119
Clinica
Chimica
0 Elsevier
Acta,
Scientific
66 (1976)
Publishing
119-123 Company,
Amsterdam
-Printed
in The Netherlands
CCA 7524
ELECTROPHORESIS OF HUMAN MULTIPLE MYELOMA AND WALDENSTRCjM’S MACROGLOBULINEMIA SERA IN SODIUM DODECYL SULFATE POLYACRYLAMIDE GELS
LINDA C. HAYES, F.V. PLAPP*, G.J. BREWER**, D. MOORE, L.L. TILZER and M. CHIGA Department of Pathology College of Health Sciences
(Received
and Oncology, and Hospital,
MEGAN
RUCKER,
University of Kansas Medical Center, Kansas City. Kansas 66103 (U.S.A.)
June 11, 1975)
Summary Sera from normal persons and patients with IgA, IgD, IgG, and IgM monoclonal gammopathies were electrophoresed in polyacrylamide gels containing sodium dodecyl sulfate. The gels were stained with Coomassie blue or were used for immunodiffusion. By this method IgG multiple myeloma and IgM Waldenstrijm’s macroglobulinemia sera were readily distinguished by electrophoresis alone, whereas IgA and IgD myeloma sera were distinguished by further immunodiffusion against anti-e-chain antibody and anti-h-chain antibody.
Introduction We have previously shown that sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis of normal human serum separated serum proteins according to molecular weight in the range from approximately 60 000 to 900 000 [l] and allowed excellent resolution of the many proteins present in serum. Because this method provided such clear separation of serum proteins according to molecular weight, we investigated whether it would allow rapid determination of which paraprotein was increased in serum from patients with multiple myeloma and Waldenstrbm’s macroglobulinemia and thus provide a rapid means for classifying these types of monoclonal sera.
* Present address: Department of Pathology, University of Chicago, Chicago, Ill., 60637, U.S.A. ** Present address: Hays Pathology Laboratory, first National Towers, Hays. Kansas, 67601, U.S.A.
120
Materials and methods Sera from patients with monoclonal gammopathies who were diagnosed as having multiple myeloma and Waldenstrom’s macroglobulinemia by the Hematology Department at the University of Kansas Medical Center and normal human sera were diluted twenty times with 0.1 M sodium phosphate buffer, pH 7.2, made 1% with respect to SDS, and incubated for 1 hour at room temperature [l]. Polyacrylamide gels were cast as previously described by Chiga et al. [ 11. The gel composition was modified from that originally described in that 3% polyacrylamide gels were used and agarose was omitted. The monomer solution contained the following components in 10 ml of 0.1 M sodium phosphate buffer, pH 7.2: 285.6 mg acrylamide; 14.4 mg NJ’-methylenebisacrylamide; 0.01 ml of N,N,N’,N’-tetramethylethylenediamine; 10 mg SDS; and 5 mg ammonium persulfate. The gels were cast in glass tubes 75 mm long and 5 mm inside diameter. The length of each gel was approximately 70 mm. 35 ~1 of distilled water were layered on top of the gel before polymerization occurred, to flatten the meniscus. Following polymerization, the water layer was removed with a Pasteur pipet and a layer of gauze was secured with a rubber band around the base of the tube to prevent gel slippage. Preliminary electrophoresis was carried out for 1 hour at 7 mA per tube to remove impurities from the gel [2]. 0.4 g of sucrose was added to each serum sample and 15 ~1 of this solution were layered on each gel (approximately 50 pg protein). Following electrophoresis of the serum at 7 mA per tube for 3 hours, the gels were stained with 0.1% Coomassie blue solution for 3 hours and destained according to Weber and Osborn [3] or were prepared for immunodiffusion. For immunodiffusion studies, gels were imbedded in 1% agar (Difco Laboratories) in double distilled water on a precleaned microscope slide, The agar was allowed to solidify, and a trough measuring 65 mm X 1 mm X 0.5 mm was cut approximately 7 mm from the gel with an LKB 6814A knife. 