Proc. Nati. Acad. Sci. USA Vol. 76, No. 8, pp. 3703-3707, August 1979
Biochemistry
Production and characterization of a monoclonal antibody to chicken type I collagen (hybridoma/collagen antibodies/immunohistochemistry)
THOMAS F. LINSENMAYER, MARY J. C. HENDRIX, AND CHARLES D. LITTLE The Developmental Biology Laboratory and the Departments of Medicine and Anatomy, Massachusetts General Hospital and Harvard Medical School,
Boston, Massachusetts 02114 Communicated by Jerome Gross, May 10, 1979
ABSTRACT We have shown that lymphocyte-myeloma cell hybridization can be used to produce large amounts of extremely high-titer specific antibodies against type I collagen, a macromolecule normally of low immunogenicity. In a passive hemagglutination assay the antibody had a high titer against chicken type I collagen but showed no activity against chicken type II or rat type I collagen. By using a two-step fluorescence histochemical procedure on sections of embryonic chicken tibia, strong fluorescence was observed in the perichondrium and surrounding connective tissue (known to contain type I collagen) but not over the cartilage (characterized by type II collagen). When used in conjunction with Staphylococcus aureus as a solid phase immunoadsorbant, the antibody was shown to bind to labeled collagen synthesized in vitro by embryonic chicken calvaria.
Extracellular matrices are composed chiefly of different genetic types of collagens, glycosaminoglycans, and proteoglycans (for reviews, see refs. 1-3). During embryonic development, individual tissues and probably even individual cells are capable of synthesizing more than one genetically distinct type of collagen, in some cases giving rise to complex, hybrid matrices. The chicken corneal epithelium, for example, synthesizes collagen of types I and II (4) which are polymerized as the primary corneal stroma (3), the embryonic neural retina synthesizes type I collagen plus a previously undescribed type (5), and individual fibroblasts produce both type I and type III collagens (6, 7). Morphological and biochemical studies of these collagenous matrices have been facilitated by the development of techniques for producing antibodies to the various genetic types of collagens differing in primary structure (8, 9). The conventional methods for producing such antibodies involve raising antisera in animals and subsequently purifying the antisera by affinity chromatography. However, even after these laborious and time-consuming purifications, the resulting antibodies may still have some degree of crossreactivity. In addition, it is thought that affinity chromatographic purifications frequently result in irreversible binding and subsequent loss of the highest-affinity antibody. Many of these problems can be avoided by use of the recently devised techniques for hybridizing antibodyproducing lymphocytes with myeloma cells (10, 11). The resulting hybridomas cultured as clones produce antibody of a single, defined specificity. Here we report the construction of a clonal line of hybridoma producing high-titer specific antibody to chicken type I collagen and describe some of the characteristics of the antibody. 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.
MATERIALS AND METHODS Collagens. Neutral-salt-soluble type I collagen was extracted from the skins of 3-week-old lathyritic chickens with 0.4 ionic strength potassium phosphate buffer at pH 7.6. Type II collagen was extracted by limited 4VC pepsinization of adult chicken sternal cartilages (12). Rat type I collagen was extracted with 0.5M HOAc from adult rat tail tendons. All of the collagens were purified by multiple salt and ethanol precipitations from neutral solutions and salt precipitations from acidic solutions. In the sternal cartilage preparation, the type II collagen was separated from type I by repeated fractional salt precipitations (13). Preparation of Mouse Lymphocytes and Cell Hybridization. The parental mouse myeloma cell line MPC 11 45.6 TG 1.7 (14), was cultured in complete medium composed of Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and antibiotics. Revertant cells were periodically removed by passage in medium supplemented with 6-thioguanine (10,gg/ml). ASW/SN mice (Jackson Laboratory) were injected subcutaneously with approximately 0.2 mg of chicken type I collagen dissolved in 0.1 M HOAc and emulsified in an equal volume of complete Freund's adjuvant. A booster injection of collagen in incomplete Freund's adjuvant was given intraperitoneally about 3 weeks later. After an additional 1-2 weeks, pairs of mice were killed by cervical dislocation and the spleens were removed. The tissue was placed in serum-free medium and minced with scissors, and the cells were liberated by agitation for 1-2 min on a vortex mixer using the most vigorous setting. The spleen cells were washed twice with serum-free medium prior to fusion. Cells were hybridized by using polyethylene glycol according to Gefter et al. (15). All manipulations were done at room temperature. Myeloma cells (106) were washed twice in serum-free medium and then were mixed with 1-3 X 107 spleen cells in 50-ml conical bottom centrifuge tubes (Falcon 2070). The cells were centrifuged at 250 X g for 5 min, and the supernatant fluid was carefully aspirated. Two-tenths milliliter of 37.5% polyethylene glycol in serum-free medium was added, and the tube was gently agitated by hand to resuspend the cells. Next, the cells were centrifuged for 3 min at 250 X g and for 2.5 min at 500 X g; they then were allowed to sit undisturbed for an additional 2.5 min. Five milliliters of serum-free medium was added to the tube; the cells were resuspended gently with a pasteur pipette and were then repelleted by centrifugation at Abbreviations: HAT medium, complete medium supplemented with
hypoxanthine, aminopterin, and thymidine; RBC, erythrocyte; Pi/
NaCl, phosphate-buffered saline. 3703
3704
Biochemistry: Linsenmayer et al.
