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Monoclonal antibodies directed against human myoglobin: Characterization and application in a bideterminant radioimmunoassay
Journal of Immunological Methods, 45 (1981) 249--254
249
Elsevier/North-Holland Biomedical Press
MONOCLONAL ANTIBODIES DIRECTED AGAINST HUMAN MYOGLOBIN: CHARACTERIZATION A N D APPLICATION IN A BIDETERMINANT RADIOIMMUNOASSAY
JOHN G.R. HURRELL 1, HUGO A. KATUS, BAN-AN KHAW, EDGAR HABER and VINCENT R. ZURAWSKI, Jr. 2
Cardiac Unit, Department of Medicine, Massachusetts General Hospital, and Harvard Medical School, Boston, MA 02114, U.S.A. (Received 20 May 1981, accepted 8 June 1981)
Monoclonal hybridoma cell lines secreting antibodies directed against h u m a n myoglobin were selected. T w o of these cell lines were grown in mouse ascitic fluid resulting in the production of large quantities of antibody. Antimyoglobin antibodies isolated from the ascitic fluids were employed in the development of the sensitive solid-phase, bideterminant radioimmunoassay for h u m a n myoglobin that uniquely recognizes two different epitopes on the same molecule.
INTRODUCTION
Monoclonal antibodies secreted by hybridoma cell lines produced by the somatic~cell hybridization technique (KShler and Milstein, 1975) have potential for applications in clinically important immunoassays. The greatest advantage offered by these antibodies is the exquisite antigenic specificity they exhibit. Following selection and propagation, stable clones of these hybrid lines produce a practically indefinite supply of weU-characterized, highaffinity antibodies. This advantage may make it attractive to produce antibodies used in current immunoassays by the hybridoma technique. The radioimmunoassay of human myoglobin has provided a model system for the application of monoclonal hybridoma antibodies to an assay which is currently performed using conventional antiserum (Jutz et al., 1975; Stone et al., 1975; Reichlin et al., 1978; Cloonan et al., 1979). 1 J.G.R.H. was the recipient of the Fulbright Fellowship while on study leave from the Commonwealth Serum Laboratories, Parkville, Australia. Present address: Commonwealth Serum Laboratories, Parkville 3 052, Victoria, Australia. 2 V.R.Z. completed a portion of this work during his tenure as a Fellow of the Research Foundation, Boston, MA. A portion of V.R.Z.'s salary support came from USPHS Grants CA-10 126-11 and CA-24 432. Present address: Centocor Inc., 3 508 Market St., Philadelphia, PA 19 104, U.S.A. 0022-1759/81/0000--0000/$02.50
© 1981 Elsevier/North-Holland Biomedical Press
250
In this paper we describe the production of hybridoma cell lines, cloning and the partial characterization of monoclonal antibodies to human myoglobin secreted by two of these clones. The application of these antibodies in a sandwich solid-phase radioimmunoassay for human myoglobins is also described. METHODS AND MATERIALS
Human myoglobin was purified to electrophoretic homogeneity from fresh cardiac tissue. Six BALB/c mice were each injected intraperitoneally (i.p.) with 100 /~g of human myoglobin homogenized in complete Freund's adjuvant (CFA). Ten weeks following the priming dose, a further 50 /~g of myoglobin were similarly administered. Three days prior to cell fusion, a final dose of 50 /~g human myoglobin was injected, in saline, into a tail vein of the animal to be sacrificed. Splenocytes from the immunized mouse were isolated, fused with hypoxanthine-aminopterin-thymidine (HAT)-sensitive (Littlefield, 1 9 6 4 ) m o u s e myeloma cells, P3-NSI/1-Ag4-1 (NS-1) (KShler and Milstein, 1976) using polyethylene glycol (Gefter et al., 1977) and seeded into 300 wells in 5 microtiter plates. Hybrid cells (hybridomas) secreting antibodies directed against human myoglobin were detected by screening cell culture supernatants in a solid-phase radioimmunometric assay as previously described (Zurawski et al., 1978). Of the cell lines secreting specific antimyoglobin antibodies, several were selected for cloning by limiting dilution on BALB/c 3T3 fibroblast feeder-layer cells. The cloning procedure was repeated twice to ensure the monoclonality of the resulting cell lines. Twice-cloned hybridoma cell lines were grown in the pristane-induced ascites fluid of syngeneic mice. Isotype analysis of the antibodies was carried in the solid-phase radioimmunometric assay using 12SI-labeled goat-(anti-mouse) isotype reagents, kindly supplied by Dr. M. Mudgett-Hunter. Antibody 4G2.2D3.1B8, isolated by 40% saturation ammonium sulfate precipitation, and human myoglobin were coupled to Sepharose 4B (Pharmacia, Pitscataway, NJ) by the CNBr method (Cuatrecasas and Anfinsen, 1971). Antibody 4E8.3C2.1F10, isolated by ion-exchange chromatography (Oi and Herzenberg, 1979), was radio-iodinated by the lactoperoxidase method (Marchalonis, 1969). For use in the radioimmunoassay the Sepharose-bound antibody was suspended in phosphate-buffered saline (PBS) containing 4% fetal calf serum (FCS), 0.2% Tween 20, and 0.02% sodium azide (RIA buffer). The samples containing myoglobin were incubated with the Sepharose-bound antibody in polystyrene tubes (11 mm × 75 mm) on a wrist-action shaker at 250 rev/min. After 16 h, 3 ml of PBS were added to each tube, the tubes centrifuged and carefully aspirated. A second wash was carried out with 2 ml of RIA buffer after which 12SI-labeled antibody 4E8.3C2.1F10 (100 pl, 35 000 cpm)was added and the tubes incubated for a further 16 h with shaking. The washing procedure was repeated and the tubes counted in a gamma counter.
251 RESULTS AND DISCUSSION
Cell hybridization The fusion of the immune splenocytes with NS-1 myeloma cells resulted in hybrids growing in 285 of the 300 microtiter wells seeded (95%). All of the wells contained hybrid cells secreting antibody specific to myoglobin. The cloning data for two of the hybrid lines are shown in Table 1. The specific antimyoglobin antibody secretion by all of the hybrids observed in the second cloning of both cell lines suggested monoclonality of the lines. Isotype analyses, shown in Table 2, for both antibodies indicated that they were of the IgG2b subclass. The two cell lines 4G2.2D3.1B8 and 4E8.3C2.1F10 were both grown successfully in mouse ascites fluids. Titration of these antibody-rich fluids in the solid-phase radioimmunometric assay gave 50% binding titers of 1 : 9 X l 0 s and 1 : 2 × l 0 s, respectively.
Radioimmunoassay development The development of a successful competitive radioimmunoassay for human myoglobin has been hampered by the difficulty in incorporating 12sI into the protein to produce a probe of high specific activity (Reichlin et al., 1978). The use of the Bolton-Hunter reagent (Bolton and Hunter, 1973) for the incorporation of ~2sI into human myoglobin was found not to be useful since reaction with this reagent completely ablated the binding of the hybridoma antibodies. Therefore, we investigated an assay for myoglobin using a sandwich