25 ~1 of either anti-y- (2.8 mg antibody/ml) or anti-a- (2.6 mg antibody/ml) chain antibody (Hyland Laboratories, Inc.) were layered in the trough to test for specific reactivity to heavy chain antibodies by the respective paraproteins. For the immunodiffusion reaction between Waldenstrom’s macroglobulinemia IgM and anti-p-chain antibody, the gel was immersed in a small test tube containing 2 ml of anti-p-chain antibody (2.5 mg antibody/ml) for 24 hours, as modified from Stumph et al. [4]. This modified procedure was necessary because the high molecular weight IgM did not leave the gel and enter the agar as readily as did IgG and IgA. Myeloma IgD was not characterized by immunodiffusion due to an insufficient amount of patient’s serum. Pictures of all gels were taken by dark field photography. Results In Fig. 1 it can be seen that normal control serum, the three myeloma sera, and the Waldenstrom’s serum all contained a prominent protein band which migrated approximately 54 mm from the top of the gel. This band represented serum albumin since it migrated in the same position as purified
b
c Fig. 1. Electrophoretic patterns of normal serum proteins and of serum protans from patients with three different classes of multiple myeloma and Waldenstrdm’s macroglobulinemia in 3% polyacrylamide gels containing 0.1% sodium dodecyl sulfate; from left to right: normal control serum and IgA. IgD. IgG, and IgM monoclonal gammopathy sera. The gels are stamed with 0.1% Coomassie blue. Fig. 2. Immunodiffusion demonstrating the specific reactivity with: a, anti--chain antibody by the myeloma IgA monomer and polymeric protein bands; b, anti-y-chain antibody by the myeloma IgG protein band; and c. antl-p-chain antibody by the Waldenstrbm’s macroglobulmemia IgM protein band in 3% polyacrylamlde gels containing 0.1% sodmm dodecyl sulfate. In (a) and (b) the gels were layered with the myeloma serum, electrophoresed according to conditions stated in the text, and embedded in 1% agar. The trough contains the respective heavy chain antibody. A gel layered with the appropriate myeloma serum was electrophoresed, stained with 0.1% Coomassie blue and photographed. This gel was superimposed upon the gel imbedded in agar for visualization of the protein bands. In (c) the lower gel was layered with IgM Waldenstrb’m’s macroglobulinemia serum, electrophoresed, and immersed in a test tube containing anti-p-chain antibody. The upper gel was layered with IgM Waldenstrbm’s macroglobulinemia serum, electrophoresed. stained with 0.1% Coomassie blue, and photographed to allow visualization of the protein bands.
bovine serum albumin monomer with a molecular weight of approximately 66 000 [ 11. It can also be observed that the IgG myeloma serum has a prominent dark staining band about 37 mm from the top of the gel, which was markedly increased in intensity compared to the corresponding band in the normal serum sample. The IgM Waldenstrom’s serum gel has a prominent band which migrated 5 mm from the top of the gel, while only a very faint band was observed at this position in normal serum. Immunodiffusion studies demonstrated that these bands were indeed IgG and IgM, respectively. Figs. 2b and c show that only the protein band from IgG myeloma serum which migrated in the 150 000 molecular weight region reacted specifically with anti-y-chain antibody and that only the protein band from IgM Waldenstrom’s serum which migrated in the 900 000 molecular weight region reacted specifically with anti-/-f-chain antibody.
122
0
I
2
Mogrotmn
3
4
d,.,ance
5
6
on cm
Fig. 3. Plot of migration distance versus logarithm of the molecular weight. The lowest x represents human serum albumin; the middle x, myeloma IgG; and the highest x, Waldenstrdm’s macroglohulinemia IgM.
Fig. 4. Electrophoretic patterns of normal serum proteins and of serum proteins from five different ~atients with IgA multiple myeloma in 3 “0 polyacrylamide gels containing 0.1% sodium dodecyl sulfate; from left to right: normal control serum and the five different IgA myeloma sera. The gels are stained with 0.1% Coomassie blue.
