All samples were transferred to their designated positions on a 96-well plate according to predetermined experimental design, that was blocked on case–control status, sex and age. Standard and blank samples were included in every batch (96-well plate) for quality control and batch correction.

The sensitivity of the method for IgG N-glycome profiling was previously determined [29] based on the minimal starting amount of IgG (µg) as well as on the proportion of its starting amount which is finally analysed chromatographically (i.e. applied to the column). Namely, the minimal starting amount of IgG is 10 µg, i.e. the minimal amount of IgG required for the reliable quantification of its released N-glycans using fluorescence detection is 0.42 µg. The precision of the method is reported with coefficients of variation (CV, %) that are calculated from the relative abundance of each glycan peak (%) of standard samples. Herein, five standard samples per plate were analysed, giving the average CV value for directly measured IgG glycan peaks of 4.28% (range 0.44–15.65%), whereas calculated derived glycan traits gave the average CV value of 1.63% (range 0.17–4.39%).

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