Bollengier, F.

Henrotin, W.

Lowenthal, A.

Clinical Chemistry and Laboratory Medicine (CCLM)

English

edoc Publication server

Bollcngier, Lowenthal and Hcnrotin: Bound and free light chains in subacutc sclerosing panencephalitis and multiple sclerosis 305
Z. Klin. Chem. Klin. Biochcm.
13. Jg. 1975,8.305-310
Bound and Free Light Chains in Subacute Sclerosing Panencephalitis and Multiple Sclerosis Serum
and Cerebrospinal Fluid
By F. Bollengier, A. Lowenthal and W. Henrotin1)
Laboratorium Fysiopathologie van het Zenuwstelsel, Vrije Universiteit Brüssel, Brussels
(Eingegangen am 22. November 1974/21. Februar 1975)
Summary: The kappa-lambda light chain ratios, the presence of free light chains and the double ring formation, with
antikappa and antilambda serum, in single radial immunodiffusion were investigated in serum and cerebrospinal fluid
of patients with subacute sclerosing panencephalitis.
Cerebrospinal fluid samples of several multiple sclerosis cases were considered simultaneously. The results obtained
suggest special immunoglobulin synthesis in both diseases.
Gebundene und freie leichte Ketten in Serum und Liquor cerebrospinalis bei Subakuter Sklerosierender Panenceplm-
litis und Multipler Sklerose
Zusammenfassung: In Serum und Liquor cerebrospinalis von Patienten mit subakuter Sklerosierender Panencephalitis
wurden die Verhältnisse von leichten Kappa- zu leichten Lambda-Ketten und die Doppelring-Bildung mit anti-Kappa-
und anti-Lambda-Serum in der einfachen radialen Immundiffusion untersucht.
Liquorproben zahlreicher Fälle von Multipler Sklerose wurden ebenfalls untersucht. Die Ergebnisse sprechen für eine
spezielle Imunnglobulinsynthese bei beiden Erkrankungen.
When examined by agar gel electrophoresis, the gamma-
globulins of the cerebrospinal fluid in multiple sclerosis
appear in the form of two or more discrete bands (1),
also called oligoclonal immunoglobulins.
Since the first description of this particular phenomenon
it is considered as the best biological diagnosis of mul-
tiple sclerosis; however it is not specific for multiple
sclerosis, and can be found in other diseases.
Several authors studied the characteristics of the
immunoglobulins in multiple sclerosis.
According to Link (2), the ratio of the kappa-lambda
light chains is increased in the cerebrospinal fluid of
multiple sclerosis patients, whereas in serum of the same
patients the values are mostly identical to those of the
controls.
More recently,Iwashita (3) reported the double ring
formation in single radial immunodiffusion for light
chains of type kappa, in unconcentrated multiple scle-
rosis and subacute sclerosing panericephalitis-cerebro-
spinal fluid.
The purpose of this paper is to investigate those parti-
cular features in 12 cases of subacute sclerosing pan-
encephalitis, a disease which is characterized by the
W. Henrotin: Hoechst Belgium - Behringwerke Department
appearance of oligoclonal gamma-globulins, both in the
serum and cerebrospinal fluid. In parallel we studied
9 cases of multiple sclerosis, in order to add possibly
new findings or to confirm earlier ones.
Furthermore, the serum and cerebrospinal fluid of sub-
acute sclerosing panencephalitis and multiple sclerosis
patients were screened for the presence of free light
chains.
Material and Methods
The diagnosis of subacute sclerosing panencephalitis was esta-
blished on the following criteria: the clinical course of the
disease, electroencephalogram, examination of the serum and
cerebrospinal fluid by agar gel electrophoresis, titration of the
measles antibodies in serum and cerebrospinal fluid and even-
tually brain biopsy. Only those cases which answered positively
to the four first criteria were accepted as certain.
For multiple sclerosis the criteria were:
clinical multifocal lesions of the central nervous system, the
typical clinical evolution of the disease and examination of the
cerebrospinal fluid by agar gel electrophoresis.
In all cases the gamma-globulins in the cerebrospinal fluid were
increased and fractionated.
Control sera were assayed directly after centrifugation, and
then stored frozen. All pathological scrum samples had been
stored frozen at least several months before use.
Cerebrospinal fluid samples, obtained by lumbar puncture,
were stored frozen for several months, except the controls,
which were used immediately. All cerebrospinal fluid samples
were concentrated twenty times when measured by single
Z. Klin. Chem. Klin. Biochem. / 13. Jahrg. 1975 / Heft 7
306 Bollengier, Lowenthal4nd Hcnrotin: Bound and free light chains in subacutc sclerosing panencephalitis and multiple sclerosis
20
19
18
17
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Control MS SSPE Control MS SSPE Control MS SSPE
n=9 n=9 n=9 n=9 n=9 n=9 n = 9 n=9 n=9
Fig. 1. IgG, kappa and lambda levels in serum from controls, multiple sclerosis and subacute sclerosing panencephalitis cases.
radial immunodiffusion (4) against total and free light chain
antiscra1).
