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Characterization of monoclonal antibodies directed against determinants on cardiac myosin heavy chain
Vol. 95, No. 4, 1980
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pages 1680-1686
August 29, 1980
CHARACTERIZATION OF MONOCLONAL ANTIBODIES DIRECTED AGAINST DETERMINANTS ON CARDIAC MYOSIN HEAVY CHAIN William A. Clark, Jr.*, Alan W. Everett*, Frank W. Fitch t, Kanitha S. Frogner~ Smilja Jakovcic~ Murray Rabinowitz*$, Ashley M. Warner*,
and Radovan Zak *§
Departments of Medicine*,Pathology t, Biochemistry ~, and Pharmacological and Physiological Sciences s, The University of Chicago, 950 East 59th St., Chicago, Illinois 60637 Received
July
7,1980
SUMMARY: Monoclonal antibodies against cardiac myosin from chicken (18d embryos) and rabbit were produced in vitro by fusion of mouse myeloma cells (P3X63-Ag8) with spleen cells from immunized BALB/c mice and Lewis rats. Three antibodies were examined and found to react with different antigenic determinants in the myosin heavy chain subunit. Antibody RCM-79 reacted specifically with heavy meromyosin. Antibodies CCM-13 and RCM-79 reacted with equal affinities to cardiac myosin from chicken, rabbit, rat, and man, but with lower affinities to skeletal muscle myosins from chicken and rabbit. Antibody CCM31A reacted only to chicken cardiac myosin and not to other cardiac or skeletal myosins tested. Our results show that monoclonal antibodies can be produced that are either specific for sequences common to all of the cardiac myosins examined or unique to the immunizing type.
INTRODUCTION:
Multiple molecular forms of myosin HC have been demonstrated in
fast, slow (1-4), and embryonic skeletal muscle (2,5), and in the atria (6) and ventricles (7-9) of the myocardium.
A single type of muscle may contain more
than one molecular form of myosin HC (3,10-12).
Evidence for heterogeneity has
been derived from analysis of peptides after limited proteolytic digestion or after chemical cleavage of the HC (2,7,13-15), electrophoretic mobility of native (7,16) and denatured (2,17) myosin and from immunological studies (1,4,9, 10,12,18).
Antibodies specific to individual forms of myosin HC have been gen-
erated by immunoadsorption of sera, a technique which is effective when pure antigens are available for adsorption.
However, myosin HC variants present in
low concentration or as mixed populations may not be detected by this technique. We therefore have elected to use the hybridoma procedure of Kohler and Milstein (19) to produce monoclonal antibodies directed against antigenic determinants on myosin HC.
With this procedure large quantities of antibodies specific to
a minor molecular form may be generated.
In this paper we have analyzed sev-
Abbreviations: HC, heavy chain; McAb, monoclonal antibody; RIA, radioimmunoassay; SDS, sodium dodecyl sulfate; PLD, posterior latissimus dorsi; ALD, anterior latissimus dorsi.
0006-291X/80/I 61680-07501 • 00/0 Copyright © 1980 by Academic Press, Inc. All rights o f reproduction in any form reserved.
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BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
eral McAbs directed against different determinants on cardiac myosin HC.
The
isolation of McAbs to cardiac (20-22) and skeletal (23) myosin has been reported in preliminary form.
