Numerous studies show that host humoral immune responses to schistosome infections are directed primarily to glycan antigens of the parasite (reviewed in Cummings and Nyame, 1996, 1999). Vaccination of animals with live, radiation-attenuated cercariae, provides a high level of resistance to challenge infection, and the protective immunity is at least partly directed against carbohydrate determinants (Hsu et al., 1981; Richter et al., 1993, 1996). Additionally, humans in endemic areas, who are resistant to schistosome infections, bear IgE antibodies to the surface glycoproteins of the parasite (Hagan et al., 1991; Rihet et al., 1991). Thus, schistosome glycans constitute some of the most important molecular targets for serodiagnosis and the development of vaccines. Many of the glycans synthesized by schistosomes have now been structurally characterized, which is facilitating the direct assessment of their antigenicity in infected animals (Cummings and Nyame, 1999).
Schistosomes synthesize a number of interesting glycan structures, many of which are related to those found in animal cells. These include glycans containing the Lewis x antigen (Lex), which were the first schistosome glycans to be structurally characterized and shown to be antigenic (Ko et al., 1990; Srivatsan et al., 1992a; van Dam et al., 1994), and specifically synthesized by schistosomes and not other trematodes or nematodes (Nyame et al., 1998). Infections with S.mansoni generate both cytolytic IgM and IgG antibodies in humans and animals (Nyame et al., 1996, 1997). Other antigenic glycans characterized from schistosomes include the O-linked glycosaminoglycan-like oligosaccharides containing GlcNAc and GlcA present on the circulating anodic antigen (CAA) (Bergwerff et al., 1994).
S.mansoni also synthesize biantennary N-glycans bearing lacdiNAc (LDN; GalNAc[beta]1->4GlcNAc-R) and the [alpha]1,3-fucosylated lacdiNAc (LDNF; GalNAc[beta]1->4[Fuc[alpha]1->3]GlcNAc-R) sequences (Srivatsan et al., 1992b) and the [beta]-1,4-N-acetylgalactosaminyltransferase and [alpha]1,3-fucosyltransferase required for the synthesis of LDN and LDNF have been characterized from adult S.mansoni (Srivatsan et al., 1994; DeBose-Boyd et al., 1996). Both LDN and LDNF sequences are especially interesting, since they are not only synthesized by parasitic helminths, but they are also found in many glycoproteins from higher animals (Baenziger, 1996; van den Eijnden et al., 1998). We now report that mice infected with S.mansoni generate IgM and IgG antibodies to LDN. The anti-LDN antibodies are very specific and react with LDN determinants presented on a variety of glycoproteins, including those from S.mansoni adults and bovine milk. Furthermore, an IgM monoclonal antibody to LDN, designated SMLDN1.1, has been generated from the spleen of S.mansoni infected mice and this monoclonal antibody displays the same specificity for LDN sequences as the antibodies in the sera of infected mice.
S.mansoni infected mice generate antibodies to LDN
The possible presence of antibodies to LDN determinants in the sera of S.mansoni infected mice was determined by ELISA using LDNT-BSA as the target (Figure
Figure 1. Structures of neoglycoproteins used in this study.
Figure 2. S.mansoni infected mice generate IgM and IgG antibodies to LDN. The presence of antibodies to LDN was determined by ELISA using LDNT-BSA as the antigenic target. Microtiter wells were coated with LDNT-BSA and incubated with pooled sera from S.mansoni infected mice or uninfected mice serially diluted 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12,800. Bound antibodies were detected by incubations with peroxidase conjugated goat anti-mouse IgM or IgG and ABTS/peroxidase substrate. Absorbance of each well was determined at 405 nm on a microtiter plate reader. The ELISAs were performed in triplicate and the results represent averages of the three determinations.
