production and characterization of monoclonal antibodies to bovine skin proteodermatan sulfate

Collagen Rel. Res. Vol. 5/1985, pp. 23-39

Production and Characterization of Monoclonal Antibodies to Bovine Skin Proteodermatan Sulfate

GORDON A. PRINGLE 1, CAROLE M. DODD\ JEFFREY W. OSBORNl,

C. HAROLD PEARSON1 and TIMOTHY R. MOSMANN2

1 Department of Oral Biology, University of Alberta, Edmonton, Alberta, Canada, T6G 2N8 and

2 DNAX Research Institute, 1450 Page Mill Road, Palo Alto, CA 94304, USA.

Abstract

To study the molecular structure and function of bovine skin proteodermatan sulfate, on a determinant by determinant basis, several monoclonal antibodies to this molecule have been produced and characterized. Based on the results of a preliminary immunogenetic analysis of 4 inbred mouse strains, SJLlJ (H-2s) mice were immunized for the fusions. Ten hybridomas were produced and the monoclonal antibodies from four of these were selected for further investigation. Employing an ELISA inhibition assay, none showed any detectable affinity for bovine collagen types I, II, III, or IV, bovine fibronectin or chondroitin or dermatan sulfate glycosaminoglycans. Each monoclonal antibody bound the chondroitinase ABC-derived protein core and none was significantly inhibited by proteinase digests of the intact molecule suggesting that the epitope of each contains a protein component. The results of competitive binding ELISA assays and immunoblots of the cyanogen bromide cleavage products of proteodermatan sulfate indicate that the 4 antibodies recognize at least 3 distinct antigenic determinants on this molecule. Immunohistochemical methods located the antigen in the dermis of bovine skin and revealed that a change in proteodermatan sulfate distribution occurs during skin development.

Key words: bovine skin, monoclonal antibodies, proteodermatan sulfate.

Introduction

Antibodies have been used extensively as probes to study the structural and functional chracteristics of connective tissue macromolecules (Furthmayr, 1982). Immunologic analyses of proteoglycans have focused almost exclusively on the high molecular weight monomer found in cartilage. Antisera have been used to visualize this proteoglycan within the cartilage matrix at the light (Poole et al., 1980) and electron microscopic (Poole et al., 1982) levels, to determine the nature of its interaction with hyaluronic acid (Thonar et al., 1982) and to help ascertain its role in joint diseases (Champion and Poole, 1981; Giant et al., 1980). With the advent of hybridoma methodology

24 G. A. Pringle, C. M. Dodd, J. W. Osborn, C. H. Pearson and T. R. Mosmann

(Kohler and Milstein, 1975), the use of monoclonal antibodies (MAbs)l to study complex molecules has become popular because, by providing more explicit and reliable data, they increase the resolution of analyses. Monoclonal antibodies to various determinants on native and enzyme-treated cartilage proteoglycan monomer have been reported by several laboratories (Dorfman et aI., 1980; Caters on et aI., 1981).

In recent years, a number of different proteoglycans have been isolated from various tissues, e. g. corneal stroma (Hassell et aI., 1979), bone (Fisher et aI., 1983), liver cell membrane (Oldberg et aI., 1979), cartilage (Shinomura et aI., 1983, Noro et aI., 1983, Rosenberg etal., 1983), follicular fluid (Yanagishita et aI., 1979) and cultured cells, e.g. ovarian granulosa cells (Yanagishita and Hascall, 1983a, 1983b), aortic smooth muscle cells (Wight and Hascall, 1983; Chang et aI., 1983)). Of interest to us are the relatively small, L-iduronate-enriched, proteodermatan sulfate (PDS) proteoglycans present in a variety of tissues, such as skin (Miyamoto and Nagase, 1980; Fujii and Nagai, 1981; Damle et aI., 1982; Pearson and Gibson, 1982), sclera (Coster and Fransson, 1981), uterine cervix (Uldbjerg et aI., 1983), tendon (Valli et aI., 1982) and periodontal ligament (Pearson and Gibson, 1982). These proteoglycans are abundant in tissues rich in non-mineralized type I collagen and have been implicated in playing a role in collagen fibrillogenesis (Obrink and Sundelof, 1973; Gelman and Blackwell, 1974; Mathews, 1975; Scott, 1980; Scott and Orford, 1981; Scott et aI., 1981; Vogel and Heinegard, 1983). We have recently isolated and characterized the proteodermatan sulfate (PDS) present in adult bovine skin (Pearson and Gibson, 1982). This proteoglycan is polydisperse by sedimentation equilibrium in the ultracentrifuge and gives a diffuse band in 7 % SDS-polyacrylamide slab gels with an average Mr ~ 80 Kda. It has only a small number of L-iduronate rich (75 % of total hexuronate) dermatan sulfate side chains, which have an average molecular weight of 16-17 Kda. The glycoprotein core is rich in asx, glx and leucine and its NHrterminal amino acid sequence has been published (Pearson et aI., 1983). To complement our biochemical studies, we have initiated an immunological study of this molecule. Nagai et al. (1983) have reported the development of a rat polyclonal antiserum to calf skin proteodermatan sulfate and its use in immunohistochemical studies. We have decided to explore the structure and function of PDS on a determinant by determinant basis and report here the production and characterization of several monoclonal antibodies capable of recognizing different epitopes on its protein core.

