Monoclonal antibodies to native noncollagenous bone ... - PNAS

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Dec 27, 1983 - BALB/cJ mice (The Jack- son Laboratory) were ..... massie blue and silver staining have shown a bandof human. BGP that comigrates with ...

Proc. Natl. Acad. Sci. USA Vol. 81, pp. 2868-2872, May 1984 Medical Sciences

Monoclonal antibodies to native noncollagenous bone-specific proteins (murine monoclonal antibody)

DEBRA D. STENNER, ROBERT W. ROMBERG, RUSSELL P. TRACY*, JERRY A. KATZMANN, B. LAWRENCE RIGGS, AND KENNETH G. MANNt Section of Hematology Research, Endocrine Research Unit, Department of Internal Medicine; and Department of Laboratory Medicine, Mayo Clinic Foundation, Rochester, MN 55905

Communicated by Robert W. Mann, December 27, 1983

Hybridoma technology was used for prepaABSTRACT ration of murine monoclonal antibodies of high titer against bone-Gla protein and osteonectin. A procedure of immunization and hybridization similar to that already described [Katzmann, J. A., Nesheim, M. E., Hibbard, L. S. & Mann, K. G. (1981) Proc. Nati. Acad. Sci. USA 78, 162-166; and Foster, W. B., Katzmann, J. A., Miller, R. S., Nesheim, M. E. & Mann, K. G. (1982) Thromb. Res. 28, 649-661] was used. However, in contrast to earlier studies, mice were immunized with an unfractionated protein mixture that had been extracted from bone under nondenaturing conditions. The extract was labeled with 125I by the chloramine-T method. After fusion and initial hybrid growth, screening was accomplished by a solid-phase radioimmunoassay with total 125I-labeled bovine bone protein extract as the tracer. The identities of antibodybound 125I-labeled proteins were assessed by dissolution of the solid-phase immune complex in NaDodSO4 and subsequent electrophoresis and autoradiography. Clones producing specific antibody to a single protein were selected by limiting dilution. The identity of the proteins against which the specific antibodies were produced was confirmed by immunoprecipitation, electrophoresis, and autoradiography. From two fusions, 30 positive hybrids to bone-Gla protein were identified; 7 of these were subcloned and 1 has been expanded as an ascites tumor. One hybrid population was positive for osteonectin, a Mr 15,000 peptide, and for bone-Gla protein. By limiting dilution, the osteonectin clone was selected and subsequently expanded as an ascites tumor. Titration curves made using the respective 1251-labeled purified proteins show the ascites tumors to be producing antibody of high titer (I50 = 10-6) for anti-bone-Gla protein and (I50 - 10-5) for antiosteonectin. Both of the antibovine antibodies are cross-reactive with the corresponding human protein. Immobilized specific antibone-Gla protein has been used to isolate human bone-Gla protein from an EDTA extract of human cortical bone. Thus, this method offers the possibility of developing a complete library of monoclonal antibodies against these and other bonespecific proteins.

cal bone have shown this noncollagenous fraction to contain at least 15 bone-specific proteins (including actin) (4). Considerable interest exists in the role of the noncollagenous bone-specific proteins in the dynamics of bone resorption and formation. The isolation and characterization of these proteins, however, is still not thoroughly understood. At present only one, bone-Gla protein (BGP) or osteocalcin, has been characterized thoroughly. It is a 49-amino acid peptide containing three y-carboxyglutamic acid residues and is known to bind selectively to hydroxyapatite; thus, it may play a role in bone mineralization (5-9) although its functional role is still not conclusively known. Another protein, osteonectin, was isolated under denaturing conditions by Termine in 1981 (10). It is a bone-specific glycoprotein with calcium-binding properties (10). Studies in our laboratory have shown osteonectin to be extractable under nondenaturing conditions with EDTA alone and thus to retain more of the native conformation and physical properties (11). Previous efforts to isolate these proteins have proceeded by classic techniques involving extensive dialysis against EDTA or EDTA/denaturant to solubilize the proteins, followed by ion exchange or gel filtration chromatography. In an effort to isolate these proteins and to develop radioimmunoassays to follow their involvement in various disease states, we have chosen an immunological approach to their isolation. This approach involves immunization of mice with unfractionated solubilized proteins and hybridoma technology to isolate antibody-producing cells specific for each protein. The isolated antibodies are then used with affinity techniques in the subsequent isolation of the corresponding proteins. This manuscript is a report of the production of two such hybridoma antibodies and the use of one of them to isolate human bone-Gla protein. A preliminary account of this work has been presented.t