Using a different gel composition we previously demonstrated that a plot of the relative mobility of serum proteins versus the logarithms of the molecular weight yielded a linear relationship [ 11. Similarly, with the present modified method, it can be seen in Fig. 3 that a plot of the logarithm of the molecular weights for albumin, myeloma immunoglobulin G and Waldenstrom’s immunoglobulin M versus the migration distance for these proteins also yielded a linear relationship. This linearity demonstrated that this SDS polyacrylamide gel electrophoresis system also separates myeloma IgG and Waldenstrom’s IgM according to their molecular weight. Referring back to Fig. 1 it can be observed that IgA and IgD myeloma sera did not exhibit a single prominent myeloma protein band. Instead many polymeric bands ranging from approximately 150 000 to 900 000 daltons can be seen. Five different IgA myeloma serum samples were obtained and their electrophoretic patterns, Fig. 4, reveal distinctly different polymeric patterns for each of the samples. In order to differentiate between the polymeric myeloma immunoglobulins A and D it was necessary to react their protein bands to the heavy chain specific antibodies, anti-a-chain and anti-b-chain. In Fig. 2a the 150 000 molecular weight region and higher molecular weight polymeric regions of the gel, in which IgA myeloma serum was electrophoresed, reacted to anti-a-chain antibody. These same protein bands failed to react to
123
anti-b-chain antibody. Due to an insufficient amount of patient’s serum the IgD myeloma serum could not be reacted to anti-cu- and 6-chain antibodies. Discussion Recent papers [7,8] have confirmed the validity of antigen-antibody precipitin reactions in the presence of sodium dodecyl sulfate. However, Green et al. [9] have challenged this validity and claimed that a nonspecific precipitin reaction occurs between specific antibody and various antigens treated with SDS. We believe our results represent a true immunologic reaction because each serum from myeloma and Waldenstrom’s macroglobulinemiapatients, diagnosed previously by conventional methods, reacted only to the appropriate antibody. Also when bovine serum albumin was treated with 1% SDS, electrophoresed, and the gel was reacted with anti-y-chain antibody no precipitin reaction was observed (Hayes, L.C., unpublished). We did note, however, that when agar for immunodiffusion studies was dissolved in buffer containing 0.1 M sodium phosphate, pH 7.2, and 0.9% sodium chloride a nonspecific precipitin band was formed during immunodiffusion. However, when the agar was dissolved in distilled water no nonspecific reaction occurred. In the present study, we have applied SDS polyacrylamide gel electrophoresis to separate serum proteins according to molecular weight [l] to study multiple myeloma and Waldenstrom’s macroglobulinemia patients’ sera. This technique, requiring only microliters of serum, is clearly useful in the clinical diagnosis and study of monoclonal gammopathies and promises to be useful in the study of other serum protein abnormalities. The heterogeneous polymers of IgA in the multiple myeloma sera demonstrated by this method are intriguing. Acknowledgement We wish to thank Juanita L. Stika for expert clerical assistance. This investigation was supported in part by NIH grant 5TOl GM 1783 and a grant from the Kaw Valley Heart Association. References 1
M. Chiga,
2
A.C.
G.J.
Peacock
3
K. Weber
4
W.E.
Stumph,
and
5
T.B.
Tomasi
6
R.M.
7
M. Kaartinen.
Chapuis
8
E. Lazarides
9
J.R.
Green,
Brewer,
E. Lopez-Corella
and
C.W.
M.
Osbom.
S.C.R. and and
Dingman,
H.M. M.E.
M.J.
K.
and and
Chem., L. Hood,
Progr.
Koshland,
Weber,
Dunn
and
Grey,
M. Hurme and
J. Biol.
Elgin
and
M.E.
Biochemistry, 244
(1969)
Proc.
Natl.
A.H.
Maddy,
14
Nature, Acad.
113
(1972)
Biochemistry, Makela,
Clin.
Sci.
Biochim.
Chin
Acta,
36
(1972)
668-674
44064412
J. Immunol.,
Allergy, 16
0.
Noelken, 7 (1968)
252
(1974)
1752-1756
81-213 (1975)
1320-1326
(1974)
U.S.A.. Biophys.
