Clinical Chemistry 46: 1326-1330, 2000;
(Clinical Chemistry. 2000;46:1326-1330.)
© 2000 American Association for Clinical Chemistry, Inc.
Clinical and Analytical Evaluation of an Enzyme Immunoassay for Myelin Basic Protein in Cerebrospinal Fluid
Mitsuhiro Ohta1,2,a,
Kiyoe Ohta1,
Jie Ma1,
Juji Takeuchi1,
Takahiko Saida1,
Masataka Nishimura1 and
Nobuyuki Itoh3
1
Clinical Research Center, Utano National Hospital, Narutaki, Ukyo-ku, Kyoto 616-8255, Japan.
2
Department of Clinical Chemistry, Kobe Pharmaceutical
University, Higashinada-ku, Kobe 658-8558, Japan.
3
Department of Genetic Biochemistry, Kyoto University
Graduate School of Pharmaceutical Science, Sakyo-ku, Kyoto 606-8304,
Japan.
a Author for correspondence. Fax 81-78-441-7559; e-mail mohta{at}kobepharma-u.ac.jp
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Abstract
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Background: RIA of myelin basic protein (MBP) in cerebrospinal
fluid (CSF) is commonly used as a biochemical marker of demyelination
in patients with multiple sclerosis (MS). Our aim was to develop a
sufficiently sensitive ELISA for MBP and evaluate it clinically in
patients with MS.
Methods: The ELISA used anti-bovine MBP antibody coated on plates
and biotinylated anti-MBP antibody. The bound antibody complex was
quantified with streptavidin-horseradish peroxidase. MBP was determined
in CSF from 84 MS patients and 55 patients with other neurological
diseases.
Results: The respective within- and between-assay CVs were 4.7%
and 7.2% at 200 ng/L, and 6.3% and 8.8% at 2000 ng/L. The detection
limit was 30 ng/L. Most of the MS patients with acute exacerbations had
markedly increased MBP in the CSF. Longitudinal studies of six MS
patients with recurrent exacerbation confirmed this observation. MBP
concentrations from 78 MS patients, as tested with our ELISA,
correlated well with those obtained by RIA (r =
0.9; P <0.01), but the detection limit of the ELISA was
much lower than that of the RIA.
Conclusions: This convenient ELISA with higher sensitivity than
the existing assays is a suitable routine assay that provides a
diagnostic indicator of myelin breakdown in the central nervous system;
moreover, it is an excellent indicator of MS disease activity.
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Introduction
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Multiple sclerosis
(MS)1
causes localized demyelination in the central nervous system. Myelin
basic protein (MBP) is the major protein component of myelin
(1), and the concentration of MBP or its fragment in
cerebrospinal fluid (CSF), as measured by a RIA, correlates closely
with the clinical activity of MS and other diseases in which there is
myelin breakdown (2)(3)(4)(5)(6)(7)(8). The clinical importance of the
quantitative determination of MBP in CSF as an index of active
demyelination has been reviewed
(7)(9)(10). We developed a
sensitive, specific, and convenient ELISA for MBP and measured MBP in
CSF from patients with MS and other neurological diseases. We also made
a serial study of six MS patients for a period of 6 to 30 months to
clarify the correlation of the clinical course of the disease and the
MBP concentration in the CSF. MBP concentrations obtained by this new
ELISA were compared with those obtained by the previously developed
RIA.
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Materials and Methods
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human subjects
All CSF samples were obtained by lumbar puncture and were stored
at -80 °C until assayed. Samples were taken from 84 Japanese MS
patients 14–68 years of age (mean age, 40 years), 55 patients 18–64
years of age (mean age, 45 years) with other neurological diseases (12
with chronic cerebrovascular disease, 14 with amyotrophic lateral
sclerosis, 12 with Parkinson disease, 5 with polyneuropathy, 4 with
Behcet disease, 5 with meningitis, and 3 with Guillain-Barré
syndrome), and 45 patients 19–67 years of age (mean age, 43 years)
with nonneurological diseases. In MS patients with exacerbations,
samples were obtained within 1 week after the onset of acute
exacerbation. CSF samples also were collected at various times from six
individuals with MS who had recurrent exacerbations for longitudinal
studies. The study was approved by the ethics committee of the Utano
National Hospital.
preparation of mbp and anti-mbp antiserum
MBP was isolated from bovine brain as described by Deibler et al.