procedure (Wide and Porath, 1966) in which one of the monoclonal antibodies was labeled with ~2sI using the lactoperoxidase method. Introduction of the label into antibody 4E8.3C2.1F10 did not affect the ability of the antibody to bind myoglobin. This is shown in Fig. 1, where it can been seen that more than 92% of the labeled antibody binds to Sepharose~oupled human myoglobin. To provide an immunoadsorbent for the assay, antibody secreted by clone 4G2.2D3.1B8 grown in ascites fluid was immobilized on Sepharose 4B. The assay consisted of incubating a sample TABLE 1 Cloning of hybridoma lines producing myosin-specific antibody. Cell line
4G2 4E8
Cloning 1
Cloning 2
Frequency of hybrids
Frequency of antimyoglobin hybrids
Frequency of hybrids
Frequency of antimyoglobin hybrids
34/120 93/180
12/34 60/93
24/117 28/60
24/24 28/28
252 TABLE 2 Binding o f isotype 12Si.labele d anti-mouse Ig antibodies to myoglobin-specific antibodies secreted by hybridoma cell lines 4G2.2D3.1B8 and 4E8.3C2.1F10; 50,000 cpm was added to each test well in the assay. Cell line
4G2.2D3.1B8 4E8.3C2.1F10
Bound [ 12 Si]anti_mous e isotype antibodies (cpm) Anti-IgG l
Anti'IgG2b
Anti-IgM
Anti-IgA
1 334 367
11 769 12 062
351 295
116 86
containing human myoglobin and the immobilized antibody (100 t~l of a 1% suspension) (see Fig. 1), at room temperature, with shaking, for 16 h. Following two washes, labeled antibody 4E8.3C2.1F10 was added to the assay tubes and incubation continued for a further 16 h. Standard curves generated by this procedure using different sample volumes are seen in Fig. 2. Determination of serum myoglobin levels has the potential for providing an early diagnostic indication of myocardial infarction (Reichlin et al., 1978; Cloonan et al., 1979); hence the radioimmunoassay of myoglobin is a valuable clinical tool. The sandwich radioimmunoassay described in this paper
! ,aNT/BODY 4G2 ON SEPHAROSE IVT/BOO Y 4E8 L A BEL ED
ANT~BODY 4 E 8 L,4BELED ~00
25,00C'~
i
8C
-4
~
2O,OOO-
~
15,000-
/..o
IO,OCO-
4C 'W
~
5000 ~
20
o~ o,1 1o 1o,o MYOGL OBIN- SEPH,4ROSE 4B (%
100 v/v)
101
102 MYOGLOBIN
103
404
Fng/rnl } .
Fig. 1. Percent binding o f iodinated antibody 4E8 to various dilutions of myoglobinSepharose 4B (v/v). A total o f 34 000 cpm were added per tube in a final reaction volume o f 300/~l. Incubation time was 16 h at 20°C. Fig. 2. The quantity of [12sI]4E8 antibody bound to Sepharose-antibody 4G2-myosin complex is plotted against free myoglobin concentration in the solution (o o). If the experiment is repeated with [12si]4G 2 and Sepharose-antibody 4G2, the results are indicated by ± ±
253
has a sensitivity which is comparable to that observed in radioimmunoassays using conventional antisera (Stone et al., 1975; Reichlin et al., 1978; Cloonan et al., 1979). The limit of detection by this assay is 10 ng/ml, which is below the average normal circulating level of myoglobin, 25 -+ 23 ng/ml (Reichlin et al., 1978). Further, the serum myoglobin levels seen following an acute myocardial infarction, that may reach 1368 -+ 1357 ng/ml (Reichlin et al., 1978), can also be readily handled by this assay without sample dilution. The monoclonality and antigenic specificity of the antibodies used in this assay were indirectly confirmed by the radioimmunoassay. If the same antibody (4E8) was utilized both coupled to the solid-phase immunoabsorbents and as 12SI-labeled second antibody, only background level of radioactivity was bound (see Fig. 2). This indicated