Monoclonal antibody to calmodulin: Development, characterization, and comparison with polyclonal anti-calmodulin antibodies

ANALYTICAL

194,369-377

BIOCHEMISTRY

(1991)

Monoclonal Antibody to Calmodulin: Development, Characterization, and Comparison with Polyclonal Anti-calmodulin Antibodies’ David

B. Sacks,*

Sharon

E. Porter,?

Jack H. Ladenson,?

and Jay M. McDonald3

*Department of Pathology, Brigham & Women’s Hospital and Harvard Medical School, 75 Francis Street, Massachusetts 02115; TDepartments of Pathology and Medicine, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110; and SDepartment of Pathology, University of Alabama at Birmingham, Lyons Harrison Research Building, 509 UAB Station, Birmingham, Alabama 35294 Received

December

6, 1990

review, see (1,2)). It belongs to a family Specific anti-calmodulin rabbit polyclonal and murine monoclonal antibodies have been produced with a thyroglobulin-linked peptide corresponding to amino acids 128-148 of bovine brain calmodulin. The monoclonal antibody is IgG-1 with K light chains. Both sets of antibodies recognize native vertebrate calmodulin, with the polyclonal antibody exhibiting an approximately fourfold higher sensitivity than the monoclonal antibody in a radioimmunoassay. The affinity of both polyclonal and monoclonal antibodies is approximately 2.5-fold higher for Ca2’-free calmodulin than for Ca2+calmodulin. Other selected members of the calmodulin family (5100, troponin, and parvalbumin) do not exhibit significant cross-reactivity with the monoclonal antibody. Troponin and SlOOfi displace some 1251-calmodulin from the polyclonal antibody, but require at least SOO-fold excess concentration. The monoclonal antibody recognizes intact vertebrate calmodulin in solution and also on solid-phase. In addition, plant calmodulin and some forms of post-translationally modified calmodulin (phosphorylated or glycated) bind the monoclonal antibody. The affinity of the monoclonal antibody is approximately 5 X 10’ liters/m01 determined by displacement of ‘2aI-calmodulin. On dot blotting the sensitivity for vertebrate calmodulin is 50 pg. The epitope for the monoclonal antibody is in the carboxy1 terminal region (residues 107-148) of calmodulin. This highly specific anti-calmodulin monoclonal antibody should be a useful reagent in elucidating the mechanism by which calmodulin regulates intracellular metabolism. 0 1991 Academic Press, Inc.

Calmodulin is a 17-kDa protein which mediates many

essential

Ca’+-dependent

physiological

processes (for

’ This study was supported in part by Research Grants DK01680 and DK25897 from the National Institutes of Health, a grant from the American Cancer Society and the Monsanto Co., St. Louis, MO. 0003-2697/91

$3.00

Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

of structurally

homologous Ca”-binding proteins, which includes troponin C, parvalbumin, and SlOO. All eukaryotic cells contain calmodulin as the product of a single gene in each organism. The amino acid sequence of calmodulin is highly conserved among species and all vertebrate calmodulins have identical structure (3). The nonidentity among species only exceeds 10% in organisms as diverse as mammals and protozoans (3), with the greatest amount of variability occurring in the third and fourth domains of the molecule. It has been difficult to raise antibodies to calmodulin due to the sequence homology among species. Polyclonal antibodies to calmodulin have been produced by a variety of techniques, almost all of which involved some modification of the antigen. These include incorporation of dinitrophenyl groups (4), performic acid oxidation (5), coupling with N-acetyl-muramyl-L-alanyl-nisoglutamine (6), or use of a methylated bovine serum albumin-calmodulin emulsion (7). Other polyclonal antibodies to calmodulin were produced with nonvertebrate calmodulin, [e.g., protozoan (8,9), trypanosome (lo), or soybean (ll)] and with synthetic peptides which correspond to part of the calmodulin molecule (12,13). In addition, native mammalian calmodulin has been used as an antigen (14-16), but calmodulin-Sepharose chromatography was required to obtain anti-calmodulin antibody from the antisera. There are very few reports in the literature of monoclonal antibodies to calmodulin. These have been produced either by immunizing mice with a mixture of calmodulin and calmodulin-binding proteins (17) or by in vitro immunization of mouse spleen cell cultures. The latter method has been performed with native calmodulin (18) or with calmodulin complexed to calmodulindependent phosphodiesterase (19). However, these previously described monoclonal antibodies are neither 369