329-330
(1974) Acta,
2268-2272 382
(1975)
457461
574-578
Clinica
Chimica
0 Elsevier
Acta,
Scientific
66 (1976)
Publishing
119-123 Company,
Amsterdam
-Printed
in The Netherlands
CCA 7524
ELECTROPHORESIS OF HUMAN MULTIPLE MYELOMA AND WALDENSTRCjM’S MACROGLOBULINEMIA SERA IN SODIUM DODECYL SULFATE POLYACRYLAMIDE GELS
LINDA C. HAYES, F.V. PLAPP*, G.J. BREWER**, D. MOORE, L.L. TILZER and M. CHIGA Department of Pathology College of Health Sciences
(Received
and Oncology, and Hospital,
MEGAN
RUCKER,
University of Kansas Medical Center, Kansas City. Kansas 66103 (U.S.A.)
June 11, 1975)
Summary Sera from normal persons and patients with IgA, IgD, IgG, and IgM monoclonal gammopathies were electrophoresed in polyacrylamide gels containing sodium dodecyl sulfate. The gels were stained with Coomassie blue or were used for immunodiffusion. By this method IgG multiple myeloma and IgM Waldenstrijm’s macroglobulinemia sera were readily distinguished by electrophoresis alone, whereas IgA and IgD myeloma sera were distinguished by further immunodiffusion against anti-e-chain antibody and anti-h-chain antibody.
Introduction We have previously shown that sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis of normal human serum separated serum proteins according to molecular weight in the range from approximately 60 000 to 900 000 [l] and allowed excellent resolution of the many proteins present in serum. Because this method provided such clear separation of serum proteins according to molecular weight, we investigated whether it would allow rapid determination of which paraprotein was increased in serum from patients with multiple myeloma and Waldenstrbm’s macroglobulinemia and thus provide a rapid means for classifying these types of monoclonal sera.
* Present address: Department of Pathology, University of Chicago, Chicago, Ill., 60637, U.S.A. ** Present address: Hays Pathology Laboratory, first National Towers, Hays. Kansas, 67601, U.S.A.
120
Materials and methods Sera from patients with monoclonal gammopathies who were diagnosed as having multiple myeloma and Waldenstrom’s macroglobulinemia by the Hematology Department at the University of Kansas Medical Center and normal human sera were diluted twenty times with 0.1 M sodium phosphate buffer, pH 7.2, made 1% with respect to SDS, and incubated for 1 hour at room temperature [l]. Polyacrylamide gels were cast as previously described by Chiga et al. [ 11. The gel composition was modified from that originally described in that 3% polyacrylamide gels were used and agarose was omitted. The monomer solution contained the following components in 10 ml of 0.1 M sodium phosphate buffer, pH 7.2: 285.6 mg acrylamide; 14.4 mg NJ’-methylenebisacrylamide; 0.01 ml of N,N,N’,N’-tetramethylethylenediamine; 10 mg SDS; and 5 mg ammonium persulfate. The gels were cast in glass tubes 75 mm long and 5 mm inside diameter. The length of each gel was approximately 70 mm. 35 ~1 of distilled water were layered on top of the gel before polymerization occurred, to flatten the meniscus. Following polymerization, the water layer was removed with a Pasteur pipet and a layer of gauze was secured with a rubber band around the base of the tube to prevent gel slippage. Preliminary electrophoresis was carried out for 1 hour at 7 mA per tube to remove impurities from the gel [2]. 0.4 g of sucrose was added to each serum sample and 15 ~1 of this solution were layered on each gel (approximately 50 pg protein). Following electrophoresis of the serum at 7 mA per tube for 3 hours, the gels were stained with 0.1% Coomassie blue solution for 3 hours and destained according to Weber and Osborn [3] or were prepared for immunodiffusion. For immunodiffusion studies, gels were imbedded in 1% agar (Difco Laboratories) in double distilled water on a precleaned microscope slide, The agar was allowed to solidify, and a trough measuring 65 mm X 1 mm X 0.5 mm was cut approximately 7 mm from the gel with an LKB 6814A knife. 