The modulation of expression of polypeptide surface antigens on developing schistosomula of Schistosoma mansoni

Five low m.w. polypeptide antigens are expressed on the surface of freshly transformed schistosomula of Schistosoma mansoni, and were reproducibly identified by surface labeling with 125I by using IODOGEN and immunoprecipitating with immune mouse sera. These molecules have approximate m.w. of 38,000, 32,000, 20,000, 17,000, and 15,000. They correspond to antigens recognized previously by lactoperoxidase-catalyzed iodination. Analysis of the surface of developing schistosomulum demonstrated that the 38,000 and 17,000 dalton antigens were lost from the parasite surface during 48 hr of in vitro culture. This process was not dependent on the presence of host serum. The two antigens were not lost due to shedding into the culture medium but were apparently sequestered to a site where they were no longer available for surface labeling. The 32,000, 20,000, and 15,000 dalton antigens, however, remained exposed on the schistosomulum surface for up to 2 days of in vitro culture. The expression of two new antigens was also induced by culture in vitro: a doublet of approximately 45,000 daltons and an antigen of approximately 11,000 daltons. The expression of the former was dependent on the presence of serum. These results demonstrate that the development of the schistosomula surface is a complex process, with events both dependent and independent of the presence of serum. In addition, the expression of polypeptide antigens is not coordinated, and antigens are lost, retained, or appear on the schistosomulum surface during the early stages of maturation.     

Three major surface antigens of Schistosoma mansoni are linked to the membrane by glycosylphosphatidylinositol

Schistosomula of Schistosoma mansoni were examined for the presence of glycosylphosphatidylinositol (GPI) anchored surface membrane Ag. Parasites were surface iodinated and cultured in the presence or absence of a crude phospholipase C (PLC) preparation or phosphatidylinositol-specific PLC (PIPLC). Culture supernatants were then analyzed: 1) by centrifugation to ascertain which molecules released from the surface were soluble or contained in membrane vesicles; 2) by immunoprecipitation with antibodies specific for the "cross-reacting determinant," an epitope revealed on some GPI-anchored proteins only after cleavage of the diacylglycerol from the protein by PIPLC, and 3) by immunoprecipitation with immune mouse sera to establish co-identity with previously described, immunologically relevant surface Ag. By using these techniques, schistosomula were shown to possess three GPI-anchored surface Ag of m.w. 38,000, 32,000 and 18,000 which are spontaneously released from the surface of schistosomula in association with membrane, but remain insoluble until cleaved by PIPLC. All three molecules were recognized by antibodies from mice vaccinated with irradiated cercariae and/or chronically infected mice. Moreover, the m.w. 38,000 component was recognized by a previously described protective mAb (E.1). A major developmental modification appears to occur in the expression of these molecules because, by the same techniques, no GPI-anchored surface Ag were detectable on 7-day-old lung stage parasites. The finding that these important parasite immunogens are GPI-anchored and released from the surface of the parasite in membrane vesicles may, in part, explain why they elicit strong immune responses capable of damaging the schistosomulum tegument.     

A major role for carbohydrate epitopes preferentially recognized by chronically infected mice in the determination of Schistosoma mansoni schistosomulum surface antigenicity

A radioimmunoassay that makes use of whole schistosomula and 125I-labeled protein A has been used to characterize and to quantify the binding of antisera to the surface of 3 hr mechanically transformed schistosomula of Schistosoma mansoni. This technique facilitates the determination of epitopes on the schistosomula in addition to those detected by surface labeling and immunoprecipitation. By using this technique, it has been demonstrated that there is a much greater binding to the parasite surface of antibodies from chronically infected mice (CMS) than of antibodies from mice infected with highly irradiated cercariae (VMS), and CMS recognizes epitopes that VMS does not. Treatment of the surface of the schistosomula with trifluoromethanesulphonic acid and sodium metaperiodate has suggested that the discrepancy of the binding between the two sera is due to the recognition of a large number of additional epitopes by CMS, which are carbohydrate in nature. Some of the carbohydrate epitopes are expressed on the previously described surface glycoprotein antigens of Mr 200,000, 38,000, and 17,000.     

