Hybridoma 1992.11:381-384. Downloaded from online.liebertpub.com by THE UNIVERSITY OF MANCHESTER on 11/19/14. For personal use only.
HYBRIDOMA Volume 11, Number 3, 1992 Mary Ann Liebert, Inc., Publishers
Production and Characterization of Mouse Monoclonal Antibodies to Native Human GERALDINE
Myeloperoxidase
CAMBRIDGE,1
BRIAN
LEAKER,1
and TONY J. HALL2
and 2Department of Immunology, University College of Medicine, London W1P 9PG, U.K.
'Department of Nephrology, Institute of Urology, and Middlesex School
Myeloperoxidase (MPO) is a hemoprotein present in the azurophilic granules neutrophils. MPO catalyses the production of potent oxidant chlorinated species that play a major role in neutrophil microbicidal activity (1). The mature enzyme is a tetrameric glycoprotein (MW ~140 Kd) of two heavy (MW ~57 Kd) and two light (MW -14 Kd) subunits (2). Antibodies to MPO have been found in the sera of patients with vasculitis; for example, pauci-immune crescentic nephritis and polyarteritis (3). It has been suggested that anti-MPO autoantibodies may play a role in the pathogenesis of vasculitic diseases (4), and this view is supported by recent observations showing that anti-MPO antibodies can stimulate TNF-alpha-primed human neutrophils to degranulate and produce oxygen radicals (5). The epitope(s) on MPO recognised by these autoantibodies have not been identified, but it is known that serum autoantibodies bind only to native MPO (6). We have generated 18 mouse monoclonal antibodies (MAbs) that recognise and define three distinct epitopes on native, but not denatured MPO, in order to try to define the epitope(s) on MPO that are recognised by autoantibodies from human sera. The production and characterization of these MAbs is described in this report. Six BALB/c mice were immunized with 50 ug of highly purified human MPO (7) in complete Freund's adjuvant (Sigma) i.p., followed by two booster injections at three weekly intervals of 50 ug MPO in incomplete Freund's (Sigma). The mice were bled 7 days after the third injection and all 6 animals had IgG antibodies of
to MPO with titers of >1 :1000 in ELISA. Three separate fusions with 2 spleens/fusion were performed 3 days after a final i.v. injection of 50 ug MPO in saline. Briefly, washed spleen cells were mixed at a ratio of 4:1 with SP2/0 cells and fused with 1 ml of 40% w/v PEG 4000 (Merck) and 5% DMSO v/v (Sigma) in RPMI 1640 medium for 1 min. The fused cells were diluted slowly with RPMI 1640 with gentle stirring and allowed to stand for 30 min at 37oC before resuspension in RPMI 1640 containing 20% fetal calf serum, 2 mM glutamine and 20 µ 2-mercaptoethanol (complete medium). The fused cells were seeded in flat bottomed 96 well plates containing 3,000 peritoneal exúdate cells/well in complete medium containing HAT (Sigma). After selection in HAT medium (10-14 days after fusion), hybridoma supernatants were screened for IgG antibodies to MPO by ELISA, and hybridomas of interest were cloned twice by the hanging drop method. For the MPO ELISA, half-area EIA plates (Costar) were coated overnight with 100 ng/well of MPO in bicarbonate buffer pH 9.6. After washing with PBS + 0.1% Tween-20 (Sigma), 50 uL of hybridoma supernatant was added for 3 hr
381
by a mixture of alkaline phosphatase-labelled rabbit anti-mouse IgG1, IgG2a, IgG2b and IgG3 antibodies (Southern Biotechnology Associates) at a final dilution of 1:500 each for 3 hr. After washing, the substrate nitrophenyl phosphate at 1 mg/ml in diethanolamine buffer pH 9.8 was added and the absorbance at 410 nM measured on a Dynatech Microplate reader, usually after 30 minutes of color development. All incubations were at room temperature. The MAbs were isotyped using the individual AP-labelled anti-IgG subclass antibodies and AP-labelled rabbit anti-mouse kappa or lambda light chain antibodies (S.B.A.). The ELISA additivity test, using hybridoma supernatants in the MPO ELISA detected by AP-labelled anti-IgG subclass antibodies, was performed as described by Friguet et al. (8) and is outlined in the legend of
Hybridoma 1992.11:381-384. Downloaded from online.liebertpub.com by THE UNIVERSITY OF MANCHESTER on 11/19/14. For personal use only.
followed
Table 1.
