PEGylation of microbead surfaces reduces unspecific antibody binding in glycan-based suspension array
| Autor: | Alexander Chinarev, Viola Heinzelmann-Schwarz, Tatiana Pochechueva, André Fedier, Francis Jacob, Nicolai V. Bovin |
|---|---|
| Rok vydání: | 2014 |
| Předmět: |
Glycan
Glycopolymer Fluoroimmunoassay Immunology Unspecific binding Biotin Polyethylene Glycols chemistry.chemical_compound Polysaccharides Neoplasms Heterobifunctional poly(ethylene glycols) PEG ratio medicine Humans Immunology and Allergy Glycan-based suspension array Anti-glycan antibodies biology medicine.diagnostic_test End-biotinylated glycopolymers Microbead (research) Microspheres 3. Good health High-Throughput Screening Assays chemistry Biochemistry Immunoglobulin M Immunoassay Immunoglobulin G biology.protein PEGylation Female Antibody Protein Binding |
| Zdroj: | Journal of Immunological Methods Journal of immunological methods |
| ISSN: | 0022-1759 |
| DOI: | 10.1016/j.jim.2014.06.015 |
| Popis: | 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 60PEG-units significantly reduced the background binding. The end-point addition of biotin-linked PEGs, especially in the case of PEG consisting from 50PEG-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. |
| Databáze: | OpenAIRE |
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