(11). In brief, an acid extract of brain delipidated with
chloroform-methanol was chromatographed on carboxymethylcellulose, pH
10.4, after which the MBP fractions were eluted with a linear salt
gradient. In a typical preparation, 
300 mg of MBP was isolated from
500 g of brain tissue. Protein homogeneity for the single band
with a molecular mass of 18 kDa was determined by polyacrylamide gel
electrophoresis at pH 4.0 (6). Amino acid analysis of the
protein showed that its composition corresponded to that of the A1
protein reported by Eylar et al. (12).
Anti-MBP antisera were raised in Japanese White rabbits, using a
schedule that favored the induction of specific anti-MBP antiserum
without producing experimental allergic encephalomyelitis
(13). Briefly, 100 µg of MBP in saline was emulsified with
Freund’s incomplete adjuvant (saline:adjuvant, 2:3, by volume), and
then injected subcutaneously at six sites on the back of a rabbit;
three booster injections were given at 2-week intervals. MBP (100 µg)
emulsified with an equal volume of Freund’s complete adjuvant was then
injected once a week for 8 weeks. After the final injection, the
rabbits were bled, and the antisera were prepared.
The anti-MBP antibody IgG fraction prepared by chromatography on
protein A-Sepharose CL-6B (Pharmacia Fine Chemicals) was further
purified by affinity chromatography on an MBP-Sepharose 4B column.
preparation of biotinylated anti-mbp antibody
Affinity-purified anti-MBP antibody IgG was biotinylated
with
5-(N-succinimidyl-oxycarbonyl)pentyl-D-biotinamide
(Dojindo) (14).
two-site elisa for mbp
A microtiter plate coated with 100 µL of anti-bovine MBP IgG (3
mg/L) diluted with 10 mmol/L carbonate buffer, pH 9.3, was incubated
for 1 h at room temperature. After the plate had been washed
twice, the unoccupied protein binding sites in each well were blocked
for 1 h at room temperature with 200 µL of 10 mmol/L carbonate
buffer, pH 9.3, containing 5 g/L bovine serum albumin. One hundred
microliters of a MBP calibrator or CSF sample was added to the wells,
and the plate was incubated for 1 h at room temperature. After
another washing with washing buffer (50 mmol/L Tris-HCl buffer, pH 7.0,
containing 0.2 mol/L NaCl, 10 mmol/L CaCl2, and 1
mL/L Triton X-100), 100 µL of biotinylated anti-MBP antibody (20
µg/L) was added, and the plate was incubated for 1 h at room
temperature. After three more washings, the plate was incubated for
1 h at room temperature with 100 µL of streptavidin-horseradish
peroxidase (Amdex) diluted 10 000-fold. After three final washings,
the plate was allowed to react for 15 min at room temperature with 100
µL of a substrate solution of 3,3',5,5'-tetramethylbenzidine and
H2O2 (Kirkegaard & Perry
Laboratories). The reaction was stopped by the addition of 100 µL of
1 mol/L phosphoric acid, after which the absorbance at 450 nm was
recorded with an ELISA reader (Labsystems). All washings and
incubations were done with gentle shaking.
ria for mbp
MBPs were measured by a double-antibody RIA described previously
(6).
statistical analysis
The following methods were used for the statistical analysis:
regression analysis for the evaluation of calibration curve; a plot for
assessment of the agreement between two methods (15); and
the Kruskal–Wallis nonparametric ANOVA. P <0.05 was
considered statistically significant.
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Results
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The calibration curve showed a quantitative relationship for human
MBP at concentrations of 30–2000 ng/L (Fig. 1
). The within- and between-assay CVs were 4.7% and 7.2%
(n = 10) at 200 ng/L and 6.3% and 8.8% (n = 5) at 2000
ng/L.