The IgG content was measured by single radial immunodiffusion,
using Tripartigen2) plates for the serum, and LC Partigen3) pla-
tes for the cerebrospinal fluid.
For the preparation of the agar plates with incorporated light
chain antiserum, the following procedure was used:
1% agar (Difco Noble) was prepared in barbital buffer pH 8.6
(μ 0.05) and made up to 4% in concentration of the particular
antibodies at 56 °C and 5 ml of each solution was poured in an
empty partigen box.
Wells were punched into the gel and filled with 5 μΐ serum or
cerebrospinal fluid.
Diffusion at room temperature was allowed for 50 hours.
For serum and cerebrospinal fluid samples, developed against
total light chain antiserum, the precipitation lines appeared
very clearly, and were read after careful washings with 0.01
mol/1 phosphate 0.15 mol/1 NaCl buffer pH 7.4.
When developed against free light chain antiserum, the agar
gel plates were either, first washed several times with distilled
water, covered with a fresh solution of 197 mg DOPA in
0.1 mol/1 phosphate buffer pH 7.2 (5), incubated overnight
and finally rewashed with distilled water, or washed with
phosphate buffered saline, covered with a 4% aqueous tannic
acid solution for 30 minutes, and washed with distilled water
for 1 hour. The diameters of the rings were measured for each
sample and the kappa, lambda concentrations calculated from
a standard curve, established with several dilutions of reference
kappa and lambda Bence Jones proteins.
Results
Table 1 and 2 give the mean values ± SEM and the signi-
ficant differences between controls and pathological
cases on the one hand, and on the other hand, between
subacute sclerosing panencephalitis and multiple sclero-
sis cases.
*) Goat serum anti Bence Jones type kappa TNN/05 —
Behringwerkc AG - Marburg/Lahn
Goat serum anti Bence Jones type lambda TNO/05 -
Behringwerke AG - Marburg/Lahn
Rabbit serum anti Bence Jones type kappa TNL/05 -
Behringwerke AG - Marburg/Lahn
Rabbit serum anti Bence Jones type lambda TNM/05 -
Behringwerke AG - Marburg/Lahn
2) Tri-Partigen - IgG - TDS/03 - Behringwerke AG -
Marburg/Lahn ·
3) LC Partigen - IgG - TCR/03 - Behringwerke AG -
Marburg/Lahn
In serum the total IgG content, when compared to the
controls, was increased in the pathological cases, but
more significantly in subacute sclerosing panencephali-
tis than in multiple sclerosis (fig. 1).
Tab. 1. Mean values ± SEM and the significant differences be-
tween control and pathological sera for p = 0.05.
8/1
Multiple
sclerosis
Control
Subacute
sclerosing
panence-
phalitis
IgG
M ± S E M 11.90 ±0.95
Kappa total
M i SEM 5,98 ± 0.60
Lambda total
M ± S E M 2,34 ±0.31
Kappa/lambda
M ± S E M 2.57 + 0.12
12.58 ±2.06 15.23 ±3.17*
7.08 ± 1.68* 5.47 ±1.61
2.61 ±0.54 2.62 ±0.56
2.74 ±0.57** 2.10±0.31*/**
* pathological values differ significantly from controls for
p 0.05
** multiple sclerosis values differ significantly from subacute
sclerosing panencephalitis values for ρ 0.05
Tab. 2. Mean values^t SEM and significant differences between
control and pathological cerebrospinal fluid for p = 0.05
mg/1 Control
Multiple
sclerosis
Subacute
sclerosing
panence-
phalitis
IgG
M ± S E M 19.5 ±11.3
Kappa total
M ± SEM 8.7 ± 4.7
Lambda total
M ± S E M 3.6 ± 1.7
Kappa/lambda
M ± SEM 2.4 ι 0.2
49.4 ± 31.6*/** 112.4 ± 40.8*/**
25.4 ± 14.0* 28.0 ±13.2*
7.4 ± 3.4* 14.5 ± 10.0*
4.3 ± 3.6 2.9 ± 1.9
* pathological values differ significantly from controls for
p 0.05
** multiple sclerosis values differ significantly from subacute
sclerosing panencephalitis values for p 0.05
Z. Klin. Chem. Klin. Biochem. / 1*3. Jahrg. 1975 / Heft 7
Bollengier, Lowenthal and Hcnrotin: Bound and free light chains in subacute sclerosing panenccphalitis and multiple sclerosis 307
Consequently, the kappa-lambda ratio was increased in
multiple sclerosis and slightly decreased in subacute
sclerosing panencephalitis with regard to the controls
(fig. 2).
When screened for the presence of free chains with the
Ouchterlony immunodiffusion technique, none were
detected, either in the control, or in pathological
samples.
In cerebrospinal fluid the IgG values were increased both
in multiple sclerosis and subacute sclerosing panencepha-
litis,, but to a more important amount in subacute
sclerosing panencephalitis.
