In this study we designed and evaluated various kinds of PEG-modifications, exploiting unique chemically synthesized end-biotinylated glycopolymer capture molecules in combination with a simple and affordable PEG-linking,

to optimize the current version of our previously “in-house” developed SGA in order to reduce the experimental background (essentially unspecific and non-target binding) of this glycan-based assay. This background reduction may minimize the risk of occurrence of false-positive/negative results and may improve the diagnostic performance (increased sensitivity and specificity) of SGA. The following conclusions may be drawn from the findings: (i) The most significant decrease of background binding was achieved when PEG molecules bearing two functional PCI-32765 cell line groups, biotin and amine (hence heterobifunctional), were covalently attached directly to microbeads. This modification may be beneficial because it decreases “experimental noise” at low detection signals and does not compromise specific binding of the cognate anti-glycan antibodies. Interestingly, the shorter version of these two heterobifunctional PEGs, biot-PEG23-NH2, exhibits a more pronounced repelling effect, namely the capacity to block binding of non-target antibodies, than the respective longer version and therefore may preferably be used

in an advanced version of SGA. (ii) The end-point addition LEE011 ic50 of biot-PEG50 can be used to repel unspecific binding caused by endogenous biotin potentially present in the analyte (e.g. plasma samples) or in secondary antibody samples and can be easily combined with bead surface PEGylation. (iii) A considerable extent of unspecific binding can be attributed

to the IgG class of the Coproporphyrinogen III oxidase antibodies whereas the contribution of IgM class antibodies to unspecific binding signals is low. It is therefore recommended to use IgM class rather than IgG class antibodies in glycan-based immunoassays. (iv) Background binding was not reduced when glycopolymers were PEG-modified at their side-chains, possibly because the PEG-chains that are attached to polyacrylamide backbone of glycopolymer preclude specific binding of anti-glycan antibodies to the glycan epitopes. Taken together, the combination of the appropriate PEG-modifications, i.e. the bead modification with PEG23 and the end-point addition of biot-PEG50 is a promising advancement in the optimization of the current version of our SGA. These or similar modifications probably could be also recommended to be included in experimental protocols of related bead-based immunoassays for the improvement of their diagnostic performance. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. The authors declare no competing financial interest. This work was supported by the Swiss National Science Foundation (Grant Number: 310030-143619).