Enzyme-linked Lectin Assay Research Articles

Galactose grafting on poly(ε-caprolactone) substrates for tissue engineering: a preliminary study

The grafting of galactose units onto poly(ε-caprolactone) (PCL) substrates by a wet chemistry two-step procedure is proposed. Even though a reduction of hardness from 0.58–0.31GPa to 0.12–0.05GPa is achieved, the chemical functionalization does not negatively affect the tensile modulus (332.2±31.3MPa and 328.5±34.7MPa for unmodified and surface-modified PCL, respectively) and strength (15.1±1.3MPa and 14.8±1.5MPa as assessed before and after the surface modification, respectively), as well as the mechanical behaviour evaluated through small punch test. XPS and enzyme-linked lectin assay (ELLA) demonstrate the presence, and also the correct exposition of the saccharidic epitope on PCL substrates. The introduction of carbohydrate moieties on the PCL surfaces clearly enhances the hydrophilicity of the substrate, as the water contact angle decreases from 82.1±5.8° to 62.1±4.2°. Furthermore, preliminary biological analysis shows human mesenchymal stem cell viability over time and an improvement of cell adhesion and spreading.

Neuraminidase-Inhibiting Antibody Response to H5N1 Virus Vaccination in Chronically Ill and Immunocompromised Patients

Neuraminidase-inhibiting (NAi) antibodies have been reported to be an independent correlate of protection from influenza disease, but the NAi antibody response to influenza vaccination has never been assessed in chronically ill or immunocompromised participants. Using an enzyme-linked lectin assay, we demonstrated that 2 immunizations with a Vero cell culture-derived, whole-virus H5N1 A/Vietnam vaccine induces NAi antibodies in 94.3% of chronically ill and 83.8% of immunocompromised participants. A booster with a heterologous A/Indonesia H5N1 vaccine induced comparable NAi antibody titers in both groups and resulted in 100% seropositivity. These data support prepandemic H5N1 vaccination strategies for these highly vulnerable risk groups.

Thiol‐ene and thiol‐yne‐based synthesis of glycodendrimers as nanomolar inhibitors of wheat germ agglutinin

ABSTRACTAlkene and alkyne functional polyester‐based dendrimers of generation 1 to 4 have been prepared and reacted under free‐radical conditions with 2‐acetamido‐2‐deoxy‐1‐thio‐β‐D‐glucose (GlcNAc‐SH). As the alkene‐dendrimers underwent the addition of one thiyl radical per ene group whereas each yne group of alkyne‐dendrimers was saturated by two thiyl radicals, a collection of glycodendrimers with glycan density ranging from six to ninety‐six GlcNAc per dendrimer was obtained. The recognition properties of the prepared glycodendrimers toward the wheat germ agglutinin (WGA) were evaluated by enzyme‐linked lectin assay (ELLA). The eight glycodendrimers were excellent ligands showing IC50 values in the nanomolar range and relative potencies per sugar unit up to 2.27 e6 when compared to monosaccharidic GlcNAc used as monovalent reference. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014, 52, 2422–2433

ChemInform Abstract: A Double Diastereoselective Michael‐Type Addition as an Entry to Conformationally Restricted Tn Antigen Mimics.

A totally stereocontrolled C-Michael addition of serine-equivalent C-nucleophiles to tri-O-benzyl-2-nitro-d-galactal was used as the key step to synthesize several pyrano[3,2-b]pyrrole structures. These scaffolds could be regarded as conformationally restricted Tn antigen mimics, as we have demonstrated by biological assays. The pyranose rings retain their 4C1 chair conformation, as shown by molecular modeling and NMR spectroscopy. The expected bioactivity was established by a competition-tailored enzyme-linked lectin assay using both soybean and Vicia villosa agglutinins as model lectins. The facile described synthetic route and the strategic combination of computational and experimental techniques to reveal conformational features and bioactivity demonstrate the prepared glycomimics to be promising candidates for further exploitation of this scaffold to give glycans for lectin blocking and vaccination.