The values of total kappa and lambda chain are consi-
derably increased in both diseases, when compared to
those of the controls. But the lambda chain concen-
3.5
10
| 2.5
"o
g-2.0
1
10
Q5
Control
n =9
MS SSPE
tration is more significantly increased in subacute
sclerosing panencephalitis than in multiple sclerosis
(fig. 3).
The kappa-lambda ratio is increased up to 11 for mul-
tiple sclerosis patients, and up to 6.5 for subacute
sclerosing panencephalitis patients. The control values
of kappa/lambda in cerebrospinal fluid are about 2.4
(fig-4).
Double ring formation with total kappa antiserum was
noticed in 8 out of 9 multiple sclerosis cases, and 11 out
of 12 subacute sclerosing panencephalitis cases (fig. 5, 6).
With total lambda antiserum 6 multiple sclerosis and 4
subacute sclerosing panencephalitis cases displayed
12
11 -
9
8
I 6
5 5
3
2
\
0.9
0.8 Control
n=9
MS
n=9
SSPE
n=12
Fig. 2. Kappa/lambda ratio in serum from controls, multiple
sclerosis and subacute sclerosing panencephalitis
Fig. 4. Kappa/lambda ratio in cerebrospinal fluid from controls,
multiple sclerosis and subacute sclerosing panencephalitis.
160
170
160
150
140
120
no
Ξ 100
J; 90
« 80
CD
- 70
60
50
40
30
20
10
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Control
n=9
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en
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MS SSPE "Control MS SSPE "Control
n=9 n=12 n=9 n=9 n=12 n=9
0<
*
·»
•
•
•
~
S
·»
•
9
MS SSPE
n=9 n=12
Fig. 3. IgG, kappa and lambda levels in cerebrospinal fluid from controls, multiple sclerosis and subacute sclerosing panencephalitis
cases.
Z. Klin. Chem. Klin. Biochem. / 13. Jahrg. 1975 / Heft 7
308 Bollcngicr, Lowcnthal |nd Hcnrotin: Bound and free light chains in subacutc sclerosing panencephalitis and multiple sclerosis
Fig. 5. Radial immunodiffusion of concentrated multiple sclerosis
cerebrospinal fluid samples; kappa chains.
1) Bence Jones kappa: 620 mg/1
2) Bence Jones kappa: 310 mg/1
3) Bence Jones kappa: 160 mg/1
4)-12) multiple sclerosis cerebrospinal fluid
Fig. 7. Radial immunodiffusion of concentrated multiple sclerosis
cerebrospinal fluid; lambda chains.
1) Bence Jones lambda: 310 mg/1
2) Bence Jones lambda: 160 mg/1
3) Bence Jones lambda: 80 mg/1
4)-12) multiple sclerosis cerebrospinal fluid
Fig. 6. Radial immunodiffusion of concentrated subacute sclero-
sing panencephalitis cerebrospinal fluid; kappa chains.
1) Bence Jones kappa: 620 mg/1
2) Bence Jones kappa: 310 mg/1
3) Bence Jones kappa: 160 mg/1
7)-12) subacute sclerosing panencephalitis cerebrospinal
fluid
Fig. 8. Radial immunodiffusion of concentrated subacute sclero-
sing panencephalitis cerebrospinal fluid; free kappa chains.
1) Bence Jones kappa: 80 mg/1
2) Bence Jones kappa: 40 mg/1
3) Bence Jones kappa: 20 mg/1
7)-12) subacute sclerosing panencephalitis cerebrospinal
fluid
Z. Klin. Chem. Klin. Biochem. /13. Jahrg. 1975 / Heft 7
Bollengier, Lowenthal and Henrotin: Bound and free light chains in subacute sclerosing panenccphalitis and multiple sclerosis 309
double rings, but they did not appear so clearly as with
total kappa antiserum (fig. 7).
Cerebrospinal fluid samples of controls were negative
when developed against free light chains antiserum.
But 8 multiple sclerosis -cerebrospinal fluid had free
kappa chains and 3 of them free lambda chains; 11 out
of 12 subacute sclerosing panencephalitis cases had both
free kappa and lambda chains (fig. 8, 9).
The kappa-lambda ratio of the free chains in subacute
sclerosing panencephalitis was below 2 for all of them.
~
is
c 4
"a
•5 3
* 2Q> Sε f
u. l
0.9
0.8
n?u./
ι
-
—
_
s ·
^ ···
-
—
Control MS
n = 9 n = 9
JH
*
•
•Μ
*
SSPE
n=12
30
20
10"
5 9
f 8
t/) 1
c. 1
<< 5
0)
3
2
11
-
7
-
-
-
-
"
~
 β ·
•
Control MS SSPE
n=9 n = 9 n=12
Fig. 9. Free kappa and lambda levels in cerebrospinal fluid from
multiple sclerosis and subacute sclerosing panencephalitis
cases.
Discussion
1. The quantitative distribution of IgG, kappa and
lambda chains, is much more even in control serum
and cerebrospinal fluid, than in the pathological
samples where the values are widely spread.