IgM antibodies recognizing carbohydrate epitopes shared between schistosomula and miracidia of Schistosoma mansoni that block in vitro killing

A series of monoclonal antibodies (mAb) was raised in mice against Schistosoma mansoni, which recognized a carbohydrate determinant on a major Mr greater than 200,000 schistosomulum surface antigen. These mAb cross-reacted with the surface of cercariae and miracidia and with schistosomula of S. haematobium and S. bovis. Other mAb were generated that only recognized a Mr 20,000 schistosomulum surface antigen; they did not cross-react with eggs or miracidia and were species specific. The anti-Mr 20,000 mAb of the IgG1 isotype exhibited high levels of complement-dependent cytotoxicity to schistosomula in vitro. IgM mAb that recognized carbohydrate epitopes of the Mr greater than 200,000 surface antigen blocked the lethal activity of the anti-Mr 20,000 mAb. The IgM anti-Mr greater than 200,000 mAb also reduced complement-dependent cytotoxicity of serum from mice vaccinated with irradiated cercariae.     

Cell-free synthesis of Schistosoma mansoni surface antigens: stage specificity of their expression

Messenger RNA has been extracted from all stages of the life cycle of the parasitic multicellular helminth Schistosoma mansoni. In vitro translation of these mRNA preparations in rabbit reticulocyte lysates yielded in each case a large number of polypeptides. Immunoprecipitation of translation products either by serum from immune mice or from human patients demonstrated that relatively few, approximately 10, polypeptides are recognised as antigens. Two of the in vitro synthesised antigens, of mol. wts. 22 000 and 14 000, were demonstrated to correspond to schistosomula surface antigens. The expression of these antigens may show stage specificity. Both are readily detected from adult and sporocyst translation products, neither from schistosomula and only the 22 000 antigen from miracidia. This is an unexpected finding since similar polypeptide antigens occur on the surface of schistosomula. These results indicate that not only are schistosomula surface antigens preformed at the preceding sporocyst stage, i.e., within the snail host, but they also remain invariant throughout the life cycle in the vertebrate host. Two other prominent schistosomula surface antigens of mol. wts. 38 000 and 32 000, were not recognised amongst cell-free translation products directed by RNA from any life cycle stage. The demonstration that at least two schistosomula surface antigens are detectable amongst adult mRNA cell-free translation products demonstrates the feasibility of identifying the genes encoding them in cDNA libraries from adult worm mRNA.     

Analysis of the anti-Schistosoma mansoni surface antibody response during murine infection and its potential contribution to protective immunity

Absorption of serum from chronically infected mice with homogenized schistosome eggs reduced antibody binding to the schistosomulum surface by 94%, indicating that almost all schistosomulum surface recognition during chronic infection is due to epitopes shared with the egg. Absorption of the serum with egg homogenate from which protein antigens had been removed by boiling and digestion with proteinase K resulted in a similar reduction of antisurface antibody demonstrating that all the shared epitopes that are recognized are carbohydrate in nature. Analysis of the time course of anticarbohydrate antibody production and the levels of antibody in mice infected with a single sex of schistosome indicated that eggs directly stimulated this response. Mouse mAb were identified that bound at very high levels to the schistosomulum surface and that recognized carbohydrate epitopes shared with the egg. Three of these had previously been demonstrated to passively transfer resistance, indicating that these surface carbohydrates are potential targets of protective immunity in the mouse. All the anticarbohydrate mAb also bound to the surface of schistosomula of other schistosome species. Thus, the strong immune response against these epitopes in chronic infection could account for the cross-specific immunity observed. Mice vaccinated with irradiated cercariae lacked high levels of anticarbohydrate antibodies and their recognition of the surface was largely due to antibody to species-specific polypeptide epitopes. With respect to the Mr greater than 200,000 and 38,000 antigens, it was demonstrated that these epitopes were present on the same antigens that bear the carbohydrate moieties recognized by antibodies from chronically infected mice. This specific polypeptide recognition is also reflected in the immunity generated by exposure to irradiated cercariae.     

Schistosoma mansoni polypeptides immunogenic in mice vaccinated with radiation-attenuated cercariae

