Schistosoma mansoni: reactivity with infected human sera and monoclonal antibody characterization of a glycoprotein in different developmental stages

Cercarial glycoproteins of Schistosoma mansoni were purified by concanavalin A affinity chromatography. The purified fraction consisted of at least 15 polypeptides when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sera of infected humans specifically immunoprecipitated all of these polypeptides. These purified glycoproteins were used as antigen for preparing monoclonal antibodies. One of these monoclonal antibodies immunoprecipitated cercarial polypeptides that were identical to polypeptides immunoprecipitated with sera of infected humans as analyzed by two-dimensional gel electrophoresis. Direct binding assays with ¹²⁵I-labeled monoclonal antibody showed that proteins sharing antigenic determinants recognized by this monoclonal antibody were present not only in cercariae (the source of the immunogen) but also in adult male and female worms and in eggs. The protein molecules expressing these antigenic determinants were glycosylated in each of the developmental stages of the larvae, but differed with respect to molecular weight. These findings indicate a role for this monoclonal antibody in serodiagnosis and immunoprophylaxis.     

Identification of Schistosoma mansoni glycolipids that share immunogenic carbohydrate epitopes with glycoproteins

The immunoreactivity of sera of infected hosts against glycolipids derived from Schistosoma mansoni eggs, adult male worms, and cercariae was analyzed by immunostaining of glycolipids resolved by high-performance thin-layer chromatography. Eggs contained the greatest number of immunogenic glycolipids and bound the largest proportion of serum antibodies. Virtually all of the immunogenic egg glycolipids were neutrally charged and contained oligosaccharide chains larger in size than five sugar residues. The glycolipids of each developmental stage were shown by use of five monoclonal antibodies to share schistosome-specific carbohydrate epitopes that were also present on glycoproteins. Several of the carbohydrate epitopes were expressed throughout the life cycle, yet the overall structures of the glycolipids were not conserved. Quantitative analyses by solid-phase binding assays indicated that the carbohydrate epitopes were differentially expressed between the glycolipids and glycoproteins of developmental stages. Sera from infected humans and mice both contained very high levels of anti-carbohydrate antibodies that were reactive with the glycolipids, irrespective of the stage or intensity of disease. Mice harboring unisexual infections of either male or female worms also recognized the egg glycolipids in a pattern indistinguishable from that of patently infected mice. A greater proportion of the humoral response against egg antigens in infected humans was directed against protein determinants, as compared with infected mice.     

Use of specific monoclonal and polyclonal antibodies to define distinct antigens of the porcine zonae pellucidae

Specific monoclonal and polyclonal antibodies to solubilized porcine and rabbit zonae pellucidae (ZP) and to purified ZP glycoprotein components have been used to define distinct ZP antigens. These studies demonstrate that the individual ZP glycoproteins contain both unique and shared determinants. One monoclonal antibody (R5) has been used to demonstrate that the major porcine ZP glycoprotein, which has multiple charge species ranging in molecular weight from 42,000 to 120,000, is composed of two distinct polypeptide antigens unique to this glycoprotein class. These distinct antigens can be differentiated by immunoblotting after high-resolution two-dimensional polyacrylamide gel electrophoretic separation of trypsin-treated or deglycosylated glycoproteins. The two polypeptides also differ in their staining properties with the silver-based color stain and in their susceptibility to proteolysis. A second monoclonal antibody (PSI) has been used to define a determinant shared by all three major porcine ZP glycoprotein classes. This determinant appears to involve either a carbohydrate moiety or some other molecular feature related to post-translational modification, since the antibody recognizes only the acidic species of each glycoprotein class, and does not recognize the deglycosylated forms of the proteins. This work demonstrates that there are both unique and shared antigenic determinants present in the individual components of the ZP, but that the immunodominant determinants appear to be unique to each glycoprotein.     

Cross-reactivity between herpes simplex virus glycoprotein B and a 63,000-dalton varicella-zoster virus envelope glycoprotein.