MATERIALS AND METHODS: Myosin was prepared from cardiac and skeletal muscle by extraction in 0.6 M NaCI, 20 mM MgS04, 0.i mM DTT, 0.i mM EGTA, 10 raM ATP, and 5 mM phosphate buffer (pH 7.0) followed by ammonium sulfate fractionation (24). H M M a n d L M M w e r e prepared from rabbit and chicken myosin according to SzentGyorgi et al. (25). Myosin and meromyosin fractions were labeled with '251 by the lactoperoxidase/glncose oxidase catalyzed reaction (Enzymobead reagent, BioRad). BALB/c mice and Lewis rats were immunized with i00 ~g of myosin emulsified in Freund's adjuvant by IP injection at intervals of 1-4 weeks. Three days before fusion the animals were boosted with i00 ~g of myosin administered IV. Spleen lymphocytes were fused with the P3-X63-Ag8 mouse myeloma line with polyethylene glycol (19,26,27). Hybridomas producing antimyosin antibodies were cloned by limiting dilution on an irradiated rat thymocyte feeder layer. Antimyosin activity in medium from hybridoma colonies was screened and characterized with a modified indirect RIA. Hybridoma medium (25-50 ~i) was incubated for i hr at 37°C with 0.1-0.5 ~g of [1251]myosin in 50 ~i of modified borate-buffered saline (pH 8.4) containing 40 mM Na pyrophosphate, 1% each of Na deoxycholate and Triton X-100 and 2% normal serum. Goat anti-rat or antimouse immunoglobulin (Cappel) was added and incubation continued for 2 hr at 37°C to precipitate the immune complexes. T h e immunoprecipitates were washed in the above buffer without normal serum and counted to determine bound [~2Sl]myosin radioactivity. Quantitative binding of antibody to homologous and heterologous myosin species was assayed in a competitive RIA. Known quantities of different unlabeled myosin was added to the standard RIA reaction to assess the degree to which they competed for antibody binding with the homologous [~251]myosin. Mixing of different proportions of the same labeled and unlabeled myosin in the competitive RIA confirmed that antibody binding to myosin was not significantly altered in freshly iodinated protein (28).
RESULTS:
Lymphocytes obtained from the spleen of rats or mice immunized with
native cardiac myosin were fused with the P3-X63-Ag8 myeloma line. colonies were screened for antibodies against myosins by RIA.
The hybridoma
Three strongly
reacting hybridoma colonies were cloned by limiting dilution and the antibodies derived from the subcloned lines were examined.
Two of the hybridoma lines were
derived from mice immunized with 18 day-old embryonic chick cardiac myosin (CCM13 and CCM-3ZA); the third line was derived from a rat immunized with rabbit cardiac myosin (RCM-79). are shown in Fig. i.
Titration curves of the three McAbs obtained by RIA
The maximum precipitation of [1251]cardiac myosin with
each of these antibodies is in the range 65-70% of the total labeled antigen. The results shown for RCM-79 and CCM-13 in Fig. i were obtained after concentration of the antibodies by ammonium sulfate precipitation, whereas that shown for CCM-31A was obtained from untreated culture medium. We established that each of the antibodies reacted with the myosin HC subunit by using SDS/polyacrylamide gel electrophoresis and radioautography of the
1681
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
70-
6
0
~
v
O
Z 50 O 40-
~ 30Q
~ 20.
10"
I
i0o
I0 -=
io-Z MONOCLONAL
10-3
10-4
10-5
ANTIBODY DILUTION
Fig. i. Titration of McAbs RCM-79 I~ CCM-13 O, and CCM-31A • by RIA. The per cent of [z25I]myosin bound was corrected for the non-specific binding observed with medium from P3 cultures. Data for RCM-79 and CCM-13 were obtained with antibodies concentrated by ammonium sulfate, while that for CCM31A were from untreated culture medium. labeled antigen in the immunoprecipitates and in the supernatant.
The radioauto
graph shown in Fig. 2 demonstrates that antibody RCM-79 reacts specifically with the myosin HC since only radioactivity corresponding to the HC is seen in the immunoprecipitate
(lane 2).
Cardiac myosin light chains which are dissociated
from the heavy subunit by the presence of deoxycholate and Triton X-IO0 in the
Fig. 2. Radioautography of 0.1% SDS/5% polyacrylamide gels after electrophoresis (30) of [z~SI]myosin from rabbit heart (i) and of the immunoprecipitate (2) and supernatant (3) after reaction with McAb RCM-79. Only radioactivity corresponding to myosin HC appears in the immunoprecipitate while myosin light chains (LC) are in both the labeled antigen and RIA supernatant.
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BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE i Binding of [1251]labeled myosin, HMM and LMM by monoclonal antibodies Per cent of total antigen bound [125I]antigen RCM-79
CCM-13
Rabbit cardiac myosin
65
46
2
Chicken cardiac myosin
65
65
54
Chicken cardiac HMM
39
8
6
0.4
0.7
1.4
Chicken cardiac LMM
CCM-31A
Rabbit and chicken myosin as well as chicken HMM and LMM were prepared and labeled with I=5I. Approximately 0.25 Dg of each iodinated antigen was reacted with each antibody in the indirect RIA. The table gives the maximum amount of immunoprecipitated antigen after correction for non-specific background precipitation with normal serum.
immunoprecipitation
reaction,
do not appear in the immunoprecipitates.
light chains can be identified, i) and in the supernatant
however,
both in the [12SI]labeled myosin
(lane 3) after electrophoresis.
obtained for CCM-13 and CCM-31A
The antigenic site to which these antibodies
A significant
sin was immunoprecipitated myosin from chicken heart.