We compared the humoral immune response of mice protected against Schistosoma mansoni by vaccination with radiation-attenuated cercariae to that of patently infected mice, and we identified antigens that elicit a greater, or unique, immune response in the vaccinated mice. These comparisons were based upon radioimmunoprecipitations and immunodepletion of [35S]methionine-labeled schistosomular and adult worm polypeptides, followed by one- and two-dimensional polyacrylamide gel analyses. The humoral responses of patently infected mice and of mice vaccinated once were remarkably similar and were directed against schistosome glycoproteins ranging in molecular size from greater than 300 to less than 10 kDa. Exposing mice to a second vaccination resulted in a marked change in the immune response, to one predominantly directed toward high molecular size glycoproteins. Sequential immunodepletion techniques identified five schistosomular and seven adult worm antigens that showed a greater or unique immunogenicity in vaccinated mice as compared with patently infected mice. These adult worm antigens were purified by preparative sequential immunoaffinity chromatography and used to prepare a polyclonal antiserum, anti-irradiated vaccine. This antiserum bound to the surface of live newly transformed and lung-stage schistosomula, as assessed by immunofluorescence assays, and was reactive with a number of 125I-labeled schistosomular surface polypeptides, including a doublet of 150 kDa that was also recognized by sera of vaccinated mice but not by sera of patently infected mice.     

Screening of murine monoclonal antibodies against living schistosomula of Schistosoma mansoni by radioimmunoassay

A radioimmunoassay was developed to screen supernatants of murine monoclonal antibodies against surface antigens of living schistosomula of Schistosoma mansoni. Of 196 clones screened, 10% bound schistosomula. Of these, 74% bound only schistosomula. The remaining molecules also reacted with soluble adult worm antigens and soluble egg antigens as determined by enzyme-linked immunosorbent assay. Immunoblot analysis demonstrated that monoclonal antibody 204-3E4 reacted with a 68 kDa protein, a glycoprotein that induces substantial resistance against S. mansoni infection. Recognition of an 18 kDa antigen by 204-3F1 antibody was stage-specific with the antigen being expressed in cercariae, 3- and 24-h-old parasites but not 4-day, lung stage or adult worms. Monoclonal antibody 204-4E3 reacted with purified S. mansoni paramyosin. These data indicate that radioimmunoassay using living schistosomula is a rapid alternative method to identify murine hybridomas that secrete antibodies which react with surface antigens of S. mansoni.     

Surface antigens on cercariae,schistosomula and adult worms of Schistosoma mansoni

Shah J. and Ramasamy R. 1982. Surface antigens on cercariae, schistosomula and adult worms of Schistosoma mansoni. International Journal for Parasitology12: 451–461. The surface protein antigens of Schistosoma mansoni were radiolabelled by lactoperoxidase catalysed I¹²⁵-iodination and analysed by immune-precipitation and polyacrylamide gel electrophoresis. The results showed that regularly labelled surface antigens of mol. wts >150,000, 78,000, 45,000 and 22,000 were present on adult worms. Common surface antigens were observed on the cercariae, schistosomula and adult worms. It is suggested that surface antigens released from living adult worms can sensitise a host to react against the invading schistosomula of a secondary infection. However, the failure to vaccinate mice using material containing adult worm surface antigens suggests that the induction of protective immunity is a complex phenomenon.     

Native species of helix destabilizing protein-1 in mouse myeloma identified by antibody probing of Western blots

Antibody probing of Western blots is a method for analyzing the apparent Mr of a protein in any given preparation (Renart, J., Reiser, J., and Stark, G., Proc. Natl. Acad. Sci. USA 76: 3116, 1979). We prepared a rabbit antiserum to purified mouse myeloma helix destabilizing protein-1 and used this antiserum in Western blotting experiments with a crude homogenate of mouse myeloma. The results indicated that the native species of helix destabilizing protein-1 can be degraded during purification. This in vitro proteolysis results in complete loss of the native species and accumulation of lower Mr proteins that represent limit digestion products. These findings have identified the true native form of mouse myeloma HDP-1 as a protein of apparent Mr = 36,000 to 38,000, instead of the Mr = 24,000 and 27,000 proteins obtained by routine purification.     

Induction of protection against Brugia malayi infection in jirds by microfilarial antigens

