An Improved Polyacrylamide Gel Electrophoretic Method for
Demonstration of Oligoclonal Bands of Immunoglobulin G in
Cerebrospinal Fluid
RITA LEE, B.Sc, W. CHEW LIM,
PH.D.,
BERNARD R. CASEY, M.B., CH.B. AND HENRY C. FORD, M.D., PH.D.
A two-step procedure is described in which analytical disc gel
electrophoresis in polyacrylamide is used to demonstrate oligoclonal bands of immunoglobulin G in cerebrospinal fluid.
The principal advantage of the procedure is that unconcentrated samples of cerebrospinal fluid as small as 200 fiL can
be assayed. The results obtained in samples from 100 patients
with various neurologic diseases show that the procedure is
sensitive and specific for multiple sclerosis. (Key words: Multiple sclerosis; Oligoclonal bands; Polyacrylamide gel electrophoresis; Cerebrospinal fluid; Immunoglobulin G) Am J Clin
Pathol 1983; 79: 112-114
Division of Chemical Pathology, Department of Laboratory
Services, Wellington Hospital, Wellington, New Zealand
Sephadex A-50 (Pharmacia Fine Chemicals, Uppsala,
Sweden) was prepared within a disposable polypropylene pipette tip (Lancer, St. Louis, MO; product No. HRI
8889-220003). Sephadex G-25, coarse, (Pharmacia) was
swollen in 50 mmol/L Tris-HCl buffer, pH 8, containing
100 mmol/L NaCl and was added to the pipette tip to
a height of 15 mm. DEAE-Sephadex A-50, swollen in
the same buffer, was added to the column to give a final
bed volume of 160 ^L, exclusive of the Sephadex G-25.
The column was washed with several milliliters of the
buffer and then 200 nL of cerebrospinal fluid was applied. The eluate was collected and was combined with
the eluate from a subsequent 100-^iL wash with 50
mmol/L Tris-HCl buffer, pH 8, that contained 150
mmol/L NaCl. The combined eluate was then subjected
to analytical disc gel electrophoresis on polyacrylamide
gels.
THE DEMONSTRATION of oligoclonal bands of immunoglobulin G (IgG) in cerebrospinal fluid9" is now
an accepted laboratory aid in the diagnosis of multiple
sclerosis.813 Of the various assay methods available,
agarose gel electrophoresis and isoelectric focusing have
received the most attention. Electrophoresis of cerebrospinal fluid in cylindrical gels of polyacrylamide 45 is a
technic with sensitivity and specificity comparable to the
more widely used assay methods, 315 and it offers the
advantage that prior concentration of the cerebrospinal
fluid is not required. The method suffers from the disadvantage that haptoglobin polymers and oligoclonal
bands of IgG migrate to similar regions in the gel, which
may lead to difficulty in the interpretation of results. 2 3 ' 4
We have developed a two-step procedure in which
haptoglobin polymers are removed from the cerebrospinal fluid sample prior to electrophoresis in polyacrylamide gels. In the present paper, we report the results
obtained using this procedure to assay 100 samples of
cerebrospinal fluid.
The electrophoretic conditions for the separation and
identification of the oligoclonal bands are described in
detail elsewhere.3 In brief, gels 12.5 cm long were cast
in glass tubes (140 X 6 mm internal diameter). Twenty
microliters of 10% (w/v) sucrose solution was added to
the combined eluate (total volume, 350-450 pL), which
was then layered onto the top of the gel. Electrophoresis
was conducted at 4 mA/gel until the marker dye (bromphenol blue) was 0.5 cm from the bottom of the tube.
The gels were removed, fixed with a sulfosalicylic acidtrichloroacetic acid solution, stained with Coomassie
brilliant blue, and destained with a weak solution of
acetic acid.
To establish the final conditions of ion exchange column chromatography, under which a separation of IgG
and haptoglobin polymers could be achieved, a sample
of cerebrospinal fluid was used that was known to contain both polyclonal IgG and haptoglobin polymers.
Column chromatography was performed as described
above, except that after application of the sample, the
column was washed successively with 100-/iL aliquots
Materials and Methods
The final conditions of ion exchange chromatography
used to remove haptoglobin polymers from a cerebrospinal fluid sample were as follows: a column of DEAEReceived April 13, 1982; received revised manuscript and accepted
for publication June 24, 1982.
