: Immunofixation: I. General principles and application to agarose gel electrophoresis. Clin Chem 22:497-499, 1976 14 Vandvik B: Oligoclonal IgG and free light chains in the cerebrospinal fluid of patients with multiple sclerosis and infectious diseases of the central nervous system. ScandJ Immunol 6:913-922, 1977 15 Vandvik B, Natvig JB, Wiger D: IgGl subclass restriction of oligoclonal IgG from cerebrospinal fluids and brain extracts in patients with multiple sclerosis and subacute encephalitides. Scand J lmmunol 5:427-436, 1976 16 Vandvik B, W e d L, Grandien M , e t al: Progressive rubella virus panencephalitis: synthesis of oligwlonal virus-specific IgG antibodies and homogeneous free light chains in the central nervous system. Acta Neurol Scand 57:53-64, 1978 17 Zettervall 0, Link H: Electrophoretic distribution of kappa and lambda immunoglobulin light chain determinants in serum and CSF in multiple sclerosis. Clin Exp Immunol 7 : 3 6 5 - 3 7 2 , 1970
Agarose gel electrophoresis and immunofixation of CSF and serum from 39 patients with multiple sclerosis (MS) revealed oligoclonal IgG in the CSF in all cases and oligoclonal IgA and IgM in 1 patient each. IgG kappa bands only were found in 10 patients, while no patient had IgG lambda bands alone. IgG kappa bands predominated in 20 patients and IgG lambda bands in 5, while 4 patients had the same number of IgG kappa and IgG lambda bands. Twenty-seven patients also displayed IgG bands with kappa and lambda present simultaneously. Bands of free lambda chains were found in 7 patients, while free kappa chain bands were not seen. One or 2 faint IgG bands in 4 patients constituted the only serum abnormality. In 4 additional MS patients selected on the basis of normal findings on agarose gel electrophoresis of the CSF, immunofixation did not reveal oligoclonal Ig, while isoelectric focusing showed bands in 1. Immunofixation is recommended for proving the presence of oligoclonal Ig in CSF and for characterizing oligoclonal Ig into classes and types of light chains. Link H, Laurenzi MA: Immunoglobulin class and light chain type of oligoclonal bands in CSF in multiple sclerosis determined by agarose gel electrophoresis and immunofixation. Ann Neurol 6: 107-1 10, 1979
The occurrence of oligoclonal immunoglobulins (Ig) in cerebrospinal fluid due to Ig synthesis within the central nervous system is a well-known phenomenon in multiple sclerosis (MS). Electrophoresis carried out on agar gel or agarose gel [ 8 ] and isoelectric focusing [ 51 are equally suitable for demonstrating oligoclonal Ig bands. At least 90% of MS patients display oligoclonal Ig when concentrated CSF is investigated with any of these methods [ 3 , 5 , 6, 9, 101. Due to its higher resolution power, isoelectric focusing also reveals oligoclonal Ig i n serum from about 40% of MS patients IS]. T h e oligoclonal Ig in MS CSF has been identified as IgG [6], and oligoclonal IgG is also present in MS brain [7].Additional evidence for the oligoclonal character of IgG in MS CSF has been obtained by demonstrating a predominance of kappa light chains [9, 12, 171 and y l heavy chains [ 151. The antibody character and function of the oligoclonal IgG in MS is still unknown, and further immunochemical characterization of the oligoclonal IgG is warranted. The aim of this investigation was to
characterize bands demonstrable in CSF and serum in MS patients with regard to Ig class, light chain type, and presence of free light chains. For this purpose, an immunofixation technique after agarose gel electrophoresis was used.
From the Department of Neurology, University Hospital, Linkiiping, Sweden.
Address reprint requests t o D r Link, Department of Neurology, University Hospital, S-58 1 85 Linkiiping, Sajeden.