The development of immunologic methods to reduce transmission of human lymphatic filariasis depends on measures that will enhance the host's ability to eliminate infective larvae, adult worms, or blood-borne microfilariae (mf). The present study was designed to assess the capacity of a crude extract of Brugia malayi mf to decrease the level of microfilaremia and adult worm burden in jirds inoculated with infective larvae, and to identify the filarial antigens that elicit antibody responses in these animals. Thirty weeks after subcutaneous inoculation with 75 infective larvae, 100% of control jirds were patent (i.e., had microfilaremia) compared with 60% of the group immunized with 10 micrograms of crude microfilarial extract (p less than 0.05). In addition, microfilaremia was lower in patent immunized animals compared with controls (p less than 0.05). The mean total number of adult female B. malayi per jird recovered at necropsy in control animals was 16.0 vs 7.0 in immunized jirds (p less than 0.05). Serum of immunized jirds contained anti-mf antibodies with an end titer of 1:8000, a value similar to that of animals with chronic B. malayi infection. Microfilarial antigens of Mr approximately 150,000, 75,000, 42,000, and 25,000 were identified in immunoblotting studies by reactivity with antibodies in sera of immunized jirds. Antibodies induced by immunization with microfilarial extract were not specific for this stage of the parasite life cycle, as jird anti-mf antibodies reacted with a Mr approximately 150,000 and several Mr 50,000 to 110,000 antigens derived from immature and mature adult parasites of both sexes. These data indicate that immunization of jirds with a water soluble microfilarial extract enhances the host's ability to eliminate adult worms and blood-borne mf. The filarial antigens that induce antibodies in immunized jirds have been identified.     

Schistosoma japonicum: protective immunity induced by schistosomulum-derived cells in a mouse model

We previously reported that immunization with intact live cells from schistosomula of Schistosoma japonicum (S.j) partially protected the Kunming strain of mice from challenge infection. In the present work, 2 immune protective experiments were designed to further validate the protective effect induced by this type of vaccine and to optimize the immunization protocol, including the number of inoculations and parasite stages from which immunogenic cells were derived. Three antigens derived from 18-day-old postinfection live (LLC) and dead (DLC) larval worm cells and from dead 42-day-old postinfection adult worm cells (DAC) were used as immunogens. Our results demonstrate that live cells from 18-day-old worms are capable of inducing significant protection in mice using a murine-Sj challenge model as shown by reduction rates of worm recoveries and egg burdens. The development of adult worms was stunted. A Th1-biased immune response was reflected in the protected groups as evidenced by the ratio of IgG2a/IgG1. A 38-kDa polypeptide was recognized by sera from LLC immunized animals. We demonstrate that live parasite cells are a source of novel protective antigens that can be exploited for vaccine development.     

Localization and identification of Schistosoma mansoni/KLH-crossreactive components in infected mice.

KLH (Keyhole limpet hemocyanin) is highly immunogenic, and crossreactive epitopes occur widely in nature. In schistosomiasis, infected hosts generate antibodies reactive with KLH. This is of diagnostic importance but we lack detailed information on the immunogen-carrying molecules and their distribution in the worm. We used anti-KLH antibodies to localize cross-reacting epitopes in the various developmental stages of the parasite in experimental Schistosoma mansoni infection. The staining results show KLH crossreactivity in the life stages of the parasite. By immunoblotting we show that KLH-crossreactive antigenic epitopes in the parasite eggs are carbohydrates, also recognized by antibodies against soluble schistosome egg antigens. The localizations in the larval stages and in adult worms suggest that crossreacting antigenic epitopes are secretory products.     

Schistosoma mansoni surface glycoproteins. Analysis of their expression and antigenicity

Surface glycoproteins from newly transformed schistosomula of Schistosoma mansoni have been identified by surface radioiodination and lectin-affinity chromatography. From the glycoconjugates bound by the three lectins used, concanavalin A, peanut agglutinin and fucose-binding protein, only in the concanavalin-A-bound fractions were glycoproteins identified. Changes in concanavalin-A-binding glycoproteins were detected after transformation and early maturation of the schistosomula. Some glycoproteins disappeared (Mr 38 000, 29 000 and 25 000), some appeared independently of host molecules (Mr 19 000), others only appeared after culture in human serum (Mr 45 000). Two major glycoproteins of Mr 32 000 and 16 000 were detected on all stages examined. Within the total set of surface glycoproteins identified on 3-h schistosomula only the strong Mr-38 000-32 000 complex was found to be antigenic. Thus many major low-molecular-mass surface glycoproteins of the parasite are not recognised as antigens by immune animals. The separation of only the Mr-38 000-32 000 antigens by concanavalin A affinity chromatography indicates the feasibility of using this method in conjunction with immunoaffinity columns to purity these molecules.     

The measurement of the relative turnover rates of proteins of the surface membranes and other fractions of Schistosoma mansoni in culture.