Eighteen MPO-specific MAbs (15 IgG1,k and 3 IgG2a,k) were produced from the 3 fusions. In the ELISA additivity test, 3 distinct epitope specificities were identified on MPO that are defined by the prototypal MAbs 16D9 (IgG1,k), 16E3 see Table 1. The epitope recognition of the 18 (IgG1,k) and 23C7 (IgG2a,k) MAbs from the 3 fusions was: 12 like 16D9, 3 like 16E3 and 3 like 23C7. These 3 prototypal MAbs retained their distinctive MPO epitope specificity after cloning and were specific for MPO, since they did not bind to human serum albumin, ovalbumin, human immunoglobulins, lactoferrin or thyroid peroxidase in -
ELISA.
The reactivity of the 3 prototypal MAbs with ethanol fixed, purified human neutrophils was assessed by indirect immunofluorescence staining as previously described (3,9), and was compared with the staining obtained with human ANCA sera as shown in Fig. 1 All 3 MAbs gave bright perinuclear staining of human neutrophils, 2 of the MAbs (16D9 and 23C7) also stained neutrophil cytoplasmic .
TABLE 1.
Some
Examples Of
ELISA
Additivity Results With Anti-MPO MAbs.
MAbs at concentrations that gave near maximal binding in the MPO ELISA, added to MPO coated EIA plates alone or in combination. If the sum of the individual MAb optical densities (O.D.) is similar to the observed O.D. with the MAbs added together, then the epitopes recognised are different. If the observed O.D. with the MAbs together is approximately half the sum of the individual O.D.s, then the same or a spatially-related epitope is recognised. were
expected if MAb epitope recognition is different/same
O.D.s
Individual MAbs
MAb O.D.
16D9
+
16E3
0.36
16D9
+
14B2
16E3
+
16D9
O.D.
observed
/ 0.56
1.37
=
DIFF.
0.36+0.81
1.17/0.59
0.52
=
SAME
14B2
0.76
+
0.81
1.57
/
0.79
1.67
=
DIFF.
+
23C7
0.76
+
0.45
1.21
/
0.62
1.28
=
DIFF.
16E3
+
23C7
0.32
+
0.45
0.77
/
0.39
0.86
=
DIFF.
23C7
+
14B2
0.45
+
0.81
1.26
/
0.63
1.14
=
DIFF.
+
0.76
1.12
Purified MPO run on reducing and non-reducing SDS-PAGE gels and Western blotted onto nitrocellulose, was tested for reactivity with the prototypal MAbs 16D9, 16E3 and 23C7. None of the MAbs bound to the MPO heavy or light subunits separated on reducing gels, but all 3 MAbs bound to native MPO (not shown). None of the MAbs inhibited the enzymic activity of MPO as determined by the guaiacol substrate method (2).
382
Hybridoma 1992.11:381-384. Downloaded from online.liebertpub.com by THE UNIVERSITY OF MANCHESTER on 11/19/14. For personal use only.
ii ^
¿,
ß?
*W'
I
.
^ « _
Figure
1.
Antibody staining
of ethanol-fixed human
3
neutrophils.
granules (Fig.
1 ; Panels C and D). This may reflect antibody binding to MPO migrated to a perinuclear site during ethanol fixation. Photomicrograph showing the typical p-ANCA staining pattern using indirect immunofluorescence of human neutrophils by human serum containing anti-MPO antibodies (A). Staining patterns of human neutrophils by MAbs 16E3, 16D9 and 23C7 which recognize 3 distinct epitopes on MPO are shown in panels B, C and D, respectively.
which has not
Other groups have also made MAbs to human MPO for use in immunophenotyping hematapoietic cells (10), for diagnosis of acute myeloid leukemias (11) and for studying MPO heterogeneity (12). We have produced MAbs that recognise 3 distinct epitopes on native human MPO, and these MAbs are currently being used to define the epitopes recognised by anti-MPO autoantibodies present in the serum of patients with vasculitis (13). Since these MAbs recognise native but not denatured MPO (i.e. separated subunits), it is likely that they bind to topographical determinants of the MPO tetramer rather than linear sequences within individual subunits. Studies are in progress to determine the nature of the sequences involved in cell recognition of MPO. ACKNOWLEDGMENTS This work was supported by The Arthritis and Rheumatism Council of Great Britain and the St. Peter's Trust. Dr. P. Banga kindly supplied thyroid peroxidase for use in the study. REFERENCES
1.
