A Monoclonal Antibody That Specifically Recognizes a Glucuronic Acid 2-Sulfate-containing Determinant in Intact Chondroitin Sulfate Chain*

Monoclonal antibodies produced against chick embryo limb bud proteoglycan (PG-M) were selected for their ability to recognize determinants on intact chondroitin sulfate chains. One of these monoclonal antibodies (IgM; designated MO-225) reacts with PG-M, chick embryo cartilage proteoglycans (PG-H, PG-Lb, and PG-Lt), and bovine nasal cartilage proteoglycan, but not with Swarm rat chondrosarcoma proteoglycan. The reactivity of PG-H to MO-225 is not affected by keratanase digestion but is completely abolished after chondroitinase digestion. Competitive binding anal-yses with various glycosaminoglycan samples indicate that the determinant recognized by MO-225 resides in a D-glucuronic acid 2-sulfate(~1+3)N-acetylgalactos-amine 6-sulfate disaccharide unit (D-unit) common to antigenic chondroitin sulfates. A tetrasaccharide trisulfate containing D-unit at the reducing end is the smallest chondroitin sulfate fragment that can inhibit the binding of the antibody to PG-H. Decreasing the size of a D-unit-rich chondroitin sulfate by hyaluronidase

In recent years a large number of apparently different chondroitin sulfate proteoglycans have been isolated. They are primarily located in extracellular matrices where they appear to help maintain tissue architecture and influence cell growth and differentiation (for reviews, see Refs. [1][2][3]. Some, such as those intercalated in the cell membrane of melanoma cells (4) or those located in the secretory granules of mouse mast cells (5) and human natural killer cells (6), appear to have different, more specialized functions. In early studies two types of chondroitin sulfate were distinguished, chondroitin 4-sulfate composed of repeating GlcA'-GalNAc 4-* This work was supported by grants-in-aid for Cancer Research, Scientific Research, and Special Project Research from the Ministry of Education, Science and Culture, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. The abbreviations used are GlcA, glucuronic acid; PG-M, the major chondroitin sulfate proteoglycan of stage 22-23 chick embryo limb bud; PG-H, PG-Lb, and PG-Lt, three distinct proteoglycan components of 12-day-old chick embryo cartilage; AlD1, large aggregating chondroitin sulfate proteoglycan monomer from bovine nasal cartilage; Agg-Dl, large aggregating chondroitin sulfate proteoglycan SO4 disaccharide units and chondroitin 6-sulfate composed of repeating GlcA4alNAc 6-S04 disaccharide units (7). However, it has since been shown that considerable chemical variability can be superimposed upon the component sugar units. Thus, the galactosamine residues can be 4,6-bissulfated either at the nonreducing terminal (8) or in the internal portion (9) of chondroitin sulfate. The glucuronic acid residues, on the other hand, can be sulfated (10) at either position 2 or position 3 (11). It appears that most if not all chondroitin sulfate proteoglycans carry copolymers of these sulfated disaccharide units in which the percentages of the disaccharides vary with the source and age of the tissue (for a review, see Ref. 12).
Recent immunological studies of proteoglycans have revealed that epitopes for antibody binding exist not only on the core proteins but also on intact keratan sulfate (13,14) and chondroitin sulfate side chains (15). The latter antibody was prepared against ventral membranes of cultured chick fibroblast and has been shown to react primarily with the chondroitin sulfate associated with cell surface membranes. However, there has been no information on the precise antigenic structure recognized by this antibody.
Recently, we have prepared a number of monoclonal antibodies against two distinct chondroitin sulfate proteoglycans, PG-H (16) and PG-M (17), isolated from 12-day-old chick embryo epiphysial cartilage and stage 22-23 chick embryo limb buds, respectively. In this paper, we report that one of these antibodies specifically recognizes a GlcA 2-S04-containing determinant contained in the chondroitin sulfate chains of proteoglycans.  Concentration (Fg/ml) giving 50% inhibition in ELISA on PG-H. Percent of total hexuronate content or total 35S activity (chick embryo cartilage preparation only) estimated from the yields of the unsaturated disaccharides produced by digestion with chondroitinases; nonsulfated unit, GlcA-+GalNAc; A-unit, GlcA-GalNAc &so4; C-unit, GlcA4alNAc 6-S04; D-unit, GlcA 2-S04-GalNAc 6-SO4; E-unit, GlcA-GalNAc 4,6-bis-SO4. e About 7% of the total hexuronate was recovered as glucose-carrying pentasaccharides and higher oligosaccharides with different sulfate contents (see the text for details).