The double isotope labelling method of Arias, Doyle & Schimke (1969) and Dehlinger & Schimke (1971) was used to determine the relative rates of turnover of proteins in various tissue fractions and in the culture medium of adult Schistosoma mansoni. The majority of proteins in the surface membrane turned over at about the same rate. This is consistent with a model for membrane assembly and degradation involving multi-protein units. The proteins in the other fractions examined, except frozen-thawed supernatant fluid, also turned over at about the same rate. In the frozen and thawed supernatant fluid, the higher molecular weight (greater than 40 000 daltons) proteins turned over at a much greater rate than the lower molecular weight proteins (less than 40 000 daltons). The antigens in the culture medium, both particulate and soluble, had a higher turnover rate than the worm tissue proteins. It is concluded that the culture medium antigens are released as the result of two distinct processes: (a) surface membrane turnover and (b) a rapid secretory process. It is possible that the high molecular weight proteins found in the forzen-thawed supernatant fluid are involved in the rapid secretory process. The culture media of 6-day and 16-day schistosomula were also examined by the double isotope method. The culture medium of the adult worm and 16-day schistosomula contains more material presumed to be secreted than does that from the 6-day schistosomula. This may explain the poor immunogenicity of young schistosomula.     

Characterization of antigens of the nematode Nippostrongylus brasiliensis by monoclonal antibodies

Hybridomas secreting monoclonal antibodies to Nippostrongylus brasiliensis antigens were generated by hybridization of IR983F myeloma cells with spleen cells from Lou/M/Wol rats infected with living third-stage larvae. Antibodies specific either for larval or worm antigens were identified by enzyme-linked immunosorbent assays with Nippostrongylus brasiliensis fragments, homogenates and secretions as antigens. The results demonstrate that all antibodies which recognized larval antigens (38 antibodies) also reacted with worm surfaces. Ten antibodies were specific only for worm antigens. Ten antibodies reacted with worm homogenate, three antibodies recognized components of worm secretion and 17 antibodies combined with acetylcholinesterase. The epitope specificity was investigated by the capacity of various glycosides, aminoacids, N-acetylneuraminic acid and phosphorylcholine to inhibit the binding to worm fragments. The analysis revealed that alpha-methylglucoside, alpha-methylmannoside, N-acetylglucosamine, N-acetylgalactosamine, fucose and the amino acids leucine, phenylalanine, tyrosine, serine, tryptophan did not combine with the antigen-binding sites of the antibodies. Proline, arginine and histidine, however, displayed inhibitory effects. With N-acetylneuraminic acid as inhibitor three groups of antibodies could be discriminated. At a concentration of 10-20 mM, phosphorylcholine was a potent inhibitor for all antibodies.     

Analysis of Clonorchis sinensis antigens and diagnosis of clonorchiasis using monoclonal antibodies.

Clonorchis sinensis is a common parasite of man in Korea. Researches on the specific antigens of C. sinensis would be valuable not only because those elucidate the molecular characteristics of this fluke but also because it is applicable to immunodiagnosis. Although many monoclonal antibodies have been used in the field of parasite immunology, few articles on monoclonal antibodies against C. sinensis have been published so far. The aim of this study was to analyze C. sinensis antigens recognized by monoclonal antibodies, and to set up ELISA-inhibition test using C. sinensis specific monoclonal antibodies for improved specificity of immunodiagnostic tests. By fusion between spleen cells of the mice immunized with C. sinensis water-soluble crude adult worm antigens and plasmacytoma cells of mouse origin, 29 hybridoma clones secreting anti-C. sinensis monoclonal antibodies were made, and 8 clones among those were found specific. After cell cloning, isotypes of 6 selected specific monoclonal antibodies were determined to be IgG1, IgG2b and IgA. Four exposed antigenic determinants of natural infection were recognized by different specific monoclonal antibodies. By enzyme-immunoelectrotransfer blot, 10 KD, 34 KD antigenic determinants were found to be reacted with CsHyb 0714-20, CsHyb 0605-10 monoclonal antibodies, respectively. The antigenic determinant recognized by CsHyb 0714-20 monoclonal antibody was revealed to be located at the surface and parenchyme of a parasite by indirect immunofluorescent antibody technique, and those reacted with CsHyb 0605-10, CsHyb 0714-25 monoclonal antibodies were found at the parenchyme and intestine. The antigenic determinant reacted with CsHyb 0605-23 monoclonal antibody was found mainly around the uterine eggs. Four antigenic determinants recognized by specific monoclonal antibodies were all found to be present in the early eluted fractions of C. sinensis antigens separated by Sephadex G-200 gel filtration. By conventional ELISA, 75% of clonorchiasis cases were found positive, but 7.1% of normal controls and 37.5% of paragonimiasis cases showed false positives. However, by ELISA-inhibition test using C. sinensis specific monoclonal antibody (CsHyb 0605-23), 77.1% of clonorchiasis cases were found positive, and there were no false positives in normal controls or paragonimiasis cases, indicating 100% specificity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Western Blot analysis of the antigens of Toxoplasma gondii recognized by human IgM and IgG antibodies