(lane
The same result was
(data not shown).
by direct binding and immunoprecipitation (Table i).
The
react was further delineated
with iodinated myosin, HMM and LMM
fraction of labeled chicken and rabbit cardiac myoby McAbs RCM-79 and CCM-13, while CCM-31A bound only RCM-79 also binds rabbit and chicken H M M w h i l e
CCM-
13 and CCM-31A bind only a small fraction of labeled chicken cardiac HMM.
None
of the antibodies bind significant
amounts of LMM.
of RCM-79 can be clearly localized
to the HMM fragment, while the binding sites
of CCM-13 and CCM-31A,
The antigenic binding site
though probably localized in ~ M ,
appear to be signifi-
cantly altered by trypsin digestion of myosin. The reactivity of the three McAbs toward several types of myosin was examined by competitive RIA.
Binding with cardiac myosins from chicken,
rabbit,
and rat as well as chicken fast (PLD), and chicken slow (ALD), and mixed bit longissimus
dorsi)
skeletal muscle myosins is shown in Fig. 3.
and CCM-13 bind all three cardiac myosin tested with equal affinity.
(rab-
Both RCM-79 RCM-79
binds mixed rabbit skeletal muscle myosin less well than any of the cardiac myosins.
Similarly,
CCM-13 has a greater affinity for cardiac myosins
than for
chicken skeletal myosin, but displays differential binding with fast (PLD) and slow (ALD) types of myosin
(Fig. 3B).
The binding of CCM-13 to ALD myosin is
clearly less than that to any cardiac myosin, binding to PLD myosin.
McAb CCM-31A,
cific for chicken cardiac myosin,
but is much greater than the
on the other hand, appears to be more spe-
since it binds neither cardiac myosin from
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Vol. 95, No. 4, 1980
.
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
.
.
.
,
,
,
,
,
IJ ,
,
,
,
.
.
.
.
.
.
.
.
.
II
A (RCM-79)
. . . .
B (CCM-13)
IOO-
75-
50" Z
~
25~
-
J
,_o, F--
8~ ~8
C (CCM-31A}
D
I00
5
I
2
3
4
5
6
7
8
9
I 0 15 2 0 2 5
COMPETING MYOSIN(~g)
Fig. 3. The antigenic specificity of RCM-79 (A), CCM-13 (B), CCM-31A (C), and mouse immune serum (D) is shown by their reaction with different types of myosin in a competitive RIA. Increasing concentrations of unlabeled myosin prepared from chicken cardiac Q; rabbit cardiac O; rat cardiac &; chicken anterior latissimus dorsi D; chicken posterior latissimus dorsi I; and rabbit longissimus dorsi [] muscles was added to the RIA reaction described in Materials and Methods. Mouse immune serum was collected after three weekly injections of 200 ~g of chicken cardiac myosin and was dilated i:i00 for assay.
rabbit and rat, nor chicken ALD and PLD myosins
(Fig. 3C).
Competition for
antibody binding has also been conducted with human cardiac myosin, which binds with a high affinity to CCM-13 and RCM-79, but not to CCM-31A (data not shown). Antibody RCM-79, which was derived from rat lymphocytes, binds to rat cardiac myosin with an affinity equal to that of rabbit and chicken cardiac myosins. The immune serum binds the immunizing type of myosin more selectively than do McAbs RCM-79 and CCM-13 (Fig. 3D). CCM-31A.
Its reactivity is similar to that of clone
Thus, McAbs may be isolated in which reactivity toward different myo-
sin types may be similar (CCM-31A) or quite different
(CCM-13 and RCM-79) from
the whole immune serum in the animal from which the lymphocytes for fusion were derived.
DISCUSSION:
Each McAb we have described reacted with an antigenic determinant
on the myosin HC.
Moreover,
the antibody produced by RCM-79 reacts specifically
with the HMM subfragment of the myosin HC.