MATERIALS AND METHODS Antigen Preparation. Tibias from freshly slaughtered cows

were scraped free of connective tissue and periosteum and the cortical bone was powdered with a vertical milling machine (Division of Textron, Bridgeport, CT). The powdered bone was placed in a sintered glass funnel and washed thoroughly with cold distilled water and then with acetone to remove lipids. The washed powder was dried under vacuum

Whole cortical bone is 69% inorganic material, 22% organic material, and 9% water. The inorganic matter consists of hydroxyapatite [Ca10(PO4)6(OH)2] and other mineral complexes; the organic material is 90% insoluble collagen, while 10% is noncollagenous protein (1). The collagen found in bone is type I and is identical to nonmineralized tissue type I collagen (2). The noncollagenous fraction, which is less well defined, represents about 2% of the total bone mass (1) and contains both specific bone proteins and serum proteins that are concentrated in bone (3). Two-dimensional electrophoretograms of EDTA and EDTA/guanidine extracts of corti-

Abbreviations: BGP, bone-Gla protein (or osteocalcin); Tris/NaCi,

0.05 M Trizma base/0.1 M NaCI/0.2% NaN3, pH 7.3; Pi/NaCl, phosphate-buffered saline. *Present address: Clinical Pathology Laboratory, University of Rochester Medical Center, Box 608, 601 Elmwood Avenue, Rochester, NY 14642. tTo whom reprint requests should be addressed. tAmerican Society of Bone and Mineral Research Meeting, San Antonio, TX, June 5-7, 1983. Stenner, D. D., Romberg, R. W., Tracy, R. P., Katzmann, J. A., Riggs, B. L. & Mann, K. G. (1983) Calcif. Tissue Int. 35, 665.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.


