Immobilization of polyacrylamide-based glycoconjugates on solid phase in immunosorbent assays (original) (raw)
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Multiplex suspension array for human anti-carbohydrate antibody profiling
The Analyst, 2011
Glycan-binding antibodies form a significant subpopulation of both natural and acquired antibodies and play an important role in various immune processes. They are for example involved in innate immune responses, cancer, autoimmune diseases, and neurological disorders. In the present study, a microsphere-based flow-cytometric immunoassay (suspension array) was applied for multiplexed detection of glycan-binding antibodies in human serum. Several approaches for immobilization of glycoconjugates onto commercially available fluorescent microspheres were compared, and as the result, the design based on coupling of end-biotinylated glycopolymers has been selected. This method requires only minute amounts of glycans, similar to a printed glycan microarray. The resulting glycomicrospheres were used for detection of IgM and IgG antibodies directed against ABO blood group antigens. The possibility of multiplexing this assay was demonstrated with mixtures of microspheres modified with six different ABO related glycans. Multiplexed detection of anti-glycan IgM and IgG correlated well with singleplex assays (Pearson's correlation coefficient r ¼ 0.95-0.99 for sera of different blood groups). The suspension array in singleplex format for A/B trisaccharide, H di and Le x microspheres corresponded well to the standard ELISA (r > 0.94). Therefore, the described method is promising for rapid, sensitive, and reproducible detection of anti-glycan antibodies in a multiplexed format.
Journal of Immunological Methods, 2014
Glycan-based suspension array (SGA) is an "in-house" developed multi-target immunoassay, employing commercially available fluorescent microbeads as a solid support for unique chemically synthesized glycopolymers which capture naturally occurring human anti-glycan antibodies. SGA is a sensitive and reliable tool for the high-throughput screening of anti-glycan antibody alterations characteristic for a vast number of human diseases including cancer. However, unspecific background binding, for instance binding of non-target antibodies, is a common obstacle in such immunoassays. In an attempt to reduce unspecific background binding of serum (or plasma) antibodies, we prepared glycosylated microbeads modified with linear poly(ethylene glycols) (PEGs) of different lengths. We compared several kinds of PEG modifications: (a) partial side-chain substitution of glycopolymers by PEGs of different lengths, (b) end-point addition of biotin-linked PEGs to glycopolymer-coupled beads, and (c) linking of heterobifunctional PEGs to the bead surface prior to glycopolymer immobilization. Among the various modifications investigated, the direct modification of the bead surface with linear heterobifunctional PEGs, consisting of 23-and 60 PEG-units significantly reduced the background binding. The end-point addition of biotin-linked PEGs, especially in the case of PEG consisting from 50 PEG-units, helped to repel non-target binding caused by endogenous biotin. We observed unspecific binding predominantly for antibodies of IgG but of IgM class. The novel design of fluorescent microbeads allows the detection of human anti-glycan antibodies with increased specificity and opens new horizons for practical application of SGA as a diagnostic tool.
Immobilization of antibodies on alginate-chitosan beads
International journal of pharmaceutics, 2000
An anti-hapten IgG was covalently immobilized on glutaraldehyde-activated alginate-chitosan gel beads. The antibody immobilization efficiency was influenced by glutaraldehyde-bead reaction time, IgG concentration and pH. In addition, immobilization conditions such as glutaraldehyde and antibody concentrations influenced antibody hapten binding affinity. The immobilized IgG on the beads was stable and no reduction in the percent binding to hapten was noticed following 25 days of storage. It was concluded that antibodies could be successfully immobilized on alginate-chitosan gel beads. Such a system can be applied for the development of immunoaffinity purification and immunoassays.
Chemistry & Biology, 2008
Glycan recognition leading to cell-cell interactions, signaling, and immune responses is mediated by various glycan-binding proteins (GBPs) showing highly diverse ligand specificities. We describe here a rapid glycan immobilization technique via 4-hydrazinobenzoic acid (HBA)-functionalized beads and its application to high-throughput screening of miniature pig kidney N-glycan-binding proteins by using a massspectrometric approach. Without any derivatization steps, the purified pig kidney N-glycans were directly immobilized on to HBA-functionalized beads and subsequently used to identify GBPs from human serum. This screening method showed remarkable performance for identifying potential GBPs closely involved in pig-to-human xenograft rejection mediated by human serum, including antibodies, cytokines, complement components, siglec, and CD antigens. Thus, these results demonstrate that the GBP screening method was firmly established by one-step immobilization of the N-glycans on to microsphere and highly sensitive mass-spectrometric analysis.
