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Immunoglobulin class and light chain type of oligoclonal bands in CSF in multiple sclerosis determined by agarose gel electrophoresis and immunofixation.

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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
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
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.
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
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
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 )
( N = 14)
CSF IgG Index
(N = 2)
(N = 3 5 )
(N = 4 )
“Normal range in our laboratory.
hBand containing free lambda chains present
specimens and a predominance of lambda type in 5 ,
while the remaining 4 samples had the same numbers
of kappa and lambda IgG bands. In addition, 1 or
more IgG bands with kappa and lambda light chains
present in the same band were found in 27 of the 27
CSF specimens. Oligoclonal IgA and IgM were of the
lambda type. No correlation was observed between
the total number of oligoclonal bands or their light
chain type and the electrophoretic mobility of the
In 7 CSF specimens, 1 or 2 bands were found
which stained with the antiserum against lambda
chains and which could not be identified as a component of IgG, IgA, o r IgM. These bands were considered to consist of free lambda chains. The electrophoretic mobility of these bands was anodic in 3
specimens, cathodic in 3, and anodic as well as
cathodic in 1. All 7 patients belonged to those 27
who also had oligoclonal IgG bands of both kappa
and lambda type in their CSF. No bands of free
kappa chains were identified.
T h e CSF kappdlambda ratio was determined in 36
CSF and 35 serum specimens (see the Table). Abnormally high CSF kappdlambda ratios were found
only in patients with IgG bands of kappa type or a
predominance of IgG bands of kappa type. Five patients had IgG bands of kappa type only in the presence of normal CSF kappahambda ratios. Two patients displayed abnormally low CSF kappdlambda
ratios, and in both of them, bands consisting of free
lambda chains were found in addition to prevalent
IgG lambda bands. Four patients (10%) had normal
CSF IgG index values in the presence of oligoclonal
IgG (see the Table).
I n 4 MS patients in whom agarose gel electrophoresis of the CSF was normal, immunofixation
using specific antisera against IgG, IgA, JgM, kappa,
and lambda did not reveal oligoclonal Ig. Isoelectric
focusing demonstrated oligoclonal bands in CSF and
serum in 1 of these patients, and the CSF IgG index
was elevated (0.83) in another.
The present results obtained with immunofixation
after agarose gel electrophoresis revealed that in addition to oligoclonal IgG, CSF from patients with MS
may contain oligoclonal IgA and IgM; this appears to
be an unusual finding, but further investigations on
larger patient series are necessary to establish the
frequencies. Oligoclonal IRA and IgM migrated on
the anodic side of the application slit, together with
the majority of CSF proteins, and were demonstrable
only by immunofixation. This was also true for 1 or 2
of the IgG bands in 16 patients and for free lambda
chain bands in 4 patients.
Determinations of the kappdlambda ratio have revealed that about 50% of MS patients have a predominance of light chains of the kappa type in the
CSF, which cannot be demonstrated in their serum
[c)]. This kappa predominance has been attributed to
an exaggerated synthesis of IgG molecules containing
kappa light chains and is proved by the present results obtained with immunofixation. A predominance
of IgG bands of kappa type was found in 30 of our 39
MS patients, while 4 displayed identical numbers of
IgG kappa and lambda bands and only 5 had a predominance of IgG lambda bands. These findings
contrast to those in aseptic meningitis [ I ] and
Guillain-Barre syndrome [ 111, in which the majority
of oligoclonal IgG synthesized within the C N S is
of lambda type. These observations need to be
confirmed in larger clinical series. If they are correct,
a selection of the oligoclonal immune response
within the CNS depending on the etiological agent
can be proposed.
Twenty-seven patients displayed, in addition to
IgG kappa and lambda bands, IgG bands with both
kappa and lambda chains present in the same band.
This is probably due to microheterogeneity within
single bands, which is corroborated by the observation of additional bands of oligoclonal IgG when CSF
from patients with MS is investigated by isoelectric
focusing [ 51. The finding indicates that such bands
Link and Laurenzi: Ig Class and Light Chain Type in MS CSF
derive from more than one cell clone. T h e higher
resolution power of isoelectric focusing enables detection of oligoclonal IgG in individual MS patients
in whom agarose gel electrophoresis is normal [>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
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
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