Cross-reactive monoclonal antibodies recognizing both herpes simplex virus (HSV) glycoprotein B and a major 63,000-dalton varicella-zoster virus (VZV) envelope glycoprotein were isolated and found to neutralize VZV infection in vitro. None of the other VZV glycoproteins was recognized by any polyclonal anti-HSV serum tested. These results demonstrate that HSV glycoprotein B and the 63,000-dalton VZV glycoprotein share antigenic epitopes and raise the possibility that these two proteins have a similar function in infection.     

Changes in cell surface glycoproteins during Dictyostelium development analysed using monoclonal antibodies

We have produced a series of monoclonal antibodies that recognize carbohydrate epitopes on cell surface glycoproteins of developing amoebae of Dictyostelium discoideum. The antibodies were found to have differential specificity for amoebae at different stages of development and were classified into types A to E on the basis of their temporal pattern of reactivity with the developing amoebal cell surface. Evidence from Western Blots and digestion of the glycoproteins with alkaline phosphatase were consistent with previous reports that the cell surface glycoproteins are extensively processed during development, leading at 16 h of development to the exposure of a highly antigenic core recognized by antibodies in group E. The nature of this core structure is indicated by the finding that antibodies in group E were found also to bind with high avidity to the plant glycoprotein horse radish peroxidase.     

Targeted glycoproteomic analysis reveals that kappa-5 is a major, uniquely glycosylated component of Schistosoma mansoni egg antigens

Glycans present on glycoproteins from the eggs of the parasite Schistosoma mansoni are mediators of various immune responses of the human host, including T-cell modulation and granuloma formation, and they are the target of glycan-specific antibodies. Here we have analyzed the glycosylation of kappa-5, a major glycoprotein antigen from S. mansoni eggs using a targeted approach of lectin purification followed by mass spectrometry of glycopeptides as well as released glycans. We demonstrate that kappa-5 has four fully occupied N-glycosylation sites carrying unique triantennary glycans composed of a difucosylated and xylosylated core region, and immunogenic GalNAcβ1-4GlcNAc (LDN) termini. Furthermore, we show that the kappa-5 specific IgE antibodies in sera of S. mansoni-infected individuals are directed against the core region of the kappa-5 glycans. Whereas two previously analyzed immunomodulatory egg glycoproteins, IPSE/alpha-1 and omega-1, both express diantennary N-glycans with a difucosylated core and one or two Galβ1-4(Fucα1-3)GlcNAc (Lewis X) antennae, the kappa-5 glycosylation appears unique among the major soluble egg antigens of S. mansoni. The distinct structural and antigenic properties of kappa-5 glycans suggest a specific role for kappa-5 in schistosome egg immunogenicity.     

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.     

Epitopes of herpes simplex virus type 1 glycoprotein gC are clustered in two distinct antigenic sites.

Epitopes of herpes simplex virus type 1 (HSV-1) strain KOS glycoprotein gC were identified by using a panel of gC-specific, virus-neutralizing monoclonal antibodies and a series of antigenic variants selected for resistance to neutralization with individual members of the antibody panel. Variants that were resistant to neutralization and expressed an antigenically altered form of gC were designated monoclonal antibody-resistant (mar) mutants. mar mutants were isolated at frequencies of 10(-3) to 10(-5), depending on the antibody used for selection. The epitopes on gC were operationally grouped into antigenic sites by evaluating the patterns of neutralization observed when a panel of 22 antibodies was tested against 22 mar mutants. A minimum of nine epitopes was identified by this process. Three epitopes were assigned to one antigenic site (I), and six were clustered in a second complex site (II) composed of three distinct subsites, IIa, IIb, and IIc. The two antigenic sites were shown to reside in physically distinct domains of the glycoprotein, by radioimmunoprecipitation of truncated forms of gC. These polypeptides lacked portions of the carboxy terminus and ranged in size from approximately one-half that of the wild-type molecule to nearly full size. Antibodies recognizing epitopes in site II immunoprecipitated the entire series of truncated polypeptides and thereby demonstrated that site II resided in the N-terminal half of gC. Antibodies reactive with site I, however, did not immunoprecipitate fragments smaller than at least two-thirds the size of the wild-type polypeptide, suggesting that site I was located in the C-terminal portion. Sites I and II were also shown to be spatially separate on the gC polypeptide by competition enzyme-linked immunosorbent assay with monoclonal antibodies representative of different site I and site II epitopes.     