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Figure 1. Calibration curve for the MBP ELISA.
There is a quantitative relationship for human MBP at concentrations of
30–2000 ng/L.
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We assayed CSF samples from 84 patients with MS, 55 patients with other
neurological diseases, and 45 patients with nonneurological diseases
(Fig. 2
). The mean CSF MBP concentration in the patients with
nonneurological diseases was 54 ± 24 ng/L (mean ± SD). A
MBP concentration >102 ng/L (mean + 2 SD) was considered positive. Of
the 55 patients with neurological diseases exclusive of MS, 5 had CSF
MBP concentrations above these reference limits: 1 each with chronic
vascular disease (110 ng/L), amyotrophic lateral sclerosis (310 ng/L),
Parkinson disease (250 ng/L), polyneuropathy (210 ng/L) and meningitis
(1000 ng/L).
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Figure 2. CSF MBP in 84 patients with MS (52 patients with acute
exacerbation, 32 in remission or the clinically inactive state), 55
patients with other neurological diseases, and 16 with nonneurological
diseases.
There are significant differences between the active MS and any other
groups (P <0.001). The dashed line shows
the cutoff for MBP.
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Patients with MS were divided into two groups composed of 52 who
suffered acute exacerbations and 32 in remission or the clinically
inactive state. The active-disease group had a markedly increased mean
CSF MBP concentration (3076 ng/L) and a high frequency of increased
results (81%), whereas most of the inactive MS patients had MBP
concentrations that were not increased (mean, 89 ng/L; frequency of
increased values, 19%).
To clarify the relationship between changes in CSF MBP and the clinical
states of MS, we also measured CSF MBP concentrations in serial samples
taken from six individuals with recurrent exacerbation before, during,
and after attacks over periods ranging from 6 to 30 months (Fig. 3
, case numbers 1–6). CSF MBP values were undetectable or low in
most samples obtained before a MS attack, with the value rising rapidly
to 500-8000 ng/L at the height of the event. After an acute attack, the
CSF MBP decreased rapidly, and within 4 weeks after the exacerbation
most patients had CSF MBP values within or below the reference limits.
Follow-up studies showed that the maximum MBP concentrations in CSF
occurred during the most acute phase of the disease. Thus, good
agreement was found between the MBP concentration and clinical symptoms
in these six patients.
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Figure 3. Changes in CSF MBP concentrations in six MS patients with
recurrent exacerbation.
CSF samples were collected over a period of 6–30 months before,
during, and after acute exacerbation. The dashed line
shows the cutoff for MBP. Arrows indicate acute
relapse.
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We compared the results obtained by ELISA and the previously developed
RIA for 78 MS samples; MBP was detected in 67% by the ELISA and in
49% by the RIA. There was a good correlation between the CSF MBP
concentrations measured by ELISA and RIA (r = 0.90;
P <0.01). Fig. 4
shows the mean of the results obtained by the two methods vs
the difference between the results obtained by the two methods. The RIA
gave higher values than ELISA at low concentrations and lower values
than ELISA at high concentrations.
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Figure 4. Comparative analysis of ELISA and RIA for MBP
determination.
Plot of the respective differences (ELISA - RIA) vs the means of
the two methods.
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Discussion
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From an immunochemical point of view, MBP concentrations measured
in CSF represent MBP-like materials, not necessarily genuine MBP
(10). RIA has been used most frequently to measure MBP,
largely because of the need for extremely high sensitivity. We
previously developed a RIA system for measuring MBP in the CSF as a
diagnostic indicator of myelin breakdown in the central nervous system
(6)(16). The lowest MBP concentration detected
by our RIA system in CSF was 0.5 µg/L, a detection limit similar
to that of the existing RIA (2)(3)(4)(5)(6)(7)(8)(9).