250 X g for 5 min. The supernatant was removed; the cells were dispersed in 20 ml of complete medium and were put into a 370C humidified incubator for 48 hr to allow recovery. The recovered cells were pelleted and resuspended in 100 ml of complete medium supplemented with 0.1 mM hypoxanthine, 0.4 liM aminopterin, 16 ,gM thymidine, and 3 ,gM glycine (HAT medium) (16) and plated out in four 24-well plates (Linbro). After the first 3 days the cells were fed with 1 ml of HAT medium per well; every 34 days subsequently, half of the medium was replaced with fresh HAT medium. The cells in wells positive for antibody were cloned by dilute plating in 96-well flat-bottom plates (Linbro) (17). Cloning was accomplished by limiting dilution in HAT medium so that one drop (approximately 0.07 ml) contained an average of either 1 or 0.5 cell when added to a well. Every 7 days an additional drop of HAT medium was added to each well, and after 2-3 weeks the wells were tested for antibody activity. Positive wells were recloned, and then clones were transferred first into HAT medium without aminopterin and finally into complete medium. Large amounts of antibody were obtained from spent culture medium harvested from 150-mm plates or roller bottles. Medium was subsequently concentrated by using a hollow-fiber concentrator (Amicon, Lexington, MA). Alternatively, antibody was obtained from the ascites fluid of athymic nude mice (nu/nu) that had been injected 2-3 weeks previously with 107 cloned hybridoma cells. The ascites fluid was diluted with saline to prevent the formation of a gel, which rapidly occurred in undiluted fluid. Then the peritoneal cavity was flushed with saline, and the total diluted fluids were pooled for each mouse. Immunological Procedures. Antibody activity was routinely assayed by passive hemagglutination performed according to Beil et al. (18). Briefly, collagens (0.3% solutions in 0.1 M CaOAc) were coupled to human type 0 erythrocytes (RBCs) (10% solution) with glutaraldehyde. Then, 100,-l aliquots of the derivatized erythrocytes (diluted 1:20) were added to 100-,ul portions of antibody-containing solution that had been serially diluted in round-bottom microtiter plates, and the plates were allowed to develop overnight at 4°C. Immunoglobulins were characterized by immunoelectrophoresis on 1% agarose-coated glass microscope slides using 0.08 ionic strength barbital buffer (pH 8.2 or 8.6) (19). Electrophoresis was performed for 1-3 hr at 6-8 mA per slide. Subsequently, 100 Al of antiserum was added to the center trough and the slides were allowed to develop for 3 days at 4°C. The slides were viewed by indirect lighting and also after staining with Coomassie blue. Immunohistochemistry. Sections were stained with antibodies by using a double-layered, sandwich technique. Tibias were removed from 14-day chicken embryos and fixed in 1% paraformaldehyde for 15 min; then free aldehyde groups were quenched in 0.15 M Tris-HCI at pH 7.4. The tissue was quickfrozen to liquid N2 temperature, and 8-,um sections were cut on a cryostat. Sections were air dried onto albumin-coated slides at room temperature for 1-2 hr and were then treated with testicular hyaluronidase in phosphate-buffered saline (4000 units/ml) for 20 min at 370C (20). Subsequently, the sections were treated with the hybridoma antibodies, either concentrated or diluted with phosphate-buffered saline (Pi/NaCl) for 1 hr at room temperature, followed by washing for 1 min each in three changes of Pi/NaCl. The secondary antibody, consisting of rhodamine-conjugated IgG fraction of rabbit anti-mouse IgG (Cappel Laboratories, Cochranville, PA, lot 10169; diluted 4: 1500 with Pi/NaCl) was applied and allowed to react with the section for 1 hr in the dark. The sections were rinsed as above
Proc. Nati. Acad. Sci. USA 76 (1979)
and were then coated with 90% (vol/vol) glycerol in Pi/NaCl, before coverslips were attached. Slides were viewed in a Zeiss photomicroscope III with the rhodamine filter set. Immunoprecipitation Reaction with Labeled Collagen. The labeled collagen was obtained from cultures (21) of whole, lathyritic 18-day embryonic calvaria that had been labeled in vitro with [3H]proline. The collagen was extracted in 0.4 ionic strength potassium phosphate buffer at pH 7.6- and, to remove noncollagenous proteins, was twice treated at 40C with pepsin (0.1 mg/ml) for 24 hr in 0.5M HOAc. Prepared S. aureus was obtained from The Enzyme Center (Boston, MA) and was used according to Kessler (22). The bacteria were reconstituted and washed twice in assay buffer (0.05 M Tris-HC1, pH 7.5/0.15 M NaCl) containing 0.5% Triton X-100; then, for use in the assay, they were resuspended at a concentration of 10% (vol/vol) in buffer containing 0.05% Triton and bovine serum albumin (1 mg/ml). The preparation was used within 1 week. For assay, 20-50 til of labeled collagen in immunological assay buffer were pipetted into 1.5-ml conical centrifuge tubes and then varying amounts of antibody that had also been dialyzed into assay buffer were added. Volumes were equalized by addition of assay buffer and the tubes were shaken for 4 hr at 240C and then left overnight at 40C. Equal volumes of S. aureus reagent were then added to each tube such that the total IgG in the tube containing the maximal amount of IgG would be quantitatively bound, and the tubes were incubated as above. After incubation, the tubes were centrifuged twice in a Beckman Microfuge-B, and the precipitate was washed twice in the Triton-containing buffer. The washings were pooled with the supernatants. The pellets were extracted twice (2% sodium dodecyl sulfate/0.0625 M Tris-HCl, pH 6.8) with each extraction being shaken for 2 hr at 370C and then for 10 min at 100°C. These extraction supernatants were pooled. In some experiments, antibodies were treated with denatured instead of native collagen and all incubations were performed at 370C to prevent renaturation. All fractions were mixed with 4 ml of Hydrofluor (National Diagnostics, Somerville, NJ) and assayed for radioactivity in a Beckman scintillation counter. RESULTS Six separate cell hybridizations were performed, and more than 400 wells of 24-well plates were tested for antibody production by passive hemagglutination. The medium from one well, I1B6, was positive when tested with RBCs coated with chicken collagen type I.* The hybridoma cells from this well were cloned in 96-well microtiter plates, and some positive clonal wells were recloned, giving rise to several hundred antibody-positive subclones. Most of these were frozen for future studies. Four clones were grown to mass culture densities and used to produce large amounts of antibody-containing medium and ascites fluid. The passive hemagglutination data on the titers (expressed as -log2) and the specificity of the antibody are summarized in Table 1. The spent culture medium from pooled I1B6 subclones (which could be obtained in liter amounts) had an average titer of 6-7 (-log2) when tested on chicken type I-coated RBCs. After an approximately 20-fold concentration by Amicon filtration, the titer increased to 11-13 (-log2). This concentrated medium had an equally high titer when tested against RBCs coated with chicken type I collagen that had been *
In more recent experiments we have been isolating clones of cells producing antibody to type II collagen. The frequency of production of antibody-producing cells was raised to 1 in 100 wells of 24-well plates by using splenic cells removed 3 days after the animals had been given a booster dose of antigen.