25 ~1 of either anti-y- (2.8 mg antibody/ml) or anti-a- (2.6 mg antibody/ml) chain antibody (Hyland Laboratories, Inc.) were layered in the trough to test for specific reactivity to heavy chain antibodies by the respective paraproteins. For the immunodiffusion reaction between Waldenstrom’s macroglobulinemia IgM and anti-p-chain antibody, the gel was immersed in a small test tube containing 2 ml of anti-p-chain antibody (2.5 mg antibody/ml) for 24 hours, as modified from Stumph et al. [4]. This modified procedure was necessary because the high molecular weight IgM did not leave the gel and enter the agar as readily as did IgG and IgA. Myeloma IgD was not characterized by immunodiffusion due to an insufficient amount of patient’s serum. Pictures of all gels were taken by dark field photography. Results In Fig. 1 it can be seen that normal control serum, the three myeloma sera, and the Waldenstrom’s serum all contained a prominent protein band which migrated approximately 54 mm from the top of the gel. This band represented serum albumin since it migrated in the same position as purified
b
c Fig. 1. Electrophoretic patterns of normal serum proteins and of serum protans from patients with three different classes of multiple myeloma and Waldenstrdm’s macroglobulinemia in 3% polyacrylamide gels containing 0.1% sodium dodecyl sulfate; from left to right: normal control serum and IgA. IgD. IgG, and IgM monoclonal gammopathy sera. The gels are stamed with 0.1% Coomassie blue. Fig. 2. Immunodiffusion demonstrating the specific reactivity with: a, anti--chain antibody by the myeloma IgA monomer and polymeric protein bands; b, anti-y-chain antibody by the myeloma IgG protein band; and c. antl-p-chain antibody by the Waldenstrbm’s macroglobulmemia IgM protein band in 3% polyacrylamlde gels containing 0.1% sodmm dodecyl sulfate. In (a) and (b) the gels were layered with the myeloma serum, electrophoresed according to conditions stated in the text, and embedded in 1% agar. The trough contains the respective heavy chain antibody. A gel layered with the appropriate myeloma serum was electrophoresed, stained with 0.1% Coomassie blue and photographed. This gel was superimposed upon the gel imbedded in agar for visualization of the protein bands. In (c) the lower gel was layered with IgM Waldenstrb’m’s macroglobulinemia serum, electrophoresed, and immersed in a test tube containing anti-p-chain antibody. The upper gel was layered with IgM Waldenstrbm’s macroglobulinemia serum, electrophoresed. stained with 0.1% Coomassie blue, and photographed to allow visualization of the protein bands.
bovine serum albumin monomer with a molecular weight of approximately 66 000 [ 11. It can also be observed that the IgG myeloma serum has a prominent dark staining band about 37 mm from the top of the gel, which was markedly increased in intensity compared to the corresponding band in the normal serum sample. The IgM Waldenstrom’s serum gel has a prominent band which migrated 5 mm from the top of the gel, while only a very faint band was observed at this position in normal serum. Immunodiffusion studies demonstrated that these bands were indeed IgG and IgM, respectively. Figs. 2b and c show that only the protein band from IgG myeloma serum which migrated in the 150 000 molecular weight region reacted specifically with anti-y-chain antibody and that only the protein band from IgM Waldenstrom’s serum which migrated in the 900 000 molecular weight region reacted specifically with anti-/-f-chain antibody.
122
0
I
2
Mogrotmn
3
4
d,.,ance
5
6
on cm
Fig. 3. Plot of migration distance versus logarithm of the molecular weight. The lowest x represents human serum albumin; the middle x, myeloma IgG; and the highest x, Waldenstrdm’s macroglohulinemia IgM.
Fig. 4. Electrophoretic patterns of normal serum proteins and of serum proteins from five different ~atients with IgA multiple myeloma in 3 “0 polyacrylamide gels containing 0.1% sodium dodecyl sulfate; from left to right: normal control serum and the five different IgA myeloma sera. The gels are stained with 0.1% Coomassie blue.