Bovine milk glycoproteins contain biantennary N-linked glycans in which one of the two outer branches are terminally substituted with LDN sequences (Girardet et al., 1995; Sato et al., 1995, 1997; Coddeville, 1998). To further confirm the presence of anti-LDN antibodies, sera from infected mice were analyzed for reactivity towards bovine milk glycoproteins. Bovine milk glycoproteins were purified by chromatography on Con A-Sepharose and subjected to Western blot analysis using sera from infected and uninfected mice. Sera from S.mansoni infected mice reacted with numerous bovine milk glycoproteins, and most of the reactivity was lost upon treatment with N-glycanase (Figure
Figure 3. Sera from S.mansoni infected mice recognize LDN sequences on N-glycans of bovine milk glycoproteins. Approximately 10 µg of bovine milk glycoproteins purified over columns of Con A-Sepharose were treated as indicated with N-glycanase, and analyzed by SDS-PAGE and Western blot using pooled sera derived from (A) infected or (B) uninfected mice and peroxidase conjugated goat anti-mouse IgM secondary antibody. Lane 1, untreated sample; lane 2, mock treated sample lacking N-glycanase; lane 3, N-glycanase treated. Anti-LDN in sera of S.mansoni infected mice are primarily of IgM class and react exclusively with LDN sequences
Figure 4. Analysis of the isotypes of the anti-LDN antibodies in sera from S.mansoni infected mice. Antibodies from sera of S.mansoni infected mice were affinity purified over a column of immobilized bovine milk glycoproteins and were diluted 1:5 and analyzed against LDNT-BSA using ELISA procedures described in Materials and methods. The isotypes of bound antibodies were determined by incubation with a 1:200 dilution of peroxidase-conjugated anti-mouse immunoglobulin isotype-specific antibodies.
To determine their isotypes and glycan specificity, the anti-LDN antibodies were affinity purified from infected mice sera over columns of immobilized bovine milk glycoproteins. Affinity purified antibodies reactive to LDN sequences were primarily of IgM class, but IgG1 was also detected (Figure
Figure 5. Anti-LDN antibodies in sera of S.mansoni infected mice react specifically with glycans containing LDN determinants. Antibodies in pooled sera from S.mansoni infected mice, purified over a column of immobilized bovine milk glycoproteins, were diluted 1:5 and tested against the indicated glycan targets by ELISA, as described in Figure 2, using peroxidase-conjugated goat anti-mouse IgM secondary antibody.
Affinity purified anti-LDN react with LDN glycans on S.mansoni glycoproteins
The affinity purified antibodies were characterized further by analyzing their reactivity towards LDN determinants on adult schistosome glycoproteins. Detergent extracts of adult S.mansoni were separated by SDS-PAGE, transferred to nitrocellulose and probed with the affinity purified antibodies as described in Materials and methods. The antibodies reacted with numerous glycoproteins from the extract and the reactivity was almost completely abolished by N-glycanase treatment (Figure
Figure 6. Western blot of affinity-purified anti-LDN against total adult S.mansoni extract. Approximately 10 µg of detergent extract of adult S.mansoni, with or without treatment with N-glycanase, were separated by SDS-PAGE and analyzed by Western blot, as described in Materials and methods, using a 1:5 dilution of antibodies to affinity purified anti-LDN and peroxidase-conjugated goat anti-mouse IgM secondary antibody. Generation of monoclonal antibodies to LDN
Because antibodies are clearly induced to LDN determinants in mice infected with S.mansoni, we used the spleens of these infected animals to generate monoclonal antibodies to LDN. BALB/c mice were infected with S.mansoni cercariae for 8 weeks and their spleens were fused with SP2/O myeloma cells to generate hybridomas. Four hybridomas secreting IgM antibodies to LDN were selected by ELISA using LDNT-BSA as targets. The four hybridomas were developed into single cell clones secreting monoclonal antibodies to LDN. One of the clones, designated as SMLDN1.1, was found to be specific in ELISA for LDN determinants, and it was used to produce ascites.
The specificity of the ascites was determined by ELISA against a panel of neoglycoproteins and glycoproteins. SMLDN1.1 specifically recognizes LDNT-BSA in ELISA (Figure
Figure 7. ELISA of monoclonal antibody SMLDN1.1 against neoglycoprotein targets. Ascites derived from SMLDN1.1 hybridomas was serially diluted 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12,800 and analyzed by ELISA against the indicated glycan targets, as described in Materials and methods, using peroxidase-conjugated goat anti-mouse IgM secondary antibody.
Figure 8. Western blot of SMLDN1.1 against various glycoproteins. Approximately 10 µg each of bovine milk glycoproteins (lane 1), keyhole limpet hemocyanin (lane 2), cholera toxin (lane 3), purified recombinant gp120 (lane 4), and fetal bovine fetuin (lane 5) were analyzed by Western blot using (A) a 1:1000 dilution of SMLDN1.1 and peroxidase-conjugated goat anti-mouse IgM secondary antibody or (B) the peroxidase-conjugated goat anti-mouse IgM secondary antibody alone.
