Blood samples were collected prospectively from 48 patients at the Department of Gynaecology, University Hospital Zurich, after written informed consent was given (Table ). Ethical approval for this study was granted by the appropriate Ethical Board in 2006 (to V.H.S., SPUK Canton of Zurich, Switzerland). Two venous blood samples (12 mL) were collected pre-operatively per patient in EDTA blood tubes (BD Vacutainer®
, 0.184M EDTA, BD Diagnostics, Franklin Lakes, US) and stored on ice until further processing. Blood samples were centrifuged at 3000g
at 4°C for 10 min, and aliquots of the supernatant plasma frozen at −80°C. All collected blood samples were processed using the same protocol and within 3 h of their collection.
Clinicopathological characteristics. Patient numbers and percentage (in brackets)
ELISA NUNC MaxiSorp 96-well immunoplates (Thermo Fisher Scientific, Roskilde, Denmark) were coated with Glyc-PAA (Lectinity Holdings, Moscow, Russia), 10 μg/mL, 100 μl per well in carbonate buffer (50 mM Na2CO3/ NaHCO3, pH 9.6) for 12 h at 4°C. Carbohydrate-free PAA was applied as negative control. Plates were blocked with 1% (w/v) BSA (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) in PBS for 40 min at 37°C and washed four times with PBS containing 0.5% (v/v) Tween-20 after incubation. Plasma samples were diluted 1:1000 in incubation buffer (PBS, 0.3% (w/v) BSA, 0.02% (v/v) Tween 20), added to plates in duplicate and incubated for 60 min at 37°. Between each of the following steps plates were washed four times with PBS containing 0.5% (v/v) Tween-20: incubation with 100 μl per well of goat anti-human Ig (IgA + IgG + IgM) conjugated to long chain biotin for 60 min at 37° (Pierce, Rockford, IL, USA, 0.16 μg/mL in incubation buffer); streptavidin horse raddish peroxidase conjugate for 60 min at 37°C (Southern Biotechnology Associates, Inc., Birmingham, AL, USA, 0.083 μg/mL in incubation buffer); and chromogen substrate 3,3′,5,5′-tetramethylbenzidine (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) for 5 min at RT. The peroxidase reaction was stopped by addition of equal volumes of 1M H2SO4. Absorbance was measured at 450 nm using a TECAN plate reader (Tecan Spectrafluor Plus, Tecan Trading AG, Männedorf, Switzerland).
Suspension array (SA) The Bio-Plex Suspension Array (Bio-Rad Laboratories, Hercules, CA, USA) is a multiplex analysis system that permits the simultaneous analysis of up to 100 different biomolecules in a single microplate well. The constituents of each well are drawn up into the flow-based Bio-Plex array reader, which quantifies each specific reaction based on its bead color using fluorescently labeled reporter molecules specific for each target protein followed by Bio-Plex Manager software data analysis. A 96-well Multiscreen HTS filter plate (Millipore Corp., Billerica, MA, USA) was soaked in 100 μl of antibody diluent for 5 min (PBS-0.05 M Tris, pH 7.2, 0.25% BSA, Sigma-Aldrich Chemie GmbH, Buchs, Switzerland). Antibody diluent incorporating 2000 beads/well (50 μl/well) was added. The plate was washed three times with 100 μl of washing buffer (PBS-0.05 M Tris, pH 7.2) using a vacuum manifold (Bio-Rad, Munich, Germany). Human samples were added in duplicates to wells (in antibody diluent 1:40 (50 μl/well)) and agitated at 1,100 rpm for 30 s on a microplate shaker before incubation on a shaker (200–300 rpm) for 1 h at RT in the dark. After incubation, the plate was washed three times using washing buffer. Secondary antibodies (R-phycoerythrin conjugated goat anti-human Ig (IgM + IgG + IgA, H + L; Southern Biotechnology Associates Inc., Birmingham, AL, USA, 25 ng/well) were added and incubated for 30 min on the plate shaker in the dark. The plate was washed three times with washing buffer, beads were then resuspended and shaken for 30 s at 1,100 rpm in 100 μl of washing buffer before being analyzed on the Bio-Plex array reader. Data were acquired in real time analyzing 100 beads by their median fluorescence intensitiy (MFI) using a computer software package (Bio-Plex Manager 4.1; Bio-Rad Laboratories, Hercules, CA, USA).