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SACKS

specific nor do they exhibit a high affinity for native calmodulin. To our knowledge, there are no published reports of a monoclonal antibody specific for native vertebrate calmodulin. Using a thyroglobulin-linked peptide corresponding to amino acids 128-148 of bovine brain calmodulin (fourth domain), we have succeeded in producing a specific monoclonal antibody to vertebrate calmodulin. In this study, we describe the production and characterization of the monoclonal antibody and compare it to polyclonal antibodies produced in rabbits with the same thyroglobulin-linked peptide. MATERIALS

AND

METHODS

Materials. Mice were obtained from Jackson Laboratories (Bar Harbor, ME). The following reagents were obtained from the manufacturers listed: microtiter plates from Dynatech (Alexandria, VA), MPL2 and TDM emulsion from RIB1 (Hamilton, MT), PVDF (Immobilon-P) from Millipore (Bedford, MA), Ca2+-free porcine calmodulin from Ocean Biologics (Edmonds, WA), 1251from DuPont-New England Nuclear (Boston, MA), iodo-beads from Pierce (Rockford, IL), Freund’s complete and incomplete adjuvant from GIBCO (Grand Island, NY), prestained molecular weight markers from Diversified Biotech (Newton, MA), 2,6,10,14-tetramethyl-pentadecane from Aldrich (Milwaukee, WI), Protein G affinity columns from Pharmacia (Piscataway, NJ), bovine thrombin from U.S. Biochemical (Cleveland, OH), goat anti-mouseand goat anti-rabbit-alkaline phosphatase conjugates and nonimmune mouse and rabbit sera from Sigma (St. Louis, MO), alkaline phosphatase substrate kit from Vector Laboratories (Burlingame, CA), and rabbit anti-mouse IgG from Pel-Freez (Rogers, AR). All reagents for polyacrylamide gel electrophoresis, P-6DG desalting gel, and nitrocellulose were from Bio-Rad (Richmond, CA). All other chemicals were from Sigma. A peptide corresponding to Preparation of antigen. amino acids 128-148 of bovine calmodulin was synthesized by the solid-phase method (20) using automatic instrumentation (Applied Biosystems Model 430A). Purification was by reverse-phase high-performance liquid chromatography (Waters, Bedford, MA) on C,, columns eluted with a linear acetonitrile gradient containing 0.5% (v/v) trifluoroacetic acid and composition confirmed by amino acid composition. The peptide was conjugated to bovine thyroglobulin with [N-ethyl-N’(3-dimethylaminopropyl)] -carbodiimide. This thyro’ Abbreviations Used: SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline (50 mM sodium phosphate, 150 mM NaCl, pH 7.5); RIA, radioimmunoassay; MPL + TDM, monophosphoryl lipid A and trehalose dimycolate; EGTA, ethylene glycol bis (@aminoethyl ether)N,N’-tetraacetic acid; BSA, bovine serum albumin; PVDF, polyvinylidene difluoride; PEG, polyethylene glycol.

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Preparation of polyclonal anti-calmodulin antiSerum. Female New Zealand White rabbits were immunized by multiple intradermal injections along the back as described by Vaitukaitus (21). Each injection site received 100 pg of conjugated peptide in an emulsion of Freund’s complete adjuvant and Dulbecco’s phosphatebuffered saline, pH 7.2. Seven and 15 weeks after the first immunization, booster injections were given with the peptide in an emulsion of equal volumes of Freund’s incomplete adjuvant and PBS.