Biochemistry: Linsenmayer et al. Table 1. IB6 hybridoma titers RBC collagen
Antibody source
type*
Proc. Natl. Acad. Sci. USA 76 (1979)
MS
Titer, -log2 6-7
I Spent medium 11-13 I Concentrated medium (20X) I (pepsinized) 11-12 Concentrated medium (20X) 0 II Concentrated medium (20X) 0 I, rat tail Concentrated medium (20X) Concentrated medium (20X), ;1o I DEAE purified 15->17 I Ascites fluid (diluted 1:4) 14 I (pepsinized) Ascites fluid (diluted 1:4) II 0 Ascites fluid (diluted 1:4) * All chicken collagen, except line 5 was rat tail collagen.
previously treated with pepsin at 4VC to remove the nonhelical telopeptide ends-thus demonstrating the antigenic determinants to be in the helical portion of the molecule. The 20X concentrated antibody-containing culture medium showed no reactivity with RBCs coated with chicken type II collagen or rat tail tendon type I collagen. An even higher-titered antibody could be obtained by growing the 11B6 cells in the peritoneal cavity of athymic nude mice and harvesting the ascites fluid. The yields of diluted ascites fluid were 2045 ml per animal. The diluted ascites fluid from eight different animals had titers of 15->17 (-log2) when tested against RBCs coated with intact or pepsin-treated chicken type I collagen. After affinity purification using Sepharose-bound type I collagen, the resulting material had an immunoglobulin concentration of 0.09 mg/ml and a titer of 11-12 (decreased by dilution during purification). No detectable activity against type II coated cells was observed. The class of immunoglobulin responsible for the antibody activity was determined by immunoelectrophoresis of concentrated medium from cultures of parental cells (MPC 11) and hybridoma cells (11B6) and whole mouse serum. Fig. 1 shows immunoelectrophoresis profiles of reactions with antibody to whole mouse serum and to mouse IgG. With anti-MS, both the MPC parent and the hybridoma gave a single, sharp arc whereas the whole mouse serum, as expected, gave numerous reaction products. Against the anti-IgG, both parental cell and hybridoma media gave single arcs as did the mouse serum. However, the arc produced by the serum was much longer than that produced by the medium of either cell line. The only detectable difference between the hybridoma and the parent was the slightly longer arc of the latter. Subsequently, Kurt Bloch (Massachusetts General Hospital) kindly performed doublegel-diffusion analyses of MPC 11 and 11B6 media to test their reactivity with antisera against IgG heavy chain subclasses y2a, y2b, y3, and -y. Both cell lines reacted only with anti-'y2b sera; no reaction was obtained with anti-IgA. The antibody showed a high degree of specificity when used in a double-antibody sandwich technique. Cryostat sections of 14-day embryonic chicken tibia, including both cartilage and noncartilagenous regions, were treated with hyaluronidase to remove proteoglycan and thus unmask the cartilage collagen (10). Then the sections were treated with the monoclonal antibody and subsequently with rhodamine-conjugated rabbit IgG directed against mouse IgG. Sections treated with anti-type I collagen ascites fluid (Fig. 2b) showed the brightest fluorescence in the perichondrial region. Loose connective tissue also showed bright staining. The matrix in the cartilaginous regions, on the other hand, was usually negative or at most had only a slight background fluorescence. No fluorescence was detected when rhodamine-conjugated antiserum to mouse IgM heavy chains was used as a second reagent.
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H FIG. 1. Immunoelectrophoretic profiles of whole mouse serum (MS) and concentrated medium from cultures of 11B6 hybridoma (H) or MPC 11 parental (MPC) cells. The top three slides were allowed to react with rabbit antiserum made against whole mouse serum (anti-MS); the bottom two were allowed to react with the IgG fraction of goat antiserum made against mouse IgG (anti-IgG).
To verify that the collagen in the cartilaginous region had been unmasked by the hyaluronidase treatment and was available to antibody binding, identical sections were treated with culture medium from another of our hybridoma lines (as yet uncloned) making antibody to chicken type II collagen (Fig. 2a). These sections showed bright fluorescence of the cartilaginous extracellular matrix but no staining of the perichondrium. In the section shown here, we used the concentrated ascites fluid (titer, 15 -log2). However, ascites fluid diluted 1:200 (the highest dilution so far tested) and culture medium concentrated 4X gave equally bright fluorescence. We tested the antibody for its ability to interact with pepsin-treated proline-labeled collagen obtained from cultures of chicken embryo calvaria. Preparatory experiments (data not shown) showed that the IgG synthesized by both the parental and hybridoma cell lines would bind to the S. aureus cells. Thus, the bacterial cells were used as a solid-phase immunoabsorbant
Biochemistry: Linsenmayer et al.
3706
Proc. Natl. Acad. Sci. USA 76 (1979)
14 .0
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FIG. 2. Sections from the cartilaginous regions of 14-day embryonic chicken tibias treated first with monoclonal antibody to type I collagen (b) or to type II collagen (a) and then with rhodamineconjugated IgG fraction of rabbit anti-mouse IgG. The antibody source was undiluted ascites fluid for type I and antibody was 4X concentrated culture medium for type II. CA, cartilage; P, perichondrium; CT, connective tissue. (A, X320; B, X 200.)
to precipitate the antigen-antibody complex. Each experiment included controls run using affinity-purified chicken type I collagen antibodies that had been raised in rabbits (20). The antibody produced in rabbits (Rab. AB I) was added in increasing concentrations until a plateau of precipitable counts was reached at about 50% of the total counts in the samples (Fig. 3 upper). The monoclonal antibody IB6 (Fig. 3 lower) behaved similarly; however, 31% of the counts was the maximum precipitated with it. No additional radioactivity was brought down when the tubes showing maximal precipitation were treated with a second quantity of monoclonal antibody. In both monoclonal antibody precipitates and supernatants, 44% of the proline was present as 4-hydroxyproline. No counts above background were precipitated when the substrate had been thermally denatured.
DISCUSSION When compared with conventional antibodies raised in animals, the monoclonal antibodies produced by hybridoma cells potentially have a number of highly desirable characteristics including a single definable specificity, reproducibility, and amounts available (see ref. 11). This technique has exceptional promise for the production of large amounts of high-titer, non-crossreacting antibodies to closely related antigens, such as the different genetic types of collagens. After cloning, the anti-type I-producing hybridoma (11B6) maintained antibody production for more than 6 months before loss of activity was detected (numerous early passages of sub-
I...... A*-
.......
10 20
.
..................