Medical Sciences: Stenner et aL and stored at -20TC. One hundred grams of dried bone was placed in approximately 2 ft of Spectrapor no. 3 dialysis tubing (Mr cutoff, 3500; Spectrum Medical Industries, Los Angeles, CA 90054) and demineralized by extensive dialysis (57 days) against 4 liters of 0.5 M EDTA/10 mM benzamidine, pH 7.4, at 40C. After dialysis, the material retained was centrifuged, and the supernatant was desalted at room temperature by gel filtration over a Sephadex G-25 column equilibrated with 50 mM ammonium bicarbonate. The material was then lyophilized. To determine the presence of bonespecific proteins, electrophoresis in two dimensions was carried out according to a modification of Anderson and Anderson (12) previously described (13), and chromatograms were compared with chromatograms of serum samples analyzed similarly. Immunization and Cell Fusion. BALB/cJ mice (The Jackson Laboratory) were injected intraperitoneally with 100 Ag of lyophilized bovine bone extract emulsified in 0.2 ml of 50% Freund's complete adjuvant and 0.01 M Na phosphate/0.15 M NaCl, pH 7.4 (P1/NaCl). Ten to fourteen days after the primary injection, a booster injection was given in either Freund's incomplete adjuvant or Pi/NaCl. Three days after the second injection, the mouse was sacrificed and the spleen was removed under sterile conditions. Spleen cells (108) were suspended in RPMI 1640 medium (GIBCO)/10% fetal calf serum. NS-1 mouse myeloma cells (107) were added to the spleen cells, pelleted, and washed in medium containing no fetal calf serum. Cells were then fused by a modification of the procedure described by Kennet et al. (14) previously described (15, 16). Radioimmunoassay. To measure antibody production, we used a solid-phase double-antibody technique utilizing immobilized rabbit antimouse IgG as the primary antibody (Cappel Laboratories, Cochranville, PA). To coat tubes with the rabbit anti-mouse IgG, -2 ,g of rabbit IgG in 0.3 ml of 0.05 M sodium carbonate buffer (pH 9.5) was placed in polystyrene test tubes (Scientific Products T1226-22) and incubated at 4°C overnight. To remove excess IgG and block unoccupied sites, tubes were washed twice with 0.5 ml of buffer containing 0.05 M Trizma base/0.1 M NaCl/0.2% NaN3, pH 7.3 (Tris/NaCl), and once with 0.5 ml of TrisINaCl/1% bovine serum albumin. Coated tubes could then be used immediately or stored at -20°C with 0.3 ml of the final wash buffer. All liquid was removed before tubes were used. A 0.2-ml aliquot of culture supernatant from each well was added to the coated tube and incubated at 4°C for 4-6 hr. For negative control, 0.2-ml aliquots of HAT medium (hypoxanthine/aminopterin/thymidine) and a 1:100 dilution of normal mouse serum were also assayed. After incubation, the supernatants were aspirated and discarded, and tubes were washed 3 times with 0.5 ml of Tris/NaCl/0.1% bovine serum albumin. To determine the presence of possible monoclonal antibodies that may have bound to the rabbit anti-mouse IgG, 0.2 ml of 125I-labeled bovine bone proteins containing 60,000 cpm in Tris/NaCl/0.1% bovine serum albumin was aliquoted to the tubes and incubated at 4°C for 4-6 hr. The labeled bovine bone extract was the same as used for immunization and was prepared by the chloramineT method of radioiodination. At the end of the final incubation, label was aspirated, and tubes were washed three times with 0.5 ml of Tris/NaCl; radioactivity was assayed with a Beckman 8000 gamma counter to determine positives. Positives are defined as those tubes containing at least twice as many cpm as tubes to which HAT medium and normal mouse serum had been added. Electrophoresis and Autoradiography. The identity of tint antigen or antigens present was determined by electrophoresis and autoradiography. The first step in this method is the addition of 0.070 ml of 1% NaDodSO4 in 0.04 M Tris borate directly to the test tubes. The tubes are then incubated at

Proc. NatL. Acad. Sci. USA 81 (1984)


