CLIN. CHEM. 25/8,
1495-1498
(1979)
IgD Myeloma Protein with “Unreactive” LightChain Determinants
Jan Cejka’ and Karel Kithier2
Serum from a patient with multiple myeloma showed
a monoclonal protein, classified by immunoelectrophoresis
as lgD. Immunofixation electrophoresis
and immunoelectrophoresis
failed to demonstrate a precipitation reaction between the paraprotein and antisera to immunoglobulin light chains. The light chains of the monoclonal
protein, immunologically
inaccessible in the intact molecule,
reacted with anti-A chain antisera only after reduction
and alkylation of the paraprotein. Moreover, interpretation
of the immunoelectrophoretic
patterns was hampered by
the presence in patient’s serum of free A chains having
about the same mobility as that of the paraprotein.
Characterization
of monoclonal immunoglobulins
of patients with multiple
myeloma
gammopathies
is usually not difficult;
trophoretic
experiment
is sufficient
in sera
or other monoclonal
a single immunoelec-
to characterize
the
as to its heavy-chain class and light-chain
type,
by using antisera specific for heavy and light immunoglobulin
Materials and Methods
Reagents
Monospecific
antisera against human IgG, IgA, 1gM, and
X chains were obtained from Meloy Laboratories,
ic and
Springfield, VA 22151 and from Kallestad
Laboratories,
Chaska, MN 55318. Antisera to free K and X chains were also
obtained from Behring Diagnostics, Somerville, NJ 08876.
Radial immunodiffusion plates for the quantitation of serum
immunoglobulins
IgG, IgA, 1gM, and IgD were purchased
from Meloy Laboratories.
Ribonuclease A, chymotrypsinogen A, and ovalbumin were
from Pharmacia (calibration
kit for molecular weight determination),
Piscataway,
NJ 08854, and bovine serum albumin
from Miles Laboratories, Elkhart, IN 46515. Sephadex G-200
(40-120 zm) was purchased from Pharmacia, and Sea-Kem
(ME) agarose from Marine Colloids, Rockland,
ME 04841.
paraprotein
Procedures
chains. In some cases, however, failure to detect or correctly
characterize the paraprotein may result from low paraprotein
concentration, superimposition of several proteins (double
myeloma, presence of light chains in serum) in the electrophoretic and immunoelectrophoretic patterns, or the absence
For agarose gel electrophoresis we used a 10 g/L agarose
solution in 75 mmol/L barbital buffer, pH 8.6, containing 2
of precipitation
of the monoclonal immunoglobulmn
with antisera to light chains. In such cases it often is necessary to
isolate the abnormal immunoglobulin
and use special techniques for its proper characterization.
The failure of some IgA myeloma proteins to react with
antisera
specific for immunoglobulin
light chains is well
documented
(1, 2). The presence
of light chains in such
paraproteins can be demonstrated
by urea-starch
gel electrophoresis at acid pH or by gel chromatography
in dissociating solutions of the reduced and alkylated purified protein
(3). A similar inaccessibility to light chains by antisera against
light chains was noted by Rowe and Fahey (4) in an IgD
myeloma protein. The present communication
concerns an
IgD monoclonal
protein that initially appeared to lack light
chains. After reduction
and alkylation,
the paraprotein was
shown to possess X-type light chains that were immunologically inaccessible in the intact molecule.
Department of Immunochemistry,
Children’s Hospital of Michigan, Detroit, MI 48201.
2 Department
of Pathology, Wayne State University School of
Medicine, Detroit, MI 48201.
Received Mar. 22, 1979; accepted June 1, 1979.
mmol of calcium lactate per liter. The electrophoresis was run
at 15-20 V/cm for 80 mm under cooling. Immunof’ixation
electrophoresis was performed after agarose gel electropho-
resis by overlaying the agarose gel with respective monospecific antisera; the details of the technique have been described
earlier (5). Immunoelectrophoresis
was performed by a
modification of the micromethod described by Scheidegger
(6). Single radial immunodiffusion
was used for the quantitation of immunoglobulins
in patients’ serum. Sephadex
G-200 chromatography was done on a 1.5 )< 90 cm column
equilibrated
taining
with a 0.1 mol/L
phosphate
1 mol of NaCI per liter.
buffer,
pH 7.3, conIgD
by a modification
of
The partly purified
paraprotein
was reduced and alkylated
the method described by Fleischman et al. (7). The reduction
performed in a 0.5 mol/L tris(hydroxymethyl)methylamine-HC1 buffer, pH 8.0, and 0.1 mol/L /3-mercaptoethanol
for 1 h at room temperature; alkylation was in 0.1 mol/L iowas
doacetamide
for 1 h at 0 #{176}C.