50 1I1B6 AB, ml
100
FIG. 3. Precipitation of [3H]proline-labeled calvaria collagen with increasing amounts of anti-type I collagen IgG purified from either rabbit serum (Rab. AB. I) (Upper) or IB6 culture medium (I1B6 AB) (Lower). *, Precipitates; 0, supernatants; &, precipitates with heat-denatured collagen.
clones have been frozen). When tested by passive hemagglutination for anti-chicken type I collagen activity, nude mouse fluid had titers of 15->17 serial dilutions. Because fluid had been diluted at least 1:4 with Pi/NaCl, the highest titers were in reality >19, thus giving detectable antibody response at a direct dilution of greater than 1:0.5 X 106. Even at the very high antibody titers obtainable in the concentrated ascites fluid, when tested by hemagglutination the antibody contained no detectable crossreactivity to either a closely related genetic type of collagen from the same species (chicken, type II) or the same genetic type from a different species (rat, type I). The antibody reacted equally well with chicken type I collagen from which the nonhelical extensions had been enzymatically removed, thus localizing the antigenic determinant to the helical domain. Because the antibody failed to bind to denatured collagen, the triple helical conformation is required to generate the antigenic site (8, 9). The affinity with which this monoclonal antibody binds to collagen has not yet been quantitatively determined. Operationally, however, the antibody remains bound during the washing procedures used for immunohistochemistry and in assay of binding to labeled collagen. The subclass of immunoglobulins responsible for antibody activity appears to be identical to that being produced by the parental myeloma cell line. In immunoelectrophoretic analysis, the similarly migrating single arcs of anti-IgG crossreacting material that had been synthesized by both parental and hybridoma cell lines suggest that each is making the same subclass of IgG previously shown to be synthesized by MPC 11 (14). In
Proc. Natl. Acad. Sci. USA 76 (1979)
Biochemistry: Linsenmayer et al.
3707
addition, protein A precipitated the same relative proportion (approximately 75%) of the medium proteins synthesized by the two different cell lines. These results suggest that the lymphocyte that fused with the MPC 11 cell to produce the anticollagen synthesizing hybridoma cell was, most probably, itself synthesizing IgG of the 'y2b class. At present we do not know why the type I monoclonal antibody interacted with only about one-third of the collagen synthesized by calvaria. Several preparations of labeled, twice-pepsinized calvaria collagen behaved similarly; because these had 44% of their prolines hydroxylated, they were free of contamination. Because the calvaria are membrane bones, only type I collagen is present. Under identical conditions, the monoclonal antibody precipitated considerably less collagen than did different batches of conventional antibodies against chicken skin type I collagen raised in rabbits. We suggest that the antibody may recognize a subclass of type I collagen, reflecting either primary sequence differences or posttranslational modifications. Such collagen subclasses could also explain why the rabbit antibodies, for which skin type I collagen served as both immunogen and affinity purification agent, reacted only partially with calvaria collagen. Evidence for such subclasses of type I collagen also comes from somatic cell genetic analysis indicating that genes for human-type I collagen are found on both chromosomes 17 (23) and 7 (24). It is also known that posttranslational modifications such as hydroxylations and glycosylations can be incomplete (25, 26). An extensive test of 11B6 monoclonal antibody on the type I collagen synthesized by a number of different tissues from various stages of embryos may provide some clarification.
19. Hudson, L. & Hay, F. C. (1976) Practical Immunology (Black-
We gratefully acknowledge the excellent technical assistance of Eileen Gibney. We thank Drs. Jerome Gross and Elizabeth Hay for encouragement and helpful discussions during the course of this work. We also thank Drs. Bryan Clark and Jerry Schwaber for helpful technical suggestions. This is publication no. 780 of the Robert W. Lovett Memorial Group for the Study of Diseases Causing Deformities. This research was supported by National Institutes of Health Research Grants EY02261, AM03564, and HD00143. T.F.L. is recipient of National Institutes of Health Research Career Development Award AM00031; M.J.C.H. is a National Institutes of Health Postdoctoral Fellow (HL05682); and C.D.L. is a Fellow of the Damon RunyonWalter Winchell Cancer Foundation.
20. von der Mark, H., von der Mark, K. & Gay, S. (1976) Dev. Biol. 48,237-249. 21. Linsenmayer, T. F., Toole, B. P. & Trelstad, R. L. (1973) Dev. Biol. 35, 232-239. 22. Kessler, S. W. (1975) J. Immunol. 115, 1617-1624. 23. Raj, C. V. S., Church, R. L., Klobutcher, L. A. & Ruddle, F. H. (1977) Proc. Natl. Acad. Sci. USA 74,4444-4448. 24. Sykes, B. & Solomon, E. (1978) Nature (London) 272, 548549. 25. Bornstein, P. (1967) Biochemistry 6,3082-3093. 26. Balian, G., Click, E. M., Hermodson, M. A. & Bornstein, P. (1972) Biochemistry 11, 3798-3806.
1. ;(Gross, J. (1974) Harvey Lect. 68, 351-432. 2. Toole, B. P. & Linsenmayer, T. F. (1977) Clin. Orthop. Rel. Res. 129,258-278. 3. Hay, E. D., Linsenmayer, T. F., Tretstad, R. L. & von der Mark, K. (1979) in Current Topics in Eye Research, eds. Zadunaisky, J. A. & Davson, H. (Academic, New York), pp. 1-35. 4. Linsenmayer, T. F., Smith, G. N. & Hay, E. D. (1977) Proc. Natl. Acad. Sci. USA 74,39-43. 5. Linsenmayer, T. F. & Little, C. D. (1978) Proc. Natl. Acad. Sci. USA 75, 3235-3239. 6. Church, R. L., Yaeger, J. A. & Tanzer, M. L. (1974) J. Mol. Biol. 86,785-799. 7. Gay, S., Martin, G. R., Muller, P. K., Timpl, R. & Kuhn, K. (1976) Proc. Natl. Acad. Sci. USA 73,4037-4040. 8. Furthmayr, H. & Timpl, R. (1976) Int. Rev. Connect. Tiss. Res.
7,61-99. 9. Timpl, R. (1976) in Biochemistry of Collagen eds. Ramachandran, G. N. & Reddi, A. H. (Plenum, New York), pp. 319-375. 10. Kohler, G. & Milstein, C. (1975) Nature (London) 256, 495497. 11. Melchers, F., Potter, M. & Warner, N. (1978) Lymphocyte hy-
bridomas (Springer, New York).
12. Miller, E. J. (1971) Biochemistry 10, 1652-1659. 13. Trelstad, R. L., Kang, A. H., Toole, B. P. & Gross, J. (1972) J. Biol. Chem. 247, 6469-6473. 14. Margulies, D. H., Kuehl, W. M. & Scharff, M. D. (1976) Cell 8, 405-415. 15. Gefter, M. L., Margulies, D. H. & Scharff, M. D. (1977) Somatic Cell Genet. 3, 231-236. 16. Littlefield, J. W. (1964) Science 145,709-710. 17. Orkin, S. H., Harosi, F. I. & Leder, P. (1975) Proc. Natl. Acad. Sci. USA 72,98-102. 18. Beil, W., Furthmayr, H. & Timpl, R. (1972) Immunochemistry
9,779-788.
well, London).