Material and Methods

Antigens-PDS from depilated adult bovine skin was extracted and purified in the presence of proteinase inhibitors essentially as described previously (Pearson and Gibson, 1982; Pearson et aI., 1983). Purity of the antigen was assessed by amino acid analysis and SDS-polyacrylamide gel electrophoresis.

1 Abbreviations used in this manuscript are: MAbs, monoclonal antibodies; PDS, proteodermatan sulfate; (ABC)core, PDS protein core produced by chondroitinase ABC; H-2, major histocompatibility gene complex of the mouse; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; ELISA, enzyme linked immunosorbent assay; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; SDS, sodium dodecyl sulfate.

Monoclonal Antibodies to Skin Proteodermatan Sulfate 25

PDS protein core ((ABC)core) was produced using chondroitinase ABC (from Proteus vulgaris, Miles Laboratories, Rexdale, Ontario) as described by Oike et al. (1980). The digestion was carried out in a stainless steel tube, in the presence of several proteinase inhibitors (Oike et al., 1980), for 45 min at 37"C using 0.2 units (manufacturer's units) of enzyme/mg PDS. To separate the protein core from the small digestion products, the digest was treated as described previously (Pearson et al., 1983) or applied to a 1 x 114 cm Sephacryl S-300 (Pharmacia, Canada) column equilibrated with a 4 M guanidinium HCI, 0.5 M sodium acetate-acetic acid buffer, pH 6.8. The fractions containing the protein core were dialyzed exhaustively against water at 6 DC and lyophilized.

Cyanogen bromide cleavage products of PDS were produced by incubation of the intact molecule (previously reduced for 22 h under N2 in a 0.2 M NH4HC03 buffer, pH 7.2 containing 25 % 2-mercaptoethanol) in 70 % formic acid for 4 h at 30 DC with constant stirring. Some of the preparation remained insoluble and was removed by centrifugation. The supernatant was diluted with 20 volumes of water and lyophilized.

Papain digests of PDS were produced by incubating 1 mg of proteoglycan in 1 mlof a 0.1 M sodium acetate-acetic acid, pH 6.0 buffer containing 5 mM EDTA, 6 mM cysteine HCI and 6.2 units of papain (Sigma Chemical Co., St. Louis, MO) for 24 hat 65 DC. For subsequent immunological assays, the papain was inactivated, to prevent it digesting the MAbs, by adding iodoacetamide (in 3-fold molar excess to cysteine HCI) and incubating the mixture overnight in the dark at room temperature. Digestion controls were performed for 24 h at 65 DC in buffer (a) lacking enzyme, (b) lacking papain and the subsequent addition of iodoacetamide and (c) lacking papain and cysteine HCI and addition of iodoacetamide. These were performed to verify that any large change in the antigenicity of PDS caused by digestion was in fact due to the action of the enzyme and not the digestion conditions or the buffer constituents.

Acid soluble bovine type I collagen was isolated from salt-extracted calf skin with 50 mM sodium citrate buffer, pH 3.7 as described by Pontz et al. (1970). Bovine type III collagen was obtained by digesting calf skin with pepsin and purified by salt precipitation (Chung and Miller, 1974). Type II collagen from bovine articular cartilage was solubilized with pepsin and purified according to the method of Herbage and Buffevant (1974). Bovine type IV collagen was isolated by pepsin digestion of anterior lens capsules (Kefalides and Denduchis, 1969).

