Profiling human serum antibodies with a carbohydrate antigen microarray

Abstract

Carbohydrate antigen arrays (glycan arrays) have been recently developed for the high-throughput analysis of carbohydrate macromolecule interactions. When profiling serum, information about experimental variability, interindividual biological variability, and intraindividual temporal variability is critical. In this report, we describe the characterization of a carbohydrate antigen array and assay for profiling human serum. Through optimization of assay conditions and development of a normalization strategy, we obtain highly reproducible results with a within-experiment coefficient of variation (CV) of 10.8% and an overall CV (across multiple batches of slides and days) of 28.5%. We also report antibody profiles for 48 human subjects and evaluate for the first time the effects of age, race, sex, geographic location, and blood type on antibody profiles for a large set of carbohydrate antigens. We found significant dependence on age and blood type of antibody levels for a variety of carbohydrates. Finally, we conducted a longitudinal study with a separate group of 7 serum donors to evaluate the variation in anti-carbohydrate antibody levels within an individual over a period ranging from 3 to 13 weeks and found that, for nearly all antigens on our array, antibody levels are generally stable over this period. The results presented here provide the most comprehensive evaluation of experimental and biological variation reported to date for a glycan array and have significant implications for studies involving human serum profiling.

Figure 1

Characterization and validation of the carbohydrate array. Within-experiment (aliquot-to-aliquot) variability plots showing (a) standard deviation as a function of mean fluorescence signal across 4 aliquots of the same sample for normalized log-transformed (base 2) data and (b) CV as a function of mean fluorescence signal for the same 4 aliquots for normalized untransformed data. Overall variability plots showing (c) standard deviation as a function of mean fluorescence signal combined over 5 different samples for normalized log-transformed (base 2) data and (d) CV as a function of mean fluorescence signal for the same 5 samples for normalized untransformed data. Box plots showing (e) distribution of the slopes from dilution experiment combined over 3 samples and distribution of carbohydrate-specific slopes from dilution experiment separately by sample (f).

Figure 2

Microarray data from 48 healthy human donors for antibodies to 122 carbohydrate antigens. Box plots of the distribution of log-transformed (base 2) normalized signals.

Figure 3

Blood type covariate association for blood groups A and B and related disaccharides. Box plots showing distribution of normalized log-transformed (base 2) signals for the blood group determinants and the related disaccharides, Adi and Bdi, by blood type.

Figure 4

Microarray data from 48 healthy human donors for antibodies to 122 carbohydrate antigens. Heat map of data clustered without median centering. Carbohydrates are indicated in the rows and subjects are indicated in the columns. Each rectangle represents the log-transformed (base 2) normalized signal. The color of the rectangle corresponds to values on the color scale to the left of the heat map. Gray rectangles represent missing values, many of which were below detectable level.