that the epitope recognized by antibody 4E8 was blocked and unavailable for reaction with the iodinated antibody. The ablation of antibody binding after iodination with the BolterHunter reagent, furthermore, suggests that a lysine residue or terminal amino group is part of, or close by, the antigenic determinant on the myoglobin molecule. The radioimmunoassay described has confirmed the utility of the sandwich radioimmunoassay technique employing two monoclonal antibodies of different antigenic specificities. While this approach appears to be useful in the determination of serum myoglobin concentrations, its unique applicability will be in circumstances where resolution may be enhanced by requiring that two epitopes rather than only one be recognized by an assay. This is of particular value when the molecular species to be differentiated are similar and share most epitopes but differ in only one or a small number. Another application is the specific assay of isozymes. Most antibodies recognize one or another subunit of a multisubunit protein. Antibodies specific for the interface between two subunits are rarely reported. Thus it is difficult to quantify a single species in a mixture of isozymes. A relevant problem in clinical chemistry is the quantitation of the MB isozyme of creatine phosphokinase in the presence of the MM and BB isozymes. Antibodies reported to date are either M- or B subunit specific. A sandwich assay of the type described here utilizing two monoclonal antibodies that were uniquely specific for the M and B subunits, respectively, would solve this problem. REFERENCES Bolton, A.E. and W.M. Hunter, 1973, Biochem. J. 133,529. Cloonan, M.J., G.A. Bishop and D.E.L. Wilcken, 1979, Pathology 11,689. Cuatrecasas, P. and C.B. Anfinsen, 1971, in: Methods in Enzymology, Vol. 22, ed. W. Jakoby (Academic Press, New York) p. 345. Gefter, M.C., D.H. Margulies and M.D. Scharff, 1977, Somat. Cell Genet. 3, 231. Jutz, R.V., G.W. Nevatt, F.J. Palmer and J.C. Nelson, 1975, Am. J. Cardiol. 35,147. KShler, G. and C. Milstein, 1975, Nature 256,495. KShler, G. and C. Milstein, 1976, Eur. J. Immunol. 6,511.
254 Littlefield, J.W., 1964, Science 145,709. Marchalonis, J.J., 1969, Biochem. J. 113,299. Oi, V.T. and L.A. Herzenberg, 1979, Mol. Immunol. 16, 1005. Reichlin, M., J.P. Visco and F.J. Klocke, 1978, Circulation 57, 52. Stone, M.J., J.T. Willerson, C.E. Gomez-Sanchez and M.R. Waterman, 1975, J. Clin. Invest. 56, 1334. Wide, L. and J. Porath, 1966, Biochim. Biophys. Acta 130, 257. Zurawski, Jr., V.R., E. Haber and P.H. Black, 1978, Science 199, 1439.
249
Elsevier/North-Holland Biomedical Press
MONOCLONAL ANTIBODIES DIRECTED AGAINST HUMAN MYOGLOBIN: CHARACTERIZATION A N D APPLICATION IN A BIDETERMINANT RADIOIMMUNOASSAY
JOHN G.R. HURRELL 1, HUGO A. KATUS, BAN-AN KHAW, EDGAR HABER and VINCENT R. ZURAWSKI, Jr. 2
Cardiac Unit, Department of Medicine, Massachusetts General Hospital, and Harvard Medical School, Boston, MA 02114, U.S.A. (Received 20 May 1981, accepted 8 June 1981)
Monoclonal hybridoma cell lines secreting antibodies directed against h u m a n myoglobin were selected. T w o of these cell lines were grown in mouse ascitic fluid resulting in the production of large quantities of antibody. Antimyoglobin antibodies isolated from the ascitic fluids were employed in the development of the sensitive solid-phase, bideterminant radioimmunoassay for h u m a n myoglobin that uniquely recognizes two different epitopes on the same molecule.