The end-biotinylated glycopolymers for coupling to fluorescent microspheres, Glyc-PAA-biot1
, were produced in-house (Laboratory of Carbohydrates, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation). Biotinylated glycopolymers were coupled to fluorescent carboxylated beads of 5.5 μm diameter with distinct spectral “addresses” (Bio-Rad Laboratories Inc., Hercules, CA, USA). Each set of beads was embedded with a precise ratio of red and infrared fluorescent dyes allowing its identification by measuring the intensities of the two classifier fluorochromes. The stock vial of microspheres (1.25
microspheres/mL) was vortexed for 30 s and sonicated for 30 s in a water bath prior its use. Bead suspension (100 μl; 1.25
microspheres, 0.2 nmol –COOH groups in total, according to the supplier’s information) was centrifuged for 4 min, 14,000 g at RT. The pellet was resuspended in bead wash buffer (100 μl; Bio-Plex amine coupling kit, Bio-Rad Laboratories Inc., Hercules, CA, USA) by vortexing and sonication, and washed by centrifugation as described above. After gentle removal of supernatant, the pellet was resuspended in 80 μl of bead activation buffer (Bio-Plex amine coupling kit, Bio-Rad Laboratories Inc., Hercules, CA, USA), vortexed and sonicated. Sulfo-N
-hydroxysuccinimide sodium salt (S-NHS) and 1-ethyl-3-[3,3-dimethylaminopropyl]carbodiimide hydrochloride (EDC; Pierce Biotechnology, Rockford, IL, USA, both 50 mg/mL in activation buffer) were prepared immediately prior to use, and 10 μl of each solution was added to the bead suspension, followed by vortexing for 30 s. Beads were incubated on a vertical rotor in the dark for 20 min at RT. The activated beads were centrifuged and supernatant removed. The pellet was resuspended in 150 μl biotin-solution (0.1 M NaHCO3
, pH 8.3, containing 1 μg (≈ 2 nmol) of biotin-NH(CH2
. Lectinity Holdings, Moscow, Russia) and incubated on a vertical rotor with medium speed for 2 h at RT in the dark. Obtained biotinylated beads were pelleted by centrifugation and resuspended in 150 μl of 50 mM ethanolamine (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) in 0.1 M NaHCO3
, pH 9.0 to quench unbound activated groups. Beads, protected from light, were agitated on a rotator for 30 min at RT and centrifuged. The pellet was washed twice with 500 μl PBS, pH 7.4 and resuspended in streptavidin-solution (400 pmol streptavidin in 150 μl PBS; Bio-Rad Laboratories Inc., Hercules, CA, USA). Suspended bead were vortexed and agitated on a rotator in the dark for 2 h at RT or 12 h at 4°C. Beads were washed twice with 500 μl PBS followed by centrifugation. Glyc-PAA-biot1
solutions in water (20 pmol end-biotin labeled Glyc-PAA, ~30 kDa, Glyc contents - 20% mol) [24
], were added with 1.25
streptavidin-coated beads in 150 μl PBS to the reaction tubes. The mixture was protected from light and agitated on a rotator for 6 h at RT. Modified microspheres were centrifuged, supernatant removed and beads washed twice with 500 μl of bead storage buffer (Bio-Rad Laboratories Inc., Hercules, CA, USA). Beads were centrifuged and resuspended in 100 μl of bead storage buffer and concentration determined using a hemocytometer (Roth AG, Karlsruhe, Germany) before storing at 4°C, protected from light.