In serum the multiple sclerosis cases we screened had
generally higher kappa-lambda ratios than the con-
trols, a fact which does not entirely correspond
with Link's results. According to Link (2) the kappa-
lambda ratio in multiple sclerosis serum is almost
identical to the controls.
On the contrary, all subacute sclerosing panencepha-
litis cases, we studied, had lower rates than controls.
In multiple sclerosis-cerebrospinal fluid, the total
kappa chains were increased, whereas in subacute
sclerosing panencephalitis-cerebrospinal fluid the
lambda chain increase dominated.
One has the impression that in both diseases there
is a synthesis of particular immunoglobulins: predomi-
nantly kappa immunoglobulins for multiple sclerosis
and lambda immunoglobulins for subacute sclerosing
panencephalitis.
The presence of free light chains in multiple sclerosis
and subacute sclerosing panencephalitis-cerebrospi-
nal fluid is a fact difficult to explain. Do those free
chains originate from a natural proteolysis of the
immunoglobulins, or are they a result of a defective
synthesis, are questions still unanswered.
2. According to Lietze (6) and Mulder (7-8), multiple
precipitation, noticed in pathological samples in single
radial immunodiffusion is either due to the blocking
of antigenic determinants by other bound proteins, or
due to the lack of antigenic determinants in the
individual immunoglobulins.
In recent studies on myeloma proteins, Mulder
suggests that double precipitation is caused by the
deficiency of certain antibody determinants. Accor-
ding to this, we can assume in our cases 2 hypothesis:
a) immunoglobulins which carry light chains with un-
complete antigenic determinants and which are
specific for the disease, are synthesized in multiple
sclerosis and subacute sclerosing panencephalitis.
b) The antisera used are deficient in certain antibodies
to the proteins studied.
3. The results we obtain for the serum are not entirely
conclusive. The kappa/lambda ratio seems to depend
on the state of the serum: frequent freezings and
thawings, conservation at 4°C, and even in a freezer,
modify the ratio at random. Spontaneous proteolysis,
due to the presence of plasmin in the serum, could
occur and influence the light chain concentration.
The kappa/lambda ratios we found in control serum
are slightly superior to values reported in the litera-
ture. This may be due to the use of Bence Jones pro-
teins as standards instead of the generally used stan-
dard serum. For the determination of free light
chains one has to use Bence-Jones proteins as refer-
ences, because the antisera used only react with free
light chains; in order to remain in the same standard
conditions, we also used the same Bence-Jones pro-
teins for the assay of the total light chains.
Conclusion
In spite of the discussed technical aspects, we think we
can assume that multiple sclerosis and subacute sclero-
sing panencephalitis synthetize immunoglobulins which
are different for both diseases by their light chain quanti-
tative distribution. The oligoclonal reaction of the
gamma-globulins in multiple sclerosis-cerebrospinal
fluid and in subacute sclerosing panencephalitis
serum and cerebrospinal fluid, although non specific,
is an important tool in the diagnostic approach
of those diseases. To gain a better insight in their
immunopathology, we thought that analysis of the
constituents of the IgG's, could be of value. Some
Z. Klin. Chem. Klin. Biochem. / 13. Jahrg. 1975 / Heft 7 23
310 Bollengicr, Lowenthal dfcd Hcnrotin: Bound and free light chains in subacute sclerosing panencephalftis and multiple sclerosis
previous results showed that subacute sclerosing panen-
cephalitis serum immunoglobulins do not differ by
the sedimentation velocity coefficients of isolated IgG,
Fab and Fc fragments from the control values. On the
other hand kappa and lambda light chains of multiple
sclerosis and subacute sclerosing panencephalitis
immunoglobulins have a different quantitative distribu-
tion in serum as well as in cerebrospinal fluid.
From our results it can be stressed that the immuno-
globulins of multiple sclerosis and subacute sclerosing
panencephalitis have characteristic features: in multiple
sclerosis kappa chains predominate, whereas lambda
chains predominate in subacute sclerosing panence-
phalitis.
References
1. Lowenthal, A. (1964), Agargel electrophoresis in Neurology
(Elsevier Publishing Company).
2. Link, H. & Zetterwali, 0. (1970) Clin. Exp. Immunol. 6,
435-438.
3. hvashita, H., Grunwald, F. & Bauer, H. (1974), J. Neurol.
207, 45-52.
4. Mancini, G. A., Carbonara, A. & Heremans, J. (1965),
Immunochemistry 2, 235-254.
5. Madhosingh, C. & Wood, J. M. (1971), Anal. Chem. 44, '
523-527.
6. Lietze, A., Sinclair, C. & Rowe, H. (1970), Clin. Biochem. 3,
335-338.
7. Mulder, J., Sloots, L. C. E. & Verhaar, M. A. T. (1972),
J. Immun. Methods /, 211-213.
8. Mulder, J. & Verhaar, M. A. T. (1973), Clin. Chim. Acta 45,
32,5—00 j.