Preparation and characterization of monoclonal antibodies. Eight-week-old female A/J mice were given intraperitoneal injections with 25 c(g of thyroglobulinlinked peptide emulsified in 200 ~1 of MPL and TDM in 0.9% (w/v) NaCl on Days 0 and 30. An additional injection (25 pg) was given without adjuvant on Day 60. Tendays later the mice were bled and serum was screened for antibody with 1251-calmodulin in a liquid-phase assay. A fourth injection (25 pg without adjuvant) was given on Day 90 and 3 days later spleens were removed aseptically from antibody-producing mice. Splenocytes were fused with the myeloma cell line P3-X-63-Ag8-653 (10’ cells) essentially as described by Kijhler and Milstein (22) with fusion mediated by PEG. Hybridomas were screened for antibodies to calmodulin by solid-phase RIA using a modification of Vaidya et al. (23). Briefly, goat anti-mouse IgG antibodies (2 pg/ml) in borate-buffered saline (100 mM boric acid, 25 mM sodium borate, 150 mM NaCl, pH 8.2) were incubated overnight at 4°C on 96-well microtiter plates. After the plates were washed and blocked with 1% BSA (w/v) in PBS, hybridoma supernate was added then incubated for 4 h at 22°C and the plates were washed again. Antibodies to calmodulin were detected by incuper well. After bating 100,000 cpm of ‘251-calmodulin washing and drying the plates, the bound radiolabeled calmodulin was quantified in a gamma counter. Wells containing counts more than two times the negative control (unrelated hybridoma supernates) were considered positive. Background counts were less than 300 cpm. Hybridomas producing antibodies to calmodulin were cloned by limiting dilution at 30 to 50 cells/well and screened. To insure monoclonality, positive wells were recloned initially at 3 to 5 cells/well and subsequently at 0.5 cells/well. CAF,/J mice primed with pristane (2,6,10,14-tetramethyl-pentadecane) were injected intraperitoneally with lo6 hybrid cells. Ascites fluid was collected and stored frozen at -20°C. Ascites was pooled and monoclonal antibody purified by Protein G affinity chromatography. The purity of the antibody was evaluated by SDS-PAGE. Iodinution of calmodulin. Porcine brain calmodulin was iodinated as previously described (24). Briefly, 100

MONOCLONAL

ANTIBODY

pg of calmodulin was incubated with two iodo-beads in 200 mM Tris-HCl, 0.5 mM CaCl,, pH 7.5, containing 1.0 to 1.5 mCi of “‘1 for 2 min at 22°C. The sample was applied to a P-6DG column, calmodulin was eluted with PBS, and 2-ml fractions were collected. The radiolabeled calmodulin was used only if trichloroacetic acidprecipitable counts exceeded 90% of the total counts; it bound to adipocyte membranes in a Ca’+-dependent manner (25) and it was precipitated by rabbit anti-calmodulin serum. The ‘251-calmodulin comigrated with unlabeled calmodulin on SDS-PAGE. Radioimmunoassay. Standard curves were generated by incubating serial dilutions of calmodulin (100 ~1) with an equal volume of rabbit serum (diluted 1:2000) in PBS containing 1.0% (w/v) BSA. This dilution of antiserum precipitated approximately 30% of B mar. Incubations were performed at 22°C for 2 h in the presence of 1 mM EGTA, 1 mM CaCl,, or neither, as indicated in the figure legends. After a 2-h incubation at 22°C with 10 ~1 of ‘251-calmodulin (100,000 cpm), 50 ~1 of goat anti-rabbit IgG was added. Samples were incubated overnight at 4’C and then PEG in PBS was added to a final concentration of 12.5% (v/v). The samples were centrifuged at 1,500g for 20 min at 4“C; the supernatant was aspirated and the precipitate washed twice with 12.5% (v/v) PEG in PBS. 1251-calmodulin remaining in the pellets was quantified in a gamma counter. RIA with the monoclonal antibody was performed in the same manner, with the following modifications. The monoclonal antibody was diluted to 10 pg/ml and the second (precipitating) antibody was goat anti-mouse IgG instead of goat anti-rabbit IgG. Specificity was determined by competition RIA. This was performed as described above, but instead of unlabeled calmodulin, competing antigens were added at the concentrations indicated in the figure legends. Western blotting. Western blotting was performed by a modification of Towbin et al. (26). After SDSPAGE, gels were equilibrated in transfer buffer (25 mM Tris, 192 mM glycine and 20% (v/v) methanol) for 15 min and then transferred to PVDF which had been prewet according to the manufacturer’s instructions. Transfer was performed at 250 mA (constant current) for 60 min and calmodulin was fixed onto the membrane by incubation in 0.2% (v/v) glutaraldehyde in PBS for 45 min at 22°C (27). Membranes were briefly rinsed in PBS and nonspecific binding sites were blocked with 2% (w/v) BSA, 0.1% (w/v) gelatin in PBS for 60 min at 37°C. Following three lo-min washes with ice-cold 0.05% (v/v) Tween 20 in PBS (PBS-Tween), the membranes were incubated with 0.5 pg/ml monoclonal antibody in PBS for 60 min at 37°C. After washing, incubation was performed for 2 h at 22°C with l/1000 dilution of goat anti-mouse IgG-alkaline phosphatase conjugate. Five lo-min washes in PBS-Tween and one lomin wash with deionized water were followed by detec-