INTRODUCTION
Monoclonal antibodies secreted by hybridoma cell lines produced by the somatic~cell hybridization technique (KShler and Milstein, 1975) have potential for applications in clinically important immunoassays. The greatest advantage offered by these antibodies is the exquisite antigenic specificity they exhibit. Following selection and propagation, stable clones of these hybrid lines produce a practically indefinite supply of weU-characterized, highaffinity antibodies. This advantage may make it attractive to produce antibodies used in current immunoassays by the hybridoma technique. The radioimmunoassay of human myoglobin has provided a model system for the application of monoclonal hybridoma antibodies to an assay which is currently performed using conventional antiserum (Jutz et al., 1975; Stone et al., 1975; Reichlin et al., 1978; Cloonan et al., 1979). 1 J.G.R.H. was the recipient of the Fulbright Fellowship while on study leave from the Commonwealth Serum Laboratories, Parkville, Australia. Present address: Commonwealth Serum Laboratories, Parkville 3 052, Victoria, Australia. 2 V.R.Z. completed a portion of this work during his tenure as a Fellow of the Research Foundation, Boston, MA. A portion of V.R.Z.'s salary support came from USPHS Grants CA-10 126-11 and CA-24 432. Present address: Centocor Inc., 3 508 Market St., Philadelphia, PA 19 104, U.S.A. 0022-1759/81/0000--0000/$02.50
© 1981 Elsevier/North-Holland Biomedical Press
250
In this paper we describe the production of hybridoma cell lines, cloning and the partial characterization of monoclonal antibodies to human myoglobin secreted by two of these clones. The application of these antibodies in a sandwich solid-phase radioimmunoassay for human myoglobins is also described. METHODS AND MATERIALS
Human myoglobin was purified to electrophoretic homogeneity from fresh cardiac tissue. Six BALB/c mice were each injected intraperitoneally (i.p.) with 100 /~g of human myoglobin homogenized in complete Freund's adjuvant (CFA). Ten weeks following the priming dose, a further 50 /~g of myoglobin were similarly administered. Three days prior to cell fusion, a final dose of 50 /~g human myoglobin was injected, in saline, into a tail vein of the animal to be sacrificed. Splenocytes from the immunized mouse were isolated, fused with hypoxanthine-aminopterin-thymidine (HAT)-sensitive (Littlefield, 1 9 6 4 ) m o u s e myeloma cells, P3-NSI/1-Ag4-1 (NS-1) (KShler and Milstein, 1976) using polyethylene glycol (Gefter et al., 1977) and seeded into 300 wells in 5 microtiter plates. Hybrid cells (hybridomas) secreting antibodies directed against human myoglobin were detected by screening cell culture supernatants in a solid-phase radioimmunometric assay as previously described (Zurawski et al., 1978). Of the cell lines secreting specific antimyoglobin antibodies, several were selected for cloning by limiting dilution on BALB/c 3T3 fibroblast feeder-layer cells. The cloning procedure was repeated twice to ensure the monoclonality of the resulting cell lines. Twice-cloned hybridoma cell lines were grown in the pristane-induced ascites fluid of syngeneic mice. Isotype analysis of the antibodies was carried in the solid-phase radioimmunometric assay using 12SI-labeled goat-(anti-mouse) isotype reagents, kindly supplied by Dr. M. Mudgett-Hunter. Antibody 4G2.2D3.1B8, isolated by 40% saturation ammonium sulfate precipitation, and human myoglobin were coupled to Sepharose 4B (Pharmacia, Pitscataway, NJ) by the CNBr method (Cuatrecasas and Anfinsen, 1971). Antibody 4E8.3C2.1F10, isolated by ion-exchange chromatography (Oi and Herzenberg, 1979), was radio-iodinated by the lactoperoxidase method (Marchalonis, 1969). For use in the radioimmunoassay the Sepharose-bound antibody was suspended in phosphate-buffered saline (PBS) containing 4% fetal calf serum (FCS), 0.2% Tween 20, and 0.02% sodium azide (RIA buffer). The samples containing myoglobin were incubated with the Sepharose-bound antibody in polystyrene tubes (11 mm × 75 mm) on a wrist-action shaker at 250 rev/min. After 16 h, 3 ml of PBS were added to each tube, the tubes centrifuged and carefully aspirated. A second wash was carried out with 2 ml of RIA buffer after which 12SI-labeled antibody 4E8.3C2.1F10 (100 pl, 35 000 cpm)was added and the tubes incubated for a further 16 h with shaking. The washing procedure was repeated and the tubes counted in a gamma counter.