Western Blot analysis revealed that both IgM and IgG antibodies present in the sera of humans infected with Toxoplasma gondii recognize three major antigens with apparent m.w. of 32,000, 22,000, and 6000, respectively. In addition, IgG antibodies recognized at least 17 other antigenic components. After subcellular fractionation, enrichment of the three major antigens recognized by IgM and IgG antibodies by the membrane fraction was observed. Solubilization of membrane-enriched preparations with a mixture of sodium dodecyl sulfate and sodium deoxycholate did not reveal any new antigenic structures that reacted with IgM or IgG antibodies. Treatment of Toxoplasma lysate preparations and various fractions obtained after differential centrifugation with NaIO4 diminished the reactivity of the antigens with both IgM and IgG antibodies. Lipase treatment had no effect on the number or nature of antigens recognized by IgM antibody. Treatment with pronase and trypsin eliminated the 32,000 and 22,000 m.w. antigenic components detected by IgM antibodies, whereas such treatment had no effect on the 6000 m.w. component. Periodic acid-Schiff staining of polyacrylamide gels of Toxoplasma sonicates revealed the presence of three components corresponding to m.w. of 62,000, 45,000, and 6000, respectively. At least 15 components, including the 6000 m.w. component, directly bound concanavalin A.     

Schistosoma mansoni: cloning of antigen gene sequences in Escherichia coli

Fischer rat protective antiserum (F-2x) prepared from Schistosoma mansoni-infected rats was used to screen an adult worm cDNA library constructed in a lambda gt11 bacteriophage expression vector. This led to the isolation of several clones yielding proteins reactive with antibodies in the infection serum. Counter-screening of these clones with Wistar-Furth rat nonprotective antiserum (W-2x) enabled identification of clones either uniquely or preferentially reacting with F-2x, in addition to clones of nearly equal reactivity with both antisera. Six clones were further characterized. Five expressed beta-galactosidase/S. mansoni fusion proteins which migrated more slowly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than beta-galactosidase and all were reactive in a Western immunoblot assay. The cDNA insert sizes in the clones ranged from 150 to 900 base pairs. Rabbit antibodies prepared against fusion proteins from three of the clones recognized biosynthetically radio-labeled 4-week worm proteins of sizes 20, 38, and 70 kDa, respectively. The 20- and 38-kDa proteins were among the protein antigens uniquely recognized by the F-2x protective antiserum. These proteins are therefore candidates for protective vaccine antigens and the recombinant lambda clones are now serving as useful reagents for obtaining the corresponding nucleotide gene sequences.     

Ontogeny and characterization of mesenchyme antigens of the sea urchin embryo

A monoclonal antibody, Sp12, binds to cortical granules, the hyaline layer, and skeletogenic, chromogenic, and blastocoelar mesenchyme of sea urchin eggs and embryos. Adult urchins also express Sp12 antigens in the dermal layer of the test and spines. Antigen is expressed on the surface of primary mesenchyme cells after they have entered the blastocoel, and by two secondary mesenchyme derivatives--the blastocoelar cells after they have been released from the tip of the archenteron, and the pigment cells in prism stage embryos. Immunogold localizations show antigen on the surfaces of mesenchyme, within membrane bounded vesicles, and associated with the Golgi apparatus. Western blots of antigens immunoprecipitated from seven developmental stages reveal twelve antigens ranging in Mr from 35 k to 240 k. Most of these antigens appear, disappear or change Mr over the first five days of development. Characterizations of this complex array of antigens show that the epitope recognized by Sp12 is eliminated by proteolytic enzymes and endoglycosidase F, while immunoreactivity is only reduced by periodate oxidation. As well, calcium magnesium free seawater extracts a subset of antigens different from that retained by crude membrane preparations. It is proposed that the mesenchyme of sea urchin embryos produces a family of developmentally regulated cell surface and extracellular matrix glycoproteins which all exhibit a carbohydrate epitope recognized by Sp12.