Our data further indicate that each
hybridoma line produces an antibody that is directed against a different anti-
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Vol. 95, No. 4, 1 9 8 0
genic determinant.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The antigenic determinants recognized by CCM-13 and CCM-31A
antibodies are clearly different since CCM-13 binds to cardiac myosin of several different species, whereas CCM-31A reacts only with chicken cardiac myosin. CCM13 and RCM-79 have similar affinities for different types of skeletal and cardiac myosin, but they differ in that the antigenic determinant recognized by CCM-13 is largely destroyed by trypsin proteolysis. Antibodies produced by two of the three clones studied reacted identically with cardiac myosins from a variety of species, including chicken, rabbit, rat and man.
This suggests that the molecular structure of the antigenic deter-
minant that reacts with antibodies of clones RCM-79 and CCM-13 are similar, if not identical, in the four cardiac myosins tested.
However, CCM-31A displayed
significant affinity only toward the original antigen, chick cardiac myosin. Affinity differences of these antibodies towards skeletal muscle myosin have also been observed.
Presumably those antibodies displaying a broad spectrum of
interaction react with antigenic sites in which there are few, if any, amino acid differences between species, whereas antibodies reacting exclusively to the stimulating antigen bind to a site in which there is sequence divergence. Our results also illustrate that it is possible, with the hybridoma technique, to isolate an antibody which may be present in only small quantities in the total immune serum.
The antigenic specificity of CCM-31A is very similar
to that of whole mouse serum in that the binding occurs predominantly with a determinant found on chick cardiac myosin HC and on no other myosin tested. The specificies of CCM-13 and RCM-79, however, are quite different from that of the immune serum.
Thus, antibodies appearing in relatively low titers in whole
immune serum can be isolated by the hybridoma technique. Antibody RCM-79 bound rabbit and rat cardiac myosins with equal affinity. The cross reactivity of RCM-79 with rat cardiac myosin indicates that an autoimmune response was stimulated after immunization of rat with rabbit cardiac myosin.
This observation is similar to the report by Urbanski and Margoliash
(29) that administration of a heterologous antigen induced antibodies not only against antigenic sequences which are different, but also against some sequences which are identical in both species.
ACKNOWLEDGMENTS: Cells of the P3-X63-Ag8 mouse myeloma line were kindly provided by Dr. C. Milstein. This study was supported in part by grants HL-09172, HL-04442, HL-16637, HL-20592 and AI14197 from the National Institutes of Health, grants from the Muscular Dystrophy Association of America and the Louis Block Fund of the University of Chicago
REFERENCES i. Gauthier, G.F., and Lowey, S. (1979) J. Cell Biol. 81, 10-25. 2. Rushbrook, J.I., and Stracher, A. (1979) Proc. Natl. Acad. Sci. USA 76, 4331-4334.
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3. Hoh, J.F.Y., and Yeoh, G.P.S. (1979) Nature (London) 280, 321-323. 4. Masaki, T., and Yoshizaki, C. (1974) J. Biochem. 76, 123-131. 5. Whalen, R.G., Schwartz, K., Bouveret, P., Sell, S.M., and Gros, F. (1979) Proc. Natl. Acad. Sci. USA 76, 5197-5201. 6. Sartore, S., Pierobon-Bormioli, S., and Schiaffino, S. (1978) Nature (London) 274, 82-83. 7. Hoh, J.F.Y., Yeoh, G.P.S., Thomas, M.A.W., and Higginbottom, L. (1979) FEBS Lett. 97, 330-334. 8. Flink, I.L., Rader, J.H., and Morkin, E. (1979) J. Biol. Chem. 254, 3105-3110. 9. Lompre, A.