Biochemistry
Production and characterization of a monoclonal antibody to chicken type I collagen (hybridoma/collagen antibodies/immunohistochemistry)
THOMAS F. LINSENMAYER, MARY J. C. HENDRIX, AND CHARLES D. LITTLE The Developmental Biology Laboratory and the Departments of Medicine and Anatomy, Massachusetts General Hospital and Harvard Medical School,
Boston, Massachusetts 02114 Communicated by Jerome Gross, May 10, 1979
ABSTRACT We have shown that lymphocyte-myeloma cell hybridization can be used to produce large amounts of extremely high-titer specific antibodies against type I collagen, a macromolecule normally of low immunogenicity. In a passive hemagglutination assay the antibody had a high titer against chicken type I collagen but showed no activity against chicken type II or rat type I collagen. By using a two-step fluorescence histochemical procedure on sections of embryonic chicken tibia, strong fluorescence was observed in the perichondrium and surrounding connective tissue (known to contain type I collagen) but not over the cartilage (characterized by type II collagen). When used in conjunction with Staphylococcus aureus as a solid phase immunoadsorbant, the antibody was shown to bind to labeled collagen synthesized in vitro by embryonic chicken calvaria.
Extracellular matrices are composed chiefly of different genetic types of collagens, glycosaminoglycans, and proteoglycans (for reviews, see refs. 1-3). During embryonic development, individual tissues and probably even individual cells are capable of synthesizing more than one genetically distinct type of collagen, in some cases giving rise to complex, hybrid matrices. The chicken corneal epithelium, for example, synthesizes collagen of types I and II (4) which are polymerized as the primary corneal stroma (3), the embryonic neural retina synthesizes type I collagen plus a previously undescribed type (5), and individual fibroblasts produce both type I and type III collagens (6, 7). Morphological and biochemical studies of these collagenous matrices have been facilitated by the development of techniques for producing antibodies to the various genetic types of collagens differing in primary structure (8, 9). The conventional methods for producing such antibodies involve raising antisera in animals and subsequently purifying the antisera by affinity chromatography. However, even after these laborious and time-consuming purifications, the resulting antibodies may still have some degree of crossreactivity. In addition, it is thought that affinity chromatographic purifications frequently result in irreversible binding and subsequent loss of the highest-affinity antibody. Many of these problems can be avoided by use of the recently devised techniques for hybridizing antibodyproducing lymphocytes with myeloma cells (10, 11). The resulting hybridomas cultured as clones produce antibody of a single, defined specificity. Here we report the construction of a clonal line of hybridoma producing high-titer specific antibody to chicken type I collagen and describe some of the characteristics of the antibody. 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.
MATERIALS AND METHODS Collagens. Neutral-salt-soluble type I collagen was extracted from the skins of 3-week-old lathyritic chickens with 0.4 ionic strength potassium phosphate buffer at pH 7.6. Type II collagen was extracted by limited 4VC pepsinization of adult chicken sternal cartilages (12). Rat type I collagen was extracted with 0.5M HOAc from adult rat tail tendons. All of the collagens were purified by multiple salt and ethanol precipitations from neutral solutions and salt precipitations from acidic solutions. In the sternal cartilage preparation, the type II collagen was separated from type I by repeated fractional salt precipitations (13). Preparation of Mouse Lymphocytes and Cell Hybridization. The parental mouse myeloma cell line MPC 11 45.6 TG 1.7 (14), was cultured in complete medium composed of Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and antibiotics. Revertant cells were periodically removed by passage in medium supplemented with 6-thioguanine (10,gg/ml). ASW/SN mice (Jackson Laboratory) were injected subcutaneously with approximately 0.2 mg of chicken type I collagen dissolved in 0.1 M HOAc and emulsified in an equal volume of complete Freund's adjuvant. A booster injection of collagen in incomplete Freund's adjuvant was given intraperitoneally about 3 weeks later. After an additional 1-2 weeks, pairs of mice were killed by cervical dislocation and the spleens were removed. The tissue was placed in serum-free medium and minced with scissors, and the cells were liberated by agitation for 1-2 min on a vortex mixer using the most vigorous setting. The spleen cells were washed twice with serum-free medium prior to fusion. Cells were hybridized by using polyethylene glycol according to Gefter et al. (15). All manipulations were done at room temperature. Myeloma cells (106) were washed twice in serum-free medium and then were mixed with 1-3 X 107 spleen cells in 50-ml conical bottom centrifuge tubes (Falcon 2070). The cells were centrifuged at 250 X g for 5 min, and the supernatant fluid was carefully aspirated. Two-tenths milliliter of 37.5% polyethylene glycol in serum-free medium was added, and the tube was gently agitated by hand to resuspend the cells. Next, the cells were centrifuged for 3 min at 250 X g and for 2.5 min at 500 X g; they then were allowed to sit undisturbed for an additional 2.5 min. Five milliliters of serum-free medium was added to the tube; the cells were resuspended gently with a pasteur pipette and were then repelleted by centrifugation at Abbreviations: HAT medium, complete medium supplemented with
hypoxanthine, aminopterin, and thymidine; RBC, erythrocyte; Pi/
NaCl, phosphate-buffered saline. 3703
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Biochemistry: Linsenmayer et al.