Lie. Sc. F. Bollengier
B-1000 Brussels
Eversstraat 2
Z. Klin. Chem. Klin. Biochem. / 13. Jahrg, 1975 / Heft 7


Bound and Free Light Chains in Subacute Sclerosing Panencephalitis and Multiple Sclerosis Serum and Cerebrospinal Fluid

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Bollcngier, Lowenthal and Hcnrotin: Bound and free light chains in subacutc sclerosing panencephalitis and multiple sclerosis 305Z. Klin. Chem. Klin. Biochcm.13. Jg. 1975,8.305-310Bound and Free Light Chains in Subacute Sclerosing Panencephalitis and Multiple Sclerosis Serumand Cerebrospinal FluidBy F. Bollengier, A. Lowenthal and W. Henrotin1)Laboratorium Fysiopathologie van het Zenuwstelsel, Vrije Universiteit Brüssel, Brussels(Eingegangen am 22. November 1974/21. Februar 1975)Summary: The kappa-lambda light chain ratios, the presence of free light chains and the double ring formation, withantikappa and antilambda serum, in single radial immunodiffusion were investigated in serum and cerebrospinal fluidof patients with subacute sclerosing panencephalitis.Cerebrospinal fluid samples of several multiple sclerosis cases were considered simultaneously. The results obtainedsuggest special immunoglobulin synthesis in both diseases.Gebundene und freie leichte Ketten in Serum und Liquor cerebrospinalis bei Subakuter Sklerosierender Panenceplm-litis und Multipler SkleroseZusammenfassung: In Serum und Liquor cerebrospinalis von Patienten mit subakuter Sklerosierender Panencephalitiswurden die Verhältnisse von leichten Kappa- zu leichten Lambda-Ketten und die Doppelring-Bildung mit anti-Kappa-und anti-Lambda-Serum in der einfachen radialen Immundiffusion untersucht.Liquorproben zahlreicher Fälle von Multipler Sklerose wurden ebenfalls untersucht. Die Ergebnisse sprechen für einespezielle Imunnglobulinsynthese bei beiden Erkrankungen.When examined by agar gel electrophoresis, the gamma-globulins of the cerebrospinal fluid in multiple sclerosisappear in the form of two or more discrete bands (1),also called oligoclonal immunoglobulins.Since the first description of this particular phenomenonit is considered as the best biological diagnosis of mul-tiple sclerosis; however it is not specific for multiplesclerosis, and can be found in other diseases.Several authors studied the characteristics of theimmunoglobulins in multiple sclerosis.According to Link (2), the ratio of the kappa-lambdalight chains is increased in the cerebrospinal fluid ofmultiple sclerosis patients, whereas in serum of the samepatients the values are mostly identical to those of thecontrols.More recently,Iwashita (3) reported the double ringformation in single radial immunodiffusion for lightchains of type kappa, in unconcentrated multiple scle-rosis and subacute sclerosing panericephalitis-cerebro-spinal fluid.The purpose of this paper is to investigate those parti-cular features in 12 cases of subacute sclerosing pan-encephalitis, a disease which is characterized by theW. Henrotin: Hoechst Belgium - Behringwerke Departmentappearance of oligoclonal gamma-globulins, both in theserum and cerebrospinal fluid. In parallel we studied9 cases of multiple sclerosis, in order to add possiblynew findings or to confirm earlier ones.Furthermore, the serum and cerebrospinal fluid of sub-acute sclerosing panencephalitis and multiple sclerosispatients were screened for the presence of free lightchains.Material and MethodsThe diagnosis of subacute sclerosing panencephalitis was esta-blished on the following criteria: the clinical course of thedisease, electroencephalogram, examination of the serum andcerebrospinal fluid by agar gel electrophoresis, titration of themeasles antibodies in serum and cerebrospinal fluid and even-tually brain biopsy. Only those cases which answered positivelyto the four first criteria were accepted as certain.For multiple sclerosis the criteria were:clinical multifocal lesions of the central nervous system, thetypical clinical evolution of the disease and examination of thecerebrospinal fluid by agar gel electrophoresis.In all cases the gamma-globulins in the cerebrospinal fluid wereincreased and fractionated.Control sera were assayed directly after centrifugation, andthen stored frozen. All pathological scrum samples had beenstored frozen at least several months before use.Cerebrospinal fluid samples, obtained by lumbar puncture,were stored frozen for several months, except the controls,which were used immediately. All cerebrospinal fluid sampleswere concentrated twenty times when measured by singleZ. Klin. Chem. Klin. Biochem. / 13. Jahrg. 