-M., Schwartz, K., d'Albis, A., Lacombe, G., Thiem, N.V., and Swynghedauw, B. (1979) Nature (London) 282, 105-107. i0. Lowey, S., Benfield, P.A., Silberstein, L., and Lang, L.M. (1979) Nature (London) 282, 522-524. ii. Pope, B.J., Wagner, P.D., and Weeds, A.G. (1977) J. Mol. Biol. 109, 470-473. 12. Rubinstein, N.A., Pepe, F.A., and Holtzer, H. (1977) Proc. Natl. Acad. Sci. USA 74, 4524-4527. 13. d'Albis, A., Pantaloni, C., and Bechet, J.-J. (1979) FEBS Lett. 106, 81-84. 14. Leger, J.J., Klotz, C., Cavaille, F., and Marotte, F. (1979) FEBS Lett. 106, 157-161. 15. Flink, I.L., Morkin, E., and Elzinga, M. (1977) FEBS Lett. 84, 261-265. 16. d'Albis, A., P@ntaloni, C., and Bechet, J.-J. (1979) Eur. J. Biochem. 99, 261-272. 17. Umeda, P.K., Zak, R., and Rabinowitz, M. (1980) Biochemistry 19, 1955-1965. 18. Arndt, I., and Pepe, F.A. (1975) J. Histochem. Cytochem. 23, 159-168. 19. Kohler, G., and Milstein, C. (1975) Nature (London) 256, 495-497. 20. Clark, W.A., Everett, A.W., Jakovcic, S., Zak, R., and Rabinowitz, M. (1980) J. Cell Biol. in press (abstract). 21. Chizzonite, R.A°, Everett, A.W., Clark, W.A., Jakovcic, S., Rabinowitz, M., and Zak, R. (1980) J. Cell Biol. in press (abstract). 22. Ehrlich, P.H., Zurawski, V.R., Khaw, B.A., and Haber, E. (1980) Circulation 60, 139 (abstract). 23. Kiehart, D.P., and Pollard, T.D. (1980) Fed. Proc. 39, 2167 (abstract). 24. Chantler, P.D., and Szent-Gyorgi, A.G. (1978) Biochemistry 17, 5440-5448. 25. Szent-Gyorgi, A°G., Cohen, C., and Phillott, D.E. (1960) J. Mol. Biol. 2, 133-142. 26. Davidson, W.F., and Parish, C.R. (1975) J. Immunol. Meth. 7, 291-300. 27. McKearn, T.J., Sarmiento, M., Weiss, A., Stuart, F.P., and Fitch, F.W. (1978) Curr. Top. Microbiol. Immunol. 81, 61-65. 28. Hunter, W.M. (1978) In Handbook of Experimental Immunology. Weir, D.M., E~, Chap. 14, pp. 14.1-14.40. Blackwell S~cientific, Oxford. 29. Urbanski, G.J., and Margoliash, E. (1977) J. Immunol. 118, 1170-1180. 30. Weber, K., and Osborne, M. (1969) J. Biol. Chem. 244, 4406-4412.
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Pages 1680-1686
August 29, 1980
CHARACTERIZATION OF MONOCLONAL ANTIBODIES DIRECTED AGAINST DETERMINANTS ON CARDIAC MYOSIN HEAVY CHAIN William A. Clark, Jr.*, Alan W. Everett*, Frank W. Fitch t, Kanitha S. Frogner~ Smilja Jakovcic~ Murray Rabinowitz*$, Ashley M. Warner*,
and Radovan Zak *§
Departments of Medicine*,Pathology t, Biochemistry ~, and Pharmacological and Physiological Sciences s, The University of Chicago, 950 East 59th St., Chicago, Illinois 60637 Received
July
7,1980
SUMMARY: Monoclonal antibodies against cardiac myosin from chicken (18d embryos) and rabbit were produced in vitro by fusion of mouse myeloma cells (P3X63-Ag8) with spleen cells from immunized BALB/c mice and Lewis rats. Three antibodies were examined and found to react with different antigenic determinants in the myosin heavy chain subunit. Antibody RCM-79 reacted specifically with heavy meromyosin. Antibodies CCM-13 and RCM-79 reacted with equal affinities to cardiac myosin from chicken, rabbit, rat, and man, but with lower affinities to skeletal muscle myosins from chicken and rabbit. Antibody CCM31A reacted only to chicken cardiac myosin and not to other cardiac or skeletal myosins tested. Our results show that monoclonal antibodies can be produced that are either specific for sequences common to all of the cardiac myosins examined or unique to the immunizing type.
INTRODUCTION:
Multiple molecular forms of myosin HC have been demonstrated in
fast, slow (1-4), and embryonic skeletal muscle (2,5), and in the atria (6) and ventricles (7-9) of the myocardium.
A single type of muscle may contain more
than one molecular form of myosin HC (3,10-12).
Evidence for heterogeneity has
been derived from analysis of peptides after limited proteolytic digestion or after chemical cleavage of the HC (2,7,13-15), electrophoretic mobility of native (7,16) and denatured (2,17) myosin and from immunological studies (1,4,9, 10,12,18).