250 X g for 5 min. The supernatant was removed; the cells were dispersed in 20 ml of complete medium and were put into a 370C humidified incubator for 48 hr to allow recovery. The recovered cells were pelleted and resuspended in 100 ml of complete medium supplemented with 0.1 mM hypoxanthine, 0.4 liM aminopterin, 16 ,gM thymidine, and 3 ,gM glycine (HAT medium) (16) and plated out in four 24-well plates (Linbro). After the first 3 days the cells were fed with 1 ml of HAT medium per well; every 34 days subsequently, half of the medium was replaced with fresh HAT medium. The cells in wells positive for antibody were cloned by dilute plating in 96-well flat-bottom plates (Linbro) (17). Cloning was accomplished by limiting dilution in HAT medium so that one drop (approximately 0.07 ml) contained an average of either 1 or 0.5 cell when added to a well. Every 7 days an additional drop of HAT medium was added to each well, and after 2-3 weeks the wells were tested for antibody activity. Positive wells were recloned, and then clones were transferred first into HAT medium without aminopterin and finally into complete medium. Large amounts of antibody were obtained from spent culture medium harvested from 150-mm plates or roller bottles. Medium was subsequently concentrated by using a hollow-fiber concentrator (Amicon, Lexington, MA). Alternatively, antibody was obtained from the ascites fluid of athymic nude mice (nu/nu) that had been injected 2-3 weeks previously with 107 cloned hybridoma cells. The ascites fluid was diluted with saline to prevent the formation of a gel, which rapidly occurred in undiluted fluid. Then the peritoneal cavity was flushed with saline, and the total diluted fluids were pooled for each mouse. Immunological Procedures. Antibody activity was routinely assayed by passive hemagglutination performed according to Beil et al. (18). Briefly, collagens (0.3% solutions in 0.1 M CaOAc) were coupled to human type 0 erythrocytes (RBCs) (10% solution) with glutaraldehyde. Then, 100,-l aliquots of the derivatized erythrocytes (diluted 1:20) were added to 100-,ul portions of antibody-containing solution that had been serially diluted in round-bottom microtiter plates, and the plates were allowed to develop overnight at 4°C. Immunoglobulins were characterized by immunoelectrophoresis on 1% agarose-coated glass microscope slides using 0.08 ionic strength barbital buffer (pH 8.2 or 8.6) (19). Electrophoresis was performed for 1-3 hr at 6-8 mA per slide. Subsequently, 100 Al of antiserum was added to the center trough and the slides were allowed to develop for 3 days at 4°C. The slides were viewed by indirect lighting and also after staining with Coomassie blue. Immunohistochemistry. Sections were stained with antibodies by using a double-layered, sandwich technique. Tibias were removed from 14-day chicken embryos and fixed in 1% paraformaldehyde for 15 min; then free aldehyde groups were quenched in 0.15 M Tris-HCI at pH 7.4. The tissue was quickfrozen to liquid N2 temperature, and 8-,um sections were cut on a cryostat. Sections were air dried onto albumin-coated slides at room temperature for 1-2 hr and were then treated with testicular hyaluronidase in phosphate-buffered saline (4000 units/ml) for 20 min at 370C (20). Subsequently, the sections were treated with the hybridoma antibodies, either concentrated or diluted with phosphate-buffered saline (Pi/NaCl) for 1 hr at room temperature, followed by washing for 1 min each in three changes of Pi/NaCl. The secondary antibody, consisting of rhodamine-conjugated IgG fraction of rabbit anti-mouse IgG (Cappel Laboratories, Cochranville, PA, lot 10169; diluted 4: 1500 with Pi/NaCl) was applied and allowed to react with the section for 1 hr in the dark. The sections were rinsed as above
Proc. Nati. Acad. Sci. USA 76 (1979)
and were then coated with 90% (vol/vol) glycerol in Pi/NaCl, before coverslips were attached. Slides were viewed in a Zeiss photomicroscope III with the rhodamine filter set. Immunoprecipitation Reaction with Labeled Collagen. The labeled collagen was obtained from cultures (21) of whole, lathyritic 18-day embryonic calvaria that had been labeled in vitro with [3H]proline. The collagen was extracted in 0.4 ionic strength potassium phosphate buffer at pH 7.6- and, to remove noncollagenous proteins, was twice treated at 40C with pepsin (0.1 mg/ml) for 24 hr in 0.5M HOAc. Prepared S. aureus was obtained from The Enzyme Center (Boston, MA) and was used according to Kessler (22). The bacteria were reconstituted and washed twice in assay buffer (0.05 M Tris-HC1, pH 7.5/0.15 M NaCl) containing 0.5% Triton X-100; then, for use in the assay, they were resuspended at a concentration of 10% (vol/vol) in buffer containing 0.05% Triton and bovine serum albumin (1 mg/ml). The preparation was used within 1 week. For assay, 20-50 til of labeled collagen in immunological assay buffer were pipetted into 1.5-ml conical centrifuge tubes and then varying amounts of antibody that had also been dialyzed into assay buffer were added. Volumes were equalized by addition of assay buffer and the tubes were shaken for 4 hr at 240C and then left overnight at 40C. Equal volumes of S. aureus reagent were then added to each tube such that the total IgG in the tube containing the maximal amount of IgG would be quantitatively bound, and the tubes were incubated as above. After incubation, the tubes were centrifuged twice in a Beckman Microfuge-B, and the precipitate was washed twice in the Triton-containing buffer. The washings were pooled with the supernatants. The pellets were extracted twice (2% sodium dodecyl sulfate/0.0625 M Tris-HCl, pH 6.8) with each extraction being shaken for 2 hr at 370C and then for 10 min at 100°C. These extraction supernatants were pooled. In some experiments, antibodies were treated with denatured instead of native collagen and all incubations were performed at 370C to prevent renaturation. All fractions were mixed with 4 ml of Hydrofluor (National Diagnostics, Somerville, NJ) and assayed for radioactivity in a Beckman scintillation counter. RESULTS Six separate cell hybridizations were performed, and more than 400 wells of 24-well plates were tested for antibody production by passive hemagglutination. The medium from one well, I1B6, was positive when tested with RBCs coated with chicken collagen type I.* The hybridoma cells from this well were cloned in 96-well microtiter plates, and some positive clonal wells were recloned, giving rise to several hundred antibody-positive subclones. Most of these were frozen for future studies. Four clones were grown to mass culture densities and used to produce large amounts of antibody-containing medium and ascites fluid. The passive hemagglutination data on the titers (expressed as -log2) and the specificity of the antibody are summarized in Table 1. The spent culture medium from pooled I1B6 subclones (which could be obtained in liter amounts) had an average titer of 6-7 (-log2) when tested on chicken type I-coated RBCs. After an approximately 20-fold concentration by Amicon filtration, the titer increased to 11-13 (-log2). This concentrated medium had an equally high titer when tested against RBCs coated with chicken type I collagen that had been *
In more recent experiments we have been isolating clones of cells producing antibody to type II collagen. The frequency of production of antibody-producing cells was raised to 1 in 100 wells of 24-well plates by using splenic cells removed 3 days after the animals had been given a booster dose of antigen.