90'C for S min. Next, a drop of bromphenol blue in 50% glycerol/water is added and either the entire solution or an aliquot containing 2000 cpm is subjected to electrophoresis on a 5-15% linear gradient polyacrylamide slab gel as described by Neville (17). Labeled compounds of known molecular weight and labeled purified bone proteins used in the radioimmunoassay were analyzed for comparison. After electrophoresis, the slab gel was dried on filter paper in a Hoefer Scientific model SE 1150 slab gel dryer (Hoefer, San Francisco). The slab gel was then transferred to a wafer radioform cassette (Halsey X-ray Products, Brooklyn, NY) and Kodak XR-5 film (Kodak) was placed on top. The wafer was placed at -70TC for 48-72 hr. Length of exposure is dependent on cpm per sample electrophoresed. The film was developed by the x-ray Department of the Mayo Clinic. Subcloning and Passage as Ascites Tumors. Parent wells (initial wells after fusion) may contain a number of hybrid populations. To separate the populations and prevent overgrowth of antibody-secreting cells by nonproducers, the wells were subcloned as soon as possible. Fresh "feeder" cells were prepared by washing the peritoneum of one 8week-old female BALB/cJ mouse (The Jackson Laboratory) with 3 ml of 0.34 M sterile sucrose. The cells recoverd were spun and suspended in 25 ml of RPMI 1640 medium/10% fetal calf serum, and one drop was added to each well of five 96-well microculture plates and incubated at 370C overnight. The cells from the parent well were dispersed as a homogenous suspension and an aliquot of each was counted on a hemacytometer. Viability counts were carried out by diluting the cells 1:1 with trypan blue. That volume containing 2 x 104 viable cells was then removed and diluted to 1 ml with medium. At this point, two dilutions were done, one that resulted in 100 cells per 5 ml and another that contained 20 cells per 5 ml. The two dilutions were then distributed to the feeder plates, each plate receiving a total of 5 ml (0.05 ml per well), yielding plates having 1 cell per well and plates having 1 cell per 5 wells. Once growth had been achieved, supernatants were again tested for antibody production by radioimmunoassay and for single antigenic specificity by autoradiography. Antibody-positive clones were expanded into 24well Costar plates and eventually into small tissue flasks before overcrowding occurred. Each transfer required the addition of new feeder cells. Seven days prior to the inoculation of mice with hybridoma clones, the mouse was primed with 0.3 ml of sterile pristane delivered intraperitoneally. One to 1.5 x 106 cells were then injected into each mouse. Cloned hybridoma cells were also frozen in culture medium/10% fetal calf serum/5% dimethyl sulfoxide under liquid nitrogen for future use. When mice begin to show symptoms of tumor growth, they can be tapped for ascites fluid. This was carried out by first wiping the abdomen of the mouse with 70% alcohol and then inserting a sterile 19-gauge needle into the peritoneum and allowing the fluid to drop into a test tube. Two to four milliliters of ascites fluid was usually obtained from each mouse, and the mice could be tapped two or three times before death occurred. The ascites fluid was then spun at approximately 350 x g for 10 min to remove cellular and particulate matter. Usually the passage of clones as ascites tumors resulted in a 103- to 104-fold increase in antibody titer, which was determined by the solid phase radioimmunoassay. A titration curve was also prepared using labeled human bone extract (prepared the same as bovine) as tracer. Isolation of Immunoglobulin from Ascites Fluid. Isolation of the specific Ig from the ascites fluids was achieved by two methods. For the fluid containing anti-BGP antibodies, gel filtration over an Ultrogel AcA 34 column at 220C was used. Elution buffer for the AcA column was 0.01 M sodium phosphate/0.15 M NaCl, pH 7.3/0.02% azide. A 70% (NH4)2SO4 cut was made on the IgG peak, and the pellet was suspended


Medical Sciences: Stenner et aLPProc. NatL Acad ScL USA 81

in PBS with 15% glycerol for storage at -20'C. A two-dimensional electrophoretogram of the concentrate showed relatively pure monoclonal IgG. Purification of the anti-osteonectin antibodies was by protein A-Sepharose chromatography (18). Elution of the various protein peaks was by a stepwise pH gradient, 0.1 M phosphate (pH 8.0), 0.1 M citrate (pH 6), and 0.1 M citrate (pH 3). All buffers for the protein A column contained 0.05% sodium azide. Both the two-dimensional electrophoretogram of the pH 6 peak and determination of the IgG subclassification by immunoelectrophoresis showed it to be pure monoclonal IgG1. The pH 6 peak was concentrated and stored in the same manner as the anti-BGP. Isolation of Human BGP Through Use of Monoclonal Antibody. Anti-BGP IgG (3.5 mg) was covalently linked to 10 ml of cyanogen bromide-activated Sepharose (19). The immunoreactive gel was washed with 0.05 M Trizma base/0.1 M NaCl/0.02% NaN3, pH 7.3, and then added to a solution containing 10.3 mg of human bone extract prepared the same as the bovine extract and dissolved in the above buffer. After overnight incubation at 40C, the entire suspension was placed into a column and 2-ml fractions were collected. When the absorbance at 280 nm of the effluents was at baseline, the column buffer was switched to 0.1 M glycine (pH 2.8), and 2-ml fractions were collected into 1 M Tris base (pH 8.5) to readjust the pH to between 7 and 8. Glycine fractions containing protein as determined by the absorbance at 280 nm were pooled and concentrated by addition of (NH4)2SO4. The protein pellet was then dialyzed against 50 mM (NH4)2CO3 to remove (NH4)2SO4 and subsequently lyophilized and stored at -20°C.