Glycoconjugate Journal, 2000
The TF, Tn, and SiaTn glycotopes are frequently expressed in cancer-associated mucins. Antibodies to these glycotopes were found in human serum. A set of polyacrylamide (PAA)-based glycoconjugates was applied to the direct and competitive enzyme-linked immunosorbent assays (ELISA) to characterize the specificity of serum IgG antibodies. The anti-TF,-Tn and-SiaTn IgG were affinity purified from serum of cancer patients and characterized using PAA-conjugates and free saccharides. The anti-TF and-Tn antibodies were shown to be specific. The anti-TF IgG bound both Galβ1-3GalNAcαand Galβ1-3GalNAcβ-PAA, the latter was three-four times more effective inhibitor of antibody binding. The anti-Tn IgG reacted only with GalNAcα-PAA. The anti-SiaTn IgG cross-reacted with Tn-PAA but SiaTn-PAA was five-six times more effective inhibitor in a competitive assay. The IC 50 values for PAA-conjugates with the corresponding antibodies typically ranged from 2 to 5 × 10 −8 M. The antibodies display a low specificity to mucin-type glycoconjugates in comparison with PAA-conjugates as was shown for mucins isolated from human malignant tumor tissues, ovine submaxillary mucin (OSM) and asialo-OSM. The unusual IgG-antibody specificity to GalNAcβ and GalNAcβ1-3GalNAcβ ligands was found in human serum.
Glycoconjugate Journal, 2011
Anti-glycan antibodies represent a vast and yet insufficiently investigated subpopulation of naturally occurring and adaptive antibodies in humans. Recently, a variety of glycan-based microarrays emerged, allowing high-throughput profiling of a large repertoire of antibodies. As there are no direct approaches for comparison and evaluation of multiglycan assays we compared three glycan-based immunoassays, namely printed glycan array (PGA), fluorescent microsphere-based suspension array (SA) and ELISA for their efficacy and selectivity in profiling anti-glycan antibodies in a cohort of 48 patients with and without ovarian cancer. The ABO blood group glycan antigens were selected as well recognized ligands for sensitivity and specificity assessments. As another ligand we selected P 1 , a member of the P blood group system recently identified by PGA as a potential ovarian cancer biomarker. All three glycoimmunoassays reflected the known ABO blood groups with high performance. In contrast, anti-P 1 antibody binding profiles displayed much lower concordance. Whilst anti-P 1 antibody levels between benign controls and ovarian cancer patients were significantly discriminated using PGA (p= 0.004), we got only similar results using SA (p=0.03) but not for ELISA. Our findings demonstrate that whilst assays were largely positively correlated, each presents unique characteristic features and should be validated by an independent patient cohort rather than another array tech-Nicolai V. Bovin and Viola Heinzelmann-Schwarz contributed equally.
Journal of Immunological Methods, 1996
In the present study, cup-shaped 1-3 microns large cell wall fragments from Thermoanaerobacter thermohydrosulfuricus L111-69 covered with a hexagonal S-layer lattice composed of glycoprotein subunits were shown to act as a matrix for the immobilization of human IgG. After cross-linking the S-layer glycoprotein lattice with glutaraldehyde (S-layer microparticles), IgG was either bound to carbodiimide activated carboxyl groups from acidic amino acids from the protein moiety or to the carbohydrate chains activated with cyanogen bromide or oxidized with periodate. After determining the binding capacity of the S-layer lattice for human IgG, the orientation of the immobilized antibody molecules was investigated using anti-human IgG peroxidase conjugates with different specificity. Attachment of S-layer microparticles with covalently bound human IgG to microplates precoated with anti-human IgG of different specificity led to clear correlations between the amount of applied human IgG and the absorption values in the immunoassays. The steepest absorption curves were obtained when human IgG was bound to the carbohydrate chains exposed on the surface of the S-layer lattice. This confirmed that the location and the accessibility of the immobilized antibodies on S-layer microparticles is of major importance for the response in immunoassays. In addition to the high reproducibility of the amount of IgG which could be bound to the S-layer lattice and the high reproducibility of the absorption curves in the immunoassays, one major advantage of using cup-shaped S-layer microparticles can be seen in the considerable increase of the actual surface available for binding processes and immunological reactions.