Biochemistry: Linsenmayer et al. Table 1. IB6 hybridoma titers RBC collagen
Antibody source
type*
Proc. Natl. Acad. Sci. USA 76 (1979)
MS
Titer, -log2 6-7
I Spent medium 11-13 I Concentrated medium (20X) I (pepsinized) 11-12 Concentrated medium (20X) 0 II Concentrated medium (20X) 0 I, rat tail Concentrated medium (20X) Concentrated medium (20X), ;1o I DEAE purified 15->17 I Ascites fluid (diluted 1:4) 14 I (pepsinized) Ascites fluid (diluted 1:4) II 0 Ascites fluid (diluted 1:4) * All chicken collagen, except line 5 was rat tail collagen.
previously treated with pepsin at 4VC to remove the nonhelical telopeptide ends-thus demonstrating the antigenic determinants to be in the helical portion of the molecule. The 20X concentrated antibody-containing culture medium showed no reactivity with RBCs coated with chicken type II collagen or rat tail tendon type I collagen. An even higher-titered antibody could be obtained by growing the 11B6 cells in the peritoneal cavity of athymic nude mice and harvesting the ascites fluid. The yields of diluted ascites fluid were 2045 ml per animal. The diluted ascites fluid from eight different animals had titers of 15->17 (-log2) when tested against RBCs coated with intact or pepsin-treated chicken type I collagen. After affinity purification using Sepharose-bound type I collagen, the resulting material had an immunoglobulin concentration of 0.09 mg/ml and a titer of 11-12 (decreased by dilution during purification). No detectable activity against type II coated cells was observed. The class of immunoglobulin responsible for the antibody activity was determined by immunoelectrophoresis of concentrated medium from cultures of parental cells (MPC 11) and hybridoma cells (11B6) and whole mouse serum. Fig. 1 shows immunoelectrophoresis profiles of reactions with antibody to whole mouse serum and to mouse IgG. With anti-MS, both the MPC parent and the hybridoma gave a single, sharp arc whereas the whole mouse serum, as expected, gave numerous reaction products. Against the anti-IgG, both parental cell and hybridoma media gave single arcs as did the mouse serum. However, the arc produced by the serum was much longer than that produced by the medium of either cell line. The only detectable difference between the hybridoma and the parent was the slightly longer arc of the latter. Subsequently, Kurt Bloch (Massachusetts General Hospital) kindly performed doublegel-diffusion analyses of MPC 11 and 11B6 media to test their reactivity with antisera against IgG heavy chain subclasses y2a, y2b, y3, and -y. Both cell lines reacted only with anti-'y2b sera; no reaction was obtained with anti-IgA. The antibody showed a high degree of specificity when used in a double-antibody sandwich technique. Cryostat sections of 14-day embryonic chicken tibia, including both cartilage and noncartilagenous regions, were treated with hyaluronidase to remove proteoglycan and thus unmask the cartilage collagen (10). Then the sections were treated with the monoclonal antibody and subsequently with rhodamine-conjugated rabbit IgG directed against mouse IgG. Sections treated with anti-type I collagen ascites fluid (Fig. 2b) showed the brightest fluorescence in the perichondrial region. Loose connective tissue also showed bright staining. The matrix in the cartilaginous regions, on the other hand, was usually negative or at most had only a slight background fluorescence. No fluorescence was detected when rhodamine-conjugated antiserum to mouse IgM heavy chains was used as a second reagent.
3705
-.,R-
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H Ms U)
2o
6
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X
_ i I-1. MPC
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MPC
H
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H FIG. 1. Immunoelectrophoretic profiles of whole mouse serum (MS) and concentrated medium from cultures of 11B6 hybridoma (H) or MPC 11 parental (MPC) cells. The top three slides were allowed to react with rabbit antiserum made against whole mouse serum (anti-MS); the bottom two were allowed to react with the IgG fraction of goat antiserum made against mouse IgG (anti-IgG).
To verify that the collagen in the cartilaginous region had been unmasked by the hyaluronidase treatment and was available to antibody binding, identical sections were treated with culture medium from another of our hybridoma lines (as yet uncloned) making antibody to chicken type II collagen (Fig. 2a). These sections showed bright fluorescence of the cartilaginous extracellular matrix but no staining of the perichondrium. In the section shown here, we used the concentrated ascites fluid (titer, 15 -log2). However, ascites fluid diluted 1:200 (the highest dilution so far tested) and culture medium concentrated 4X gave equally bright fluorescence. We tested the antibody for its ability to interact with pepsin-treated proline-labeled collagen obtained from cultures of chicken embryo calvaria. Preparatory experiments (data not shown) showed that the IgG synthesized by both the parental and hybridoma cell lines would bind to the S. aureus cells. Thus, the bacterial cells were used as a solid-phase immunoabsorbant
Biochemistry: Linsenmayer et al.
3706
Proc. Natl. Acad. Sci. USA 76 (1979)
14 .0
vtp
CA;
UP, s
4 .j
x
E Rab. AB. I,Iul ._
0~ I
16
..-_ __
12 8 4 ':
. rV. 'il:
:i
CT
tW.
I.
3.-
.11
'S'
:R
.:
FIG. 2. Sections from the cartilaginous regions of 14-day embryonic chicken tibias treated first with monoclonal antibody to type I collagen (b) or to type II collagen (a) and then with rhodamineconjugated IgG fraction of rabbit anti-mouse IgG. The antibody source was undiluted ascites fluid for type I and antibody was 4X concentrated culture medium for type II. CA, cartilage; P, perichondrium; CT, connective tissue. (A, X320; B, X 200.)
to precipitate the antigen-antibody complex. Each experiment included controls run using affinity-purified chicken type I collagen antibodies that had been raised in rabbits (20). The antibody produced in rabbits (Rab. AB I) was added in increasing concentrations until a plateau of precipitable counts was reached at about 50% of the total counts in the samples (Fig. 3 upper). The monoclonal antibody IB6 (Fig. 3 lower) behaved similarly; however, 31% of the counts was the maximum precipitated with it. No additional radioactivity was brought down when the tubes showing maximal precipitation were treated with a second quantity of monoclonal antibody. In both monoclonal antibody precipitates and supernatants, 44% of the proline was present as 4-hydroxyproline. No counts above background were precipitated when the substrate had been thermally denatured.
DISCUSSION When compared with conventional antibodies raised in animals, the monoclonal antibodies produced by hybridoma cells potentially have a number of highly desirable characteristics including a single definable specificity, reproducibility, and amounts available (see ref. 11). This technique has exceptional promise for the production of large amounts of high-titer, non-crossreacting antibodies to closely related antigens, such as the different genetic types of collagens. After cloning, the anti-type I-producing hybridoma (11B6) maintained antibody production for more than 6 months before loss of activity was detected (numerous early passages of sub-
I...... A*-
.......
10 20
.
..................