ND, not determined because 35S would not label nonsulfated disaccharide units.
old chick embryo epiphysial cartilages; AlDl from bovine nasal cartilage (20); and Agg-Dl from Swarm rat chondrosarcoma (21). Glycosaminoglycans-Shark fin chondroitin sulfates (Fraction I, 11, and 111) were prepared as follows. About 120 g of the fin of shark (Glyphis glaucus) was minced and digested at 55 "C for 18 h with 60 mg of Prolisin in 60 ml of water. The digest was made 0.5 M in NaOH and kept at 40 "C for 1 h. After neutralization with HCl, insoluble materials were removed by filtration through Celite. From the filtrate, chondroitin sulfate was precipitated with 1.5 volumes of 95% (v/v) ethanol containing 1.3% (w/v) sodium acetate, washed successively with ethanol and ether, and dried in a vacuum over P205; yield, 5 g. The chondroitin sulfate preparation was dissolved in 100 ml of 1 M NaCl and applied to a Diaion HPA-10 (anion exchange resin) column (2.6 X 55 cm) that had been equilibrated with 1 M NaCl. The column was washed with 600 ml of 1 M NaCl and then eluted stepwise with 400 ml of 1.5 M NaCl, 180 ml of 1.5 M NaCl, and 450 ml of 2.5 M NaCl. Effluents were dialyzed free of NaCl and evaporated at reduced pressure to a concentration near 10 mg of polysaccharide/ml. The chondroitin sulfate was precipitated with ethanol, washed, and dried as above: yield, 1.9 g (the first 1.5 M NaCl fraction, designated Fraction I), 1.21 g (the second 1.5 M NaCl fraction, designated Fraction II), and 0.83 g (the 2.5 M NaCl fraction, designated Fraction 111). The composition of disaccharide repeat units of these preparations is given in Table I Oligosaccharides-A mixture of tetrasaccharides from whale cartilage chondroitin sulfate was prepared by digestion with testicular hyaluronidase followed by gel chromatography as described by Flodin et al. (26). ADi-OS, ADi-4S, ADi-6S, ADi-diSD, and ADi-diSE (9) were the products of Seikagaku Kogyo Co., Tokyo.
The tetrasaccharides AGlcA((31-3)GalNAc 4-S0&31+4)GlcA 2-S04(pl-3)GalNAc 6-SO4 and AGlcA(B1-3)GalNAc 4,6-bis-SOa(p1+4)GlcA 2-S04(p14)GalNAc 6-SO4 were prepared from shark fin chondroitin sulfate Fraction 111 (see above) by taking advantage of the fact that chondroitinase AC I Flavobacterium heparinurn* acts much more slowly on the hexosaminidic linkages to GlcA %so4 residues in chondroitin sulfate than on the hexosaminidic linkages to nonsulfated glucuronosyl residues (29). Briefly, a solution of 100 mg of shark fin chondroitin sulfate Fraction 111 in 2 ml of 0.025 M Tris buffer, pH 7.2, containing 0.4 mg of calcium acetate, 2.5 mg of bovine serum albumin, and 10 units of chondroitinase AC I F. heparinum (27) was incubated at 37 "C for 16 h. The reaction was stopped by placing the tube in a boiling water bath for 2 min. After centrifugation at 900 X g for 5 min, the clear supernatant was concentrated to a small volume at reduced pressure and then chromatographed on a Cellulofine GCL-90-m column (3 X 96 cm) equilibrated and eluted with 0.5 M NaC1. The eluates were screened for hexuronic acid-positive material. Three peak materials corresponding in Kd to hexasaccharide, tetrasaccharide, and disaccharide were obtained in the hexuronate ratio of 1:1617. The tetrasaccharide fractions were pooled and subjected to ion exchange chromatography on an AG 1-X4 column (2.2 X 30 cm) eluted with a linear NaCl concentration gradient (1-3.5 M). The column yielded two peak materials (hexuronate ratio = 3.2:l) eluting at about 2 M NaCl and 3 M NaCl, respectively. The identification of the 2 and 3 M NaCl fraction as the tetrasaccharide tri-and tetrasulfate, respectively, was based on ( a ) an absorption maximum at 232 nm, (b) the molar ratios of total hexuronate, galactosamine, and sulfate to AGlcA, (c) the formation of equimolar amounts of ADi-4S and ADi-diSD (from the trisulfate) or ADi-diSE and ADi-diSD (from the tetrasulfate) on chondroitinase ABC digestion, and (d) the susceptibility of the GlcA 2-SOd-GalNAc 6-SO4 disaccharide unit (in both tetrasaccharides) to reduction with NaBH4 (see Ref. 30 for the analytical methods based on the selective reduction with NaBH4).