Figure 9. LDN sequences are expressed on numerous glycoproteins from different schistosome species. Approximately 10 µg of detergent extracts of S.mansoni, S.haematobium, and S.japonicum were separated by SDS-PAGE and the presence of LDN glycans were determined by Western blot using a 1:1000 dilution of SMLDN1.1 and peroxidase-conjugated goat anti-mouse IgM secondary antibody. Extracts of COS7 cells were analyzed as controls.
We have shown that infection of mice with S.mansoni leads to the generation of antibodies to the terminal carbohydrate structure known as the lacdiNAc or LDN sequence. The antibodies against LDN determinant, which were immunoaffinity purified from the sera of infected mice, and the monoclonal antibody SMLDN1.1, derived from the spleens of infected mice, specifically recognize LDN determinants. Furthermore, the anti-LDN response in mice is not influenced significantly by genetic background, since identical responses were generated in both infected outbred Swiss Webster mice and inbred BALB/c mice.
The presence of LDN sequences in glycoproteins of S.mansoni were originally identified in our studies on the overall structures of N-glycans synthesized by the parasite (Nyame et al., 1989; Srivatsan et al., 1992b). It was found that the LDN determinant, and their [alpha]1,3-fucosylated derivative now termed LDNF, is abundantly present in complex-type biantennary N-glycans from S.mansoni (Srivatsan et al., 1992b). Preliminary work had suggested that some of the LDN-containing N-glycans might be recognized by antibodies in the sera of infected animals (Srivatsan et al., 1992a), but the current study represents the first formal demonstration of the immunogenicity of the LDN sequence in any animal and the generation of a monoclonal antibody to this determinant.
LDN sequences are also synthesized by other trematodes and nematodes (Dell et al., 1999), but in many cases these sequences are modified by other sugars and may occur in subterminal positions. For example, the dog heartworm Dirofilaria immitis, synthesizes complex-type N-glycans containing terminal LDN sequences and many are [alpha]1,3-fucosylated to make an LDNF structure (Kang et al., 1993). Trichinella spiralis, a parasitic gut nematode, synthesizes N-glycans containing LDN sequences that are modified with the unusual sugar d-tyvelose (3,6-dideoxy-d-arabinohexose), which itself it highly antigenic (Reason et al., 1994). LDN sequences occur in internal positions of glycosphingolipids of the parasitic pig nematode Ascaris suum (Lochnit et al., 1997). Antibodies to LDN and different modifications of LDN sequences in other helminths, coupled with antibodies to other types of glycan antigens, such as Lex (Ko et al., 1990; Srivatsan et al., 1992a; Nyame et al., 1997), might allow the immunological discrimination of different parasitic glycoconjugates, which could be highly useful in developing specific immunodiagnostic assays for parasites. There are reasons for optimism in this direction, since serodiagnosis of schistosomiasis using antibodies to carbohydrate antigens of CCA and CAA are showing promise in the diagnosis and treatment of schistosomiasis (De Clercq et al., 1997; van Lieshout et al., 1997).