1975 / Heft 7306 Bollengier, Lowenthal4nd Hcnrotin: Bound and free light chains in subacutc sclerosing panencephalitis and multiple sclerosis201918171ΐ5Ξ u~ 131211109_— /--_··ι ·- 0-./**-*-.•j••••••·— · 9§8c. 7Ό c-5 6i 5O> /O> Η"3«.•-*~··'---·φ··M<= 7•••,·*••σ> 6g 5I 40^ 3S 2u_ ^---""" β"~ ·1 ·**" ··*-r···· ..·*'—Control MS SSPE Control MS SSPE Control MS SSPEn=9 n=9 n=9 n=9 n=9 n=9 n = 9 n=9 n=9Fig. 1. IgG, kappa and lambda levels in serum from controls, multiple sclerosis and subacute sclerosing panencephalitis cases.radial immunodiffusion (4) against total and free light chainantiscra1).The IgG content was measured by single radial immunodiffusion,using Tripartigen2) plates for the serum, and LC Partigen3) pla-tes for the cerebrospinal fluid.For the preparation of the agar plates with incorporated lightchain antiserum, the following procedure was used:1% agar (Difco Noble) was prepared in barbital buffer pH 8.6(μ 0.05) and made up to 4% in concentration of the particularantibodies at 56 °C and 5 ml of each solution was poured in anempty partigen box.Wells were punched into the gel and filled with 5 μΐ serum orcerebrospinal fluid.Diffusion at room temperature was allowed for 50 hours.For serum and cerebrospinal fluid samples, developed againsttotal light chain antiserum, the precipitation lines appearedvery clearly, and were read after careful washings with 0.01mol/1 phosphate 0.15 mol/1 NaCl buffer pH 7.4.When developed against free light chain antiserum, the agargel plates were either, first washed several times with distilledwater, covered with a fresh solution of 197 mg DOPA in0.1 mol/1 phosphate buffer pH 7.2 (5), incubated overnightand finally rewashed with distilled water, or washed withphosphate buffered saline, covered with a 4% aqueous tannicacid solution for 30 minutes, and washed with distilled waterfor 1 hour. The diameters of the rings were measured for eachsample and the kappa, lambda concentrations calculated froma standard curve, established with several dilutions of referencekappa and lambda Bence Jones proteins.ResultsTable 1 and 2 give the mean values ± SEM and the signi-ficant differences between controls and pathologicalcases on the one hand, and on the other hand, betweensubacute sclerosing panencephalitis and multiple sclero-sis cases.*) Goat serum anti Bence Jones type kappa TNN/05 —Behringwerkc AG - Marburg/LahnGoat serum anti Bence Jones type lambda TNO/05 -Behringwerke AG - Marburg/LahnRabbit serum anti Bence Jones type kappa TNL/05 -Behringwerke AG - Marburg/LahnRabbit serum anti Bence Jones type lambda TNM/05 -Behringwerke AG - Marburg/Lahn2) Tri-Partigen - IgG - TDS/03 - Behringwerke AG -Marburg/Lahn ·3) LC Partigen - IgG - TCR/03 - Behringwerke AG -Marburg/LahnIn serum the total IgG content, when compared to thecontrols, was increased in the pathological cases, butmore significantly in subacute sclerosing panencephali-tis than in multiple sclerosis (fig. 1).Tab. 1. Mean values ± SEM and the significant differences be-tween control and pathological sera for p = 0.05.8/1MultiplesclerosisControlSubacutesclerosingpanence-phalitisIgGM ± S E M 11.90 ±0.95Kappa totalM i SEM 5,98 ± 0.60Lambda totalM ± S E M 2,34 ±0.31Kappa/lambdaM ± S E M 2.57 + 0.1212.58 ±2.06 15.23 ±3.17*7.08 ± 1.68* 5.47 ±1.612.61 ±0.54 2.62 ±0.562.74 ±0.57** 2.10±0.31*/*** pathological values differ significantly from controls forp 0.05** multiple sclerosis values differ significantly from subacutesclerosing panencephalitis values for ρ 0.05Tab. 2. Mean values^t SEM and significant differences betweencontrol and pathological cerebrospinal fluid for p = 0.05mg/1 ControlMultiplesclerosisSubacutesclerosingpanence-phalitisIgGM ± S E M 19.5 ±11.3Kappa totalM ± SEM 8.7 ± 4.7Lambda totalM ± S E M 3.6 ± 1.7Kappa/lambdaM ± SEM 2.4 ι 0.249.4 ± 31.6*/** 112.4 ± 40.8*/**25.4 ± 14.0* 28.0 ±13.2*7.4 ± 3.4* 14.5 ± 10.0*4.3 ± 3.6 2.9 ± 1.9* pathological values differ significantly from controls forp 0.05** multiple sclerosis values differ significantly from subacutesclerosing panencephalitis values for p 0.05Z. Klin. Chem. Klin. Biochem. / 1*3. Jahrg. 