Macrolides Attenuate Phorbol Ester-Induced Tumor Necrosis Factor-α and Mucin Production from Human Airway Epithelial Cells

Background/Aims: Macrolide antibiotics are effective drugs in chronic bronchiolitis and chronic rhinosinusitis with mucus hypersecretion. However, the mechanism of action is unclear. This study was designed to investigate the effect of azithromycin (AZM; 15-membered) and midecamycin acetate (MDM; 16-membered) on MUC5AC and MUC2 gene expression and secretion from human airway epithelial cells. The effects of the two macrolides on tumor necrosis factor-α (TNF-α) release were also examined. Methods: Confluent NCI-H292 human mucoepidermoid airways epithelial cells were pretreated with AZM or MDM for 2 h and then stimulated with 200 nmol/l phorbol 12-myristate 13-acetate (PMA) for 8 h. The MUC5AC and MUC2 gene expression was measured by real-time quantitative RT-PCR. Total mucin in culture supernatants was measured using enzyme-linked lectin assay. Enzyme-linked immunosorbent assay was used to determine MUC5AC, MUC2 and TNF-α released by the cells. Results: AZM and MDM attenuated PMA-induced MUC5AC and MUC2 gene and protein expression in NCI-H292 cells. They also suppressed PMA-mediated TNF-α in the cells. Conclusion: The present study demonstrates that AZM and MDM suppress the synthesis of mucin and TNF-α from human airway epithelial cells.

Modulation of Mucin mRNA (MUC5AC and MUC5B) Expression and Protein Production and Secretion in Caco-2/HT29-MTX Co-cultures Following Exposure to Individual and Combined Fusarium Mycotoxins

Intestinal epithelial cells (IECs) are a critical component of the innate local immune response. In order to reduce the risk of pathogen infection or xenobiotic intoxication, different host defense mechanisms have been evolved. Evidence has shown that upon ingestion of food or feed contaminated with toxins (e.g., mycotoxins), IECs respond by regulating mucin secretions, which act as a physical barrier inhibiting bacterial attachment and subsequent infection-related processes. However, the effect of Fusarium mycotoxins on mucin production remains unclear. Consequently, the aim of this study was to evaluate individual and interactive effects of four common Fusarium mycotoxins, deoxynivalenol, nivalenol, zearalenone, and fumonisins B1 on mRNA expression and secretion of mucins, MUC5AC, and MUC5B, as well as total mucin-like glycoprotein secretion, using Caco-2 (absorptive-type) and HT29-MTX (secretive-type) cells and their co-cultures (initial seeding ratios Caco-2/HT29-MTX: 90/10 and 70/30). Our results showed that individual and mixtures of mycotoxins significantly modulated MUC5AC and MUC5B mRNA and protein, and total mucin-like glycoprotein secretion as measured by quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and enzyme-linked lectin assay, respectively. Additive effects were not always observed for mixtures. Also, the present study showed that in co-cultures, lower MUC5AC and MUC5B mRNA, protein and total mucin production occurred following exposure, which might suggest higher intestinal permeability and susceptibility to toxin exposure. This study demonstrates the importance of selecting an appropriate cell model for the in vitro investigation of Fusarium mycotoxin effects either alone or in combinations on the immunological defense mechanisms of IECs, and will contribute to improved toxin risk assessments.

Thiol–ene Mediated Neoglycosylation of Collagen Patches: A Preliminary Study

Despite the relevance of carbohydrates as cues in eliciting specific biological responses, the covalent surface modification of collagen-based matrices with small carbohydrate epitopes has been scarcely investigated. We report thereby the development of an efficient procedure for the chemoselective neoglycosylation of collagen matrices (patches) via a thiol-ene approach, between alkene-derived monosaccharides and the thiol-functionalized material surface. Synchrotron radiation-induced X-ray photoelectron spectroscopy (SR-XPS), Fourier transform-infrared (FT-IR), and enzyme-linked lectin assay (ELLA) confirmed the effectiveness of the collagen neoglycosylation. Preliminary biological evaluation in osteoarthritic models is reported. The proposed methodology can be extended to any thiolated surface for the development of smart biomaterials for innovative approaches in regenerative medicine.