Methods
Immunization, Fusion, and Cloning-Three 6-week-old female BALB/c mice were injected intraperitoneally with either PG-M or PG-H solutions (50 pg of the antigen/mouse, emulsified in complete Freund's adjuvant). Injection of immunogen in incomplete Freund's adjuvant was repeated twice in %week intervals and the formation of antibodies was monitored by ELISA (see below). Two days after the final injection, the spleens of two mice showing high antibody titers were removed and the cells from spleen were fused with NS-1 myeloma line (8-azaguanine-resistant) at a lymphocyte to myeloma ratio of 101, using 20% (w/v) polyethylene glycol 4000 (32). The fusion products were suspended in hypoxanthine/aminopterin/thymidinecontaining medium (RPMI 1640, 10% fetal calf serum). The cell suspension was aliquoted (1 X 10' cells/well) into 96-well plates supplemented with feeder cells from a peritoneal lavage of a pristanetreated BALB/c mouse and the plates were cultured at 37 "C in an incubator in the presence of 5% COP. After 2 weeks of culture, hybridomas showing positive reaction in ELISA on PG-M or PG-H were cloned by limiting dilution. From these, a clone that reacted with PG-M but not with chondroitinase ABC-treated PG-M and a clone that reacted with PG-H but not with keratanase-treated PG-H were selected. Selected hybridoma clones were stored under liquid nitrogen or grown up in larger tissue culture dishes for large scale preparations of monoclonal antibodies. In some experiments, 1 X 10s cloned hybridoma cells from confluent culture dishes were injected intraperitoneally into male BALB/c mouse injected with 0.5 ml of pristane and the ascites fluid was recovered 2 weeks later as a sample of monoclonal antibody. Isotype of monoclonal antibody was determined with the mouse monoclonal isotyping kit (Zymed Laboratories).
ELISA for Characterization of the Monoclonal Antibodies-ELISA was done as described by Rennard et al. (33) with a slight modification. Briefly, antigens (5 pg of protein/ml in carbonate buffer/azide, 200 pllwell) were coated on the plastic surface of the microtiter well by passive adsorption overnight at 4 "C. The plates were then rinsed three times with phosphate-buffered saline, pH 7.4, containing 0.05% (w/v) Tween 20 and appropriate culture supernatant (50 pl diluted with 100 p1 of phosphate-buffered saline, pH 7.4) or ascites fluids (0.1 pl diluted with 100 pl of phosphate-buffered saline, pH 7.4) were added and incubated for 2 h at room temperature. The plates were then rinsed as above and horseradish peroxidase-conjugated goat anti-mouse IgM (or IgG + IgM) diluted 1:500 into phosphate-buffered saline/Tween 20 was added as the second antibody. After 1 h of incubation, o-Phenylenediamine (0.1 mg/ml in methanol diluted 1:100 into 0.03% (v/v) H,Oz) was added and color allowed to generate for 15 min, after which the reaction was stopped by adding 50 p1 of 8 M H,SO,. The brown color produced was measured spectrophotometrically at 480 nm.