Materials and Methods Cerebrospinal fluid and serum specimens were obtained simultaneously from 43 patients 2 3 to 59 years old (mean, 37 years) with clinically confirmed MS. Thirty-nine were consecutive patients visiting the department; the remaining 4 were selected on the basis of normal findings on agarose gel electrophoresis of CSF. CSF leukocytes were counted and differentiated by phase contrast microscopy. Albumin and IgG were determined simultaneously in previously unfrozen, unconcentrated CSF and in serum, mostly within 24 hours after lumbar puncture, by an automatic immunoprecipitation techn i q u e utilizing nephelometric analyses of antigen-antibody complexes in a flow system (Auto-analyzer 11, Technicon, New York, NY). T h e CSF IgG index [lo], equal t o (CSF IgGiserum IgG):(CSF albuminiserum albumin), was calculated; an index value above 0 . 7 0 , which constitutes the
Accepted for publication Feb 17, 1979.
0364-5134/79/080107-04%01.25 @ 1078 by Hans Link
107
u p p e r limit of normal in our laboratory, is considerecl to indicate I g G synthesis within t h e C N S . K a p p a and lambda light chains were quantitated in previously frozen, unconcentrated (:SF a n d s e r u m by single radial immunodiffusion, and the k a p p d l a m b d a ratio 112, 171 was determined. T h e 95% confidence limits in our laboratory a r e 0.7 t o 1.7 f o r CSF and 0 . 7 to I .3 for serum. Agarose gel electrophoresis was performed o n CSF concentrated by ultrafiltration at 4°C in collodion bags (Sartorius Membranfilter, Giittingen, W e s t Germany). Four microliters of concentrated CSF with an IgG level o f 2 t o 4 gm p e r liter ~ v a srun i n parallel with t h e corresponding serum, diluted \vith physiological saline to a similar IgG level. J’reformvd agarose gels (Panagel slide, Millipore Biomedica, Acton, M A ) and a Panagel electrophoresis apparatus (Millipore Biomedica) w e r e used as previously d e scribed I 1 , 2 I. Twenty-six previously unfrozen samples of CSF and associated s e r u m w e r e investigated within o n e k after lumbar puncture, while 17 CSF and serum samples had b e e n previously frozen and stored a t -20°C for u p t o five months. T h e occurrence of 1 o r m o r e homogeneous bands in the g a m m a globulin region in addition t o those normally seen mas considered abnormal. For immunofixation, five aliquots from t h e same C S F specimen and five of s e r u m f r o m each patient were subjected simultaneously t o agarose gel electrophoresis. For demonstration of IgG, C S F and s e r u m with an IgG concentration of 1.5 t o 2.0 g m p e r liter w e r e used; CSF and serum Tvith an 1gG conc.entration of 3 to 4 g m per liter was utilized for kappa and lambda light chains. For demonstration of IgA and IgM, CSF was concentrated 100 to 200 times, and undiluted s e r u m was used. Electrophoresis was carriecl o u t as described in the preceding paragraph. For subsequent characterization of t h e Ig class and light chain type of oligoclonal Ig, cellulose acetate strips (Sepraphore 111, G e l m a n Instrunient Company, Ann Arbor, MI) w e r e clipped in the corresponding antiserum and applieci t o the gel surface immediately after electrophoresis I I , 1.211. D u r i n g incubation in a moist chamber for o n e hour, specific i m m u n e complexes a r e formed as insoluble precipitates. Proteiiis not precipitated w e r e then removed by washing in 1 t o 2 liters of physiological saline during o v e r night stirring. This Lvas followed by absorption o n filter paper moistened in physiological saline, whereupon t h e plate was dried, stained with a m i d o black diluted in 5‘’f acetic acid, destained in 57f’ acetic acid, and clried. T h e band pattern o n the gel was compared with that obtained o n agarose gel electrophoresis without immunofixation. Isoelec rric focusing was carried o u t as previously described 141. All antisera were purchased from Dakopatts (Copenhag e n , D e n m a r k ) . The antisera against IgG, IgA, and I g M were specific for t h e corresponding heavy chains, while t h e antisera against kappa and lambda light chains w e r e monospecific against human Bence J o n e s protein and directed against surface as well as hidden determinants.