Fucosyltransferase 1 mediates angiogenesis, cell adhesion and rheumatoid arthritis synovial tissue fibroblast proliferation

IntroductionWe previously reported that sialyl Lewisy, synthesized by fucosyltransferases, is involved in angiogenesis. Fucosyltransferase 1 (fut1) is an α(1,2)-fucosyltransferase responsible for synthesis of the H blood group and Lewisy antigens. However, the angiogenic involvement of fut 1 in the pathogenesis of rheumatoid arthritis synovial tissue (RA ST) has not been clearly defined.MethodsAssay of α(1,2)-linked fucosylated proteins in RA was performed by enzyme-linked lectin assay. Fut1 expression was determined in RA ST samples by immunohistological staining. We performed angiogenic Matrigel assays using a co-culture system of human dermal microvascular endothelial cells (HMVECs) and fut1 small interfering RNA (siRNA) transfected RA synovial fibroblasts. To determine if fut1 played a role in leukocyte retention and cell proliferation in the RA synovium, myeloid THP-1 cell adhesion assays and fut1 siRNA transfected RA synovial fibroblast proliferation assays were performed.ResultsTotal α(1,2)-linked fucosylated proteins in RA ST were significantly higher compared to normal (NL) ST. Fut1 expression on RA ST lining cells positively correlated with ST inflammation. HMVECs from a co-culture system with fut1 siRNA transfected RA synovial fibroblasts exhibited decreased endothelial cell tube formation compared to control siRNA transfected RA synovial fibroblasts. Fut1 siRNA also inhibited myeloid THP-1 adhesion to RA synovial fibroblasts and RA synovial fibroblast proliferation.ConclusionsThese data show that α(1,2)-linked fucosylated proteins are upregulated in RA ST compared to NL ST. We also show that fut1 in RA synovial fibroblasts is important in angiogenesis, leukocyte-synovial fibroblast adhesion, and synovial fibroblast proliferation, all key processes in the pathogenesis of RA.

Analyzing swine sera for functional antibody titers against influenza A neuraminidase proteins using an enzyme-linked lectin assay (ELLA).

Neuraminidase (NA) is an envelope glycoprotein of influenza viruses, including swine-lineage influenza A viruses. NA possesses sialidase activity, which is functionally important at multiple points in viral replication, counter-balancing the sialic acid receptor binding activity of the hemagglutinin (HA), the other major envelope glycoprotein. The NA proteins of influenza A viruses have been classified into nine serological subtypes, and they undergo antigenic drift variation similar to that of HA. Antibodies to NA are analyzed much less often than antibodies to HA. The conventional assay for NA inhibition (NI) antibody titration, established decades ago, is widely considered unwieldy and inefficient for routine use. In recent years, a few new formats have been developed which still measure inhibition of NA enzymatic function, but more efficiently and with less chemical waste produced. Described here is the enzyme-linked lectin assay (ELLA), which is performed in 96-well plates and analyzed on a spectrophotometric plate reader. An important factor in adoption of the ELLA technique for animal studies, such as swine, is the choice of NA antigen, which may be purified protein or whole virus containing an antigenically irrelevant HA protein. This NI assay, in conjunction with the hemagglutination inhibiting (HI) antibody assay, offers a practical way to characterize viral isolates more fully and to quantify antibodies induced by infection or vaccination.

Aberrant sialylation and fucosylation of intracellular proteins in cervical tissue are critical markers of cervical carcinogenesis

Numerous studies have suggested that increased sialylation and fucosylation levels are signs of cancer progression. The majority of studies have focused on cell surface and bloodstream glycosylation changes associated with cancer progression, while little attention has been paid to changes in the glycosylation of cytosolic proteins. We compared the mannosylation, sialylation and fucosylation levels of cytosolic proteins obtained from human cervical tissues without neoplastic lesions vs. with cancer, using lectin blot and enzyme-linked lectin assay (ELLA) systems. There were no quantitative differences in mannosylation levels between the cytosolic proteins of normal and cancer tissues. However, we found markedly reduced sialylation (P<0.001) and fucosylation (P<0.01) in the proteins of cancer tissues. The ELLA system for detecting sialylation had extremely high sensitivity (91-100%) and specificity (82-100%) in distinguishing normal and cancer tissues. Thus, the changes in the glycosylation of cytosolic proteins during carcinogenesis of the cervix are quite different from previous observations concerning glycoconjugates in the bloodstream or on the cell surface. We suggest that changes in the glycosylation of intracellular proteins may be useful markers of the development of cervical cancer.