Reduced
and alkylated
protein
evaluated by sodium dodecyl sulfate-polyacrylamide
gel
electrophoresis in a phosphate buffer (0.1 mol/L, pH 7.1)
containing 1 g of sodium dodecyl sulfate per liter, according
to Ahmad-Zadeh et al. (8).
was
Case History
The patient, an 85-year-old man, was first admitted to the
hospital for evaluation of an anemia and a large bowel mass.
CLINICAL CHEMISTRY, Vol. 25, No. 8, 1979
1495
1
2
3
elictrophoresis of patient’s serum, partly
purified lgD paraprotein, and serum free-A chains
Slot 1, patIent’s serum(diluted sixfold);slot 2, patIent’s serum (diluted 12-fold);
Fig. 3. Immunofixation
slot
3,
IgO fraction
from Sephadex G-200 (1 g/L solution); and slot
G-200 (2 g/L solution)
4, serum
free-Achainsfrom Sephadex
Fig. 1. Agarose gel electrophoresis
1, Normal human serum; 2, patient’s serum (diluted twofold); 3, patIent’s urine
(concentrated lOX). IgO myeloma protein (1) and urinary Bence Jonesprotein
(II) show slight differences In mobility
This was diagnosed as adenocarcinoma
of the colon, and
transverse
colectomy
and splenectomy were performed.
Laboratory studies on admission were as follows: hemoglobin
63 g/L, hematocrit
19.8%, serum urea nitrogen 310 mg/L,
creatinine 25 mg/L, total protein 65 g/L, and albumin 30 g/L.
Serum protein electrophoresis on cellulose acetate showed
increased /3-globulin fraction. The urine gave a 1+ reaction
for protein, with protein excretion of 1.8 g/24 h (2.3 L).
About eight months after this initial admission, the patient
was re-admitted to the hospital for repair of a large epigastric
ventral hernia. At this time, serum urea nitrogen and creatinine values reached 660 and 58 mg/L, respectively. Serum
protein electrophoresis
showed a homogeneous band in the
/32 region, and bone-marrow aspirate revealed 19.8% plasma
cells, most of them abnormal. Quantitation
of individual immunoglobulins showed normal concentration of IgG (8.2 g/L;
normal limits 8-18 g/L), decreased concentrations of IgA (0.51
g/L; normal limits 0.9-4.5 g/L) and 1gM (0.26 gIL; normal
limits 0.65-2.65 gIL), and highly increased IgD (11.4 gIL;
normal limit up to 0.4 gIL). Increased amounts of free light
chains of the lambda type were found in the patient’s urine
by immunoelectrophoresis. Total urinary protein was 1.1 g/24
h (2.17 L).
On the last admission five months later, the patient’s laboratory results were essentially unchanged, except for still
higher urea nitrogen and creatinine concentrations (1100 and
61 mg/L, respectively). Final diagnosis at the present time is
IgD myeloma with chronic anemia and renal failure secondary
to multiple
myeloma.
Results
A serum specimen obtained at the time of the last admission
subjected to agarose gel electrophoresis.
The electrophoretic pattern (Figure 1) showed a homogeneous M-component in the $2 region and a marked decrease in the intensity
of the y band, corresponding to the decreased concentration
of the three principal immunoglobulmns.
The immunoelectrophoretic analysis of patient’s serum with antisera specific
for IgD and light chains is shown in Figure 2. Precipitin arcs
of restricted electrophoretic mobility, typical for monoclonal
proteins,
were formed with antisera against 5chains (Figure
2a) and A chains (Figure 2c), suggesting the presence of an
was
IgD(A) paraprotein
in patient’s serum. However, close inspection of the immunoelectrophoretic
patterns revealed that
..Ons.
the mobilities of the two precipitin
lines were not the same,
the precipitin
line obtained with anti-A antiserum
being
shifted slightly to the anode. Subsequent
immunoelectrophoresis of the patient’s serum with an antiserum to free A
chains demonstrated
the presence of free A chains in the same
position. It became apparent that the abnormal precipitin line
developed with the anti-A antiserum
(Figure
2c) did not correspond to the IgD M-component but rather to free A chains
present in serum. A Bence Jones protein of A type found in the
patient’s urine also had a slightly higher anodic mobility than
that of the IgD myeloma protein (Figure 1).
Immunofixation
electrophoresis
of the patient’s serum
confirmed the findings of immunoelectrophoresis.