Serum antibody screening by surface plasmon resonance using a natural glycan microarray
Glycoconjugate Journal, 2008
A surface plasmon resonance (SPR) based natural glycan microarray was developed for screening of interactions between glycans and carbohydrate-binding proteins (CBPs). The microarray contained 144 glycan samples and allowed the real-time and simultaneous screening for recognition by CBPs without the need of fluorescent labeling. Glycans were released from their natural source and coupled by reductive amination with the fluorescent labels 2-aminobenzamide (2AB) or anthranilic acid (AA) followed by high-performance liquid chromatography (HPLC) fractionation making use of the fluorescent tag. The released and labeled glycans, in addition to fluorescently labeled synthetic glycans and (neo)glycoproteins, were printed on an epoxide-activated chip at fmol amounts. This resulted in covalent immobilization, with the epoxide groups forming covalent bonds to the secondary amine groups present on the fluorescent glycoconjugates. The generated SPR glycan array presented a subset of the glycan repertoire of the human parasite Schistosoma mansoni. In order to demonstrate the usefulness of the array in the simultaneous detection of glycan-specific serum antibodies, the anti-glycan antibody profiles from sera of S. mansoni-infected individuals as well as from non-endemic uninfected controls were recorded. The SPR screening was sensitive for differences between infection sera and control sera, and revealed antibody titers and antibody classes (IgG or IgM). All SPR analyses were performed with a single SPR array chip, which required regeneration and blocking of the chip before the application of a serum sample. Our results indicate that SPR-based arrays constructed from glycans of natural or synthetic origin, pure or as mixture, can be used for determining serum antibody profiles as possible markers for the infection status of an individual.
Effects of Neighboring Glycans on Antibody-Carbohydrate Interaction
Angewandte Chemie International Edition, 2011
Carbohydrate recognition is a crucial event in many biological processes, including the progression of diseases such as AIDS, influenza, and cancer. Thus, characterization and reconstruction of carbohydrate epitopes to mimic authentic composition and presentation have become one of the goals in glycoscience that may greatly influence the strategy of drugs design. For example, carbohydrate epitopes on virus or cancer cells represent attractive targets for development of carbohydrate-based vaccines. Understanding the presentation of carbohydrate epitopes on cell surface allows us to more closely mimic the natural setting in the context of vaccine design. For instance, the HIV envelope glycoprotein gp120 contains high-mannose clusters on its surface to shield peptides from recognition by the host immune system and facilitate invasion by binding to the C-type lectin DC-SIGN on dendritic cells. Therefore, mimicking the high-mannose clusters on the HIV surface has become a promising approach to develop carbohydrate-based vaccines. However, clustered expression patterns of carbohydrates are complicated. Incomplete mimicry of carbohydrate epitopes on cell surface could lead to a failed vaccine design. Recently, Danishefsky and co-workers have shown that the Man 9 GlcNAc 2 -based vaccine elicited a high-titer antibody response that recognizes the Man 9 GlcNAc 2 epitope but fails to neutralize HIV, thereby suggesting that it is not an optimal mimic of the epitope of gp120. To reach an optimal presentation of the carbohydrate epitope, several research groups have attempted to address the question by modulating antigen density and flexibility, but an optimal method has not yet emerged. Furthermore, there is a concern that carbohydrate-protein interactions may be either enhanced by multivalency or suppressed by steric hindrance. In addition, though not well understood, the proximity effect by other molecules may be significant. Herein, we investigated the effects of neighboring glycans on carbohydrate-antibody interaction using glycan microarrays. Interestingly, we found that heterogeneous glycans, which were prepared by mixing two distinct oligosaccharides and spotted onto glass slides, provide the superior binding affinity compared to the individual components in the microarray experiments. These results additionally suggest that heterogeneous-ligand glycans can serve as a novel strategy for the development of carbohydrate-based vaccine design.