RESULTS The first step in clonal selection involved screening the initial 96 culture wells by radioimmunoassay and resulted in a total of 30 hybrids from two fusions that were positive for antibody production. Dissolution of the antibody-125I-labeled antigen complex contained on the test tube wall with 1% NaDodSO4 followed by electrophoresis and subsequent autoradiography showed the positive supernatants to contain at least three distinct antibody populations. The initial autoradiographic analysis of the antibody-producing wells is shown in Fig. 1. Culture supernatant from parent well IIIA3 bound bone-Gla protein, an unidentified peptide with molecular weight of -15,000, and osteonectin to the test tube (lanes 1 and 2). The remaining culture supernatants bound only BGP and the Mr 15,000 peptide (lanes 3 and 4). When cells from the parent antibody-producing well, IIIA3, were distributed to yield one clone per five wells, we were able to separate the antibody-producing populations (Fig. 2) into those directed against BGP, those against osteonectin, and possibly a third against a Mr 15,000 peptide and BGP; this third population has not been characterized. Continued culturing and eventual passage of these specific cell suspensions as ascites tumors resulted in the production of high titer monoclonal antibodies in 2-4 wk. Two of the ascites fluids, IIIA3A8 and IIIA3A1O, were positive for antiosteonectin antibodies and one, IA520IVG-12, was positive for BGP antibodies. Titration curves for the anti-BGP and the anti-osteonectin ascites fluids were made by using the respective labeled purified bovine proteins. Half-maximal binding for the ascites fluids was 10-5 for both the antiosteonectin tumors and 10-6 for the one anti-BGP tumor. The ascitic fluid titer for the anti-osteonectin antibodies is shown in Fig. 3. Specificity and cross-reactivity of the antibody-containing fluids was tested by comparing the reactivity of the ascites fluids with labeled bovine bone extract, purified labeled bo-



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FIG. 1. Autoradiograph of antibody-positive material from the initial screening. To each of the tubes containing material that bound 1251I-labeled bovine bone extract, 0.070 ml of 1% NaDodSO4 in Tris borate was added. The mixtures were incubated at 900C for 5 min and the solution was then applied to a 5-15% linear gradient polyacrylamide slab gel. After electrophoresis, the slab gel was dried onto filter paper, exposed to Kodak film, and developed to identify relative molecular weights of the complexed 1251-labeled antigen. Lanes: 1 and 2 represent well IIIA3, which contains three antibodyproducing populations; 3 and 4, a representative sample of the remaining 12 positive wells; 5-8, background radioactivity bound to test tubes containing medium but not antibody; 9 and 10, aliquots of 115I-labeled bovine bone EDTA extract and 1251I-labeled standards of known molecular weight, respectively.

vine bone proteins, and labeled human bone extract. For all experiments, the ascites fluid antibody bound only the appropriate protein. These data are shown in the autoradiographs of Figs. 4 and 5. Fig. 4 shows the cross-reactivity of



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FIG. 2. Autoradiograph from the subclone screening of parent well IIIA3. A suspension of 20 hybrid cells was divided among % wells, yielding 1 cell per 5 wells. Culture supernatants from these % wells were screened by using the solid-phase radioimmunoassay with '25I-labeled bovine bone EDTA extract as tracer. Subsequent electrophoresis and autoradiography of the complexed antigens showed separation of the three antibody-producing populations. lanes 1 and 2 are 1251I-labeled standards of known molecular weight and 1251I-labeled bovine bone EDTA extract, respectively; lanes 3 and 4 are from the initial screening. The remaining lanes show the separation of the three antibody populations by their ability to complex only one antigen from the bone mixture. There were two clones that bound osteonectin (lanes 5, 6 and 9, 10) and one that bound the M, 15,000 peptide and BGP (lanes 7 and 8); the remaining clones bound only BGP (lanes 11-13).