As demonstrated in Figure 3, the IgD paraprotein reacted with anti-#{246}
antiserum
(slot 1); however, no precipitin band was detected
in the same position when antiserum against A chains was used
(slot 2). Instead, the immunofixation
pattern showed a slightly
faster migrating band, the mobility
that of free A chains found in urine.
ns, normal human serum; ps, patient’s serum. Anode to the right
1496 CLINICAL CHEMISTRY, Vol. 25, No. 8, 1979
to
To separate the free A chains from the IgD paraprotein for
immunochemical
study, we gel-chromatographed
the
patient’s serum on Sephadex G-200. As shown in Figure 4, the
IgD protein was eluted in fractions midway between the first
and second peaks, as determined by double immunodiffusion
further
Fig. 2. Immunoelectrophoretic analysis of patient’s serum with
antisera to lgD and K and A chains
of which was similar
a
sJ
871
b
a
is
ii
24
30
C
l9Dred+aik
FraU,,n no. (4n)
Fig. 4. Gel chromatography
of patient’s
serum on Sephadex
G-200
The bars desIgnate the fractions
BJ
corresponding to lgD paraprotein (lgD) and free
A light chains (BJA)
Fig. 5. lmmunoelectrophoretic
of the individual
column fractions. Fractions of the middle
part of the IgD peak were pooled, dialyzed against distilled
water, and lyophilized. On immunoelectrophoresis,
this material (concentration 10 g/L) showed minor impurities when
tested with antiserum to human serum proteins but no reaction with antisera specific for IgG, IgA, and 1gM. The free
A chains were eluted from the column in the descending portion of the third peak (designated BJA) as determined by
immunoelectrophoresis
with antisera to free A chains. The
individual fractions containing A chains were pooled and
concentrated by ultrafiltration
with a Minicon-B15 concentrator.
Figure 3 shows immunofixation electrophoresis of the partly
purified IgD paraprotein; no precipitin band was detected
with an anti-A antiserum (slot 3). The same negative result
was obtained
with several other antisera against A chains,
suggesting
the presence of “unreactive”
on Sephadex
analysis of fractions separated
0-200 with antisera to lgD and A chains
IgO. partly puif led IgO myeloma pitSein (2.5 gIL); BJ, partly purIfied serum free-A
chains (10 gIL); lgOr,,,+,,,,,, reduced and alkylated lgD myeiomaprotein(1 gIL).
Anode to the right
chains in serum have the same electrophoretic
mobility,
dif-
ferentiation of the two entities might be difficult. The use of
specific antisera against free light chains is useful in these
cases, although no definite conclusions can be drawn even
from such experiments. IgD paraprotein was separated from
free A chains by immunofixation
electrophoresis; this technique has been shown to provide a better separation than
immunoelectrophoresis
and easy immunochemical characterization of homogeneous proteins of similar mobilities (5,
12, 13). The difference in mobility of the bands corresponding
IgD
(hidden) light-chain
determinants
in the intact IgD molecule. On the other hand,
the free light chains isolated from serum reacted readily with
antiserum to A chains (slot 4). The results of immunofixation
electrophoresis were confirmed by immunoelectrophoresis
(Figure 5), run for an extended period of time to show the
mobility differences between the isolated IgD protein and free
A chains. Again, no reaction was detected between the isolated
paraprotein and antiserum to A chains (Figure Sb). Only a
RN
red.alk
i*nr
CTOV
cz
BSA
zz
H
faint precipitin
line was detected with some anti-A antisera
when we used high concentrations
of IgD paraprotein
(>10
g/L). After reduction and alkylation, the IgD protein reacted
readily with anti-A chain antiserum (Figure 5c).
The presence of light chains in the IgD paraprotein was also
demonstrated by electrophoresis of the reduced and alkylated
protein in sodium dodecyl sulfate-polyacrylamide
gel (Figure
6). Using marker proteins of known molecular mass, we estimated the relative molecular mass of A chains in the IgD
paraprotein
to be 22 000, a value that agrees well with that
(23 000) established for light chains in immunoglobulins of
all classes.
Discussion
The case reported here has some features in common with
other IgD myelomas,
namely
the presence
of Bence Jones
protein in urine and occurrence of free light chains in serum
(9-11). Free light chains can easily be detected in serum by
immunoelectrophoresis as a separate precipitin band formed
with antisera specific for immunoglobulin
light chains.
However, in cases where the myeloma protein and free light
Fig. 6. Results of electrophoresis on sodium dodecyl sulfatepolyacrylamide
gel
lgDr,(j+d,, reduced andalkylated gOmyelomaprotein;RN, ribonuclease A (M,
13700); CT, ch)mOtrypslnOgon A (P425 000); OV, ovalb,nln (P445 000); BSA,
bovine serum albumin (1468 000). H and L designate heavy and light chains,
respectively
CLINICAL CHEMISTRY, Vol. 25, No. 8, 1979
1497
to the paraprotein and free light chains is quite obvious from
the immunofixation pattern (Figure 3, slots 1 and 2). We finally proved the presence of free light chains in this patient’s
serum by gel chromatography;
A light chains were eluted from
the column
in the descending
part of the albumin
peak. The
elution
volume suggested that the free light chains were
present
in serum as dimers.
chains in the urine is sufficient
disorder.