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CALMODULIN

371

tion with Vector Laboratories Alkaline Phosphatase Substrate Kit II, used as instructed by the manufacturer. In order to ensure adequate transfer, duplicate lanes on the PVDF were stained after glutaraldehyde fixation with 0.1% (w/v) amido black in 45% (v/v) methanol and 10% (v/v) acetic acid and destained in 90% (v/v) methanol containing 2% (v/v) acetic acid. Western blotting with the polyclonal antibodies was performed as described above using a 116250 dilution (in PBS) of affinity-purified antibody. Detection of antigen-antibody complexes was carried out with goat anti-rabbit IgG-alkaline phosphatase conjugate. Dot blotting. Serial dilutions containing 5 ~1 of antigen were applied to nitrocellulose or PVDF, allowed to dry completely, and then incubated in 0.2% (v/v) glutaraldehyde in PBS. Blocking, incubation with primary and secondary antibodies, and color development were performed as described above for Western blotting. Duplicates of 5 ~1 of carrier buffer were spotted onto the membranes to distinguish protein from artifacts. Thrombin digestion of calmodulin. Thrombin digestion of calmodulin was performed by incubating 200 pg of calmodulin with 20 units of bovine thrombin in 50 mM Tris (pH 8.0) containing 1 mM dithiothreitol and 5 mM EGTA at 30°C. After incubation for various times, aliquots containing 20 pg of calmodulin were removed and the reaction was stopped by adding an equal volume of ice-cold 10% (w/v) trichloroacetic acid. Fragments were separated by SDS-PAGE, transferred to PVDF, and probed with polyclonal or monoclonal antibodies as described above. Post-translational modification of calmodulin. Various species of phosphocalmodulin were prepared with insulin receptor from human placenta (24) or rat hepatocytes (28) or with casein kinase II as described (29). Calmodulin was glycated by incubating 60 PM calmodulin in PBS containing 1 mM CaCl, and 100 mM glucose at 22°C for 20 days (30). Glycated calmodulin was separated from nonglycated on a Glyc-Affin column (Isolab, Akron, OH) as instructed by the manufacturer. Other methods. The isotype of the monoclonal antibody was determined with the Bio-Rad Mouse Typer subisotyping panel kit. Protein concentrations were determined by the method of Bradford (31) using bovine serum albumin as standard. SDS-PAGE was performed as previously described (24) in 15% polyacrylamide gels, with prestained molecular weight markers when performing Western blotting. RESULTS

Production of anti-calmodulin monoclonal antibodies. Preliminary experiments with the thyroglobulin-linked peptide indicated that A/J mice developed much higher serum antibody titers to calmodulin than C57BL or BALB/c mice. Initial fusions of spleen cells

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from antibody-producing A/J mice with the myeloma cell line P3-X-63-Ag8-653 generated few hybridomas that produced antibodies to calmodulin. Despite repeated attempts, however, most of these anti-calmodulin antibody-producing hybridomas failed to survive when the wells were expanded. Those that survived did not grow in soft agar nor did they survive routine limiting dilution conditions of approximately 0.5 cells/well. Only by cloning as soon as positive hybridomas were detected, and performing initial limiting dilution at 30 to 50 cells/well, were we able to obtain hybridomas that secreted specific anti-calmodulin antibodies. Subsequent subcloning was performed at three to five cells/ well, followed by 0.5 cells/well to ensure monoclonality. Characterization showed the monoclonal antibody to be IgG-1 subclass with K light chains. Comparison of polyclonal and monoclonal antibodies. Comparison of the polyclonal and monoclonal antibodies in a RIA is demonstrated in Fig. 1. Binding of ‘261-calmodulin to both sets of antibodies is progressively inhibited by increasing amounts of nonlabeled calmodulin. Half-maximum inhibition is observed at 425 and 1700 ng of calmodulin for the polyclonal and monoclonal antibodies, respectively (Fig. 1). The polyclonal antibody thus exhibits an approximately fourfold higher sensitivity than that of the monoclonal. The background counts per minute precipitated in the absence of antibody were always less than 5%. The affinity of the monoclonal antibody for calmodulin is approximately 5 X 10’ liters/m01 determined by displacement of ‘261-calmodulin with unlabeled calmodulin. The two anti-calmodulin monoclonal antibodies developed by Hansen and Beavo (17) were purchased from Chemicon (Temecula, CA) and evaluated for their abil-