50 1I1B6 AB, ml
100
FIG. 3. Precipitation of [3H]proline-labeled calvaria collagen with increasing amounts of anti-type I collagen IgG purified from either rabbit serum (Rab. AB. I) (Upper) or IB6 culture medium (I1B6 AB) (Lower). *, Precipitates; 0, supernatants; &, precipitates with heat-denatured collagen.
clones have been frozen). When tested by passive hemagglutination for anti-chicken type I collagen activity, nude mouse fluid had titers of 15->17 serial dilutions. Because fluid had been diluted at least 1:4 with Pi/NaCl, the highest titers were in reality >19, thus giving detectable antibody response at a direct dilution of greater than 1:0.5 X 106. Even at the very high antibody titers obtainable in the concentrated ascites fluid, when tested by hemagglutination the antibody contained no detectable crossreactivity to either a closely related genetic type of collagen from the same species (chicken, type II) or the same genetic type from a different species (rat, type I). The antibody reacted equally well with chicken type I collagen from which the nonhelical extensions had been enzymatically removed, thus localizing the antigenic determinant to the helical domain. Because the antibody failed to bind to denatured collagen, the triple helical conformation is required to generate the antigenic site (8, 9). The affinity with which this monoclonal antibody binds to collagen has not yet been quantitatively determined. Operationally, however, the antibody remains bound during the washing procedures used for immunohistochemistry and in assay of binding to labeled collagen. The subclass of immunoglobulins responsible for antibody activity appears to be identical to that being produced by the parental myeloma cell line. In immunoelectrophoretic analysis, the similarly migrating single arcs of anti-IgG crossreacting material that had been synthesized by both parental and hybridoma cell lines suggest that each is making the same subclass of IgG previously shown to be synthesized by MPC 11 (14). In
Proc. Natl. Acad. Sci. USA 76 (1979)
Biochemistry: Linsenmayer et al.
3707
addition, protein A precipitated the same relative proportion (approximately 75%) of the medium proteins synthesized by the two different cell lines. These results suggest that the lymphocyte that fused with the MPC 11 cell to produce the anticollagen synthesizing hybridoma cell was, most probably, itself synthesizing IgG of the 'y2b class. At present we do not know why the type I monoclonal antibody interacted with only about one-third of the collagen synthesized by calvaria. Several preparations of labeled, twice-pepsinized calvaria collagen behaved similarly; because these had 44% of their prolines hydroxylated, they were free of contamination. Because the calvaria are membrane bones, only type I collagen is present. Under identical conditions, the monoclonal antibody precipitated considerably less collagen than did different batches of conventional antibodies against chicken skin type I collagen raised in rabbits. We suggest that the antibody may recognize a subclass of type I collagen, reflecting either primary sequence differences or posttranslational modifications. Such collagen subclasses could also explain why the rabbit antibodies, for which skin type I collagen served as both immunogen and affinity purification agent, reacted only partially with calvaria collagen. Evidence for such subclasses of type I collagen also comes from somatic cell genetic analysis indicating that genes for human-type I collagen are found on both chromosomes 17 (23) and 7 (24). It is also known that posttranslational modifications such as hydroxylations and glycosylations can be incomplete (25, 26). An extensive test of 11B6 monoclonal antibody on the type I collagen synthesized by a number of different tissues from various stages of embryos may provide some clarification.
19. Hudson, L. & Hay, F. C. (1976) Practical Immunology (Black-
We gratefully acknowledge the excellent technical assistance of Eileen Gibney. We thank Drs. Jerome Gross and Elizabeth Hay for encouragement and helpful discussions during the course of this work. We also thank Drs. Bryan Clark and Jerry Schwaber for helpful technical suggestions. This is publication no. 780 of the Robert W. Lovett Memorial Group for the Study of Diseases Causing Deformities. This research was supported by National Institutes of Health Research Grants EY02261, AM03564, and HD00143. T.F.L. is recipient of National Institutes of Health Research Career Development Award AM00031; M.J.C.H. is a National Institutes of Health Postdoctoral Fellow (HL05682); and C.D.L. is a Fellow of the Damon RunyonWalter Winchell Cancer Foundation.
20. von der Mark, H., von der Mark, K. & Gay, S. (1976) Dev. Biol. 48,237-249. 21. Linsenmayer, T. F., Toole, B. P. & Trelstad, R. L. (1973) Dev. Biol. 35, 232-239. 22. Kessler, S. W. (1975) J. Immunol. 115, 1617-1624. 23. Raj, C. V. S., Church, R. L., Klobutcher, L. A. & Ruddle, F. H. (1977) Proc. Natl. Acad. Sci. USA 74,4444-4448. 24. Sykes, B. & Solomon, E. (1978) Nature (London) 272, 548549. 25. Bornstein, P. (1967) Biochemistry 6,3082-3093. 26. Balian, G., Click, E. M., Hermodson, M. A. & Bornstein, P. (1972) Biochemistry 11, 3798-3806.
1. ;(Gross, J. (1974) Harvey Lect. 68, 351-432. 2. Toole, B. P. & Linsenmayer, T. F. (1977) Clin. Orthop. Rel. Res. 129,258-278. 3. Hay, E. D., Linsenmayer, T. F., Tretstad, R. L. & von der Mark, K. (1979) in Current Topics in Eye Research, eds. Zadunaisky, J. A. & Davson, H. (Academic, New York), pp. 1-35. 4. Linsenmayer, T. F., Smith, G. N. & Hay, E. D. (1977) Proc. Natl. Acad. Sci. USA 74,39-43. 5. Linsenmayer, T. F. & Little, C. D. (1978) Proc. Natl. Acad. Sci. USA 75, 3235-3239. 6. Church, R. L., Yaeger, J. A. & Tanzer, M. L. (1974) J. Mol. Biol. 86,785-799. 7. Gay, S., Martin, G. R., Muller, P. K., Timpl, R. & Kuhn, K. (1976) Proc. Natl. Acad. Sci. USA 73,4037-4040. 8. Furthmayr, H. & Timpl, R. (1976) Int. Rev. Connect. Tiss. Res.
7,61-99. 9. Timpl, R. (1976) in Biochemistry of Collagen eds. Ramachandran, G. N. & Reddi, A. H. (Plenum, New York), pp. 319-375. 10. Kohler, G. & Milstein, C. (1975) Nature (London) 256, 495497. 11. Melchers, F., Potter, M. & Warner, N. (1978) Lymphocyte hy-
bridomas (Springer, New York).
12. Miller, E. J. (1971) Biochemistry 10, 1652-1659. 13. Trelstad, R. L., Kang, A. H., Toole, B. P. & Gross, J. (1972) J. Biol. Chem. 247, 6469-6473. 14. Margulies, D. H., Kuehl, W. M. & Scharff, M. D. (1976) Cell 8, 405-415. 15. Gefter, M. L., Margulies, D. H. & Scharff, M. D. (1977) Somatic Cell Genet. 3, 231-236. 16. Littlefield, J. W. (1964) Science 145,709-710. 17. Orkin, S. H., Harosi, F. I. & Leder, P. (1975) Proc. Natl. Acad. Sci. USA 72,98-102. 18. Beil, W., Furthmayr, H. & Timpl, R. (1972) Immunochemistry
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