Results Oligoclonal bands were found by agarose gel electrophoresis i n CSF from 39 MS patients. In 4 additional MS patients, no abnormality was recognized by 108 Annals of Neurology
Vol 6 No 2
August
197‘)
1
2
3
4
5
6
Agarose gel e1rr.trophorr.ti.rpattern of CSF fvoni I hIS patient ( 1) and patterns obtained after immunofixationof this CSF usinguntisera against IgG (2). kappa ( 3 ) ,lambda (4).IgA ( 5 ) . and 1gM (6). The horizontal line m m p n n d s to areas id .raniph applitztion; thr anode is in the ripper part. the cathode in the lort,rr. H e a y arroii8.r denote the quantitatioely most iinportant oligoi.lowall g and the slender arroiis additional, i ~ a k oligoclonal bands. This patient had one strong and one weak band in the cathodir-area cansisting of I g G kappa and IgG lanibda simultaneous[y: one strong cathodic band coitsisting of IgG kappa; one uvuk anodiir-band 1-onsistiwi:of IgG lambda; and. jinally, one iireak anodic band ionsistiizg offret lanibda light chains not clearlj~disceriiible in the pattern of total CSF.
agarose gel electrophoresis; this group is discussed separately later. Immunofixation (Figure) revealed IgG bands in all 39 CSF specimens and in 4 of the serum specimens. The oligoclonal CSF IgG formed 1 to 10 bands (mean, 5 bands), while thc oligoclonal serum IgG formed 1 or 2 bands with the same mobility as bands in the corresponding CSF. In addition, 1 IgA band was found in 1 CSF sample and 1 IgM band in another. Cathodic oligoclonal bands-i.e., bands migrating on the cathodic side of the application slitwere found i n 39 CSF and 3 serum specimens. CSF specimens from I6 patients also displayed 1 or 2 bands migrating on the anodic side of the slit, while the remaining serum had only 1 anodic band. Oligoclonal IgA and IgM migrated on the anodic side. IgG bands of kappa type only were found in 10 CSF and 3 serum specimens, while no specimen displayed IgG bands of lambda type only (Table). In the remaining 1 serum and 29 CSF specimens with oligoclonal bands, IgG bands of both kappa and lambda type were found. A predominance of IgG bands of the kappa type was observed in 20 CSF
Relationship between Light Chain Type of Olrgoclonal CSF IgG Bands, CSF KabbdLamb‘ta Ratio, and CSF IgG Index in 39 MS Patients CSF KamdLambda Ratio Oligoclonal IgG Kappa only (N = 10) Predominance of IgG kappa bands (N = 20) Predominance of IgG lambda bands ( N = 5 ) Same number of IgG kappa and IgG lambda bands ( N = 4 )
>1.7 ( N = 14)
CSF IgG Index
0.7-1.7‘‘
I; this was also the case in 1 of our 4 MS patients with normal findings on agarose gel electrophoresis of the CSF. This increase i n positive findings is unimportant from a clinical standpoint and does not diminish the importance of agar gel or agarose gel electrophoresis as a routine method for CSF investigation. Oligoclonal IgG bands in serum were found in only a few o f the MS serum specimens studied by agarose gel electrophoresis and immunofixation. Using isoelectric focusing, we previously found that no fewer than 4 1‘;i of MS serum specimens revealed oligoclonal Ig bands 151. Free light chains are regularly present in normal CSF as well as in the CSF in MS [6]. Using crossed immunoelectrophoresis, Vandvik 141 described free light chains migrating as bands in CSF in 4 of his 2 0 MS patients. These bands were of kappa type in 1 patient, lambda type in 2, and both kappa and lambda type in 1. Bands of free light chains have also been demonstrated in CSF in acute aseptic meningitis [ 11, subacute sclerosing panencephalitis [ 141, varicella zoster virus myelitis [ 141, and progressive rubella virus panencephalitis [ 161. O u r findings of bands consisting of free lambda light chains only in 7 of 39 MS patients is remarkable in view of the predominance of oligoclonal IgG of kappa type synthesized within the CNS in this disorder. The relevance of the occurrence of electrophoretically restricted populations of free light chains is not clear, but this may reflect a by-product during antibody production under intense immunogenic stimulation. Commercially available agarose gel plates and electrophoresis equipment, as recommended by Johnson et a1 [2], were used in the present investigation. T h e choice makes electrophoresis an easy, quick, and reliable procedure that is extremely useful for routine work. Subsequent immunofixation did not detect oligoclonal Ig in 4 MS patients with a normal agarose gel electrophoresis pattern. Experience with other patients (Link H: unpublished observations) has indicated, however, that in individuals with indeterminate oligoclonal bands, subsequent immunofixation, as carried out in the present investigation, may prove the presence of oligoclonal Ig. Another advantage of immunofixation is that it allows a thorough characterization of oligoclonal Ig in CSF regarding class and light chain type as well as, probably, subclass. T h e method can be recommended for further characterization of the humoral immune response in other neurological disorders involving an inflammatory reaction within the nervous system.