Antibodies specific to individual forms of myosin HC have been gen-
erated by immunoadsorption of sera, a technique which is effective when pure antigens are available for adsorption.
However, myosin HC variants present in
low concentration or as mixed populations may not be detected by this technique. We therefore have elected to use the hybridoma procedure of Kohler and Milstein (19) to produce monoclonal antibodies directed against antigenic determinants on myosin HC.
With this procedure large quantities of antibodies specific to
a minor molecular form may be generated.
In this paper we have analyzed sev-
Abbreviations: HC, heavy chain; McAb, monoclonal antibody; RIA, radioimmunoassay; SDS, sodium dodecyl sulfate; PLD, posterior latissimus dorsi; ALD, anterior latissimus dorsi.
0006-291X/80/I 61680-07501 • 00/0 Copyright © 1980 by Academic Press, Inc. All rights o f reproduction in any form reserved.
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BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
eral McAbs directed against different determinants on cardiac myosin HC.
The
isolation of McAbs to cardiac (20-22) and skeletal (23) myosin has been reported in preliminary form.
MATERIALS AND METHODS: Myosin was prepared from cardiac and skeletal muscle by extraction in 0.6 M NaCI, 20 mM MgS04, 0.i mM DTT, 0.i mM EGTA, 10 raM ATP, and 5 mM phosphate buffer (pH 7.0) followed by ammonium sulfate fractionation (24). H M M a n d L M M w e r e prepared from rabbit and chicken myosin according to SzentGyorgi et al. (25). Myosin and meromyosin fractions were labeled with '251 by the lactoperoxidase/glncose oxidase catalyzed reaction (Enzymobead reagent, BioRad). BALB/c mice and Lewis rats were immunized with i00 ~g of myosin emulsified in Freund's adjuvant by IP injection at intervals of 1-4 weeks. Three days before fusion the animals were boosted with i00 ~g of myosin administered IV. Spleen lymphocytes were fused with the P3-X63-Ag8 mouse myeloma line with polyethylene glycol (19,26,27). Hybridomas producing antimyosin antibodies were cloned by limiting dilution on an irradiated rat thymocyte feeder layer. Antimyosin activity in medium from hybridoma colonies was screened and characterized with a modified indirect RIA. Hybridoma medium (25-50 ~i) was incubated for i hr at 37°C with 0.1-0.5 ~g of [1251]myosin in 50 ~i of modified borate-buffered saline (pH 8.4) containing 40 mM Na pyrophosphate, 1% each of Na deoxycholate and Triton X-100 and 2% normal serum. Goat anti-rat or antimouse immunoglobulin (Cappel) was added and incubation continued for 2 hr at 37°C to precipitate the immune complexes. T h e immunoprecipitates were washed in the above buffer without normal serum and counted to determine bound [~2Sl]myosin radioactivity. Quantitative binding of antibody to homologous and heterologous myosin species was assayed in a competitive RIA. Known quantities of different unlabeled myosin was added to the standard RIA reaction to assess the degree to which they competed for antibody binding with the homologous [~251]myosin. Mixing of different proportions of the same labeled and unlabeled myosin in the competitive RIA confirmed that antibody binding to myosin was not significantly altered in freshly iodinated protein (28).
RESULTS:
Lymphocytes obtained from the spleen of rats or mice immunized with
native cardiac myosin were fused with the P3-X63-Ag8 myeloma line. colonies were screened for antibodies against myosins by RIA.
The hybridoma
Three strongly
reacting hybridoma colonies were cloned by limiting dilution and the antibodies derived from the subcloned lines were examined.
Two of the hybridoma lines were
derived from mice immunized with 18 day-old embryonic chick cardiac myosin (CCM13 and CCM-3ZA); the third line was derived from a rat immunized with rabbit cardiac myosin (RCM-79). are shown in Fig. i.
Titration curves of the three McAbs obtained by RIA
The maximum precipitation of [1251]cardiac myosin with
each of these antibodies is in the range 65-70% of the total labeled antigen. The results shown for RCM-79 and CCM-13 in Fig. i were obtained after concentration of the antibodies by ammonium sulfate precipitation, whereas that shown for CCM-31A was obtained from untreated culture medium. We established that each of the antibodies reacted with the myosin HC subunit by using SDS/polyacrylamide gel electrophoresis and radioautography of the
1681
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
70-
6
0
~
v
O
Z 50 O 40-
~ 30Q
~ 20.