ity to bind 1251-calmodulin. Serial twofold dilutions of each antibody were incubated in the presence and absence of Ca2+ with 1251-calmodulin, precipitated with goat anti-mouse IgG, washed, and quantified in a gamma counter as described above. Neither antibody bound more than 3% of the ‘251-calmodulin present, which was equivalent to the binding by nonimmune mouse IgG. In contrast, under the same conditions our anti-calmodulin monoclonal antibody bound up to 67% of the ‘251-calmodulin added (data not shown). Effect of Ca” on antibody binding. In the presence of Ca2+, calmodulin undergoes a conformational change to a more helical structure. The effect of Ca2’ on the affinity of the monoclonal antibody for calmodulin was therefore determined by direct RIA (Fig. 2). Ca2’ decreases to the monoclonal antithe binding of ‘251-calmodulin body. Chelation of Ca2+ with 1 mM EGTA results in a 2.6-fold (n = 10) increase in the amount of calmodulin bound to the monoclonal antibody (Fig. 2). Half-maximum inhibition is observed at 1292 ng and 500 ng in the presence of Ca2+ and EGTA, respectively. Similarly, 1 mM EGTA increases calmodulin binding to the polyclonal antibody 2.4-fold (n = 3, each experiment in triplicate) (data not shown). Therefore, the higher sensitivity of the polyclonal antibodies compared to that of the monoclonal antibody demonstrated in Fig. 1 is observed both in the presence and absence of Ca2+. The corresponding half-maximum inhibition values for the polyclonal antibodies are 345 and 125 ng of calmodulin in the presence of Ca2+ and EGTA, respectively. Heating calmodulin at 95°C for 5 min has been shown to increase the affinity of calmodulin for some polyclonal antibodies (New England Nuclear, calmodulin RIA kit). No effect of such heating was observed in the pres-

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ence of Ca2+ or EGTA with any of the antibodies we developed (data not shown). Determination of antibody specificity. Although polyclonal antibodies usually exhibit greater sensitivity than monoclonal antibodies, the latter are often more specific. To determine the specificity of the antibodies, a variety.of Ca2+-binding proteins were examined. These proteins have significant sequence homology with calmodulin, especially troponin C which is 42% identical and 26% of the other sequences are similar in charge or hydrophobicity (3). The binding of troponin C, bovine brain SlOOa and Sloop, and rabbit muscle parvalbumin to the antibodies was determined by competitive binding assays with ‘261-calmodulin (Fig. 3). No significant displacement of ‘251-calmodulin from the polyclonal antibody is observed with up to 100,000 ng of SlOOcw and parvalbumin (Fig. 3A). Some cross-reactivity is obtained at 50,000 ng of SlOO@. Troponin C exhibits crossreactivity with the polyclonal antibody, but approximately 900 times as much troponin as calmodulin is required for equivalent inhibition (Fig. 3A). In contrast, up to 50,000 ng of parvalbumin and 100,000 ng of SlOOcy, SlOO@, or troponin produced no significant displacement of ‘261-calmodulin from the monoclonal antibody (Fig. 3B). All the assays illustrated in Fig. 3 were performed in the presence of 1 mM EGTA. Addition of 1 mM Ca2+ to the assays instead of EGTA did not significantly alter the binding of parvalbumin, SlOOa, SlOO& or troponin C to either polyclonal or monoclonal antibodies (data not shown). The peptide fragment (corresponding to amino acids 128-148 of calmodulin) used to produce the antibodies exhibited 100% cross-reactivity with intact calmodulin on a mole per mole basis in a competition RIA using the monoclonal antibody (data not shown).