110 Annals of Neurology
Vol 6
No 2
August 1979
Supported by the Swedish Medical Research Council (Project N o . 3381) and by the Swedish Institute through a scholarship to D r Laurenzi.
References 1. Frydi-n A, Link H: Predominance of oligoclonal IgG type lambda in CSF in aseptic meningitis. Arch Neurol (in press) 2. Johnson KP, Arrigo SC, Nelson BJ, et al: Agarose electrophoresis of cerebrospinal fluid in multiple sclerosis. Neurology (Minneap) 27:273-277,1977 3. Iaterre EC, Callewaert A , Heremans JF: Electrophoretic morphology of gamma globulins in cerebrospinal fluid of multiple sclerosis and other diseases of the nervous system. Neurology (Minneap) 20:982-990, 1970 4 Laurenzi M A , Link H: Localization of the immunoglobulins G , A and M, beta trace protein and gamma trace protein o n isoelectric focusing of serum and cerebrospinal fluid. Acta Neurol Scand 58:141-147, 1978 5 Laurenzi M A , Link H: Comparison of agarose gel electrophoresis and isoelectric focusing in the demonstration of oligoclonal immunoglobulins in CSF and serum. Acta Neurol Scand 58:148-156. 1978 6 Link H: Immunoglobulin G and low molecular weight proteins in human cerebrospinal fluid: chemical and immunological characterization with special reference t o multiple sclerosis. Acta Neurol Scand 43:suppl 28: 1-136, 1967 7 Link H: Oligoclonal immunoglobulin G in multiple sclerosis brains. J Neurol Sci 16:103-114, 1972 8 Link H: Comparison of electrophoresis on agar gel and agarose gel in the evaluation of gamma globulin abnormalities in cerebrospinal fluid and serum in multiple sclerosis. Clin Chim Acta 46:383-389, 1973 9 Link H , Muller R: Immunoglobulins in multiple sclerosis and infections of the nervous system. Arch Neurol 25:326-344, 197 I 1 0 Link H, Tibbling G: Principles of albumin and IgG analyses in neurological disorders: 111. Evaluation of IgG synthesis within the central nervous system in multiple sclerosis. Stand J Clin Lab Invest 37:397-401, 1977 11 Link H, Wahren B, Norrby E: Prolonged pleocytosis and immunoglobulin changes in the CSF of patients with GuillainBar& syndrome and acute and reactivated C M V and EBV infections. J Clin Microbiol (in press) 12 Link H, Zettervall 0: Multiple sclerosis: disturbeci kappa/ lambda chain ratio of immunoglobulin G in cerebrospinal fluid. Clin Exp Immunol 6:435-438, 1970 13 Rirchie RF, Smith R: Immunofixation: I. General principles and application to agarose gel electrophoresis. Clin Chem 22:497-499, 1976 14 Vandvik B: Oligoclonal IgG and free light chains in the cerebrospinal fluid of patients with multiple sclerosis and infectious diseases of the central nervous system. ScandJ Immunol 6:913-922, 1977 15 Vandvik B, Natvig JB, Wiger D: IgGl subclass restriction of oligoclonal IgG from cerebrospinal fluids and brain extracts in patients with multiple sclerosis and subacute encephalitides. Scand J lmmunol 5:427-436, 1976 16 Vandvik B, W e d L, Grandien M , e t al: Progressive rubella virus panencephalitis: synthesis of oligwlonal virus-specific IgG antibodies and homogeneous free light chains in the central nervous system. Acta Neurol Scand 57:53-64, 1978 17 Zettervall 0, Link H: Electrophoretic distribution of kappa and lambda immunoglobulin light chain determinants in serum and CSF in multiple sclerosis. Clin Exp Immunol 7 : 3 6 5 - 3 7 2 , 1970