Hyaluronic acid, derma tan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, and bovine fibronectin were purchased from Miles Laboratories. Bovine elastin was purchased from Elastin Products Co., Pacific, MO.

Protein content of antigen samples was determined by the method of Lowry et al. (1951).

Animals - Male SJLlJ (H-2s) and A.CA/Sn (H-2f) mice were purchased from Jackson Laboratories, Bar Harbor, ME. Male CBA/CaJ (H-2k) and C3H.SW/Sn (H-2b) and female Balb/cCr mice were purchased from the MSB Animal Unit, University of Alberta.

Immunogenetic Analysis - Five mice from each of the four inbred strains were primed with 0.1 ml of a 0.5 mg/ml solution of PDS emulsified in an equal volume of Freund's complete adjuvant (Difco) via 2 subcutaneous injections in the back. Mice were boosted intraperitoneally with PDS in freund's incomplete adjuvant (Difco) on days 10, 20 and 36. To ascertain the response to continued boosting, 1 mouse from each group was exsanguinated by cardiac puncture on days 20, 30 and 47 and their antisera titered by ELISA. Three non-immune mice from each strain provided normal sera.

3 Collagen 5/1


Hybridoma Production - The fusion protocol was based on the method of Galfre et al. (1977) as modified by Mosmann and coworkers (Longenecker et al., 1979) using the plasmacytoma cell line, 315.43, derived from MOPC 315 (Mosmann et al., 1979). Spleen cells from mice, immunized with PDS as described above, were mixed 5 to 1 with the 315.43 cells and cell fusion initiated by suspending the cells in a 40 % polyethylene glycol solution and centrifuging at 600 X g for 10 min. The cells, in HAT selection medium supplemented with 1 mM ouabain and 107 Balb/cCr blood cells/ml, were dispensed into 24-well Linbro culture plates (Flow Laboratories) and incubated in 7.5 % CO2 at 37°C. When clusters of growing hybrid cells were macroscopically visible, 50 ~l of medium from each well was screened for anti-PDS activity by ELISA. Hybrid cell clusters in positive wells were transferred individually to 96-well plates containing fresh growth medium, hypoxanthine, thymidine and blood cells. Those responsible for the production of anti-PDS antibody were identified by a second ELISA screening and their cells were subsequently recloned in the presence of blood cells by the method of limiting dilution. Ascites fluid (produced by intraperitoneal injection of 2 x 106 hybrid cells into pristane-primed, irradiated SJLlJ X Balb/cCr mice) and pooled supernatant fluids were obtained from each hybridoma.

Enzyme-Linked Immunosorbent Assay (ELISA) - The ELISA technique used was based on the method of Rennard et al. (1980). Solutions tested for anti-PDS antibody were diluted in PBS, 0.05 % Tween 20 and incubated in Immulon I microtiter plates (Dynatech Laboratories, Alexandria, VA) coated with 200 ng PDS per well. Peroxidase conjugated rabbit anti-mouse IgG (Cappel Laboratories, West Chester, PA), diluted 1000-fold, was ordinarily used as the developing antibody and an aqueous solution of 0.01 % o-phenylenediamine and 3 X 10-3 % H20 2 was the substrate. The final color reaction was quantitated with a Titertek Multiskan (Flow Laboratories, Mississauga, Ontario) equipped with a 492 nm interference filter. Titration end points represented the point on the titration curve where the absorbance was twice the control (background) level.

Peroxidase conjugated goat anti-mouse IgG, IgA and IgM specific antisera (Cappel) were substituted as the developing antibodies to determine the class of each MAb. Similarly, goat anti-mouse IgG1, IgG2a, IgG2b (Meloy Laboratories, Springfield, VA) and rabbit anti-mouse IgG3 (Miles Laboratories) were used in conjugation with peroxidase conjugated rabbit anti-goat or goat anti-rabbit IgG antisera (Cappel), respectively, to determine the IgG subclass of each MAb.

Competitive binding experiments were carried out using Immulon plates coated with 50 ng of PDS per well. The maximum absorbance generated by each MAb alone was compared with that generated by mixing it with another MAb. Lack of a significant increase in absorbance as a result of mixing was interpreted as competition between the respective antibodies for the same, adjacent or closely related determinants.