Weiss,
S.J.
(1989) Tissue destruction by neutrophils.
320:365-376. 2.
3.
N.
Engl.
J. Med.
Pember, S.O., Shapira, R. and Kinkade, J.M. (1983) Multiple forms of myeloperoxidase from human neutrophilic granulocytes: Evidence for differences in compartmentalization, enzymatic activity and subunit structure. Archs. Biochem. Biophys. 221:391-403. Falk, R.J. and Jennette, J.C. (1988) Anti-neutrophil cytoplasmic
383
4.
Hybridoma 1992.11:381-384. Downloaded from online.liebertpub.com by THE UNIVERSITY OF MANCHESTER on 11/19/14. For personal use only.
5.
6.
7. 8.
9.
10.
11.
12.
13.
autoantibodies with specificity for myeloperoxidase in patients with systemic vasculitis and idiopathic necrotizing and crescentic glomerulonephritis. N. Engl. J. Med. 318:1651-1657. Cohen-Tervaert, J.W., Goldschmeding, R. and Elema, J.D. (1990) Association of autoantibodies to myeloperoxidase with different forms of vasculitis. Arth. Rheum. 33:1264-1272. Falk, R.J., Terrell, R.S., Charles, L.A. and Jennette, J.C. (1990) Anti-neutrophil cytoplasmic autoantibodies induce neutrophils to degranulate and produce oxygen radicals in vitro. Proc. Nati. Acad. Sci. 87:4115-4119. Roberts, D.E., Peebles, C, Curd, J.G., Tan, E.M. and Rubin, R.L. (1991) Autoantibodies to native myeloperoxidase in patients with pulmonary hemorrhage and acute renal failure. J.Clin.Immunol. 11:389-397. Mathieson, N.R., Wong, P.S. and Travis, J. (1981) Isolation and properties of human myeloperoxidase. Biochem. 20:325-330. Friguet, B., Djavadi-Ohaniance, L., Pages, J., Bussard, A. and Goldberg, M. (1983) A convenient enzyme-linked immunosorbant assay for testing whether monoclonal antibodies recognize the same antigenic site. Application to hybridomas specific for the B2-subunit of Escherichia coli tryptophan synthase. J. Immunol. Methods 60:351-358. Venning, M.C., Quinn, ., Broomhead, V. and Bird, A.G. (1990) Antibodies directed against neutrophils (c-ANCA and p-ANCA) are of distinct diagnostic value in systemic vasculitis. Quart. J. Med. 77:1287-1296. Zaki, S., Chan, W.C., McKolans, J. and Austin, G.E. (1989) Production and characterization of monoclonal antibodies to human myeloperoxidase. Clin.Immunol.Immunopathol. 50 :283-297. Van Der Schoot, C.E., Daams, G.M., Pinkster, J., Vet, R. and Von Dem Borne, A.E.G. (1990) Monoclonal antibodies against myeloperoxidase are valuable immunological reagents for the diagnosis of acute myeloid leukaemia. Br.J.Haematol. 74:173-178. Homma, T., Suzuki, K., Kudo, Y., Inagawa, M., Mizuno, S., Yamaguchi, K. and Tagawa, M. (1989) Preparation and characterization of monoclonal antibodies against human myeloperoxidase. Archs. Biochem. Biophys. 273:189-196. Cambridge, G., Hall, T.J., Leaker, B. (1991) Heterogeneity of antibodies to myeloperoxidase in sera from patients with vasculitis. J. Am. Soc. Nephrol.
(in press).
Address all correspondence to:
Cambridge, Immunology Department (A.S.H.),
Dr. G.
U.C. and Middlesex School of London W1P 9PG, U.K.