Determination of the immunoreactive site. Since the antibodies were produced with a peptide corresponding to the most distal 21 amino acids in the C-terminal of calmodulin, it was hypothesized that the antibodies bind to this region on the calmodulin molecule. In order to determine whether this is indeed the recognition site, calmodulin was digested with thrombin, the fragments were separated by SDS-PAGE, and Western blotting was performed. In the presence of EGTA thrombin cleaves calmodulin between amino acids 106 and 107, producing two peptides-a fragment consisting of residues l-106 and a smaller fragment containing residues 107-148 (32). Western blotting with the polyclonal and monoclonal

antibodies

after thrombin

digestion

is illus-

trated in Fig. 4. Both the polyclonal (Fig. 4A) and the monoclonal (Fig. 4C) antibodies recognize intact calmodulin and the 107-148 fragment, but do not bind to the fragment consisting of amino acids l-106 of calmodulin. Effect of post-translational modification of calmodulin on antibody recognition. Calmodulin has been demonstrated to undergo a variety of post-translational modifications, including glycation (30) and phosphorylation (24,33). Therefore, we determined the effect of phosphorylation and glycation of calmodulin on antibody recognition (Fig. 5). Three different species of phosphocalmodulin-phosphotyrosylcalmodulin (lane 2), calmodulin phosphorylated on serine and threonine residues by casein kinase II (lane 3), and calmodulin phosphorylated on serine/threonine residues by the insulin receptor kinase after prephosphorylation by casein kinase II (lane 4)-were examined. The broader bands seen in lane 2 and especially lanes 3 and 4 are characteristic of the migration of the various forms of phosphocalmodulin on SDS-PAGE. Incorporation of

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Effect of various Cal+-binding proteins on the binding of ‘261-calmodulin to polyclonal or monoclonal antibody. Radioimmunoassays were performed as described under Materials and Methods in the presence of 1 mM EGTA. Various concentrations of porcine brain calmodulin (0, 0), SlOOa! (6, 0), SlOO@ (A, A), parvalbumin (m, q ), or troponin C (v, V) were incubated with polyclonal or monoclonal antibody before adding 100,000 cpm 12SI-calmodulin. (A) Polyclonal antibody. (B) Monoclonal antibody. Data are shown as explained in the legend to Fig. 1. Representative experiments are shown with each point performed in triplicate. For each protein, 10,000 ng is equivalent to the following: calmodulin, 0.598 nmol; troponin, 0.549 nmol; SlOOa, 0.962 nmol; SlOOfl, 0.962 nmol; and parvalbumin, 0.665 nmol.

phosphate, however, does not interfere with the ability of the monoclonal antibody to recognize calmodulin (Fig. 5). Similarly, the monoclonal antibody binds to glycated calmodulin (lane 5). In addition, wheat germ calmodulin, which differs from vertebrate calmodulin in 14 amino acids, was recognized on immunoblotting by the monoclonal antibody (data not shown). Serial dilutions ofphosphotyrosylcalmodulin (exhibiting a stoichiometry of 1 mol of phosphate/mol of calmodulin) exhibit a sensitivity of 50 pg on dot blotting, which is identical to the sensitivity observed with native calmodulin (data not shown). The presence or absence of Ca2’ does not appear to significantly alter the sensitivity of detection of calmodulin immobilized on a solid phase by the monoclonal antibody (data not shown).

DISCUSSION

Polyclonal antisera contain many different types of antibodies specific for a variety of different antigens and exhibit a wide range of affinities, thereby creating problems with interpretation of experiments in which they are used. Monoclonal antibodies have the advantages of homogeneity, reproducible specificity, recognition of generally only one site, and the ability to be produced in large quantities. Despite these advantages, monoclonal antibodies to native vertebrate calmodulin have not been made previously. This is in part due to the fact that calmodulin is one of the most highly conserved proteins studied (34), resulting in difhculty in producing antibodies to mammalian calmodulin in rabbits or mice.