10"
I
i0o
I0 -=
io-Z MONOCLONAL
10-3
10-4
10-5
ANTIBODY DILUTION
Fig. i. Titration of McAbs RCM-79 I~ CCM-13 O, and CCM-31A • by RIA. The per cent of [z25I]myosin bound was corrected for the non-specific binding observed with medium from P3 cultures. Data for RCM-79 and CCM-13 were obtained with antibodies concentrated by ammonium sulfate, while that for CCM31A were from untreated culture medium. labeled antigen in the immunoprecipitates and in the supernatant.
The radioauto
graph shown in Fig. 2 demonstrates that antibody RCM-79 reacts specifically with the myosin HC since only radioactivity corresponding to the HC is seen in the immunoprecipitate
(lane 2).
Cardiac myosin light chains which are dissociated
from the heavy subunit by the presence of deoxycholate and Triton X-IO0 in the
Fig. 2. Radioautography of 0.1% SDS/5% polyacrylamide gels after electrophoresis (30) of [z~SI]myosin from rabbit heart (i) and of the immunoprecipitate (2) and supernatant (3) after reaction with McAb RCM-79. Only radioactivity corresponding to myosin HC appears in the immunoprecipitate while myosin light chains (LC) are in both the labeled antigen and RIA supernatant.
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TABLE i Binding of [1251]labeled myosin, HMM and LMM by monoclonal antibodies Per cent of total antigen bound [125I]antigen RCM-79
CCM-13
Rabbit cardiac myosin
65
46
2
Chicken cardiac myosin
65
65
54
Chicken cardiac HMM
39
8
6
0.4
0.7
1.4
Chicken cardiac LMM
CCM-31A
Rabbit and chicken myosin as well as chicken HMM and LMM were prepared and labeled with I=5I. Approximately 0.25 Dg of each iodinated antigen was reacted with each antibody in the indirect RIA. The table gives the maximum amount of immunoprecipitated antigen after correction for non-specific background precipitation with normal serum.
immunoprecipitation
reaction,
do not appear in the immunoprecipitates.
light chains can be identified, i) and in the supernatant
however,
both in the [12SI]labeled myosin
(lane 3) after electrophoresis.
obtained for CCM-13 and CCM-31A
The antigenic site to which these antibodies
A significant
sin was immunoprecipitated myosin from chicken heart.
(lane
The same result was
(data not shown).
by direct binding and immunoprecipitation (Table i).
The
react was further delineated
with iodinated myosin, HMM and LMM
fraction of labeled chicken and rabbit cardiac myoby McAbs RCM-79 and CCM-13, while CCM-31A bound only RCM-79 also binds rabbit and chicken H M M w h i l e
CCM-
13 and CCM-31A bind only a small fraction of labeled chicken cardiac HMM.
None
of the antibodies bind significant
amounts of LMM.
of RCM-79 can be clearly localized
to the HMM fragment, while the binding sites
of CCM-13 and CCM-31A,
The antigenic binding site
though probably localized in ~ M ,
appear to be signifi-
cantly altered by trypsin digestion of myosin. The reactivity of the three McAbs toward several types of myosin was examined by competitive RIA.
Binding with cardiac myosins from chicken,
rabbit,
and rat as well as chicken fast (PLD), and chicken slow (ALD), and mixed bit longissimus
dorsi)
skeletal muscle myosins is shown in Fig. 3.
and CCM-13 bind all three cardiac myosin tested with equal affinity.
(rab-
Both RCM-79 RCM-79
binds mixed rabbit skeletal muscle myosin less well than any of the cardiac myosins.
Similarly,
CCM-13 has a greater affinity for cardiac myosins
than for
chicken skeletal myosin, but displays differential binding with fast (PLD) and slow (ALD) types of myosin
(Fig. 3B).
The binding of CCM-13 to ALD myosin is
clearly less than that to any cardiac myosin, binding to PLD myosin.
McAb CCM-31A,
cific for chicken cardiac myosin,
but is much greater than the
on the other hand, appears to be more spe-
since it binds neither cardiac myosin from
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.
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
.
.
.
,
,
,
,
,
IJ ,
,
,
,
.
.
.
.
.
.
.
.
.
II
A (RCM-79)
. . . .