The terminal sequences of O-glycans and many complex-type N-glycans in most mammalian glycoproteins contain the typical N-acetyllactosaminyl structure Gal[beta]1->4GlcNAc[beta]1-R (lacNAc) that is further modified by sialylation, fucosylation, galactosylation, sulfation, and other types of modifications (Kornfeld and Kornfeld, 1985). However, there are increasing reports on the occurrence of the LDN sequence in vertebrate glycoproteins (reviewed in van den Eijnden et al., 1995, 1998). For example, LDN is found in bovine milk glycoproteins, where it occurs terminally and in a sialylated form (Girardet, 1995; Sato, 1995, 1997; Coddeville, 1998), human pituitary glycohormones, where it is sulfated (SO4-GalNAc[beta]1->4GlcNAc[beta]1-R) (Baenziger, 1996), human glycodelin, where it occurs terminally and in sialylated and fucosylated forms (LDNF) (Dell et al., 1995), human urokinase, where is sulfated, sialylated and fucosylated (LDNF) (Bergwerff et al., 1995), and in recombinant human protein C expressed in human 293 cells, where it is fucosylated (LDNF) (Grinnell et al., 1994). It should be emphasized that SMLDN1.1 is specific for terminal LDN determinants and will not recognize these modified LDN structures. It should be possible to expose underlying LDN determinants by treatment of glycoconjugates with glycosidases and/or sulfatases and use SMLDN1.1 to identify the presence of the LDN structures. Such approaches might be useful in evaluating the prevalence and distribution of LDN structures in mammalian glycoconjugates. It is hoped that the availability of the monoclonal antibody SMLDN1.1 to LDN will be useful in future studies to address these issues and generally assess the distribution and possible functions of LDN determinants in animals and their parasites. Sera
Pooled sera from infected mice was derived by cardiac puncture from 10 Swiss Webster mice infected each for 8 weeks with 250 S.mansoni cercariae. Pooled sera from 10 sex and age matched uninfected Swiss Webster mice were used as controls. Antibodies
Peroxidase conjugated goat anti-mouse IgM and IgG (µ and [gamma] chain-specific, respectively) were obtained from Kirkegaard and Perry (Gaithersburg, MD). The peroxidase-conjugated goat anti-mouse immunoglobulin subclass typing kit, Clonotyping System-HRP, was purchased from Southern Biotechnology Associates, Inc. (Birmingham, AL). Enzymatic synthesis of LacdiNAc tetraose (LDNT)
LDNT was synthesized from LNnT by replacing the terminal [beta]1-4 linked galactose with a [beta]1->4-linked GalNAc. The LNnT was first degalactosylated by treatment with [beta]-galactosidase to generate the trisaccharide, GlcNAc[beta]1->3Gal[beta]1->4Glc (triose). This was used as an acceptor to synthesize GalNAc[beta]1->4GlcNAc[beta]1->3Gal[beta]1->4Glc (LDNT), using bovine milk [beta]1,4-galactosyltransferase and UDP-GalNAc as the donor. The reaction mixture consisted of 5 µmol of triose, 20 µmol UDP-GalNAc, and 5 U of [beta]1,4-galactosyltransferase in 100 µl of 50 mM sodium cacodylate buffer, pH 7.4, containing 20 mM MnCl2, 0.02% NaN3, and 10 mg/ml [alpha]-lactalbumin. The reaction was carried out at 37°C and aliquots were analyzed daily by HPAE-PAD chromatography to monitor conversion of the triose to tetrasaccharide. The reaction was stopped after 3 days when it had proceeded to ~95% completion. The LDNT product was purified from the reaction mixture by chromatography on a Bio Gel P-2 column (1.5 cm × 160 cm) in H2O and 3 ml fractions were collected. The chromatographic profile was monitored by measuring the absorbance of the fractions at 214 nm. Fractions containing the tetrasaccharide product were pooled and the yield was determined by absorbance at 214 nm using GlcNAc as standard. The reaction yielded 4.8 µmol of LDNT. Aliquots of the tetrasaccharide product were hydrolyzed by strong acid and analyzed by HPAE-PAD chromatography to confirm its monosaccharide composition.