MONOCLONAL

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FIG. 4. Thrombin digestion of calmodulin and detection of fragments on immunoblots. Calmodulin was digested with thrombin as described under Materials and Methods for the times indicated. Fragments were separated by SDS-PAGE and transferred to polyvinylidene difluoride. (A) Immunoblot with polyclonal antibody. (B) Amido black staining of proteins after transfer to membrane. (C) Immunoblot with monoclonal antibody. Antigen-antibody complexes were detected with goat anti-rabbit (A) or anti-mouse (C) IgG-alkaline phosphatase conjugate. The positions of migration of intact calmodulin (CaM) and the fragments consisting of amino acids l-106 and 107-148 of calmodulin are indicated on the left. A representative experiment is shown.

We modified the standard techniques of monoclonal antibody production to obtain an anti-calmodulin monoclonal antibody. First, we used A/J mice. Although not well appreciated, the selection of the mouse strain can be very important for producing monoclonal antibodies (23). Neither BALB/c, the strain traditionally used, nor C57BL mice generated a significant serum response to the thyroglobulin-linked peptide we used. In contrast, the serum of almost all the immunized A/J mice contained antibodies to calmodulin. Second, the antigen was emulsified in MPL and TDM rather than Freund’s adjuvant. Third, cloning was performed as soon as screening of cell fusions indicated that anti-calmodulinproducing hybridoma cells were present. These hybridoma cells did not grow in soft agar nor did they survive routine limiting dilution conditions of 0.5 cells/well. It is not clear why anti-calmodulin antibody-producing cells remained viable only when grown initially at a concentration of 30 to 50 cells per well. Presumably, the hybrid cells were not surviving until their density was expanded or other components relatively decreased. We also utilized a number of modifications of calmodulin in an attempt to produce monoclonal antibodies to calmodulin. These included performic acidtreated calmodulin, calmodulin complexed to polylysine, phosphocalmodulin, and a thyroglobulinlinked peptide corresponding to amino acid residues 81108 of vertebrate calmodulin (the third domain). None of these was successful. Antibodies to native calmodulin were detected in the serum of some A/J mice immunized with the 81-108 peptide, but hybridomas did not produce anti-calmodulin antibodies. The other antigens failed to generate anti-calmodulin antibodies in mouse

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serum. Performic acid oxidation of calmodulin has been reported to make the molecule sufficiently antigenic to produce antiserum in rabbits (5,7), but not all investigators have been able to reproduce these observations (6,35). None of the mouse strains we used developed an immune response to performic acid-treated calmodulin, nor were we able to produce polyclonal antibodies to calmodulin in rabbits with this antigen. Chemically synthesized peptides of part of the surface of a protein can elicit antibodies reactive with the native molecule (for review, see (36)). Higher antigenic activity has been observed in the N- and the C-termini of different proteins (37-39) which may be due to the fact that these regions are usually located at the surface and exhibit high flexibility (40). There is a good correlation between the ability of anti-peptide antibodies to recognize antigenic determinants on a native protein and the mobility of that region of the protein (41). In addition, it appears that peptide immunization can generate antibody specificities that cannot be elicited by immunization with the whole protein (36). Antisera which recognize native calmodulin have been produced in rabbits with synthetic peptides corresponding to helical regions of calmodulin (12,13). These include the N-terminal helix (residues g-19), the central helix (residues 68-79 and 80-92), and the C-terminal helix (residues 141-148 and 134-148). A shorter peptide corresponding to residues 137-143 has been shown to be immunoreactive, but not

FIG. 5. Immunoblotting of post-translationally modified calmodulin. The various forms of post-translationally modified calmodulin were prepared, separated by SDS-PAGE, and transferred to polyvinylidene difluoride as described under Materials and Methods. The blot was reacted with monoclonal antibody followed by goat antimouse IgG-alkaline phosphatase conjugate. Each sample contains 5 pg of calmodulin. Lane 1 represents native calmodulin, lane 2 represents calmodulin phosphorylated on tyrosine residues by the insulin receptor kinase, lane 3 represents calmodulin phosphorylated on serine and threonine residues by casein kinase II, lane 4 represents calmodulin phosphorylated initially by casein kinase II and subsequently on serine and threonine residues by the insulin receptor kinase, and lane 5 represents glycated calmodulin. A representative experiment is illustrated.