Received for
publication: 3/3/92
Accepted: 3/10/92
384
Medicine,
HYBRIDOMA Volume 11, Number 3, 1992 Mary Ann Liebert, Inc., Publishers
Production and Characterization of Mouse Monoclonal Antibodies to Native Human GERALDINE
Myeloperoxidase
CAMBRIDGE,1
BRIAN
LEAKER,1
and TONY J. HALL2
and 2Department of Immunology, University College of Medicine, London W1P 9PG, U.K.
'Department of Nephrology, Institute of Urology, and Middlesex School
Myeloperoxidase (MPO) is a hemoprotein present in the azurophilic granules neutrophils. MPO catalyses the production of potent oxidant chlorinated species that play a major role in neutrophil microbicidal activity (1). The mature enzyme is a tetrameric glycoprotein (MW ~140 Kd) of two heavy (MW ~57 Kd) and two light (MW -14 Kd) subunits (2). Antibodies to MPO have been found in the sera of patients with vasculitis; for example, pauci-immune crescentic nephritis and polyarteritis (3). It has been suggested that anti-MPO autoantibodies may play a role in the pathogenesis of vasculitic diseases (4), and this view is supported by recent observations showing that anti-MPO antibodies can stimulate TNF-alpha-primed human neutrophils to degranulate and produce oxygen radicals (5). The epitope(s) on MPO recognised by these autoantibodies have not been identified, but it is known that serum autoantibodies bind only to native MPO (6). We have generated 18 mouse monoclonal antibodies (MAbs) that recognise and define three distinct epitopes on native, but not denatured MPO, in order to try to define the epitope(s) on MPO that are recognised by autoantibodies from human sera. The production and characterization of these MAbs is described in this report. Six BALB/c mice were immunized with 50 ug of highly purified human MPO (7) in complete Freund's adjuvant (Sigma) i.p., followed by two booster injections at three weekly intervals of 50 ug MPO in incomplete Freund's (Sigma). The mice were bled 7 days after the third injection and all 6 animals had IgG antibodies of
to MPO with titers of >1 :1000 in ELISA. Three separate fusions with 2 spleens/fusion were performed 3 days after a final i.v. injection of 50 ug MPO in saline. Briefly, washed spleen cells were mixed at a ratio of 4:1 with SP2/0 cells and fused with 1 ml of 40% w/v PEG 4000 (Merck) and 5% DMSO v/v (Sigma) in RPMI 1640 medium for 1 min. The fused cells were diluted slowly with RPMI 1640 with gentle stirring and allowed to stand for 30 min at 37oC before resuspension in RPMI 1640 containing 20% fetal calf serum, 2 mM glutamine and 20 µ 2-mercaptoethanol (complete medium). The fused cells were seeded in flat bottomed 96 well plates containing 3,000 peritoneal exúdate cells/well in complete medium containing HAT (Sigma). After selection in HAT medium (10-14 days after fusion), hybridoma supernatants were screened for IgG antibodies to MPO by ELISA, and hybridomas of interest were cloned twice by the hanging drop method. For the MPO ELISA, half-area EIA plates (Costar) were coated overnight with 100 ng/well of MPO in bicarbonate buffer pH 9.6. After washing with PBS + 0.1% Tween-20 (Sigma), 50 uL of hybridoma supernatant was added for 3 hr
381
by a mixture of alkaline phosphatase-labelled rabbit anti-mouse IgG1, IgG2a, IgG2b and IgG3 antibodies (Southern Biotechnology Associates) at a final dilution of 1:500 each for 3 hr. After washing, the substrate nitrophenyl phosphate at 1 mg/ml in diethanolamine buffer pH 9.8 was added and the absorbance at 410 nM measured on a Dynatech Microplate reader, usually after 30 minutes of color development. All incubations were at room temperature. The MAbs were isotyped using the individual AP-labelled anti-IgG subclass antibodies and AP-labelled rabbit anti-mouse kappa or lambda light chain antibodies (S.B.A.). The ELISA additivity test, using hybridoma supernatants in the MPO ELISA detected by AP-labelled anti-IgG subclass antibodies, was performed as described by Friguet et al. (8) and is outlined in the legend of
Hybridoma 1992.11:381-384. Downloaded from online.liebertpub.com by THE UNIVERSITY OF MANCHESTER on 11/19/14. For personal use only.
followed
Table 1.