The pentasaccharide GalNAc[beta]1->4(Fuc[alpha]1->3)GlcNAc[beta]1->3Gal[beta]1->4Glc (lacdiNAcfucopentaose or LDNFP) was synthesized from LDNT using GDP-Fuc as donor and recombinant human [alpha]1,3-fucosyltransferase VI (Calbiochem, La Jolla, CA). The reaction was carried out in eight aliquots at 37°C in a total reaction volume 200 µl/aliquot. Each reaction mixture contained 740 nmol of LDNT, 1 µmol GDP-Fuc, and 5 mU of enzyme in 50 mM sodium cacodylate, pH 7.5, containing 20 mM MnCl2, 0.02% NaN3, and 0.5 U alkaline phosphatase. (Aliquots of the reaction mixtures were analyzed daily, as described above, to monitor conversion of LDNT to LDNFP.) The reaction was stopped after 72 h and a 2 nmol sample of the reaction mixture was analyzed by HPAE-PAD chromatography, which showed that all acceptor was converted to product. The product was purified by chromatography on a Bio Gel P-2 column, as described above. The total yield of LDNFP, as determined by absorbance at 214 nm, was 5.4 µmol per reaction. Neoglycoproteins
The oligosaccharides LDNT, LDNFP, LNnT, and triose were derivatized to BSA by the reductive amidation procedure (Gray, 1974). Briefly, 1 mg of each oligosaccharide was mixed with of 670 µg BSA and 520 µg NaBH3CN in a total volume of 50 µl of 0.2M KH2PO4 buffer, pH 7.0. The mixture was incubated at room temperature in the dark for 14 days and was stopped by adding 2 ml of water. The mixture was dialyzed against water and the protein content was determined by BCA assay (Pierce, Rockford, IL). Moles of sugar derivatized to BSA was determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and found to be an average of 3-4 mol of sugar per mole of BSA. LNFPIII-BSA, LNFPII-BSA, and sialyl-Lex-BSA were purchased from V-Labs (Covington, LA). Parasite extract
The parasites were suspended in ice cold PBS, pH 7.4, containing protease inhibitor cocktail (Boehringer-Mannheim, Indianapolis, IN) and disrupted by sonication on ice using a Branson sonifier and 30 sec bursts. The homogenate was adjusted to 1% Triton X-100 and kept on ice for 30 min to allow total solubilization of proteins. The homogenate was spun at 3000 r.p.m. for 30 min at 4°C and the supernatant fractions were centrifuged further at 50,000 r.p.m. for 30 min at 4°C. The supernatant fraction was recovered and the protein content was determined by BCA assay. The extract was aliquoted and stored at -80°C until used. Purification of bovine milk glycoproteins bearing LDN sequences
Approximately 100 g of bovine nonfat dry milk powder (Nestle) was dissolved in 1 l of Con A-Sepharose column wash buffer (0.1 M Tris, pH 8.0, 0.15 M NaCl, 1 mM MgCl2, 1 mM CaCl2) and applied to a column (5 × 3 cm) of 13 mg/ml Con A-Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ) at 4°C. The column was washed extensively with wash buffer until no protein could be detected in the wash fractions by absorbance measurements at 280 nm. Bound glycoproteins were eluted in 20 batches, and at each batch the column was incubated at room temperature for 10 min with 10 ml of a saturated solution of [alpha]-methylglucoside in wash buffer before elution. Fractions containing protein were pooled, concentrated, dialyzed against water, and protein content was determined by BCA assay. Purification of anti-LDN polyclonal antibodies
Bovine milk glycoproteins purified over Con A-Sepharose (40 mg) were mixed with an equal volume 0.2 M MOPS/1.2 M sodium citrate buffer, pH 7.4 and derivatized to UltraLink biosupport medium (Pierce, Rockford, IL) using instructions provided by the manufacturer. Coupling was carried out at room temperature for 4 h with rotation followed by overnight rotation at 4°C. The final coupling density was ~15 mg milk glycoproteins per ml of biosupport medium. Pooled sera from S.mansoni infected mice (1 ml) was mixed with 7 ml of PBS and applied to a 2 ml milk glycoproteins/UltraLink column. The column was rotated at room temperature for 1 h and washed with PBS until no protein could be detected in the column wash by absorbance measurements at 280 nm. The column was washed with 10 ml of water and bound antibodies were eluted with 100 mM glycine (pH 2.8) and 2 ml fractions were collected. The eluted fractions were neutralized by adding 200 µl of 1 M Tris (pH 8.0). Fractions containing protein were pooled and protein content was determined by absorbance at 280 nm. The affinity purified antibodies were stabilized by adding BSA to a final concentration of 3 mg/ml. ELISA