Antigen samples were also analyzed by ELISA inhibition essentially as described (Rennard et aI., 1980). Two-fold serial dilutions of the inhibiting antigen were made in PBS/0.05 % Tween 20 and incubated with an appropriate concentration of hybridoma supernatant or ascites fluid overnight at 4°C. The MAb-inhibitor solutions were then incubated in washed PDS-coated wells for 30 min followed by incubation with a 1000-fold dilution of rabbit anti-mouse immunoglobulin (DAKO, Cedarlane Laboratories, Hornby, Ontario) for 30 min; peroxidase conjugated goat anti-rabbit IgG (Cappel) for 30 min; and finally substrate. Uninhibited antibody solutions and solutions without a specific antibody (controls) were used to set the upper and lower limits of color

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Fig. 1. Antibody response of inbred mice to bovine skin proteodermatan sulfate. Antisera were obtained from mice of each strain approximately 10 days after each booster injection and titered by ELISA using serial3-fold dilutions. The antiserum titers of the SJLlJ mice used for fusions are also indicated.

production. Normal mouse serum, supernatant fluid from myeloma parent cells or mouse IgG (Sigma Chemical Co.) solutions were used as controls.

Immunochemical Staining of Electrophoretically Transferred Antigens-SDS/polyacrylamide slab gel electrophoresis (pH 7.2) of antigen samples was performed by the method of Weber and Osborn (1975). Prior to electrophoresis, all samples were heated to 45 DC for at least 30 min in the presence of 2 % 2-mercaptoethanol. Antigens were transferred to nitrocellulose paper using a Transblot cell (Biorad, Canada) filled with a 50 mM sodium phosphate buffer, pH 7.2 and employing a 9 volt, 375 milliampere current for 16 h. The gel was then stained with Coomassie Blue R250 (Mechanic, 1979) while selected lanes of the nitrocellulose paper were stained with Amido Black. The remainder of the nitrocellulose paper was cut into as many strips as there were different control and MAb solutions used and stained immunochemically. The staining procedure was based on the method of Towbin et al. (1979) using peroxidase-conjugated rabbit anti-mouse IgG as the developing antibody (diluted 1 : 600) and 0.05 % diaminobenzidine tetrahydrochloride, 0.01 % H20 2 in TBS (0.15 M NaCl, 50 mM Tris-HCI, pH 7.6) as the substrate. To increase the contrast of the stain, the blots were soaked briefly in an aqueous solution of 0.02 % OS04, rinsed in water and dried.

Immunohistochemistry - Samples of skin were obtained from various species and, after gently scraping with a scalpel to remove hair or fat as necessary, cut into small pieces perpendicular to the skin surface. Individual pieces were either embedded in OCT Compound (Tissue Tek) and frozen at -22 DC or fixed in 10 % buffered formalin at room temperature for at least 4 days prior to embedding in paraffin. All sections were pretreated for 30 min in 2 % H20 2 (v/v) in absolute methanol to eliminate endogenous peroxidase activity. Sections were then stained for immunoglobulin-bound


peroxidase as detailed by Sternberger (1979) using MAb and control solutions, goat anti-mouse IgG F(abh fragments (Cappel), mouse peroxidase anti-peroxidase (Sternberger Meyer Immunocytochetrticals, Jarrettsville, MO) and diaminobenzidine tetrahydrochloride/H20 2•

Results

An immunogenetic analysis of four inbred mouse strains was carried out to ascertain which strain was capable of mounting the most potent antibody response to intact POS. SJLlJ (H-2s) mice demonstrated the highest antibody titers after both the second and third booster injections (Fig. 1), although the response of A.CA/Sn (H-2f) mice ultimately rose to the same level. The relative response of CBAlCaJ (H-2k) mice decreased with continued boosting while that of C3H.SW/Sn (H-2b) mice remained the lowest by an order of magnitude. Based on these results, two more SJLlJ mice were primed with PDS and, after receiving 2 and 3 booster injections respectively, their spleen cells were fused with the 315.43 plasmacytoma cells. Antisera, obtained from each mouse at the time of fusion, were titered to check the immunization procedures and to help verify the results of the immunogenetic analysis (Fig. 1).