Little evidence of human infection with equine influenza during the 2007 epizootic, Queensland, Australia

BackgroundEquine influenza virus (EIV) is considered enzootic in Europe (except Iceland), Asia, North Africa, and North and South America. When EIV outbreaks occur they may severely impact the equine and tourist industries. Australia faced its first EIV outbreak beginning in August of 2007. The outbreak was concentrated in New South Wales and Queensland, with more than 1400 confirmed EIV infections in horses during the first month. Rapid response from the equine industry and the federal government was successful and Australia was declared free from EIV by the end of 2007. ObjectivesThis cross-sectional study was designed to examine associations between exposure to EIV-infected horses and evidence of EIV infection in humans. Study designEmploying informed consent, between October 2007 and April 2008, 100 subjects (89 with horse exposures and 11 non-exposed) were enrolled during equine events and at the University of the Sunshine Coast. All subjects provided a blood sample and were asked to complete an online questionnaire including health history, animal exposure and demographic information. Sera samples were tested for the presence of antibodies against two H3N8 EIV strains using microneutralization, hemagglutination inhibition, and enzyme-linked lectin assays. ResultsEvidence for H3N8 infection was sparse, with only 9 study participants having any indication of H3N8 infection and the seroreactivity seen was low and easily explained by cross-reactions against human influenza strains or vaccines. ConclusionsThese data provide little evidence to support the premise that EIV infections occurred among humans exposed to EIV-infected horses during the 2007 Australian epizootic.

No evidence for zoonotic transmission of H3N8 canine influenza virus among US adults occupationally exposed to dogs

ObjectivesThe zoonotic potential of H3N8 canine influenza virus (CIV) has not been previously examined; yet considering the popularity of dogs as a companion animal and the zoonotic capabilities of other influenza viruses, the public health implications are great. This study aimed to determine the seroprevalence of antibodies against CIV among a US cohort.DesignA cross-sectional seroepidemiological study was conducted between 2007 and 2010.SettingRecruitments primarily occurred in Iowa and Florida. Participants were enrolled at dog shows, or at their home or place of employment.SampleThree hundred and four adults occupationally exposed to dogs and 101 non-canine-exposed participants completed a questionnaire and provided a blood sample.Main outcome measuresMicroneutralization and neuraminidase inhibition assays were performed to detect human sera antibodies against A/Canine/Iowa/13628/2005(H3N8). An enzyme-linked lectin assay (ELLA) was adapted to detect antibodies against a recombinant N8 neuraminidase protein from A/Equine/Pennsylvania/1/2007(H3N8).ResultsFor all assays, no significant difference in detectable antibodies was observed when comparing the canine-exposed subjects to the non-canine-exposed subjects.ConclusionWhile these results do not provide evidence for cross-species CIV transmission, influenza is predictably unpredictable. People frequently exposed to ill dogs should continually be monitored for novel zoonotic CIV infections.

A Double Diastereoselective Michael-Type Addition as an Entry to Conformationally Restricted Tn Antigen Mimics

A totally stereocontrolled C-Michael addition of serine-equivalent C-nucleophiles to tri-O-benzyl-2-nitro-d-galactal was used as the key step to synthesize several pyrano[3,2-b]pyrrole structures. These scaffolds could be regarded as conformationally restricted Tn antigen mimics, as we have demonstrated by biological assays. The pyranose rings retain their (4)C1 chair conformation, as shown by molecular modeling and NMR spectroscopy. The expected bioactivity was established by a competition-tailored enzyme-linked lectin assay using both soybean and Vicia villosa agglutinins as model lectins. The facile described synthetic route and the strategic combination of computational and experimental techniques to reveal conformational features and bioactivity demonstrate the prepared glycomimics to be promising candidates for further exploitation of this scaffold to give glycans for lectin blocking and vaccination.

High Affinity Glycodendrimers for the Lectin LecB from Pseudomonas aeruginosa

Following an iterative oxime ligation procedure, cyclopeptide (R) and lysine-based dendron (D) were combined in all possible arrangements and successively functionalized with α-fucose and β-fucose to provide a new series of hexadecavalent glycosylated scaffolds (i.e., scaffolds RD16, RR16, DR16, and DD16). These compounds and smaller analogs (tetra- and hexavalent scaffolds R4 and R6) were used to evaluate the influence of the ligand valency and architecture, and of the anomer configuration in the binding to the αFuc-specific lectin LecB from Pseudomonas aeruginosa . Competitive enzyme-linked lectin assays (ELLA) revealed that only the RD16 architecture displaying αFuc (9A) reaches strong binding improvement (IC50 of 0.6 nM) over αMeFuc, and increases the α-selectivity of LecB. Dissociation constant of 28 nM was measured by isothermal titration micorcalorimetry (ITC) for 9A, which represents the highest affinity ligand ever reported for LecB. ITC and molecular modeling suggested that the high affinity observed might be due to an aggregative chelate binding involving four sugar head groups and two lectins. Interestingly, unprecedented binding effects were observed with β-fucosylated conjugates, albeit being less active than the corresponding ligands of the αFuc series. In particular, the more flexible lysine-based dendritic structures (15B and 18B) showed a slight inhibitory enhancement in comparison with those having cyclopeptide core.