Eighteen MPO-specific MAbs (15 IgG1,k and 3 IgG2a,k) were produced from the 3 fusions. In the ELISA additivity test, 3 distinct epitope specificities were identified on MPO that are defined by the prototypal MAbs 16D9 (IgG1,k), 16E3 see Table 1. The epitope recognition of the 18 (IgG1,k) and 23C7 (IgG2a,k) MAbs from the 3 fusions was: 12 like 16D9, 3 like 16E3 and 3 like 23C7. These 3 prototypal MAbs retained their distinctive MPO epitope specificity after cloning and were specific for MPO, since they did not bind to human serum albumin, ovalbumin, human immunoglobulins, lactoferrin or thyroid peroxidase in -
ELISA.
The reactivity of the 3 prototypal MAbs with ethanol fixed, purified human neutrophils was assessed by indirect immunofluorescence staining as previously described (3,9), and was compared with the staining obtained with human ANCA sera as shown in Fig. 1 All 3 MAbs gave bright perinuclear staining of human neutrophils, 2 of the MAbs (16D9 and 23C7) also stained neutrophil cytoplasmic .
TABLE 1.
Some
Examples Of
ELISA
Additivity Results With Anti-MPO MAbs.
MAbs at concentrations that gave near maximal binding in the MPO ELISA, added to MPO coated EIA plates alone or in combination. If the sum of the individual MAb optical densities (O.D.) is similar to the observed O.D. with the MAbs added together, then the epitopes recognised are different. If the observed O.D. with the MAbs together is approximately half the sum of the individual O.D.s, then the same or a spatially-related epitope is recognised. were
expected if MAb epitope recognition is different/same
O.D.s
Individual MAbs
MAb O.D.
16D9
+
16E3
0.36
16D9
+
14B2
16E3
+
16D9
O.D.
observed
/ 0.56
1.37
=
DIFF.
0.36+0.81
1.17/0.59
0.52
=
SAME
14B2
0.76
+
0.81
1.57
/
0.79
1.67
=
DIFF.
+
23C7
0.76
+
0.45
1.21
/
0.62
1.28
=
DIFF.
16E3
+
23C7
0.32
+
0.45
0.77
/
0.39
0.86
=
DIFF.
23C7
+
14B2
0.45
+
0.81
1.26
/
0.63
1.14
=
DIFF.
+
0.76
1.12
Purified MPO run on reducing and non-reducing SDS-PAGE gels and Western blotted onto nitrocellulose, was tested for reactivity with the prototypal MAbs 16D9, 16E3 and 23C7. None of the MAbs bound to the MPO heavy or light subunits separated on reducing gels, but all 3 MAbs bound to native MPO (not shown). None of the MAbs inhibited the enzymic activity of MPO as determined by the guaiacol substrate method (2).
382
Hybridoma 1992.11:381-384. Downloaded from online.liebertpub.com by THE UNIVERSITY OF MANCHESTER on 11/19/14. For personal use only.
ii ^
¿,
ß?
*W'
I
.
^ « _
Figure
1.
Antibody staining
of ethanol-fixed human
3
neutrophils.
granules (Fig.
1 ; Panels C and D). This may reflect antibody binding to MPO migrated to a perinuclear site during ethanol fixation. Photomicrograph showing the typical p-ANCA staining pattern using indirect immunofluorescence of human neutrophils by human serum containing anti-MPO antibodies (A). Staining patterns of human neutrophils by MAbs 16E3, 16D9 and 23C7 which recognize 3 distinct epitopes on MPO are shown in panels B, C and D, respectively.
which has not
Other groups have also made MAbs to human MPO for use in immunophenotyping hematapoietic cells (10), for diagnosis of acute myeloid leukemias (11) and for studying MPO heterogeneity (12). We have produced MAbs that recognise 3 distinct epitopes on native human MPO, and these MAbs are currently being used to define the epitopes recognised by anti-MPO autoantibodies present in the serum of patients with vasculitis (13). Since these MAbs recognise native but not denatured MPO (i.e. separated subunits), it is likely that they bind to topographical determinants of the MPO tetramer rather than linear sequences within individual subunits. Studies are in progress to determine the nature of the sequences involved in cell recognition of MPO. ACKNOWLEDGMENTS This work was supported by The Arthritis and Rheumatism Council of Great Britain and the St. Peter's Trust. Dr. P. Banga kindly supplied thyroid peroxidase for use in the study. REFERENCES
1.