1975 / Heft 7Bollengier, Lowenthal and Hcnrotin: Bound and free light chains in subacute sclerosing panenccphalitis and multiple sclerosis 307Consequently, the kappa-lambda ratio was increased inmultiple sclerosis and slightly decreased in subacutesclerosing panencephalitis with regard to the controls(fig. 2).When screened for the presence of free chains with theOuchterlony immunodiffusion technique, none weredetected, either in the control, or in pathologicalsamples.In cerebrospinal fluid the IgG values were increased bothin multiple sclerosis and subacute sclerosing panencepha-litis,, but to a more important amount in subacutesclerosing panencephalitis.The values of total kappa and lambda chain are consi-derably increased in both diseases, when compared tothose of the controls. But the lambda chain concen-3.510| 2.5"og-2.0110Q5Controln =9MS SSPEtration is more significantly increased in subacutesclerosing panencephalitis than in multiple sclerosis(fig. 3).The kappa-lambda ratio is increased up to 11 for mul-tiple sclerosis patients, and up to 6.5 for subacutesclerosing panencephalitis patients. The control valuesof kappa/lambda in cerebrospinal fluid are about 2.4(fig-4).Double ring formation with total kappa antiserum wasnoticed in 8 out of 9 multiple sclerosis cases, and 11 outof 12 subacute sclerosing panencephalitis cases (fig. 5, 6).With total lambda antiserum 6 multiple sclerosis and 4subacute sclerosing panencephalitis cases displayed1211 -98I 65 532\0.90.8 Controln=9MSn=9SSPEn=12Fig. 2. Kappa/lambda ratio in serum from controls, multiplesclerosis and subacute sclerosing panencephalitisFig. 4. Kappa/lambda ratio in cerebrospinal fluid from controls,multiple sclerosis and subacute sclerosing panencephalitis.160170160150140120noΞ 100J; 90« 80CD- 70605040302010Λ---------_-——— *-_ **-··Controln=9-..-•···*•φ.•5040=C 30en.§20.iioo•5 9X 8Ό „c 7m 65/__ ·-fτ ·1 .·— 0-_ *0^.·s* 3000«··»*.20^ 1Q:σ>^ 91 θO•5 71«< 61 5& 43o--'—~———- ·*—"".«MS SSPE "Control MS SSPE "Controln=9 n=12 n=9 n=9 n=12 n=90<*·»•••~S·»•9MS SSPEn=9 n=12Fig. 3. IgG, kappa and lambda levels in cerebrospinal fluid from controls, multiple sclerosis and subacute sclerosing panencephalitiscases.Z. Klin. Chem. Klin. Biochem. / 13. Jahrg. 1975 / Heft 7308 Bollcngicr, Lowcnthal |nd Hcnrotin: Bound and free light chains in subacutc sclerosing panencephalitis and multiple sclerosisFig. 5. Radial immunodiffusion of concentrated multiple sclerosiscerebrospinal fluid samples; kappa chains.1) Bence Jones kappa: 620 mg/12) Bence Jones kappa: 310 mg/13) Bence Jones kappa: 160 mg/14)-12) multiple sclerosis cerebrospinal fluidFig. 7. Radial immunodiffusion of concentrated multiple sclerosiscerebrospinal fluid; lambda chains.1) Bence Jones lambda: 310 mg/12) Bence Jones lambda: 160 mg/13) Bence Jones lambda: 80 mg/14)-12) multiple sclerosis cerebrospinal fluidFig. 6. Radial immunodiffusion of concentrated subacute sclero-sing panencephalitis cerebrospinal fluid; kappa chains.1) Bence Jones kappa: 620 mg/12) Bence Jones kappa: 310 mg/13) Bence Jones kappa: 160 mg/17)-12) subacute sclerosing panencephalitis cerebrospinalfluidFig. 8. Radial immunodiffusion of concentrated subacute sclero-sing panencephalitis cerebrospinal fluid; free kappa chains.1) Bence Jones kappa: 80 mg/12) Bence Jones kappa: 40 mg/13) Bence Jones kappa: 20 mg/17)-12) subacute sclerosing panencephalitis cerebrospinalfluidZ. Klin. Chem. Klin. Biochem. /13. Jahrg. 1975 / Heft 7Bollengier, Lowenthal and Henrotin: Bound and free light chains in subacute sclerosing panenccphalitis and multiple sclerosis 309double rings, but they did not appear so clearly as withtotal kappa antiserum (fig. 7).Cerebrospinal fluid samples of controls were negativewhen developed against free light chains antiserum.But 8 multiple sclerosis -cerebrospinal fluid had freekappa chains and 3 of them free lambda chains; 11 outof 12 subacute sclerosing panencephalitis cases had bothfree kappa and lambda chains (fig. 8, 9).The kappa-lambda ratio of the free chains in subacutesclerosing panencephalitis was below 2 for all of them.~isc 4"a•5 3* 2Q> Sε fu. l0.90.8n?u./ι-—_s ·^ ···-—Control MSn = 9 n = 9JH*••Μ*SSPEn=12302010"5 9f 8t/) 1c. 1<< 50)3211-7----"~ β ·•Control MS SSPEn=9 n = 9 n=12Fig. 9. Free kappa and lambda levels in cerebrospinal fluid frommultiple sclerosis and subacute sclerosing panencephalitiscases.Discussion1. The quantitative distribution of IgG, kappa andlambda chains, is much more even in control serumand cerebrospinal fluid, than in the pathologicalsamples where the values are widely spread.In serum the multiple sclerosis cases we screened hadgenerally higher kappa-lambda ratios than the con-trols, a fact which does not entirely correspondwith Link's results. According to Link (2) the kappa-lambda ratio in multiple sclerosis serum is almostidentical to the controls.On the contrary, all subacute sclerosing panencepha-litis cases, we studied, had lower rates than controls.In multiple sclerosis-cerebrospinal fluid, the totalkappa chains were increased, whereas in subacutesclerosing panencephalitis-cerebrospinal fluid thelambda chain increase dominated.One has the