Elaboration of Glycopolymer-Functionalized Micelles from anN-Vinylpyrrolidone/Lactide-Based Reactive Copolymer Platform

Glycopolymer-corona-based micelles are obtained in a one-pot procedure, through reaction of D-mannosamine or D-glucosamine with the N-succinimidyl (NS) esters of a poly(D,L-lactide)-block-poly(N-acryloxysuccinimide-co-N-vinylpyrrolidone) (PLA-b-P(NAS-co-NVP)) amphiphilic copolymer (presenting quasi-alternating NAS/NVP units) in dimethyl sulfoxide (DMSO), followed by nanoprecipitation and dialysis against water. The glycopolymer micelles exhibit a higher CMC and size than those obtained from unmodified copolymer, due to increased hydrophilicity of the external block as a result of sugar derivatization, and the availability of the sugars at the micelle surface is evidenced through interactions with Concanavalin A (Con A) lectin, as attested by turbidimetric measurements and enzyme-linked lectin assay (ELLA). Interestingly, the glycopolymer micelles can be further used for hydrophobic molecule encapsulation and release, as shown with imiquimod, while keeping their interactions with con A intact. It is concluded that the PLA-based amphiphilic/reactive copolymer represents a versatile platform for glycopolymer-based micelle constructs for drug/vaccine delivery.

Human colonic mucus is a reservoir for antimicrobial peptides

Background and aimsTo prevent bacterial adherence and translocation, the colonic mucosa is covered by a protecting mucus layer and the epithelium synthesizes antimicrobial peptides. The present qualitative study investigated the contents and interaction of these peptides in and with rectal mucus. MethodsRectal mucus extracts were analyzed for antimicrobial activity and screened with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, Dot blot and immunohistochemistry for antimicrobial peptides. In addition, binding of AMPs to mucins was investigated by Western blot and enzyme-linked lectin assays. ResultsIn functional tests the mucus layer exhibited a strong antimicrobial activity. We detected 11 antimicrobial peptides in mucus extracts from healthy persons including the defensins HBD-1 and -3, the cathelicidin LL-37, ubiquitin, lysozyme, histones, high mobility group nucleosome-binding domain-containing protein 2, ubiquicidin and other ribosomal proteins. AMPs were bound by mucins but this was demonstrated to be reversible and inhibition of antibacterial activity was limited. ConclusionThese findings indicate that epithelial antimicrobial peptides are retained in the intestinal mucus layer without losing their efficacy. Thus, the mucus layer and its composition provide an attractive drug target to restore antimicrobial barrier function in intestinal diseases.

Synthesis of Multivalent Carbohydrate‐Centered Glycoclusters as Nanomolar Ligands of the Bacterial Lectin LecA from Pseudomonas aeruginosa

A family of fifteen glycoclusters based on a cyclic oligo-(1→6)-β-D-glucosamine core has been designed as potential inhibitors of the bacterial lectin LecA with various valencies (from 2 to 4) and linkers. Evaluation of their binding properties towards LecA has been performed by a combination of hemagglutination inhibition assays (HIA), enzyme-linked lectin assays (ELLA), and isothermal titration microcalorimetry (ITC). Divalent ligands displayed dissociation constants in the sub-micromolar range and tetravalent ligands displayed low nanomolar affinities for this lectin. The influence of the linker could also be demonstrated; aromatic moieties are the best scaffolds for binding to the lectin. The affinities observed in vitro were then correlated with molecular models to rationalize the possible binding modes of these glycoclusters with the bacterial lectin.