This work was supported
National
Institutes
by research
evidence of a monoclonal
grant
no. 05384-17
from the
of Health.
References
structurally
1. Osterland, C. K., and Chaplin, H., Atypical antigenic properties
of a ‘yA myeloma protein. J. Immunol. 96,842-848 (1966).
2. Seligmann, M., Mihaesco, E., and Frangione, B., Studies on alpha
chain disease.Ann. NY Acad. Sci. 190,487-500 (1971).
3. Seligmann, M., Immunochemical,
clinical and pathological features
of alpha-chain disease. Arch. Intern. Med. 135, 78-82 (1975).
4. Rowe, D. S., and Fahey, J. L., A new class of human immunoglobulins. J. Exp. Med. 121, 185-199 (1965).
5. Cejka, J., and Kithier, K., A simple method for the classification
and typing of monoclonal immunoglobulins. Immunochemistry
13,
629-631 (1976).
6. Scheidegger, J. J., Une micro-m#{233}tode
de l’immuno-electrophorese.
mt. Arch. Allergy AppI. Immunol. 7, 103-112 (1955).
7. Fleischman, J. B., Pain, R. H., and Porter, R. R., Reduction of
‘y-globulin. Arch. Biochem. Biophys., Suppl. 1, 174-179 (1962).
8. Ahmad-Zadeh, C., Piguet, J. D., and Colli, L., Molecular weight
estimation of immunoglobulin subunits on polyacrylamide
gel. Immunology 21, 1065-1071 (1971).
9. Fahey, J. L., Carbone,
P. P., Rowe, D. S., and Bachmann, R.,
Plasma cell myeloma with D-myeloma protein (IgD myeloma). Am.
J. Med. 45, 373-380 (1968).
10. Hobbs, J. R., Slot, G. M. J., Campbell, C. H., et al., Six cases of
gamma-D myelomatosis.
Lancet i, 614-618 (1966).
11. Jancelewicz,Z., Takatsuki, K., Sugai,S., and Pruzanski, W., IgD
multiple myeloma. Review of 133 cases.Arch. Intern. Med. 135,87-93
(1975).
12. Cawley, L. P., Minard, B. J., Tourtellotte,
W. W., et al., Immunofixation electrophoresis
techniques applied to identification of
proteins in serum and cerebrospinal fluid. Clin. Chem. 22,1262-1268
light-chain
(1976).
A more important finding derived from the immunofixation
electrophoresis
was that the IgD paraprotein did not react
with antisera against immunoglobulin light chains. The failure
to detect light chains in myeloma proteins, especially those
of A type, is mostly due to the use of low-avidity antisera. This
does not seem to be the case in our study, because the reactivity with antiserum could be demonstrated
after reduction
and alkylation of the paraprotein. A similar difficulty in
identifying light-chain types has been observed in the case of
some IgA myeloma proteins, particularly those with A chains.
(1, 14, 15). Also, Rowe and Fahey (4) in their study on IgD
found that some IgD proteins could be typed only after reduction, alkylation,
and separation of the heavy and light
chains. The possibility that the paraprotein we studied lacked
light chains seemed very unlikely. In gel chromatography on
Sephadex G-200 the protein was eluted in a region between
the 1gM and IgG peaks, i.e., in the position corresponding to
the known elution volume of normal serum IgD; this suggested
that the pathological protein had the same molecular size as
normal IgD. The presence of light chains was finally demonstrated in the reduced and alkylated paraprotein by immunoelectrophoresis and electrophoresis on sodium dodecyl
sulfate-polyacrylamide
gel. The explanation for the difficulty
in demonstrating
light-chain
antigenicity
Seemingly, the light chains of the intact
is not obvious.
paraprotein
were
inaccessible and therefore unable to react with
antibodies. Another explanation would be that the
light chains of the paraprotein lack the “exposed” determinants present
in normal
immunoglobulin.
We would like to acknowledge the fact that the techniques
and relatively time-consuming procedures such as those used
in this study for light-chain typing might be beyond the scope
of some clinical laboratories. Also, the use of such procedures
might not be fully justified if the cost and actual benefit to the
patient are considered; the demonstration
of monoclonal light
1498 CLINICAL CHEMISTRY, Vol. 25, No. 8, 1979
13. Ritehie, R. F., and Smith, R., Immunofixation.
I. General principles and application to agarose gel electrophoresis. Clin. Chem. 22,
497-499 (1976).
14. Seligmann, M., Mihaesco, E., Hurez, D., et al., Immunochemical
studies in four casesof alpha-chain disease. J. Clin. Invest. 48,
2374-2389
(1969).
15. Franklin,
of the untypable
(1966).
E. C., Feinstein, D., and Fudenberg,H. H., The problem
M protein.
Acta Med.
Scand.
179, Suppl. 455,80-83