Screening of the fusions revealed 15 positive wells and from these 10 clones were isolated, recloned once and frozen. Culture medium from clones lXA, 3B3, 606 and 7B 1 demonstrated strong ELISA reactions on POS-coated plates and were subsequently recloned at least twice. Ascites and supernatant fluid were obtained from each. All ascites titered in excess of 5 X 105, with several titering beyond 2 X 106

• Supernatant fluids of 6D6 and 7B1 have produced titers of 2 x 104

• Using immunoglobulin class and subclass specific antisera in the ELISA, all 4 MAbs were found to be IgG 1.

In competitive binding ELISA experiments, designed to detect the proximity of the epitopes of different pairs of MAbs, only the combination of lXA with 606 resulted in competitive binding (Fig. 2d). All other combinations produced an additive effect (Fig. 2).

The specificity of each MAb was tested against other dermal connective tissue components using ELISA inhibition analyses. The conditions of the analysis were established beforehand with intact POS used as the standard inhibitor. We found that 100,000-fold dilutions of 3B3, 606 and 7Bl ascites fluids gave sensitive, reproducible results as they were routinely inhibited to the 50 % level by 20 to 40 ng of POS protein/ml depending upon the MAb used. 1XA ascites fluid, at the same dilution, required 5 to 10 times more PDS protein/ml to be inhibited to the same degree and was used at a higher dilution. Supernatant fluids were occasionally employed and were similarly standardized beforehand. Using this analysis none of the ;mtibodies was inhibited by bovine fibronectin (Fig. 3) or bovine types I, III or IV collagen (Fig. 4). Bovine type II collagen was also tested and gave similar results (Fig. 4). None of the antibodies was inhibited by 1,000 ng of bovine elastin nor was any inhibited by 5000 ng/ml of commercially obtained hyaluronate, chondroitin 6-sulfate, chondroitin 4-sulfate or derma tan sulfate.

To explore the chemical nature of the epitope of each MAb further, PDS was degraded by various enzymes and the antigenicity of the fragments compared with that of the undigested molecule. In ELISA inhibition analyses, the purified (ABC)core of PDS was found to be as effective an inhibitor for 3B3, 6D6 and 7Bl as the intact

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Fig. 2. Competitive binding ELISA assays. Hybridoma supernatant fluids were serially diluted (2-fold) individually, or pooled in pairs and diluted as mixtures, and incubated overnight at 4°C in POS coated wells to achieve saturation. After warming, the ELISA was continued to determine whether mixtures of MAbs from 2 different hybridomas could bind to the antigen in an additivefashion. lXA (0), 3B3 (,6.),606 (0) 7B 1 (e) and the corresponding mixture of each pair (+).

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, were incubated overnight at 4°C with intact POS (,6. and 0, respectively) or bovine fibronectin (.a. and ., respectively) and then transferred to POS coated wells. The points represent the proportion of uninhibited MAb at each inhibitor concentration. lXA and 3B3 ascites fluids were not inhibited by 5000 ng/ml of fibronectin.


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Fig. 4. ELISA inhibition assay with bovine collagens. 6D6 supernatant fluid (diluted 1: 2800) and 7Bl supernatant fluid (diluted 1 : 800) were incubated overnight at 4°C with PDS (6 and 0, respectively) or bovine types I (0), II ( ... ), III (_) or IV (e) collagen and then transferred to PDS-coated wells. lXA and 3B3 monoclonal antibodies were also uninhibited by 5000 nglml of the collagens.

molecule (Fig. 5). For lXA, the (ABC)core was a somewhat less effective inhibitor and generated an inhibition curve with a slope significantly different from that of intact PDS (Fig. 5). When PDS was digested with papain the resultant breakdown of the protein component of the molecule caused a dramatic loss of antigenicity for all 4 MAbs when compared with untreated PDS (Fig. 6). Of interest was the finding that PDS that had been incubated for 24 h at 65 °C in buffer lacking papain became a more effective inhibitor for lXA and 6D6 than untreated PDS while it proved to be less effective for 3B3 (Fig. 6). 7Bl was inhibited by both PDS preparations to the same extent.

The specificity of each MAb and the nature and location of their epitopes on PDS were also analyzed by immunoblotting methods. As exemplified by lXA, all 4 MAbs bound intact PDS as well as the major (Mr-55,000) and minor (Mr-52,000) compo-

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Fig. 5. ELISA inhibition assay with purified (ABC)core. The antigenicity of the protein core of PDS (e), produced by chondroitinase ABC digestion and purified by chromatography on a Sephacryl S-300 column, was compared to undigested PDS (0) using ascites fluid from each of the 4 hybridomas.