impression that in both diseases thereis a synthesis of particular immunoglobulins: predomi-nantly kappa immunoglobulins for multiple sclerosisand lambda immunoglobulins for subacute sclerosingpanencephalitis.The presence of free light chains in multiple sclerosisand subacute sclerosing panencephalitis-cerebrospi-nal fluid is a fact difficult to explain. Do those freechains originate from a natural proteolysis of theimmunoglobulins, or are they a result of a defectivesynthesis, are questions still unanswered.2. According to Lietze (6) and Mulder (7-8), multipleprecipitation, noticed in pathological samples in singleradial immunodiffusion is either due to the blockingof antigenic determinants by other bound proteins, ordue to the lack of antigenic determinants in theindividual immunoglobulins.In recent studies on myeloma proteins, Muldersuggests that double precipitation is caused by thedeficiency of certain antibody determinants. Accor-ding to this, we can assume in our cases 2 hypothesis:a) immunoglobulins which carry light chains with un-complete antigenic determinants and which arespecific for the disease, are synthesized in multiplesclerosis and subacute sclerosing panencephalitis.b) The antisera used are deficient in certain antibodiesto the proteins studied.3. The results we obtain for the serum are not entirelyconclusive. The kappa/lambda ratio seems to dependon the state of the serum: frequent freezings andthawings, conservation at 4°C, and even in a freezer,modify the ratio at random. Spontaneous proteolysis,due to the presence of plasmin in the serum, couldoccur and influence the light chain concentration.The kappa/lambda ratios we found in control serumare slightly superior to values reported in the litera-ture. This may be due to the use of Bence Jones pro-teins as standards instead of the generally used stan-dard serum. For the determination of free lightchains one has to use Bence-Jones proteins as refer-ences, because the antisera used only react with freelight chains; in order to remain in the same standardconditions, we also used the same Bence-Jones pro-teins for the assay of the total light chains.ConclusionIn spite of the discussed technical aspects, we think wecan assume that multiple sclerosis and subacute sclero-sing panencephalitis synthetize immunoglobulins whichare different for both diseases by their light chain quanti-tative distribution. The oligoclonal reaction of thegamma-globulins in multiple sclerosis-cerebrospinalfluid and in subacute sclerosing panencephalitisserum and cerebrospinal fluid, although non specific,is an important tool in the diagnostic approachof those diseases. To gain a better insight in theirimmunopathology, we thought that analysis of theconstituents of the IgG's, could be of value. SomeZ. Klin. Chem. Klin. Biochem. / 13. Jahrg. 1975 / Heft 7 23310 Bollengicr, Lowenthal dfcd Hcnrotin: Bound and free light chains in subacute sclerosing panencephalftis and multiple sclerosisprevious results showed that subacute sclerosing panen-cephalitis serum immunoglobulins do not differ bythe sedimentation velocity coefficients of isolated IgG,Fab and Fc fragments from the control values. On theother hand kappa and lambda light chains of multiplesclerosis and subacute sclerosing panencephalitisimmunoglobulins have a different quantitative distribu-tion in serum as well as in cerebrospinal fluid.From our results it can be stressed that the immuno-globulins of multiple sclerosis and subacute sclerosingpanencephalitis have characteristic features: in multiplesclerosis kappa chains predominate, whereas lambdachains predominate in subacute sclerosing panence-phalitis.References1. Lowenthal, A. (1964), Agargel electrophoresis in Neurology(Elsevier Publishing Company).2. Link, H. & Zetterwali, 0. (1970) Clin. Exp. Immunol. 6,435-438.3. hvashita, H., Grunwald, F. & Bauer, H. (1974), J. Neurol.207, 45-52.4. Mancini, G. A., Carbonara, A. & Heremans, J. (1965),Immunochemistry 2, 235-254.5. Madhosingh, C. & Wood, J. M. (1971), Anal. Chem. 44, '523-527.6. Lietze, A., Sinclair, C. & Rowe, H. (1970), Clin. Biochem. 3,335-338.7. Mulder, J., Sloots, L. C. E. & Verhaar, M. A. T. (1972),J. Immun. Methods /, 211-213.8. Mulder, J. & Verhaar, M. A. T. (1973), Clin. Chim. Acta 45,32,5—00 j.Lie. Sc. F. BollengierB-1000 BrusselsEversstraat 2Z. Klin. Chem. Klin. Biochem. / 13. Jahrg, 1975 / Heft 7

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Bound and Free Light Chains in Subacute Sclerosing Panencephalitis and Multiple Sclerosis Serum and Cerebrospinal Fluid

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