Medical Sciences: Stenner et aL

Proc. NatL. Acad. Sci. USA 81 (1984) I







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FIG. 3. Titer curve of the anti-osteonectin ascitic fluids. Both the I11A3A8 clone (o) and the I11A3A10 clone (o) exhibit half-maximal binding at a i:iO0 dilution of ascites fluid. Results have been corrected for nonspecific binding.

the anti-bovine BGP ascites fluid, and Fig. 5, the anti-bovine osteonectin ascitic fluid cross-reactivity. Immobilization of the anti-BGP immunoglobulin onto Sepharose 4B by covalent linkage through cyanogen bromide activation resulted in an affinity resin with 0.35 mg of specific IgG per ml of resin. Use of this affinity column allowed a one-step purification of human BGP from an EDTA extract of human cortical bone. As shown in Fig. 6, the human BGP isolated by this method was free of contaminating substances and comigrated with bovine BGP isolated by the procedure of Price et al. (9). The higher molecular weight band in lane 3 is a 2-mercaptoethanol-induced artifact of the silver stain that was present in other lanes not shown on the figure and can be faintly seen in the bovine BGP lane (20).

DISCUSSION Demineralization of cortical bone with EDTA results in a mixture of at least 15 (including actin) bone-specific proteins in their presumed native state (4). This extract, when used in conjunction with hybridoma technology, theoretically has the capacity to raise monoclonal antibodies against any and all of the proteins. When coupled with clonal selection, this approach should result in a pure monoclonal population I2.






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FIG. 5. Autoradiograph showing cross-reactivity of a-bovine osteonectin. Aliquots of antibody were incubated with '25I-labeled bovine bone extract (BBEX), 125I-labeled human bone extract (HBEX), and '251-labeled bovine osteonectin (BOst) in separate assays. In all cases, the antibody bound only the appropriate protein. Lanes: 2, 5, and 8, aliquots of the BBEX, HBEX, and BOst labels; 3 and 4, 6 and 7, and 9 and 10, the immunocomplexes formed by incubation of the antibody with the label; 1, 125I-labeled standards of known molecular weight.

against any one specific protein without prior purification of the protein. Antibodies produced by this method can then be covalently linked to a solid-phase resin and subsequently used to isolate the respective proteins, resulting in a one-step protein purification method requiring only initial demineralization. Proteins obtained by this affinity chromatography will be free of contaminating peptides and therefore ideal for physical characterization experiments. Application of this approach has allowed us to generate monoclonal antibodies against two bovine bone-specific proteins, BGP and osteonectin. From a total of two fusions, seven specific clones have been obtained that produce anti-BGP antibodies and two, that produce anti-osteonectin antibodies. Of the seven anti-BGP clones, one has been expanded as


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FIG. 4. Autoradiograph showing cross-reactivity of anti-bovine BGP. Aliquots of antibody were incubated with 125I-labeled bovine bone extract (BBEX), 125I-labeled human bone extract (HBEX), and 125I-labeled bovine BGP (BBGP) in separate experiments. In all cases, the antibody bound only the corresponding protein. Lanes: 2, 5, and 7, aliquots of the BBEX, HBEX, and BBGP labels; 3 and 4, 6, and 8 the immunocomplexes formed by incubation of the antibody with the label; 1, 1251-labeled standards of known moelcular weight.

FIG. 6. Silver-stained 15% polyacrylamide slab gel showing isolation of human BGP (HBGP) by affinity purification using monoclonal anti-bovine BGP. Lanes: BBGP, classically isolated bovine BGP, which comigrates with affinity-purified HBGP; HBGP, affinity-purified HBGP. The high molecular weight contaminant in lane HBGP is a 2-mercaptoethanol-induced artifact of the silver stain.


Medical Sciences: Stenner et al.