Identification of herpes simplex virus type 1 (HSV-1) glycoprotein gC as the immunodominant antigen for HSV-1-specific memory cytotoxic T lymphocytes

The frequency and fine specificity of herpes simplex virus (HSV)-reactive cytotoxic T lymphocytes (CTL) of C57BL/6 mice was investigated in limiting dilution culture. The reactivity patterns of virus-specific CTL were assayed on target cells infected with HSV type 1, strain KOS, HSV type 2, strain Mueller, and mutants of HSV-1 (KOS) antigenically deficient or altered in glycoproteins gC or gB, two of the four major HSV-1-encoded cell surface glycoprotein antigens. Most CTL clones recognized type-specific determinants on target cells infected with the immunizing HSV serotype. In addition, the majority of HSV-1-specific CTL did not cross-react with cells infected with syn LD70, a mutant of HSV-1 (KOS) deficient for the presentation of cell surface glycoprotein gC. These data are the first demonstration of the clonal specificity of HSV-1-reactive CTL, and they identify gC as the immunodominant antigen. The fine specificity of gC-specific CTL clones was analyzed on target cells infected with mutant viruses altered in the antigenic structure of gC. These mutants were selected by resistance to neutralization with monoclonal antibodies, referred to as monoclonal antibody-resistant (mar) mutants. Most mar mutations in gC did not affect recognition by the majority of CTL clones. This indicated that most epitopes recognized by CTL are distinct from those defined by antibodies. The finding, however, that one mar mutation in gC affected both CTL and antibody recognition of this antigen may help to define antigenic sites important to both humoral and cell-mediated immunity to herpesvirus infection.     

Immunochemical characterization of related families of glycoproteins in desmosomes

Using several biochemical approaches, we have characterized the relatedness of the various glycoprotein components of the bovine epidermal desomosome. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of purified epidermal desmosomes reveals 12 proteins, of which 8 are glycosylated. Analysis with monoclonal antibodies indicates that the 8 glycoproteins comprise 3 antigenically distinct protein families. Members of the highest molecular weight glycoprotein family (a triplet of Mr = 150,000) were not distinguishable by partial proteolytic peptide mapping. At least 6 different monoclonal antibodies have been identified that recognize unique antigenic determinants shared by these proteins. Members of a 97,000-118,000-dalton glycoprotein family (about 4 bands) generate very similar but not identical partial proteolytic peptide maps. At least 3 different monoclonal antibodies have been identified that recognize unique antigenic determinants shared by these proteins. A Mr = 22,000 glycoprotein is immunologically unrelated to either of the high molecular weight glycoprotein families. Lectin-binding profiles indicate that within each immunologically related family the glycoproteins are similar in their oligosaccharide composition. Some lectins distinguish among the families. These glycoproteins probably mediate the specific intercellular recognition and adhesive functions of the desmosome.     

Micromere Differentiation in the Sea Urchin Embryo: Immunochemical Characterization of Primary Mesenchyme Cell-Specific Antigen and Its Biological Roles

Primary mesenchyme cell (PMC)-specific antigens in developing sea urchin embryos of five different species have been studied by using two different monoclonal antibodies, P4 and B2C2. Like B2C2 in Strongylocentrotus purpuratus (Anstrom et al. , 1987) P4 reacted with the N-linked carbohydrate in Strongylocentrotus intermedius embryo. Although both antibodies recognize the same group of glycoproteins in S. intermedius , P4 epitopes appeared earlier than B2C2 epitopes in Clypeaster japonicus embryo. PMCs of Anthocidaris crassispina blastulae raised in sulfate-deficient sea water were immuno-reactive with P4 but not with B2C2, although the embryos raised in normal sea water reacted with both antibodies at similar intensity. These results suggest that the epitopes of P4 and B2C2 are formed by glycosylation and sulfation, respectively. PMCs may display differential modification in their surface glycoprotein synthesis during differentiation. Furthermore, P4 inhibited cultured micromere descendant cells of Hemicentrotus pulcherrimus from attaching to the plastic dishes and forming spicules in vitro without detectable cytotoxic effect. P4-reactive glycoproteins may play important roles in cell-substrate interaction and spicule formation.     