B (CCM-13)
IOO-
75-
50" Z
~
25~
-
J
,_o, F--
8~ ~8
C (CCM-31A}
D
I00
5
I
2
3
4
5
6
7
8
9
I 0 15 2 0 2 5
COMPETING MYOSIN(~g)
Fig. 3. The antigenic specificity of RCM-79 (A), CCM-13 (B), CCM-31A (C), and mouse immune serum (D) is shown by their reaction with different types of myosin in a competitive RIA. Increasing concentrations of unlabeled myosin prepared from chicken cardiac Q; rabbit cardiac O; rat cardiac &; chicken anterior latissimus dorsi D; chicken posterior latissimus dorsi I; and rabbit longissimus dorsi [] muscles was added to the RIA reaction described in Materials and Methods. Mouse immune serum was collected after three weekly injections of 200 ~g of chicken cardiac myosin and was dilated i:i00 for assay.
rabbit and rat, nor chicken ALD and PLD myosins
(Fig. 3C).
Competition for
antibody binding has also been conducted with human cardiac myosin, which binds with a high affinity to CCM-13 and RCM-79, but not to CCM-31A (data not shown). Antibody RCM-79, which was derived from rat lymphocytes, binds to rat cardiac myosin with an affinity equal to that of rabbit and chicken cardiac myosins. The immune serum binds the immunizing type of myosin more selectively than do McAbs RCM-79 and CCM-13 (Fig. 3D). CCM-31A.
Its reactivity is similar to that of clone
Thus, McAbs may be isolated in which reactivity toward different myo-
sin types may be similar (CCM-31A) or quite different
(CCM-13 and RCM-79) from
the whole immune serum in the animal from which the lymphocytes for fusion were derived.
DISCUSSION:
Each McAb we have described reacted with an antigenic determinant
on the myosin HC.
Moreover,
the antibody produced by RCM-79 reacts specifically
with the HMM subfragment of the myosin HC.
Our data further indicate that each
hybridoma line produces an antibody that is directed against a different anti-
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The antigenic determinants recognized by CCM-13 and CCM-31A
antibodies are clearly different since CCM-13 binds to cardiac myosin of several different species, whereas CCM-31A reacts only with chicken cardiac myosin. CCM13 and RCM-79 have similar affinities for different types of skeletal and cardiac myosin, but they differ in that the antigenic determinant recognized by CCM-13 is largely destroyed by trypsin proteolysis. Antibodies produced by two of the three clones studied reacted identically with cardiac myosins from a variety of species, including chicken, rabbit, rat and man.
This suggests that the molecular structure of the antigenic deter-
minant that reacts with antibodies of clones RCM-79 and CCM-13 are similar, if not identical, in the four cardiac myosins tested.
However, CCM-31A displayed
significant affinity only toward the original antigen, chick cardiac myosin. Affinity differences of these antibodies towards skeletal muscle myosin have also been observed.
Presumably those antibodies displaying a broad spectrum of
interaction react with antigenic sites in which there are few, if any, amino acid differences between species, whereas antibodies reacting exclusively to the stimulating antigen bind to a site in which there is sequence divergence. Our results also illustrate that it is possible, with the hybridoma technique, to isolate an antibody which may be present in only small quantities in the total immune serum.
The antigenic specificity of CCM-31A is very similar
to that of whole mouse serum in that the binding occurs predominantly with a determinant found on chick cardiac myosin HC and on no other myosin tested. The specificies of CCM-13 and RCM-79, however, are quite different from that of the immune serum.
Thus, antibodies appearing in relatively low titers in whole
immune serum can be isolated by the hybridoma technique. Antibody RCM-79 bound rabbit and rat cardiac myosins with equal affinity. The cross reactivity of RCM-79 with rat cardiac myosin indicates that an autoimmune response was stimulated after immunization of rat with rabbit cardiac myosin.
This observation is similar to the report by Urbanski and Margoliash
(29) that administration of a heterologous antigen induced antibodies not only against antigenic sequences which are different, but also against some sequences which are identical in both species.
ACKNOWLEDGMENTS: Cells of the P3-X63-Ag8 mouse myeloma line were kindly provided by Dr. C. Milstein. This study was supported in part by grants HL-09172, HL-04442, HL-16637, HL-20592 and AI14197 from the National Institutes of Health, grants from the Muscular Dystrophy Association of America and the Louis Block Fund of the University of Chicago
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