For ELISA to test the effects of chondroitinase or keratanase treatment on proteoglycan antigens, appropriate enzyme (5 units of chondroitinase ABC, 0.5 unit of chondroitinase AC I1 A. aurescens, or 1 unit of keratanase/ml in enriched buffer containing protease inhibitors (16); 100 pllwell) was added to the antigen-coated plates and incubated for 1 h at 37 "C. The plates were then rinsed and subjected to ELISA as above.
For competitive inhibition tests, test samples were serially diluted into phosphate-buffered saline, pH 7.4, and 100-pl aliquots were added to the antigen-coated plates. Culture supernatants containing antibodies were then added and allowed to react with the antigens as above. The concentration of inhibitor required for 50% inhibition ( I C d was calculated from plots of A d s o nm against inhibitor concentration. Radioimmuoprecipitation" 100-pl solution of [36S]sulfate-labeled PG-M or PG-H in phosphate-buffered saline/Tween 20 (see above) was mixed with appropriate volumes of ascites fluids containing MO-225 or HM-110 and the mixtures were incubated for 12 h at 4 "C. Then, 10 pl of goat anti-mouse IgG antibody (y-and L-chain specific) was added as the second antibody and incubation continued for an additional 12 h at 4 "C. The solution was mixed with 50 pl (bed volume) of Protein A-Sepharose in 100 pl of phosphate-buffered saline/?'ween 20 and the mixture was allowed to stand at room temperature for 1 h. The resultant immunoprecipitates were pelleted by centrifugation at 1200 X g for 15 min at 4 "C. The pellets were washed three times with phosphate-buffered saline/Tween 20. Radioactivity of the washed precipitates was measured in a liquid scintillation spectrometer. For preparation of small-size chondroitin sulfate, shark cartilage chondroitin sulfate was digested with testicular hyaluronidase for 3 h as above, and the supernatant from the digest was chromatographed on a Sephadex G-100 column (calibrated with standard chondroitin sulfates of the known molecular weights) equilibrated and eluted with phosphate-buffered saline. Fractions corresponding in position to a M, = 10,000 chondroitin sulfate were pooled.
Other Methods-Hexuronic acid was measured by the method of Bitter and Muir (34) with glucuronolactone as a standard. The galactosamine content of oligosaccharides was determined, after hydrolysis with 6 M HCI at 100 'C for 8 h, by the Elson-Morgan method as modified by Strominger et al. (35). The sulfate content of oligosaccharides was determined, after hydrolysis as above, by the method of Dodgson as modified by Picard et al. (36). Disaccharide products formed by cleavage of chondroitin sulfates with chondroitinases were identified by chromatographic comparisons with previously characterized standards (9). Their amounts were measured spectrophotometrically at 232 nm (9). Chemical desulfation of whale cartikage chondroitin sulfate was carried out by the method of Kantor and Schubert (37).

Characteristics of Monoclonal Antibodies Produced against PG-M and PG-H-
The monoclonal antibody (MO-225) characterized in this paper originates from a group of hybridoma clones prepared against PG-M, the chondroitin sulfate proteoglycan isolated from stage 22-23 chick embryo limb buds (17). Of a number of hybridoma culture supernatants with significant reactivity to the immunogen in ELISA, at least two did not cross-react with chondroitinase ABC-digested PG-M, suggesting that the chondroitin sulfate chains of PG-M are antigenic. From these, we cloned a hybridoma producing an IgM ( p , K ) and injected the clone into mice. The resulting ascites fluid contained the same monoclonal antibody as that found in the culture supernatant, as judged by specificity toward various proteoglycans and glycosaminoglycans (see below). In the present study the antibody in culture supernatant was used unless otherwise indicated.
