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

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.

Prolonged immunomodulation in inflammatory arthritis using the selective Kv1.3 channel blocker HsTX1[R14A] and its PEGylated analog

“…Peptides such as HsTX1 and its analogs are known to adsorb onto surfaces, such as the polystyrene in the microplates used in proliferation assays. PEGylation has been proposed as a means for reducing peptide and protein adsorption onto surfaces [55; 56; 57; 58]. We therefore assayed the recovery of HsTX1[R14A] and PEG-HsTX1[R14A] after a 24 h incubation in the microplates used for our proliferation assays and, indeed, we observed a much lower recovery of HsTX1[R14A] than of its PEGylated analog.…”

“…In contrast, both peptides inhibited the proliferation of human and rat T EM cells with similar potencies. Toxin-derived peptides are prone to adsorption onto plastics, such as polystyrene in tissue culture microplates, and PEGylation of peptides and proteins can significantly reduce their adsorption [55; 56; 57; 58]. We therefore tested whether a 24-h incubation of HsTX1[R14A] or PEG-HsTX1[R14A] at room temperature in a tissue culture microplate affected peptide recovery, assessed by its ability to block Kv1.3 channels.…”

PEGylation of protein-imprinted nanocomposites sandwiching CdTe quantum dots with enhanced fluorescence sensing selectivity

“…35,36 The proteinrepelling effect of PEG is attributed to the low free energy at PEGwater interface and the absence of hydrogen bonding and electrostatic interactions with proteins. 37 Surface modication of the non-cavity regions of the protein MIPs in the presence of the template with a PEG-like protein-resistant polymer, so-called a post-imprinting modication strategy, is a promising method for lowering nonspecic protein adsorption. For the rst time, Luan et al 38 graed PEG chains from the surface of proteinimprinted siloxane copolymers before template removal by reaction of the surface silanol groups with methoxy-PEG-silane for reducing nonspecic binding.…”

Romantic surfaces : Zwitterionic polymer brushes for biomedical applications

“…57 The structure of a CB monomer is similar to that of glycine betaine, which is vital for the osmotic regulation of living organisms. 58 As a result, materials derived from SB or CB are considered as biomimetic. 1 Their biomimetic nature along with their excellent antifouling characteristics make them interesting for a wide range of applications, 13,59 such as biosensing, [60][61] drug delivery, 12,62 filtration, 51,[63][64] as well as for antibacterial 65 and marine coatings.…”

“…49,[55][56] The development towards improved bead-based assays follows the same trajectory as for other applications that face the implications of fouling. There are various studies that show improved performances of the microbeads by coating them with PEG, [57][58] and several examples that incorporate zwitterionic moieties have also been reported. [59][60] However, coatings using zwitterionic polymer brushes installed via the grafting-from approach are scarce.…”