Supported by a Grant from the New Zealand Neurological Foundation.
Address reprint requests to Dr. Ford: Division of Chemical Pathology, Department of Laboratory Services, Wellington Hospital,
Wellington 2, New Zealand.
0002-9173/83/0100/0112 $00.95 © American Society of Clinical Pathologists
112
BRIEF SCIENTIFIC REPORTS
Vol. 79 • No. 1
113
of the eluent buffer (50 mmol/L Tris-HCl, pR 8) that
contained increasing concentrations of NaCl, and the
eluate from each wash was collected separately. Proteins
in each eluate were assessed after electrophoresis and
staining as described.
All specimens of cerebrospinal fluid were obtained
from patients with neurologic complaints. In each instance, the attending physician was questioned concerning the likelihood of multiple sclerosis in his patient.
Usually, this information was obtained before the clinician was aware of the assay result.
Results and Discussion
Under the final conditions chosen for the ion exchange chromatography (100-150 mmol/L NaCl eluent),
the cerebrospinal fluid IgG passed unretarded through
the column, whereas all detectable haptoglobin polymers were retained (Table 1). In other experiments using
samples of cerebrospinal fluid known to contain oligoclonal bands, it was shown that the bands also passed
unretarded through the column under the final conditions of chromatography. Typical results are depicted
in Figure 1.
The results obtained when the procedure was applied
to cerebrospinal fluid samples from 100 patients are
given in Table 2. Oligoclonal bands were demonstrated
in 12 of 13 patients with definite multiple sclerosis. It
is well-recognized that there is a substantial, although
small, group of patients with clinically definite multiple
sclerosis in whom oligoclonal bands cannot be demonstrated in the cerebrospinal fluid. It has been suggested12 that such patients may follow a more benign
course than those in whom oligoclonal bands are observed. Cerebrospinal fluid samples were received from
a number of sources. Therefore, it was not possible to
apply a rigorous classification scheme for the diagnosis
of multiple sclerosis to the patients in this study. The
application of such a scheme to our patients most likely
would change the diagnosis in those individuals in
Table 1. Ion Exchange Column Chromatography
of Cerebrospinal Fluid
Protein Eluted
NaCl Concn in
Eluent Buffer
(mmol/L)
IgG
Haptoblobin
polymers
100
125
150
175
200
250
500
Present
Present
Present
Absent
Absent
Absent
Absent
Absent
Absent
Absent
Present
Present
Present
Absent
f
«*
Q
FIG. 1. Results of polyacrylamide disc gel electrophoresis of cerebrospinal fluid from a patient with definite multiple sclerosis (right)
and from a patient in whom multiple sclerosis was not present {left).
Before electrophoresis, the cerebrospinalfluidsamples were subjected
to ion exchange chromatography as described in the Methods section
of the text. The arrows point to oligoclonal bands.
whom multiple sclerosis was considered to be unlikely,
possible, or probable.
Most samples submitted for assay were obtained from
patients in whom multiple sclerosis was thought not to
be present. In only one of these cases, a patient with
hysterical paraplegia, was an oligoclonal band observed.
A possible relationship between hysteria and multiple
sclerosis has been noted.'
It is with those patients in whom the clinical diagnosis
of multiple sclerosis is uncertain that the assay may be
of greatest potential value. Long-term follow-up of patients with possible and probable multiple sclerosis may
114
LEE ET AL.
Table 2. Oligoclonal Bands in Cerebrospinal Fluid
and the Clinical Likelihood of Multiple Sclerosis
Clinical Impression
(Multiple Sclerosis)
Number of
Cases
Number of Cases with
Oligoclonal Bands
Not present
Unlikely
Possible
Probable
Definite
56
9
11
11
13
1
1
4
6
12
help to clarify the meaning of the positive and negative
assay results that were obtained.
The principal advantage of analytical disc gel electrophoresis on polyacrylamide gels as a method of demonstrating oligoclonal bands of IgG in cerebrospinal
fluid is that unconcentrated samples as small as 200
nL can be assayed. The two-step method described in
this paper exhibits sensitivity and specificity for multiple
sclerosis comparable to the levels reported for electrophoresis in agarose and isoelectric focusing 6710 and also
compares favorably to these other procedures with regard to the degree of difficulty and the time required.
10.