Weiss,
S.J.
(1989) Tissue destruction by neutrophils.
320:365-376. 2.
3.
N.
Engl.
J. Med.
Pember, S.O., Shapira, R. and Kinkade, J.M. (1983) Multiple forms of myeloperoxidase from human neutrophilic granulocytes: Evidence for differences in compartmentalization, enzymatic activity and subunit structure. Archs. Biochem. Biophys. 221:391-403. Falk, R.J. and Jennette, J.C. (1988) Anti-neutrophil cytoplasmic
383
4.
Hybridoma 1992.11:381-384. Downloaded from online.liebertpub.com by THE UNIVERSITY OF MANCHESTER on 11/19/14. For personal use only.
5.
6.
7. 8.
9.
10.
11.
12.
13.
autoantibodies with specificity for myeloperoxidase in patients with systemic vasculitis and idiopathic necrotizing and crescentic glomerulonephritis. N. Engl. J. Med. 318:1651-1657. Cohen-Tervaert, J.W., Goldschmeding, R. and Elema, J.D. (1990) Association of autoantibodies to myeloperoxidase with different forms of vasculitis. Arth. Rheum. 33:1264-1272. Falk, R.J., Terrell, R.S., Charles, L.A. and Jennette, J.C. (1990) Anti-neutrophil cytoplasmic autoantibodies induce neutrophils to degranulate and produce oxygen radicals in vitro. Proc. Nati. Acad. Sci. 87:4115-4119. Roberts, D.E., Peebles, C, Curd, J.G., Tan, E.M. and Rubin, R.L. (1991) Autoantibodies to native myeloperoxidase in patients with pulmonary hemorrhage and acute renal failure. J.Clin.Immunol. 11:389-397. Mathieson, N.R., Wong, P.S. and Travis, J. (1981) Isolation and properties of human myeloperoxidase. Biochem. 20:325-330. Friguet, B., Djavadi-Ohaniance, L., Pages, J., Bussard, A. and Goldberg, M. (1983) A convenient enzyme-linked immunosorbant assay for testing whether monoclonal antibodies recognize the same antigenic site. Application to hybridomas specific for the B2-subunit of Escherichia coli tryptophan synthase. J. Immunol. Methods 60:351-358. Venning, M.C., Quinn, ., Broomhead, V. and Bird, A.G. (1990) Antibodies directed against neutrophils (c-ANCA and p-ANCA) are of distinct diagnostic value in systemic vasculitis. Quart. J. Med. 77:1287-1296. Zaki, S., Chan, W.C., McKolans, J. and Austin, G.E. (1989) Production and characterization of monoclonal antibodies to human myeloperoxidase. Clin.Immunol.Immunopathol. 50 :283-297. Van Der Schoot, C.E., Daams, G.M., Pinkster, J., Vet, R. and Von Dem Borne, A.E.G. (1990) Monoclonal antibodies against myeloperoxidase are valuable immunological reagents for the diagnosis of acute myeloid leukaemia. Br.J.Haematol. 74:173-178. Homma, T., Suzuki, K., Kudo, Y., Inagawa, M., Mizuno, S., Yamaguchi, K. and Tagawa, M. (1989) Preparation and characterization of monoclonal antibodies against human myeloperoxidase. Archs. Biochem. Biophys. 273:189-196. Cambridge, G., Hall, T.J., Leaker, B. (1991) Heterogeneity of antibodies to myeloperoxidase in sera from patients with vasculitis. J. Am. Soc. Nephrol.
(in press).
Address all correspondence to:
Cambridge, Immunology Department (A.S.H.),
Dr. G.
U.C. and Middlesex School of London W1P 9PG, U.K.
Received for
publication: 3/3/92
Accepted: 3/10/92
384
Medicine,