251 RESULTS AND DISCUSSION
Cell hybridization The fusion of the immune splenocytes with NS-1 myeloma cells resulted in hybrids growing in 285 of the 300 microtiter wells seeded (95%). All of the wells contained hybrid cells secreting antibody specific to myoglobin. The cloning data for two of the hybrid lines are shown in Table 1. The specific antimyoglobin antibody secretion by all of the hybrids observed in the second cloning of both cell lines suggested monoclonality of the lines. Isotype analyses, shown in Table 2, for both antibodies indicated that they were of the IgG2b subclass. The two cell lines 4G2.2D3.1B8 and 4E8.3C2.1F10 were both grown successfully in mouse ascites fluids. Titration of these antibody-rich fluids in the solid-phase radioimmunometric assay gave 50% binding titers of 1 : 9 X l 0 s and 1 : 2 × l 0 s, respectively.
Radioimmunoassay development The development of a successful competitive radioimmunoassay for human myoglobin has been hampered by the difficulty in incorporating 12sI into the protein to produce a probe of high specific activity (Reichlin et al., 1978). The use of the Bolton-Hunter reagent (Bolton and Hunter, 1973) for the incorporation of ~2sI into human myoglobin was found not to be useful since reaction with this reagent completely ablated the binding of the hybridoma antibodies. Therefore, we investigated an assay for myoglobin using a sandwich procedure (Wide and Porath, 1966) in which one of the monoclonal antibodies was labeled with ~2sI using the lactoperoxidase method. Introduction of the label into antibody 4E8.3C2.1F10 did not affect the ability of the antibody to bind myoglobin. This is shown in Fig. 1, where it can been seen that more than 92% of the labeled antibody binds to Sepharose~oupled human myoglobin. To provide an immunoadsorbent for the assay, antibody secreted by clone 4G2.2D3.1B8 grown in ascites fluid was immobilized on Sepharose 4B. The assay consisted of incubating a sample TABLE 1 Cloning of hybridoma lines producing myosin-specific antibody. Cell line
4G2 4E8
Cloning 1
Cloning 2
Frequency of hybrids
Frequency of antimyoglobin hybrids
Frequency of hybrids
Frequency of antimyoglobin hybrids
34/120 93/180
12/34 60/93
24/117 28/60
24/24 28/28
252 TABLE 2 Binding o f isotype 12Si.labele d anti-mouse Ig antibodies to myoglobin-specific antibodies secreted by hybridoma cell lines 4G2.2D3.1B8 and 4E8.3C2.1F10; 50,000 cpm was added to each test well in the assay. Cell line
4G2.2D3.1B8 4E8.3C2.1F10
Bound [ 12 Si]anti_mous e isotype antibodies (cpm) Anti-IgG l
Anti'IgG2b
Anti-IgM
Anti-IgA
1 334 367
11 769 12 062
351 295
116 86
containing human myoglobin and the immobilized antibody (100 t~l of a 1% suspension) (see Fig. 1), at room temperature, with shaking, for 16 h. Following two washes, labeled antibody 4E8.3C2.1F10 was added to the assay tubes and incubation continued for a further 16 h. Standard curves generated by this procedure using different sample volumes are seen in Fig. 2. Determination of serum myoglobin levels has the potential for providing an early diagnostic indication of myocardial infarction (Reichlin et al., 1978; Cloonan et al., 1979); hence the radioimmunoassay of myoglobin is a valuable clinical tool. The sandwich radioimmunoassay described in this paper
! ,aNT/BODY 4G2 ON SEPHAROSE IVT/BOO Y 4E8 L A BEL ED
ANT~BODY 4 E 8 L,4BELED ~00
25,00C'~
i
8C
-4
~
2O,OOO-
~
15,000-
/..o
IO,OCO-
4C 'W
~
5000 ~
20
o~ o,1 1o 1o,o MYOGL OBIN- SEPH,4ROSE 4B (%
100 v/v)
101
102 MYOGLOBIN
103
404
Fng/rnl } .