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sufficient to elicit antibodies that react with native calmodulin (12). Our monoclonal antibody recognizes native calmodulin in solution and bound to a solid phase (PVDF or nitrocellulose). It also recognizes Ca2+-calmodulin and Ca2+-free calmodulin. On binding Ca2+, calmodulin adopts a more helical conformation and certain hydrophobic regions become exposed. The conformation of calmodulin plays a role in the strength of antibody binding as Ca2+-calmodulin exhibits approximately 2.5-fold lower affinity than Ca 2f-free calmodulin for both sets of antibodies. Some antibodies have been reported which only recognize Ca2+ -calmodulin (17), while others reportedly exhibit higher affinity for the Ca2+-free form (4,5). The affinity of our monoclonal antibody of appfoximately 5 X lo8 liters/m01 is in the range of other monoclonal anti-peptide antibodies (42). In addition, the monoclonal antibody recognizes calmodulin linked to certain calmodulin-binding proteins. For example, the affinity of the antibody for the calmodulin-trifluoperazine complex is the same as for native calmodulin (Sacks et al., unpublished observations). Incorporation of a phosphate group on Tyr-99 or on Thr-79, Ser-81, Ser-101, and Thr-117 of calmodulin does not prevent the recognition of calmodulin by the monoclonal antibody (see Fig. 5). In addition, phosphorylation of Tyr-99 does not alter the sensitivity of detection of calmodulin. None of these phosphorylation sites is in the C-terminal region of calmodulin where the antibody binds. It can be inferred that incorporation of phosphate groups at these sites does not significantly alter the conformation of calmodulin in the region of the fourth domain. Direct comparison of all the antibodies that have been produced to calmodulin is difficult as the reports do not all perform the same characterization of their antibodies. Most of the polyclonal antibodies previously produced are nonprecipitating (4,5,15) and none is specific. The antisera produced with nonvertebrate calmodulin either do not bind (8,9) or bind with a 20,000-fold lower avidity (11) calmodulin from higher organisms. Similarly, the monoclonal antibodies previously described (17-19) do not have a high affinity for native calmodulin. Hansen and Beavo described an anti-calmodulin monoclonal antibody that recognizes both the Ca’+-calmodulin-phosphodiesterase complex and Ca2+calmodulin, but has a low affinity for the latter (17). The antibody does not recognize Ca2+-free calmodulin (17). Furthermore, certain calmodulin-binding proteins are reported to produce interference and samples for calmodulin quantitation in crude fractions require boiling prior to assay with this antibody (17). The failure of the antibody produced by Hansen and Beavo to bind “‘Icalmodulin in our assay may be due to an alteration in the binding properties of calmodulin produced by iodination. Similarly, the monoclonal antibodies produced by

ET

AL.

Winkler et al. (19) have a higher affinity for certain calmodulin-binding proteins (such as phosphodiesterase and phosphatase) than for calmodulin. The monoclonal antibody to calmodulin we have produced is highly specific for calmodulin. In view of the characteristics described of specificity, ability to recognize calmodulin in the presence and absence of Ca2+, and the ability to recognize calmodulin in solution and on a solid-phase, this antibody should be a most useful reagent with a wide variety of possible applications. These include: (i) investigating the sites of interaction of calmodulin and its target proteins; (ii) purifying calmodulin from tissue or cell extracts (Calmodulin is most frequently purified from brain, which also contains high levels of SlOO. The lack of cross-reactivity of the monoclonal antibody with SlOOa or SlOO/l should prevent contamination of calmodulin with the SlOO proteins.); (iii) identifying calmodulin in intact tissue or extracts; (iv) immunoprecipitating calmodulin from complex mixtures; and (v) inserting the antibody into cells to investigate calmodulin function. This monoclonal antibody should contribute significantly to the elucidation of the mechanism by which calmodulin regulates intracellular metabolism. ACKNOWLEDGMENTS We are indebted to the following: Dr. Hemant Vaidya and Dr. David Silva for methodological guidance and suggestions, Dr. Moon Nahm and Dr. Mitch Scott for advice and helpful discussions, Dr. Kam Fok and Joe Bulock for peptide synthesis and preparation of performic acid-treated calmodulin, Dr. Kevin Glenn and Ed Rowald for preparation of rabbit antiserum, Dr. Jolinda Traugh for generously donating casein kinase II, Dr. John Williams for providing phosphotyrosylcalmodulin, Shirley Hanna for skillfully performing fusions, Erika Sheehan and Dr. Jun Yao for expert technical assistance, and Margarita Rosado for preparing the manuscript.

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