ELISAs were performed using reagents and procedures described previously (Nyame et al., 1997). Microtiter wells were coated with 50 µl of either 1 µg/ml (monoclonal antibody analysis) or 5 µg/ml of neoglycoconjugate (for sera analysis) solutions in bicarbonate buffer, blocked with a solution of 5% BSA in PBS and incubated with 50 µl of diluted sera or antibody. Bound antibodies were detected by incubation with 50 µl of either 1:15,000 dilution of goat anti-mouse IgM-peroxidase or 1:10,000 dilution of goat anti-mouse IgG-peroxidase followed by incubation with 100 µl of ABTS/peroxidase substrate for 10 or 20 min. Absorbance of each well was determined at 405 nm using a microtiter plate reader (Molecular Devices, Sunnydale, CA). All incubations were performed at room temperature for 1 h. The microtiter wells were washed 6× after each incubation with PBS-Tween 20 (0.3% Tween 20) using a microtiter plate washer (Dynatech, Chantilly, VA). Antibody dilutions were carried out in PBS-Tween containing 1% BSA. The assays were performed in triplicate. Western blots
Proteins and extracts were separated by SDS-PAGE on 5-20% acrylamide gradient gels under reducing conditions and blotted onto nitrocellulose filters. The filters were blocked by incubation for 2 h in a 5% solution of BSA in TBS (10 mM Tris, 150 mM NaCl, pH 7.5,) and incubated with antibodies diluted in TBS containing 1% BSA and 0.05% Tween 20 for 1 h. The filters were subsequently incubated with a 1:15,000 dilution of goat anti-mouse IgM-peroxidase conjugate for 1 h and reactive bands were revealed by 20 sec incubation with SuperSignal chemiluminescence substrate (Pierce, Rockford, IL) followed by 10 sec exposure to BioMax MR-1 film (Eastman Kodak Co, Rochester, NY). After each incubation, the filters were rinsed 3× and washed 1× for 15 min and 3× for 10 min. The washes were carried out room temperature using a wash buffer of 10 mM Tris, 300 mM NaCl, 0.05% Tween-20, pH 7.5. Enzyme digestion
Peptide N-glycosidase F digestions were performed exactly as described previously (Ausubel et al., 1997). Approximately 100 µg of protein was mixed with 25 µl of 0.2 M [beta]-mercaptoethanol/1% SDS solution in an Eppendorf tube, and the volume was adjusted to 45 µl with PBS. The mixture was boiled for 5 min and left at room temperature to cool. The cooled extracts were mixed with 25 µl 0.5 M Tris, pH 8.0, 10 µl of 10% solution of Triton X-100 in water and 3 µl of peptide N-glycosidase F (a kind gift from Dr. Robert Haltiwanger, State University of New York at Stonybrook, New York). The mixture was incubated at 37°C for 18 h and treated for analysis by SDS-PAGE. Production monoclonal antibodies to LDN
Spleen cells used in generating monoclonal antibodies to LDN were derived from BALB/c mice infected for 8 weeks with 250 S.mansoni cercariae. The spleen cells were fused with SP2/O mouse myeloma cells according to standard protocols (Harlow and Lane, 1988). Fused cells were plated into eight 96-well plates and maintained in Iscove's medium containing 20% fetal bovine serum, 10 ng/ml recombinant human interleukin-6, 2× HAT and OPI for 14 days. After 14 days, the media was changed to Iscove's medium containing HT instead of HAT. Hybridomas secreting antibodies to LDN were selected on day 14 by ELISA using 50 ml of culture supernatants. Single cell clones secreting anti-LDN antibodies were generated by limiting dilution, grown in Iscove's containing 20% fetal bovine serum and 10 ng/ml recombinant human interleukin-6 and injected into BALB/c mice primed with pristane to produce ascites.
Schistosome life stages and materials for this work were supplied through the NIH-NIAID contract NO1-AI-55270. We gratefully acknowledge the kind gift of PNGase F from Dr. Robert S.Haltiwanger. This work was supported by RO1 AI42272-01 and a grant from the Oklahoma Center for the Advancement of Science and Technology to R.D.C.
LDN, lacdiNAc, GalNAc[beta]1->4GlcNAc; LDNF, fucosylated lacdiNAc, GalNAc[beta]1->4[Fuc[alpha]1->3]GlcNAc; LDNT, lacdiNac tetraose, GalNAc[beta]1->4GlcNAc[beta]1->3Gal[beta]1->4Glc; LDNFP, lacdiNAc fucopentaose, GalNAc[beta]1->4(Fuc[alpha]1->3)GlcNAc[beta]1->3Gal[beta]1->4Glc; LNFPIII, lacto-N-fucopentaoseIII, Gal[beta]1->4(Fuc[alpha]1->3)GlcNAc[beta]1->3Gal[beta]1->4Glc; LNFPII, lacto-N-fucopentaoseII, Gal[beta]1->3(Fuc[alpha]1->4)GlcNAc[beta]1->3Gal[beta]1->4Glc; Lex, Lewis x, Gal[beta]1->4(Fuc[alpha]1->3)GlcNAc; HPAE-PAD, high pH anion exchange chromatography-pulsed amperometric detection; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; BCA, bicinchoninic acid.
Discussion
Materials and methods
Acknowledgments
Abbreviations
References
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