Fig. 1. Percent binding o f iodinated antibody 4E8 to various dilutions of myoglobinSepharose 4B (v/v). A total o f 34 000 cpm were added per tube in a final reaction volume o f 300/~l. Incubation time was 16 h at 20°C. Fig. 2. The quantity of [12sI]4E8 antibody bound to Sepharose-antibody 4G2-myosin complex is plotted against free myoglobin concentration in the solution (o o). If the experiment is repeated with [12si]4G 2 and Sepharose-antibody 4G2, the results are indicated by ± ±
253
has a sensitivity which is comparable to that observed in radioimmunoassays using conventional antisera (Stone et al., 1975; Reichlin et al., 1978; Cloonan et al., 1979). The limit of detection by this assay is 10 ng/ml, which is below the average normal circulating level of myoglobin, 25 -+ 23 ng/ml (Reichlin et al., 1978). Further, the serum myoglobin levels seen following an acute myocardial infarction, that may reach 1368 -+ 1357 ng/ml (Reichlin et al., 1978), can also be readily handled by this assay without sample dilution. The monoclonality and antigenic specificity of the antibodies used in this assay were indirectly confirmed by the radioimmunoassay. If the same antibody (4E8) was utilized both coupled to the solid-phase immunoabsorbents and as 12SI-labeled second antibody, only background level of radioactivity was bound (see Fig. 2). This indicated that the epitope recognized by antibody 4E8 was blocked and unavailable for reaction with the iodinated antibody. The ablation of antibody binding after iodination with the BolterHunter reagent, furthermore, suggests that a lysine residue or terminal amino group is part of, or close by, the antigenic determinant on the myoglobin molecule. The radioimmunoassay described has confirmed the utility of the sandwich radioimmunoassay technique employing two monoclonal antibodies of different antigenic specificities. While this approach appears to be useful in the determination of serum myoglobin concentrations, its unique applicability will be in circumstances where resolution may be enhanced by requiring that two epitopes rather than only one be recognized by an assay. This is of particular value when the molecular species to be differentiated are similar and share most epitopes but differ in only one or a small number. Another application is the specific assay of isozymes. Most antibodies recognize one or another subunit of a multisubunit protein. Antibodies specific for the interface between two subunits are rarely reported. Thus it is difficult to quantify a single species in a mixture of isozymes. A relevant problem in clinical chemistry is the quantitation of the MB isozyme of creatine phosphokinase in the presence of the MM and BB isozymes. Antibodies reported to date are either M- or B subunit specific. A sandwich assay of the type described here utilizing two monoclonal antibodies that were uniquely specific for the M and B subunits, respectively, would solve this problem. REFERENCES Bolton, A.E. and W.M. Hunter, 1973, Biochem. J. 133,529. Cloonan, M.J., G.A. Bishop and D.E.L. Wilcken, 1979, Pathology 11,689. Cuatrecasas, P. and C.B. Anfinsen, 1971, in: Methods in Enzymology, Vol. 22, ed. W. Jakoby (Academic Press, New York) p. 345. Gefter, M.C., D.H. Margulies and M.D. Scharff, 1977, Somat. Cell Genet. 3, 231. Jutz, R.V., G.W. Nevatt, F.J. Palmer and J.C. Nelson, 1975, Am. J. Cardiol. 35,147. KShler, G. and C. Milstein, 1975, Nature 256,495. KShler, G. and C. Milstein, 1976, Eur. J. Immunol. 6,511.
254 Littlefield, J.W., 1964, Science 145,709. Marchalonis, J.J., 1969, Biochem. J. 113,299. Oi, V.T. and L.A. Herzenberg, 1979, Mol. Immunol. 16, 1005. Reichlin, M., J.P. Visco and F.J. Klocke, 1978, Circulation 57, 52. Stone, M.J., J.T. Willerson, C.E. Gomez-Sanchez and M.R. Waterman, 1975, J. Clin. Invest. 56, 1334. Wide, L. and J. Porath, 1966, Biochim. Biophys. Acta 130, 257. Zurawski, Jr., V.R., E. Haber and P.H. Black, 1978, Science 199, 1439.