11
12
Acknowledgment. We thank the neurologists of Christchurch, Dunedin. and Wellington Hospitals for providing clinical information on
the patients. Mrs. J. Mooney typed the manuscript.
13.
References
1. Caplan LR, Nadelson T: Multiple sclerosis and hysteria. Lessons
learned from their association. JAMA 1980; 243:2418-2421
2. Casey BR, Mason AJ: Oligoclonal immunoglobulins and multiple
sclerosis. Br Med J 1979: 1:1149.
14,
15
A.J.C.P.-January 1983
Casey BR, Wong ST, Mason AJ, et al: The electrophoretic demonstration of unique oligoclonal immunoglobulins in cerebrospinalfluidas a diagnostic test for multiple sclerosis. Clin Chim
Acta 1981; 114:187-194
Cunningham VR: Analysis of 'native' cerebrospinalfluidby the
polyacrylamide disc gel electrophoresis technique. J Clin Pathol
1964; 17:143-148
Davis BJ: Disc electrophoresis. II. Method and application to human serum proteins. Ann NY Acad Sci 1964; 121:404-427
Hershey LA, Trotter JL: The use and abuse of cerebrospinal fluid
IgG profile in the adult: A practical evaluation. Ann Neurol
1980; 8:426-434
Hosein ZZ, Johnson K.P: Isoelectric focusing of cerebrospinal fluid
proteins in the diagnosis of multiple sclerosis. Neurology (Minneapolis) 1981; 31:70-76
Keshgegian AA (ed.): Oligoclonal immunoglobulins in cerebrospinalfluidin multiple sclerosis. Case conference. Clin Chem
1980; 26:1340-1345
Laterre EC, Callewaert A, Heremans JF, et al: Electrophoretic
morphology of gamma globulins in cerebrospinalfluidof multiple sclerosis and other diseases of the nervous system. Neurology (Minneapolis) 1970; 20:982-990
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 1973;46:383-389
Link H, Miiller, R: Immunoglobulins in multiple sclerosis and
infections of the nervous system. Arch Neurol 1971; 25:326344
Stendahl-Brodin L, Link H: Relation between benign course of
multiple sclerosis and low-grade humoral immune response in
cerebrospinal fluid. J Neurol Neurosurg Psychiatry 1980;
43:102-105
Thompson EJ: Laboratory diagnosis of multiple sclerosis: Immunological and biochemical aspects. Br Med Bull 1977;
33:28-33
Thompson EJ: Oligoclonal immunoglobulins and multiple sclerosis. Br Med J 1979; 1:1489
Thompson EJ, Kaufmann P, Shortman RC, et al: Oligoclonal
immunoglobulins and plasma cells in spinal fluid of patients
with multiple sclerosis. Br Med J 1979; 1:16-17
Evaluation of a Monoclonal Antibody-based
Immunoradiometric Assay for Prostatic Acid Phosphatase
SALLY N. DAVIES, M.B., B.S. AND NATHAN GOCHMAN, PH.D.
This report evaluates a new immunoradiometric assay for prostatic acid phosphatase in serum, based on a dual monoclonal
antibody reaction system (Hybritech-TANDEM). A solidphase antibody binds the acid phosphatase molecule and a
second monoclonal antibody to a different antigenic site serves
as the l25I-radiolabel. The method was tested on 67 patients
Received April 15, 1982; received revised manuscript and accepted
for publication May 3, 1982.
Presented in part at the 34th Annual Meeting of the American
Association for Clinical Chemistry, Anaheim, California, August 813, 1982.
Address reprint requests to Dr. Gochman: Clinical Systems Division, Beckman Instruments, Inc., Brea, California 92121.
Laboratory Service, Veterans Administration Medical Center,
San Diego and Department of Pathology, University of
California, San Diego, La Jolla, California
with various stages of prostatic carcinoma and 134 patients
without the disease. It also was compared with a conventional
polyclonal radioimmunoassay (NEN) and an enzymatic activity
method (duPont aca). The upper limit for the TANDEM assay
on nondiseased male patients was found to be 2.0 Mg/L. Based
on this upper limit of normal, the diagnostic sensitivity of the
method for all cases of prostatic carcinoma was 60%. We could
not distinguish the enzyme released in abnormal amounts due
0002-9173/83/0100/0114 $01.10 © American Society of Clinical Pathologists