Using a different gel composition we previously demonstrated that a plot of the relative mobility of serum proteins versus the logarithms of the molecular weight yielded a linear relationship [ 11. Similarly, with the present modified method, it can be seen in Fig. 3 that a plot of the logarithm of the molecular weights for albumin, myeloma immunoglobulin G and Waldenstrom’s immunoglobulin M versus the migration distance for these proteins also yielded a linear relationship. This linearity demonstrated that this SDS polyacrylamide gel electrophoresis system also separates myeloma IgG and Waldenstrom’s IgM according to their molecular weight. Referring back to Fig. 1 it can be observed that IgA and IgD myeloma sera did not exhibit a single prominent myeloma protein band. Instead many polymeric bands ranging from approximately 150 000 to 900 000 daltons can be seen. Five different IgA myeloma serum samples were obtained and their electrophoretic patterns, Fig. 4, reveal distinctly different polymeric patterns for each of the samples. In order to differentiate between the polymeric myeloma immunoglobulins A and D it was necessary to react their protein bands to the heavy chain specific antibodies, anti-a-chain and anti-b-chain. In Fig. 2a the 150 000 molecular weight region and higher molecular weight polymeric regions of the gel, in which IgA myeloma serum was electrophoresed, reacted to anti-a-chain antibody. These same protein bands failed to react to
123
anti-b-chain antibody. Due to an insufficient amount of patient’s serum the IgD myeloma serum could not be reacted to anti-cu- and 6-chain antibodies. Discussion Recent papers [7,8] have confirmed the validity of antigen-antibody precipitin reactions in the presence of sodium dodecyl sulfate. However, Green et al. [9] have challenged this validity and claimed that a nonspecific precipitin reaction occurs between specific antibody and various antigens treated with SDS. We believe our results represent a true immunologic reaction because each serum from myeloma and Waldenstrom’s macroglobulinemiapatients, diagnosed previously by conventional methods, reacted only to the appropriate antibody. Also when bovine serum albumin was treated with 1% SDS, electrophoresed, and the gel was reacted with anti-y-chain antibody no precipitin reaction was observed (Hayes, L.C., unpublished). We did note, however, that when agar for immunodiffusion studies was dissolved in buffer containing 0.1 M sodium phosphate, pH 7.2, and 0.9% sodium chloride a nonspecific precipitin band was formed during immunodiffusion. However, when the agar was dissolved in distilled water no nonspecific reaction occurred. In the present study, we have applied SDS polyacrylamide gel electrophoresis to separate serum proteins according to molecular weight [l] to study multiple myeloma and Waldenstrom’s macroglobulinemia patients’ sera. This technique, requiring only microliters of serum, is clearly useful in the clinical diagnosis and study of monoclonal gammopathies and promises to be useful in the study of other serum protein abnormalities. The heterogeneous polymers of IgA in the multiple myeloma sera demonstrated by this method are intriguing. Acknowledgement We wish to thank Juanita L. Stika for expert clerical assistance. This investigation was supported in part by NIH grant 5TOl GM 1783 and a grant from the Kaw Valley Heart Association. References 1
M. Chiga,
2
A.C.
G.J.
Peacock
3
K. Weber
4
W.E.
Stumph,
and
5
T.B.
Tomasi
6
R.M.
7
M. Kaartinen.
Chapuis
8
E. Lazarides
9
J.R.
Green,
Brewer,
E. Lopez-Corella
and
C.W.
M.
Osbom.
S.C.R. and and
Dingman,
H.M. M.E.
M.J.
K.
and and
Chem., L. Hood,
Progr.
Koshland,
Weber,
Dunn
and
Grey,
M. Hurme and
J. Biol.
Elgin
and
M.E.
Biochemistry, 244
(1969)
Proc.
Natl.
A.H.
Maddy,
14
Nature, Acad.
113
(1972)
Biochemistry, Makela,
Clin.
Sci.
Biochim.
Chin
Acta,
36
(1972)
668-674
44064412
J. Immunol.,
Allergy, 16
0.
Noelken, 7 (1968)
252
(1974)
1752-1756
81-213 (1975)
1320-1326
(1974)
U.S.A.. Biophys.
329-330
(1974) Acta,
2268-2272 382
(1975)
457461
574-578
