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J Immunol Methods. 2014 Oct;412:42-52. doi: 10.1016/j.jim.2014.06.015. Epub 2014 Jul 1.

PEGylation of microbead surfaces reduces unspecific antibody binding in glycan-based suspension array.

Author information

1
Gynecological Research Group, Department of Biomedicine, University Hospital Basel, University of Basel, Hebelstrasse 20, 4031 Basel, Switzerland. Electronic address: tatiana.pochechueva@unibas.ch.
2
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. MIklukho-Maklaya, 16/10, 117997 Moscow, Russian Federation.
3
Gynecological Research Group, Department of Biomedicine, University Hospital Basel, University of Basel, Hebelstrasse 20, 4031 Basel, Switzerland.
4
Gynecological Research Group, Department of Biomedicine, University Hospital Basel, University of Basel, Hebelstrasse 20, 4031 Basel, Switzerland; Ovarian Cancer Group, Adult Cancer Program, Lowy Cancer Research Centre, University of New South Wales, Prince of Wales Clinical School, Building C25 Kensington Campus, 2052 Sydney, Australia; Gynecological Cancer Centre, Royal Hospital for Women, School of Women's and Children's Health, Barker St., 2031 Randwick, Australia.

Abstract

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.

KEYWORDS:

Anti-glycan antibodies; End-biotinylated glycopolymers; Glycan-based suspension array; Heterobifunctional poly(ethylene glycols); Unspecific binding

PMID:
24995712
DOI:
10.1016/j.jim.2014.06.015
[Indexed for MEDLINE]
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