We now have successfully established a sensitive, specific sandwich
ELISA for MBP that uses a polyclonal anti-MBP antibody. This sensitive,
two-site ELISA system, based on a biotin-streptavidin system capable of
measuring a concentration as low as 30 ng/L, gives a genuine,
immunologically more highly specific reaction than the RIA. The
dilution curves obtained by serially diluting MBP-like material in CSF
and purified MBP were parallel. Our RIA is a competitive assay between
125I-labeled MBP and CSF MBP. In this assay
system, protein concentrations influence the RIA values. Therefore, the
RIA gave higher values than the ELISA at low concentrations as shown in
Fig. 4
. Protein concentrations do not influence the ELISA results. The
major advantages of our ELISA are that it does not use radioactive
chemicals and that its sensitivity and specificity are much better than
the existing RIA. This new assay is simple to perform, requires only a
small sample volume, does not require pretreatment of samples before
assay, and is highly reproducible. Moreover, all of the required
reagents are stable indefinitely.
Increased MBP concentrations have rarely been detected in the CSF of
patients with a wide variety of neurological diseases:
leukodystrophies, severe anoxia, myelopathies, and encephalopathies
caused by irradiation or chemotherapy (5)(17);
the acute active state in patients with myelopathy, cerebrovascular,
and neuro-Behcet diseases (6)(16); and brain
tumors (18). We, however, found increased MBP in the CSF of
five patients; one each with chronic cerebrovascular disease,
amyotrophic lateral sclerosis, Parkinson disease, polyneuropathy, and
meningitis. The RIA and ELISA for MBP in CSF, therefore, are useful for
making a clinical assessment of the various neurological diseases in
which myelin breaks down acutely. van Engelen et al. (19)
reported that the concentration of MBP in CSF increases with age.
Therefore, we used age-matched references.
The study reported here was performed on MS patients to clarify the
relation of clinical status to active myelin destruction by monitoring
the MBP in the CSF at sequential times during the course of the
disease. Longitudinal studies of individual patients showed a strict
temporal relationship between the presence of MBP in the CSF and the
peak of the disease. Severe symptoms and dramatic clinical states,
however, always were accompanied by the release of MBP into the CSF.
Detection of MBP in the CSF should be considered indicative of myelin
destruction. Most patients who had increased MBP concentrations in
their CSF during the acute phase of disease seemed to be in remission
several months later.
Antigenic material that cross-reacts with MBP, or a peptide thereof,
may be present in the blood of individuals who have had recent injury
to the myelin in the central nervous system. Palfreyman et al.
(20) detected MBP in sera from patients with head injury.
Jacque et al. (17) infrequently found MBP in sera from
cephalitic patients. Indeed, even when the blood-brain barrier has been
broken, the dilution effect caused by the larger blood volume lowers
the MBP concentration below the detection limit of most assays. MBP is
also a minor component of peripheral nervous systems.
Our new ELISA for MBP in serum samples does not require pretreatment of
samples to remove the interfering material usually present in serum.
Serum MBP could be determined at concentrations >30 ng/L. In
unpublished studies, we examined MBP concentrations in the CSF and
matched serum samples from 15 MS patients who had acute exacerbation.
Increased MBP concentrations were detected in all CSF samples tested,
whereas in serum, MBP was found in only three patients, and at very low
concentrations (180, 389, and 200 ng/L). Most serum MBP was not
detectable, even in the patients with the highest CSF MBP
concentrations. MBP therefore could be detected only rarely in the
serum and at low concentrations. The presence of serum MBP does not
necessarily correlate with the highest concentration of MBP in the CSF.
Several magnetic resonance imaging methods have been developed recently
that may provide an objective measure of MS disease activity
(21). More attention needs to be paid to MBP in the CSF and
serum as a biochemical marker of MS disease activity. This new ELISA is
convenient and simple and can be used routine assays for that purpose.
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Acknowledgments
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This work was supported, in part, by Kobe Pharmaceutical University
Collaboration Fund and The Science Research Promotion Fund of The Japan
Private School Promotion Foundation.
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Footnotes
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1 Nonstandard abbreviations: MS, multiple sclerosis; MBP, myelin basic protein; and CSF, cerebrospinal fluid. 
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Abstract
Introduction