When a purified PG-H preparation from 12-day-old chick embryo epiphysial cartilage (16) was used as an immunogen, a number of positive hybridomas producing antibodies to the immunogen were obtained but all the hybridoma culture supernatants so far tested recognized either the core protein or a keratanase-sensitive part of PG-H (note that PG-H differs from PG-M in possessing keratan sulfate side chains (16,17)). It appears that the keratan sulfate moiety of PG-H is more immunogenic than the chondroitin sulfate moiety. In this study a monoclonal IgGl (yl, K) (designated HM-110) prepared from one of these keratan sulfate-positive clones was used as a control.
In control experiments, HM-110 was examined for its reactivity to the above six proteoglycans. The keratan sulfatecontaining proteoglycans, PG-H and AlD1, showed strong reaction but the other four (lacking keratan sulfate) showed no significant reaction, consistent with the notion that the antigenic determinant for HM-110 resides in the keratan sulfate chains. The difference between MO-225 and HM-110 in specificity was further illustrated by radioimmunoprecipitation analyses. Thus, when 5000 cpm each of [35S]sulfatelabeled PG-M and PG-H were subjected to immunoprecipitation with either MO-225 or HM-110, about 25% of the added PG-H was recovered in both of the MO-225 and HM-110 immunoprecipitate, whereas the labeled PG-M was found only in the MO-225 immunoprecipitate.
As Fig. 2 shows, enzymatic removal of the keratan sulfate side chains from PG-H did not alter its immunoreactivity to  have arisen from an absence in the chondroitin sulfate of this particular proteoglycan of the determinant to be recognized by the antibody (see below for further evidence).
Competitive Binding Analysis with Glycosaminoglycans-Various glycosaminoglycans and related anionic polymers from different sources were tested as competitive inhibitors of the binding of MO-225 to PG-H. Of these, the following compounds were inactive at the highest concentration tested, 3.3 mg/ml; heparin (porcine intestinal mucosa), heparan sulfate (porcine lung), dermatan sulfate (pig skin), keratan sulfate (shark cartilage), keratan sulfate (bovine cornea), hyaluronic acid (cockscomb), dextran sulfate, and DNA. In contrast, many if not most chondroitin sulfate preparations gave significant inhibition (Table I). It appears that the inhibitory activity of chondroitin sulfate, expressed as a concentration (microgram/ml) giving 50% inhibition (ICbo), reflected the content of GlcA2-S04+GalNAc 6-SO4 disaccharide unit (designated D-unit), i.e. the higher the D-unit content the higher the inhibitory activity. Thus, the activity of shark fin chondroitin sulfate with the highest D-unit content (21.4% of the total hexuronate) was about 42,500-fold higher than that of chick embryo cartilage chondroitin sulfate (the side chains of PG-H) with a low D-unit content (2% of the total hexuronate). None of the D-unit-lacking polysaccharides, i.e. sturgeon notochord chondroitin sulfate, Swarm rat chondrosarcoma chondroitin sulfate, and squid skin chondroitin, gave any significant inhibition at the highest concentration tested, 3.3 mg/ml. Since the chondroitin sulfate from Swarm rat chondrosarcoma represents the chondroitin sulfate moiety of Agg-Dl, its failure to inhibit the binding of MO-225 may explain why the antibody did not bind to Agg-Dl (Fig. 1). It is also noteworthy that the inhibitory activity of whale cartilage chondroitin sulfate, which contains 3% D-units, was completely abolished after chemical desulfation (data not shown).
In contrast, squid cartilage chondroitin sulfate, the preparation that did not release ADi-diSD after chondroitinase ABC (or AC I F. heparinum) digestion, gave significant inhibition (ICbo = 30 wg/ml). The structural basis for this apparently divergent finding is not known. Since, however, this chondroitin sulfate has been shown to differ from ordinary chondroitin sulfates in containing disaccharide repeat units with glucose branches linked by fi-~-(l-S) to the hexosamine residues (30), the observed inhibition activity may reflect the existence of GlcA 2-so4 residues in some of those glucose carrying repeat units. The available data (30) suggest that many glucose carrying repeat units are adjacent to each other to form glucose branch-rich domains which are released after chondroitinase digestion as sulfated oligosaccharides larger than pentasaccharides.