Adhesion of enterotoxigenic Escherichia coli strains to neoglycans synthesised with prebiotic galactooligosaccharides

Enterotoxigenic (ETEC) Escherichia coli (E. coli) causes traveller’s diarrhoea and high mortality among baby animals. ETEC adhesion is mediated by lectins (adhesins) that bind to glycoconjugates on the surface of host cells. Glycans that compete for adhesion could be used for disease prevention. Neoglycans of porcine albumin (PSA) that were conjugated with prebiotic galactooligosaccharides (GOS) were synthesised using the Maillard reaction. PSA glycation was confirmed by a reduction in the number of available free amino groups, decreased tryptophan intrinsic fluorescence, increased molecular mass and Ricinus communis lectin recognition. The adhesion of four ETEC strains (E. coli H10407, CFA+, K99 and K88) to PSA–GOS was examined by an enzyme-linked lectin assay. E. coli K88 bound to PSA–GOS with greater affinity (P<0.05) than did E. coli H10407, CFA+ and K99. In addition, PSA–GOS partially inhibited the adherence of the K88 strain to intestinal mucins. Pig ETEC strain was unable to ferment galactooligosaccharide–neoglycans. These results suggest that neoglycans obtained by the Maillard reaction may serve in the prophylaxis of ETEC K88 diarrhoea.

Effect of VIP on Intracellular [Ca2+], Extracellular Regulated Kinase 1/2, and Secretion in Cultured Rat Conjunctival Goblet Cells

To determine the intracellular signaling pathways that vasoactive intestinal peptide (VIP) uses to stimulate high molecular weight glycoconjugate secretion from cultured rat conjunctival goblet cells. Goblet cells from rat bulbar and forniceal conjunctiva were grown in organ culture. Presence and localization of VIP receptors (VPAC1 and 2) were determined by RT-PCR, immunofluorescence microscopy and Western blot analysis. Intracellular [Ca(2+)] ([Ca(2+)]i) was measured using fura-2. Extracellular signal-regulated kinase (ERK)-1/2 activity was determined by Western blot analysis. High molecular weight glycoconjugate secretion was measured with an enzyme-linked lectin assay on cultured goblet cells that were serum-starved for 2 hours before stimulation with VIP, VPAC1-, or VPAC2-specific agonists. Inhibitors were added 30 minutes prior to VIP. Activation of epidermal growth factor receptor (EGFR) was measured by immunoprecipitation using an antibody against pTyr followed by Western blot analysis with an antibody against EGFR. Both VIP receptors were present in rat conjunctiva and cultured goblet cells. VIP- and VPAC-specific agonists increased [Ca(2+)]i and secretion in a concentration-dependent manner. VIP also increased ERK1/2 activity, VIP-stimulated increase in [Ca(2+)]i. Secretion, but not ERK1/2 activity, was inhibited by the protein kinase A inhibitor, H89. VIP-stimulated secretion was inhibited by siRNA for ERK2 but not by siRNA for EGFR. VIP did not increase the phosphorylation of the EGFR. In conclusion, in cultured rat conjunctival goblet cells, VPAC1 and 2 receptors are functional. VIP stimulates a cAMP-dependent increase in [Ca(2+)]i and glycoconjugate secretion, but not ERK1/2 activation. VIP does not activate with EGFR.

Thiyl Glycosylation of Propargylated Octasilsesquioxane: Synthesis and Lectin‐Binding Properties of Densely Glycosylated Clusters on a Cubic Platform

AbstractA new polyhedral oligomeric silsesquioxane (POSS) derivative with a periphery of eight PEGylated chains functionalized with terminal propargyl groups was synthesized starting from commercially available octavinyl‐POSS. The photoinduced free‐radical coupling of this octapropargyl POSS derivative with various sugar thiols enabled the preparation of globular hexadecavalent glycoclusters. Thus, it appears that according to the alkyne hydrothiolation mechanism, two thiyl radicals were added across each triple bond of the POSS scaffold side‐chains. The affinities of some of the densely glycosylated clusters towards certain lectins were measured by the Enzyme‐Linked Lectin Assay (ELLA). The binding selectivity of Concanavalin A between the hexadecavalent mannosylated and glucosylated clusters was much higher than the selectivity observed for the corresponding octavalent glycoclusters (ref.4). Moreover, the affinity of the N‐acetylglucosamine‐based cluster towards wheat germ agglutinin (WGA) revealed a remarkable glycoside cluster effect with up to a 9.0 × 105‐fold increase in binding compared to monovalent GlcNAc. As a multivalent effect of the same order of magnitude was reported for an octavalent GlcNAc cluster towards the same lectin (ref.4), it is concluded that increasing the number of sugar units around the cubic platform does not lead systematically to an enhancement of binding affinity.