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Fig. 6. ELISA inhibition assay with proteolytically degraded PDS. Using hybridoma ascites fluids of each monoclonal antibody, the antigenicity of PDS that had been digested with papain for 24 h at 65°C (0) was compared to that of PDS that had been incubated under the same conditions in buffer lacking papain (e) and to untreated PDS (X).

nents produced after its digestion with chondroitinase ABC (Fig. 7a). Blotted enzyme did not stain with any of the MAbs (results not shown). Staining of the blotted cyanogen bromide fragments of PDS with each MAb (Fig. 7b) resulted in at least 3 distinct patterns; one defined by 3B3, one defined by 7Bl, which did not stain any of the blotted fragments, and the last defined by lXA/6D6, although 6D6 recognized several bands of greater mobility that lXA did not.

To further verify the specificity of the MAbs as well as locate PDS in sections of bovine skin, an immunohistochemical analysis of each was performed using the peroxidase anti-peroxidase method. After application of diaminobenzidine, stain was present throughout the connective tissue regions of skin but absent from the epithelial structures such as hair follicles, sweat and sebaceous glands and their ducts (Fig. 8a). The stain was most prominent in the papillary (superficial) layer and between and around the large fiber bundles of the reticular (deep) layer. Using controls containing mouse IgG or normal mouse serum staining was faint and confined to the sebaceous glands and hair follicles (Fig. 8b). Using supernatants fluid derived from the myeloma parent cell line, staining was completely absent. In contrast with adult skin, fetal skin stained minimally in the subepidermal zone (Fig. 8c) and pretreatment with testicular hyaluronidase did not enhance the staining within this region (results not shown). The capability of each MAb to stain sections of skin from several other species was also investigated and is summarized in Table 1.

Discussion

We wished to develop a library of MAbs to PDS in order to monitor its tissue distribution at various stages of development, to evaluate its structural relationship

32 G. A. Pringle, C. M. Dodd, ]. W. Osborn, C. H. Pearson and T. R. Mosmann

with other proteoglycans and to study the structural basis of its biological activities such as collagen fiber binding. In particular, we sought to produce MAbs against its protein determinants. Therefore, intact PDS was used as the immunogen, rather than the (ABC)core, in order to minimize generating numerous clones to artifactual carbohydrate determinants that result from chondroitinase ABC digestion (Christner et aI., 1980). To ensure that the intact molecule was sufficiently antigenic to induce a strong antibody response, thereby increasing the number of antigen specific cells available for

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Fig. 7. Immunoblots of PDS and its fragments. (a) Intact PDS (lanes 2 and 5) and its (ABC)core (lanes 3 and 4) were transferred from 5 % gels to nitrocellulose paper and stained with Amido Black (lanes 2 and 3) or immunochemically using 1XA ascites fluid (1: 1000) (lanes 4 and 5).10!!g PDS protein was applied to lanes 2 and 3; 1 !!g PDS protein to lanes 4 and 5. (b) Intact PDS (lanes 2 and 9) and its cyanogen bromide cleavage products (lanes 3-8) were similarly blotted from a 10 % gel and stained with Amido Black (lanes 2 and 3), normal mouse serum diluted 1 : 100 (lane 4) or 1XA (lane 5), 6D6 (lane 6), 7B1 (lane 7) and 3B3 (lanes 8 and 9) ascites fluids diluted 1 : 1000.


Fig. 8. Immunohistochemical localiza tion of proteodermatan sulfate in bovine skin. (a) Formalin fixed 5 l-lm section of adult bovine skin stained for peroxidase after an overnight incubation at 4 °C with 606 supernatant fluid (diluted 1: 10), linking antibody (45 min) and mouse peroxidase anti-peroxidase (30 min). (b) Serial section to (a) using normal mouse serum (diluted 1: ] 00). (c) POS localization in a fixed section of fetal calf skin using 606 supernatant fluid diluted 1 : 10. (d) Fetal calf skin control using normal mouse serum diluted 1 : 100. The horizontal bar in each figure represents 100 l-lm.