Printed glycan array (PGA)
Printed glycan array slide fabrication and high-throughput profiling was performed as previously described [6
]. Briefly, monomeric glycans as ω-aminopropyl glycosides of 95–98% purity (Lectinity Holdings, Moscow, Russia) were diluted in 300 mM phosphate buffer pH 8.5, containing 0.005% Tween 20 and printed by robotic pin deposition on N-
hydroxysuccinimide activated glass slides (Nexterion Slide H, Schott, Jena, Germany). Glycans were printed at a 50 μM concentration in eight replicates. Free N-
hydroxysuccinimide activated groups were blocked with 50 mM ethanolamine in 50 mM borate buffer at a final pH of 9.2. Slides were then rinsed with deionized water, dried and stored at room temperature in a desiccator. Each plasma sample was diluted 1:15 with PBS containing 0.1% v/v
Tween20 and 3% w/v
BSA, thoroughly vortexed for 15 s and incubated at 37°C for 15 min to dissolve potential lipid aggregates. Samples were transferred to the array slides and gently rocked in a sealed humidified incubator for 2 h at 37°C. Unbound sample components were washed with a series of 0.1% and 0.001% Tween-20 in PBS. Antibody solution and goat anti-human IgA + IgG + IgM conjugated to long chain biotin (Pierce Biotechnology, Rockford, IL, USA, 1:100 (20 μg/mL) in PBS containing 0.1% Tween-20 and 3% BSA) was added. Slides were incubated at RT in a humidified chamber for 45 min and then washed. Bound antibodies were visualized by incubating slides with fluorescent dye streptavidin solution (Alexa Fluor555
, Molecular Probes, Invitrogen, Carlsbad, CA, USA, 1:1000 in PBS/0.1% Tween-20) at RT for 30 min. Fluorescence signals corresponding to glycan-bound antibodies were measured and quantified using ImaGene analysis software version 6.1 and 7.5 (BioDiscovery, El Segundo, USA). Signals were measured as total signal intensity (medTSI) per glycan and were expressed as median across eight intra-array replicates. In a prior discovery approach we screened plasma of non-mucinous ovarian cancer patients in comparison to healthy controls and revealed anti-P1
antibodies to be the most specific differentiating anti-glycan antibody. Apart from the standard ABO blood group system we have therefore used anti-P1
in this comparative analysis as an example of a new biomarker identified by PGA only.
In the combination of three different immuno-assays one of the major problems is the absence of a ‘gold’ standard method, which means there is no experimental approach available, which can be taken as a norm of measuring anti-glycan antibodies. Neither has an internal standard nor a background threshold been established , and the data values achieved in each method are independent from each other, which make the direct comparison impossible: (A) ELISA values reflect the oxidized product of the chromogen TMB (3, 3′, 5, 5′-tetramethylbenzidine) and is a substrate conversion based on an enzymatic reaction; (B) printed glycan array (PGA) and suspension array (SA) (R-phycoerythrin) results are achieved due to fluorescence measurements. These differences lead to data values, which require data mining before method comparison. When dealing with parameters of different units and scales it is very important to standardize or normalize data in order to allow for a reliable comparison. Here we used standardization of data as it transforms datasets into a mean zero and a unit variance and keeps ranges similar and variables different. This is also the most commonly used method for normalization and comparison of methods incorporating differing datasets. Whilst PGA data were pre-processed [6
], SA raw data were log-transformed to improve interpretability and visualization. To solve the problem of different data values, all data sets were standardized as follows: z
Standardized data (z) were generated for each vector data set (xi
) by subtraction by their mean (xmean
) and division of their standard deviation (xsd
), and called ‘standardized antibody measurements’ (SAM). Combined graphical and statistical interpretations of method-comparison studies were performed and included scatter plots combined with correlation and regression analysis [30
]. Data analysis, including calculation of mean, median, standard deviation and coefficient of variation was performed using the open source statistical programming language R (http://CRAN.R-project.org/
, version 2.8.1). Statistically significant differences were proved for each method by the Wilcoxon rank sum test or student t
test. Concordance correlation coefficients (rCCC
], which evaluate the degree to which pairs of observations fall on the 45° line through the origin, were calculated and compared within all independent methods (R package epiR). Direct comparison of two methods was performed using parametric linear regression [32
]. Non-parametric testing of median signals among all known blood groups was measured using the Kruskal-Wallis rank sum test. For comparisons of high versus
low anti-glycan antibody levels in correlation with known blood groups, sensitivity, specificity and area under the curve (AUC) were calculated for each method (R package ROCR [34
]). The best cut-off between observed false negative and false positive values was described as the “precision-recall break-even point” [34
], the point at which precision equals recall and predictions are made due to the prevalence within the data given. All p
0.05 were taken as significant.