Platelet membrane glycoproteins: role in primary hemostasis and component antigens

The biochemical details of the platelet surface as they relate to normal platelet function have been elucidated through study of labeled membranes from both normal platelets and those with congenitially defective function. Several cytoadhesive glycoprotein complexes which are integral components of the platelet membrane have been demonstrated to act as important receptors for extracellular matrix macromolecules. Glycoproteins Ia/IIa (collagen receptor), Ic/IIa (fibronectin receptor), and IIb/IIIa (fibrinogen receptor) belong to a family of cytoadhesive complexes called the integrins, while glycoprotein Ib/IX, the major von Willebrand receptor, has different features. These same major glycoproteins comprise all of the alloantigens and most of the autoantigens that have been characterized. Glycoprotein IIb/IIIa contains the alloantigens, PlA (Zw), Bak (Lek), and Pen (Yuk), as well as the most frequent target antigenic sites for anti-platelet autoantibodies. Because a number of platelet alloantigens were discovered independently by more than one group, nomenclature is confusing at present, although a system analogous to that used for histocompatibility antigens has been proposed. Precise identification of the antigenic epitopes has not yet been accomplished for all of the platelet antigens. Current research efforts include characterization of antigenic epitopes, elucidation of mechanisms by which platelet immunization occurs, and determination of the clinical implications of the presence of various platelet antibodies.     

Distribution of proteoglycans antigenically related to corneal keratan sulfate proteoglycan

Three antibodies reacting with corneal keratan sulfate proteoglycan were used to detect antigenically related molecules in 11 bovine and 13 embryonic chick tissues. Two monoclonal antibodies recognized sulfated epitopes on the keratan sulfate chain and a polyclonal antibody bound antigenic sites on the core protein of corneal keratan sulfate proteoglycan. Competitive immunoassay detected core protein and keratan sulfate antigens in guanidine HCl extracts of most tissues. Keratan sulfate antigens of most bovine tissues were only partially extracted with guanidine HCl, but the remainder could be solubilized by CNBr treatment of the guanidine-extracted residue. Keratan sulfate and core protein antigens co-eluted with purified corneal keratan sulfate proteoglycan on ion exchange high-performance liquid chromatography (HPLC). Endo-beta-galactosidase digestion of the HPLC-purified keratan sulfate antigens eliminated the binding of monoclonal anti-keratan sulfate antibodies in enzyme-linked immunosorbent assay. Extracts of all 11 bovine tissues, except those from brain and cartilage, could bind both anti-keratan sulfate monoclonal antibodies and anti-core protein polyclonal antibody simultaneously. Binding was sensitive to competition with keratan sulfate and to digestion with endo-beta-galactosidase. These results suggest widespread occurrence of a proteoglycan or sulfated glycoprotein bearing keratan sulfate-like carbohydrate and a core protein resembling that of corneal keratan sulfate proteoglycan.     

Differential expression of LacdiNAc,fucosylated LacdiNAc,and Lewis x glycan antigens in intramolluscan stages of Schistosoma mansoni

We report the expression of 3 well-characterized adult Schistosoma mansoni glycan antigens among molluscan stages of the parasite. These antigens are LacdiNAc (LDN; GalNAcbeta1-4GlcNAc-R), fucosylated LacdiNAc (LDNF; GalNAc[Fucal-3]beta1-4GlcNAc-R), and Lewis x (Le(x); Gal[Fucalpha1-3]beta1-4GlcNAc-R). The presence of the glycans was determined by both immunoblot and immunohistological methods using monoclonal antibodies that specifically recognize each glycan epitope. Immunoblot analyses reveal that LDN and LDNF epitopes are expressed on many different glycoproteins, including eggs, mother sporocysts, daughter sporocysts, and cercariae, although LDN expression among daughter sporocysts is greatly reduced. LDN and LDNF epitopes are localized on the tegument and in the intrasporocyst cell masses of both in vitro-derived and in vivo-derived mother sporocysts and in the daughter sporocysts derived on day 16 after infection. Unexpectedly, high levels of LDN and LDNF glycans were detected in the infected, but not in the uninfected, snail hemolymph, suggesting that the infecting larvae secrete LDN and LDNF glycoconjugates into the snail hosts. In contrast, the expression of Le(x) antigen among the molluscan stages is highly restricted. Le(x) is present on a few high-molecular weight glycoproteins in eggs and cercariae but is undetectable in mother and daughter sporocysts. Taken together with our earlier studies on vertebrate stages of S. mansoni, these results show that LDN and LDNF glycans are conserved during schistosome development. The study further extends the evidence that Le(x) is a developmentally regulated antigen in schistosomes.     