Table I. Crossreactivity of Monoclonal Antibodies with PDS in Skin from Various Species

Tissue Control lXA 3B3 6D6 7Bl

adult bovine skin, frozen ++ ++ ++ ++ adult bovine skin, fixed ± ++ ++ + fetal calf skin, frozen ++ ++ ++ ++ fetal calf skin, fixed ± ++ ++ ++ human gingiva, frozen ++ ++ ++ human skin, fixed ± + ++ porcine skin, frozen ++ ++ ++ + porcine skin, fixed + ++ chicken skin, frozen ++ + ++ chicken skin, fixed + +

Two-fold dilutions of supernatant fluid from hybridomas lXA, 3B3, 6D6 and 7Bl grown in culture were tested for their ability to specifically locate PDS in skin sections from several different species. The location of stain in adult bovine skin was used as the specific reference (Fig. 8 a). The pattern of staining seen in Fig. 8 b represented nonspecific staining. Normal mouse serum diluted 100-fold was used as a control. Explanation of Symbols: (-), very faint nonspecific staining or no staining at all; (±), weak nonspecific and specific staining present; (+ ), weak to moderate specific staining; (+ + ), moderate to strong specific staining.

fusion, a high responder mouse strain (SJLlJ) was used. A.CAlSn mice also responded well to PDS (Fig. 1) and their use in subsequent fusions could yield antibody specificities unobtainable from mice possessing the H-2s haplotype.

Ten hybridomas were produced in this study, and the antibodies from 4 clones were selected for further characterization. All 4 MAbs recognized purified, intact PDS in ELISA assays and in immunoblots (Fig. 7) and none was inhibited by any of the other skin components tested (Figs. 3 and 4). Each of the MAbs was also inhibited by the (ABC)core ofPDS (Fig. 5). On immunoblots (Fig. 7a), each stained the (ABC)core (Mr ~ 55,000; although the true molecular weight is probably lower (Pearson et al., 1983)) as well as a minor component of slightly greater mobility which, on the basis of its recognition by all 4 MAbs, would appear to be a structural variant of the protein core. Its greater mobility may be due to a difference in carbohydrate content or loss of a small peptide in vivo, during isolation of the PDS or during digestion with chondroitinase, although proteinase inhibitors were used. Giossl et al. (1983) also observed what they interpreted to be 2 protein cores after chondroitinase ABC digestion of proteodermatan sulfate produced by cultured human skin fibroblasts. However, they found the cores to be present in more equal amounts than we find with bovine skin PDS.

Several observations suggest at least 3 different antigenic determinants were recognized by the 4 MAbs (Figs. 2, 6 and 7b), one by 3B3, one by 7B1 and one by both 1XA and 6D6. We concluded that each of the determinants contains protein because each MAb interacted with the (ABC)core (Fig. 5) and their binding was ndt inhibited by 5000 ng/ml of derma tan sulfate chains or by papain digested PDS (Fig. 6). Papain digestion would be expected to leave the glycosaminoglycan and oligosaccharide side chains intact and attached to several amino acids. The lack of interaction between the


MAbs and the papain digests argues that protein forms an essential part of each epitope. These experiments do not rule out the possibility that a carbohydrate component may also be important.

The changes in the antigenicity of PDS shown in Fig. 6 merit further discussion. While it is quite obvious that incubation of PDS with papain resulted in the loss of its ability to inhibit the MAbs (an effect that was not produced by incubation with buffer alone), it is also apparent that the papain was sufficiently inactivated by the iodoacetamide to prevent possible proteolysis of the MAbs because the absorbance was maintained close to 100 % of the range (top curves in Fig. 6). However, it is also evident that for 3 of the 4 MAbs the inhibition curve produced after PDS was incubated in buffer lacking papain was not the same as the standard curve for untreated PDS (see Fig. 6). And, in keeping with other results, the differences fall into 3 categories; i. e. 3B3, 7B1 and 1XA/6D6. For lXA and 6D6, the incubation conditions caused PDS to become a more effective inhibitor than untreated PDS. By eliminating components from the digestion buffer (cysteine Hel, iodoacetamide) this increased effectiveness as an inhibitor was reproduced by simply prolonged incubation at 65°C. Heat denaturation, and possibly fragmentation, of the molecule under these conditions would appear to have exposed their determinants to a greater degree thereby increasing antibody affinity. In contrast, the incubation conditions caused PDS to become a less effective inhibitor for 3B3 and was shown to be due to the presence of cysteine Hel in the buffer as high temperature alone resulted in a curve superimposed on the standard curve. This suggests that the integrity of the epitope recognized by 3B3 depends in part on the normal arrangement of disulfide bonds in PDS. This proteoglycan has a significant content of half-cysteine residues (Pearson et aI., 1983) but the number and position of disulfide bonds is not yet known. Alternatively, the cysteine Hel may have induced an abnormal array of disulfide bonds in PDS through disulfide interchange thereby obsuring some of its epitopes. The determinant recognized by 7Bl was unaffected by the digestion conditions. Thus, while the papain digestion of PDS indicated that the epitope of each MAb consists of protein, the control digests helped distinguish 3B3, 7B1 and 1XA/6D6 through the effect of different influences on their respective determinants.