an ascites tumor resulting in 40 mg of high-titer specific antibody with half-maximal binding at a 10-6 dilution. Both of the anti-osteonectin clones have been expanded to ascites tumors yielding a total of 28 mg of specific antibody. Halfmaximal binding for both the anti-osteonectin ascites fluids and the purified IgG is at at 10-5 dilution. Each of these antibodies exhibits cross-reactivity with the corresponding human proteins. Exploitation of this cross-reactivity has enabled rapid and simple purification of human BGP by affinity chromatography. NaDodSO4/PAGE and subsequent Coomassie blue and silver staining have shown a band of human BGP that comigrates with bovine BGP and contains no contaminating peptides. The human BGP isolated by this method reacts equivalently with classically isolated bovine BGP in our polyclonal-based double radioimmunoassay (21). All nine clones, plus cells recovered from the three populations of ascites tumors, have been frozen in dimethyl sulfoxide under liquid nitrogen and have been successfully thawed, cultured, and injected into mice. The successful freezing and thawing of these cell lines give them an immortality that allows for an endless supply of specific antibody. Immunological depletion of BGP and osteonectin from the immunization mixture should provide the opportunity to produce antibodies against and subsequently isolate other specific bone proteins by analogous procedures. We thank Randall Miller for his excellent technical assistance and Jeanne Nemitz for her patience and assistance in the preparation of this manuscript. The work was supported by Grants AM07147, AM20605, HL01035, and HL17430-D from the National Institutes of Health and by the Mayo Foundation. 1. Triffitt, J. T. (1980) in Fundamental and Clinical Bone Physiology, ed. Urist, M. R. (Lippincott, Philadelphia, PA), pp. 4582. 2. Gay, S. & Miller, E. J. (1978) in Collagen in the Physiology and Pathology of Connective Tissue, (Gustav-Fischer Stuttgart, Germany).

Proc. NatL Acad Sci. USA 81


3. Triffitt, J. T. & Owen, M. 0. (1977) Calcif. Tissue Res. 23, 303-305. 4. Delmas, P. D., Tracy, R. P., Riggs, B. L. & Mann, K. G. (1983) Calcif. Tissue Int. 35, 661 (abstr.). 5. Price, P. A., Poser, J. W. & Kaman, N. (1976) Proc. Natl. Acad. Sci. USA 73, 3374-3375. 6. Price, P. A., Otsuka, A. S. & Poser, J. W. (1977) in Calcium Binding Proteins and Calcium Function, ed. Wasserman, R. H. (Elsevier, Amsterdam), pp. 333-337. 7. Poser, J. W., Esch, F. S., Ling, N. C. & Price, P. A. (1980) J. Biol. Chem. 255, 8685-8691. 8. Hauschka, P. V., Lian, J. B. & Gallop, P. M. (1975) Proc. Natl. Acad. Sci. USA 72, 3925-3929. 9. Price, P. A., Otsuka, A. S., Poser, J. W., Kristaponis, J. & Raman, N. (1976) Proc. Natl. Acad. Sci. USA 73, 1447-1451. 10. Termine, J. D., Kleinman, H. K., Whitson, S. W., Conn, K. M., McGarvey, M. L. & Martin, G. R. (1981) Cell 26, 99105. 11. Romberg, R. W., Werness, P. G., Riggs, B. L. & Mann, K. G. (1983) Calcif. Tissue lnt. 35, 664 (abstr.). 12. Anderson, N. L. & Anderson, N. G. (1977) Proc. Natl. Acad. Sci. USA 74, 5421-5425. 13. Tracy, R. P., Currie, R. M. & Young, D. S. (1982) Clin. Chem. 28, 890-899. 14. Kennett, R. H., Denis, K. A., Tung, A. S. & Klinman, N. R. (1978) in Current Topics in Microbiology and Immunology, eds. Melchers, F., Potter, M. & Warner, N. L. (Springer, New York), Vol. 81, pp. 77-91. 15. Katzmann, J. A., Nesheim, M. E., Hibbard, L. S. & Mann, K. G. (1981) Proc. Natl. Acad. Sci. USA 78, 162-166. 16. Foster, W. B., Katzmann, J. A., Miller, R. S., Nesheim, M. E. & Mann, K. G. (1982) Thromb. Res. 28, 649-661. 17. Neville, D. M. (1971) J. Biol. Chem. 246, 6328-6334. 18. Ey, P. L., Prowse, S. J. & Jenkin, C. R. (1978) Immunochemistry 15, 429-436. 19. March, S. C., Parikh, I. & Cuatrecasas, P. (1974) Anal. Biochem. 60, 149-152. 20. Tasheva, B., Dessev, G. (1983) Anal. Biochem. 129, 98-102. 21. Delmas, P. D., Stenner, D. D., Wahner, H. W., Mann, K. G. & Riggs, B. L. (1983) J. Clin. Invest. 71, 1316-1321.

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