to Intact Chondroitin Sulfate Inhibition of Binding of MO-225 to PG-H with Oligosaccharides Derived from Chondroitin Sulfate-As Table 11 shows, the tetrasaccharide trisulfate and tetrasaccharide tetrasulfate obtained by enzymatic digestion of shark fin chondroitin sulfate Fraction I11 gave significant inhibition with MO-225, whereas a tetrasaccharide fraction obtained by digestion of whale cartilage chondroitin sulfate was inactive at the highest concentration tested, 10,000 pg/ml. Also inactive were a series of unsaturated disaccharides (ADi-OS, ADi-4S, ADi-6S, ADi-diSE, and ADi-diSD) obtained by chondroitinase ABC digestion of shark cartilage chondroitin sulfate. In control experiments, neither of the shark fin tetrasaccharides inhibited the binding of HM-110 to PG-H at 10,000 pg/ml. Between the shark fin tetrasaccharides, the disaccharide sequence, GlcA2-S04-GalNAc 6-S04, is common. Thus, the results are compatible with the notion that MO-225 specifically recognizes D-unit. The inability of ADi-diSD to inhibit the binding may be due to the structural difference in hexuronosyl residue. Since glycosides or disaccharides consisting of D-glucuronic acid 2-sulfate and an aglycone other than N-acetylgalactosamine 6-sulfate were not available for testing, it is not yet known whether the N-acetylgalactosamine 6-sulfate residue in D-unit is an obligate requirement for recognition by the antibody.
Factors Affecting the Binding of PG-H to MO-225"Physicochemical studies (38, 39) have shown that divalent cations have significant effects on the conformation of sulfated glycosaminoglycans and proteoglycans. Using the ascites fluid containing MO-225, effects of 5 mM CaC12, 5 mM MgC12, 5 mM KCl, 1 mM EDTA, and 1 mM EGTA on the binding of the antibody to PG-H were tested. In no case, however, was the binding activity significantly influenced by these added reagents.
When shark cartilage chondroitin sulfate (D-unit content = 8.4%) was digested with testicular hyaluronidase, its ability to inhibit the binding of MO-225 to PG-H was progressively decreased with digestion time (Fig. 3). The results indicate that not only the D-unit content but also the chain length is important in determining the binding activity of antigenic chondroitin sulfate.
The binding of MO-225 to the PG-H substrate in ELISA was competitively inhibited by PG-H itself added to the medium (Fig. 4). The inhibitory activity of PG-H was considerably reduced after treatment of the proteoglycan with either TPCK trypsin or Pronase, indicating that the epitope has a requirement for protein core for a better fit of chondroitin sulfate to the paratope of the antibody. bearing A*-glucuronic acid, were also reported in preimmune rabbits by Poole et al. (41). Some of these sera reacted with intact hyaluronic acid and chondroitin but never with intact chondroitin sulfate. It could be speculated then that chondroitin sulfate side chains of native proteoglycans are nonantigenic. In two other studies, however, monoclonal antibodies directed to either intact chondroitin 4/6-sulfates (15) or saturated chondroitin 6-sulfate oligosaccharides (42) have been prepared by immunization with ventral membranes of cultured chick fibroblasts or testicular hyaluronidase-treated chick embryo cartilage proteoglycans, respectively. This would indicate that GlcA+GalNAc 4 -s o 4 and/or GlcA-GalNAc 6-SO4, the common repeat units of many avian and mammalian chondroitin sulfates, can be the focus of an immune response in mice. Alternatively, the apparent antigenicity of the avian chondroitin sulfate preparations could be due to the fact that the preparations contained a small proportion of some other structural units (such as D-unit) which are foreign to mice. reported here, we feel a need of reliable information on the fine structure of the chondroitin sulfates used for specificity studies.