The probability that the antibodies secreted by clones 1XA and 6D6 possess different specificities and also recognize different determinants is suggested by the results in Fig. 5. The change in slope of the inhibition curve of lXA by the (ABC)core was unique and probably represented a change in the nature of the antigenic determinant (i. e. it became more heterogeneous) as a result of enzyme treatment. lXA may require the presence of intact glycosaminoglycan side chains, or a portion of them, to stabilize its determinant while 6D6 does not. Further evidence that they and their determinants differ is provided by the immunoblots of the cyanogen bromide fragments of PDS (Fig. 7b) in which 6D6 recognized an additional fragment that lXA did not. Also, the increase in antigenicity of PDS by heat denaturation was noticeably greater for lXA than 6D6 (Fig. 6) and, in Table I, it can be seen that lXA reacted poorly with fixed tissue sections while 6D6 reacted very well. It may be concluded therefore that each MAb recognized a different determinant and that 1XA and 6D6 recognize adjacent determinants or, quite possibly, 2 determinants that overlap.

Immunohistochemical methods were also used to corroborate the specificity of the antibodies. All 4 MAbs stainded the same areas of adult bovine skin sections (Fig. 8a); although the degree of staining by each varied with fixation and species differences in the antigen (Table I). Stain was present throughout the dermis in close association with the numerous collagen fibers and resembles the pattern of immunofluorescence obtain-

36 G. A. Pringle, C. M. Dodd, ]. W. Osborn, C. H. Pearson and T. R. Mosmann

ed by Caterson et al. (1982) on sections of rat skin using an antiserum specific for remnants of glycosaminoglycan remaining after chondroitinase ABC digestion.

Fetal calf skin, estimated to be 130 days old (Lyne and Heideman, 1959), stained differently from adult bovine skin, the stain being confined to the deeper dermis (Fig. 8c). During the embryological development of skin, there is a transition in the proportions of its constituent glycosaminoglycans, in which hyaluronic acid and chondroitin sulfate generally decrease and iduronate rich-dermatan sulfate increases (Davidson and Small, 1963; Breen et al., 1970; Nakamura and Nagai, 1980; Parry et al., 1982) in the form of proteodermatan sulfate (Fujii and Nagai, 1981; Nakamura et al., 1983). This increase in dermatan sulfate is associated with an increase in collagen (Nakamura and Nagai, 1980) and Parry et al. (1982) proposed that it is necessary for the development of large diameter collagen fibrils such as those found in mature skin. Furthermore, Tajima and Nagai (1980) provided evidence to suggest that the transition to higher levels of collagen and derma tan sulfate is more obvious in the deeper layers of developing calf dermis. All these data were based on analysis of the glycosaminoglycans. By directly staining the protein core we have obtained independent evidence to suggest a similar change in the distribution of the whole PDS molecule with skin development.

We are currently endeavouring to determine whether the core protein which reacts with our 4 MAbs could carry glycosaminoglycans other than L-induronate rich dermatan sulfate. We also wish to determine whether other proteoglycans share epitopes with the protein core of PDS, although it seems unlikely that they would contain all 4 determinants. And, as we have recently undertaken amino acid sequencing of PDS (Pearson et al., 1983), we hope to locate and characterize the determinant of each MAb more precisely.

Acknowledgements

The authors wish to thank Sophie Lehocky and Dennis Carmel for their excellent technical assistance. This work was supported by grants from the Medical Research Council of Canada (MT 3540) and the Alberta Heritage Fund for Medical Research.

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Dr. Gordon A. Pringle, Department of Oral Biology, University of Alberta, Edmonton, Alberta, Canada T6G 2N8.

production and characterization of monoclonal antibodies to bovine skin proteodermatan sulfate

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