Marked differences in the antigenic structure of human respiratory syncytial virus F and G glycoproteins.

Monoclonal antibodies directed against the glycoproteins of human respiratory syncytial virus were used in competitive enzyme-linked immunosorbent assays for topological mapping of epitopes. Whereas epitopes of the F glycoprotein could be ascribed to five nonoverlapping antigenic sites, anti-G antibodies recognized unique epitopes, many of whose competition profiles overlapped extensively. Variant viruses selected with a neutralizing (47F) anti-F antibody lost the binding for only 47F and 49F antibodies, which mapped in the same antigenic area. In contrast, viruses selected with an anti-G antibody lost the capacity to bind most of the anti-G antibodies, and their G protein was not recognized by an anti-virus antiserum, indicating major changes in the antigenic structure of the G molecule. Finally, we found great antigenic variation of the G protein among viral isolates. This occurred even within viruses of the same subtype with only limited divergence of amino acid sequence between strains. All of these data indicate marked differences in the antigenic organization of the G and F glycoproteins of respiratory syncytial virus; we discuss these differences in terms of the chemical structure of the glycoproteins.     

Study of antigenic epitopes recognized by monoclonal antibodies to recombinant interferon-gamma

Seven hybridomas (BG 1-7) which secreted monoclonal antibodies against recombinant interferon-gamma were produced. The ascites fluids containing four of the seven monoclonal antibodies (BG 1-4) neutralized the antiviral activity of both natural and recombinant interferon-gamma. Competition between labeled and unlabeled monoclonal antibodies for interferon-gamma in a solid phase immunoassay showed that BG 1 was competed by both BG 3 and BG 4 but not by BG 2; BG 2 was competed by BG 3 but not by BG 1 nor by BG 4. These results suggest that human interferon-gamma has at least two antigenic epitopes; one of the epitopes reacted with BG 1 & BG 4 while the other reacted with BG 2; BG 3 either binds to a region overlapping with the other two epitopes or reacts with both epitopes. The antigenic epitopes recognized by these four neutralizing monoclonal antibodies are likely at or closely related to the active sites of interferon-gamma.     

Monoclonal antibodies to rabbit and pig zonae pellucidae distinguish species-specific and shared antigenic determinants

Monoclonal antibodies against rabbit or porcine zonae pellucidae (ZP) demonstrate species-specific and shared antigenic determinants. In addition, these antibodies are used to characterize the biochemical nature of these determinants. All of six monoclonal antibodies developed against porcine ZP react with porcine but not with rabbit ZP. Only one of seven monoclonal antibodies developed against rabbit ZP cross-reacts with porcine ZP. None of these antibodies recognized antigens associated with other tissues tested. High-resolution, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) followed by immunoblotting was used to demonstrate that the cross-reactive antibody recognizes an antigenic determinant which is associated with the major low molecular weight glycoprotein of both the pig and rabbit ZP. Since this antibody recognizes all charge species of this glycoprotein, it is apparent that the antigenic determinant recognized by this antibody involves protein. Further studies demonstrate that proteolytic digestion of ZP will destroy the antigenic determinant while glycosidic digestion of ZP has no effect on antibody binding. Although polyclonal antibodies to this glycoprotein inhibit sperm from binding to the zona pellucida, this monoclonal antibody does not affect sperm binding. None of the species-specific antibodies recognize ZP glycoproteins following 2D-PAGE. This is a property typical of antibodies directed against conformational antigenic determinants. The presence of common as well as unique zona antigenic determinants could explain why ZP proteins induce heteroantibodies which result in infertility while alloimmunization has no effect on fertility.     