The occurrence of sulfated glucuronosyl residues in shark chondroitin sulfate was demonstrated many years ago (9,10) and the position of sulfate determined to be C-2 (11). Since, however, this residue is present at low levels in most of the chondroitin sulfate preparations so far isolated from avian and mammalian sources, the significance of such a residue in the structure and function of proteoglycan glycosaminoglycan chains has been discounted. Recently an unusual species of heparan sulfate has been found in the nuclei of rat hepatocytes which contains a high proportion of GlcA % s o 4 residues (43). Furthermore, a sulfatase which specifically hydrolyzes the sulfate group from GlcA % s o 4 but not from L-iduronic acid %sulfate has been observed in human skin fibroblasts and chick embryo chondrocytes (44), suggesting that GlcA % s o 4 residues in glycosaminoglycans might be of greater significance than previously belie~ed. ~ We previously showed that a dermatan sulfate preparation from pig skin contains IdoA2-S04+GalNAc 4-so4 units (5-6% of the total disaccharide units) (9). In the present study, this dermatan sulfate preparation was shown to give no inhibition to the binding of MO-225 to PG-H. Also shown to be inactive were porcine intestinal mucosa heparin and porcine lung heparan sulfate which contain IdoA % s o 4 residues linked to Nand/or 0-sulfated glucosamine units at considerably high levels. The results indicate that the IdoA2-S04containing disaccharide sequences involved in these glycosaminoglycans cannot be recognized by MO-225.
The occurrence of GlcA3-S04+GalNAc 4-so4 disaccharide units has been suggested in king crab chondroitin sulfate (11). In view of its high sulfate to glucuronic acid ratio (-1.64), this polysaccharide must contain a high proportion of GlcA % s o 4 residues. It was of interest therefore to test the antigenicity of this polysaccharide to MO-225. Our competitive inhibition tests have shown that the inhibitory activity of a king crab chondroitin sulfate preparation5 is far lower (IC5o = 5.0 pg/ml) than the activities of shark cartilage chondroitin sulfate preparations. Although the precise structure of king crab chondroitin sulfate is unknown, the results suggest that MO-225 may distinguish between GlcA2-S04+GalNAc 6430, and GlcA3-S04+GalNAc 4-so4 residues. GlcA4alNAc 4,6-bis-S04, the disulfated disaccharide unit frequently found in chondroitin sulfate chains of avian and mammalian proteoglycans (8) and designated "E-unit," is unlikely to be recognized by MO-225, since squid cartilage chondroitin sulfate with a high proportion of E-units (61% of the total hexuronate) was far less inhibitory than shark fin chondroitin sulfates with only 0.8-6.7% E-units (Table I).
There may be differences in average chain length among the chondroitin sulfate samples used for competitive inhibition tests, but this could not explain the observed difference in their inhibitory activity. Thus, a M , = 10,000 fragment prepared from shark scapular cartilage chondroitin sulfate was much higher (IC50 = 170 p(g/ml)'j in inhibitory activity than sturgeon notochord chondroitin sulfate (IC5o = >3,300 pg/ml, see Table I) which has a comparable or slightly longer chain length (average M, = 12,000).
Well characterized antibodies are now available which rec-' A small amount of the nuclear heparan sulfate was kindly given by Dr. H. E. Conrad (University of Illinois) and was tested for its effect on the binding of MO-225 to PG-H. At a concentration of 10 pg/ml, the sample had no effect on the binding.
Kindly donated by Dr. N. Seno (Ochanomizu University, Tokyo). M. Yamagata, unpublished observation. ognize epitopes present in chondroitin sulfate, keratan sulfate, and core protein structures characteristic of different subtypes of chondroitin sulfate proteoglycans. The use of monoclonal antibodies directed to the different parts of proteoglycans may offer great potential in the immunohistochemical characterization of the proteoglycan subtypes distributed in various tissues. Furthermore, it can be surmised that glycosaminoglycan chains of native proteoglycans from various sources may contain different antigenic structures to which specific monoclonal antibodies can be prepared. It is certainly desirable to have monoclonal antibodies which can detect differences in fine structures among glycosaminoglycan chains.
Observations on the changes in their fine structure during biological changes in health and disease may help to understand the biological roles of glycosaminoglycan chains.