Schistosoma mansoni and Schistosoma haematobium: identification and characterization of glycoconjugate antigens in the hemolymph of infected vector snails

Two carbohydrate epitopes were identified by monoclonal antibodies (KCS and E2) and characterized with respect to their immunoreactivity, monosaccharide structure, and location. Immunofluorescence demonstrated the presence of both epitopes on the surfaces of sporocysts, cercariae, and miracidia of Schistosoma mansoni, Schistosoma haematobium, and Schistosoma japonicum. However, spatial distribution and density of expression varied among species and developmental stages, and neither epitope was detectable on adult worm surfaces. Both glycans were found in the hemolymph of infected, but not uninfected, intermediate snail hosts. The presence of epitopes in hemolymph, as well as in schistosome eggs, is species-specific for KCS, recognizing only S. mansoni, and partly specific for E2, which reacted predominantly with S. haematobium. Immunoaffinity purification of target antigens for KCS and E2 from hemolymph of infected Biomphalaria and Bulinus, respectively, followed by carbohydrate composition analysis revealed a high content of fucose in both glycans. Methylation analysis demonstrated exclusively terminal fucose for the target antigen of KCS and terminal as well as internal fucose for the one of E2. Removal of terminal fucose abolished reactivity with both monoclonal antibodies. Both glycans are different from previously characterized schistosome carbohydrates. Their biological function(s) remain to be defined.     

Use of bacterial expression cloning to define the amino acid sequences of antigenic determinants on the G2 glycoprotein of Rift Valley fever virus.

Four distinct antigenic determinants along the G2 glycoprotein encoded by the M segment RNA of the Phlebovirus Rift Valley fever virus were localized. These epitopes were defined by four monoclonal antibodies, three of which were capable of neutralizing virus infectivity; one was nonneutralizing. Immunoprecipitation by these monoclonal antibodies of either denatured or native antigen characterized the epitopes as having linear or higher order structure. Molecular cloning of G2 glycoprotein-coding sequences into a bacterial expression plasmid utilizing a beta-galactosidase fusion protein system was employed for epitope localization. A nuclease BAL 31 plasmid expression library, in which processive regions of the 3' end of the G2 glycoprotein coding sequences were deleted, allowed for approximation of the carboxy-terminal limit of the antigenic determinants. Further subcloning of limited G2 polypeptide sequences into the bacterial expression vector permitted more refined localization of the epitopes. The characteristics of the immunoreactivity of these small peptide regions (between 11 and 34 amino acids) produced in bacteria as G2-beta-galactosidase fusion proteins were similar to those of the authentic Rift Valley fever virus G2 glycoprotein. These defined antigenic determinants and their importance in virus infectivity are discussed.     

Coexpression of cancer-associated carbohydrate antigens,Tn and sialyl Tn

The expression of cancer-associated antigens, Tn and sialyl Tn, was examined using monoclonal antibodies, MLS 128 and MLS 102, recognizing these two antigens, respectively. A cell lysate from a human carcinoma cell line, LS 180 cells, was analysed by Western blotting using these two antibodies. Three glycoprotein bands were discernible with each antibody, of which two, corresponding to 250 and 210 kDa, were reactive with both the antibodies. LS 180 cells were metabolically labelled with³H-glucosamine and then the lysate from these cells was applied to two immunoaffinity columns. Sixty-five per cent of the Tn antigenic glycoproteins, based on radioactivity, bound to the MLS 102 affinity column. On the other hand, 45% of the sialyl Tn antigenic glycoproteins bound to the MLS 128 affinity column. These results indicate that some Tn and sialyl Tn antigens were expressed on the same polypeptide chains.The presence of non-sialylated GalNAc residues on the polypeptide chain with many Sia-GalNAc residues appears to be due to the incapability of three consecutive moieties of GalNAc-Ser/Thr to accept sialic acid.Abbreviations PSMF phenylmethylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - GalNAc N-acetylgalactosamine - Sia sialic acid