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Comparison of printed glycan array, suspension array and ELISA in the detection of human anti-glycan antibodies.

ABSTRACT: Anti-glycan antibodies represent a vast and yet insufficiently investigated subpopulation of naturally occurring and adaptive antibodies in humans. Recently, a variety of glycan-based microarrays emerged, allowing high-throughput profiling of a large repertoire of antibodies. As there are no direct approaches for comparison and evaluation of multi-glycan assays we compared three glycan-based immunoassays, namely printed glycan array (PGA), fluorescent microsphere-based suspension array (SA) and ELISA for their efficacy and selectivity in profiling anti-glycan antibodies in a cohort of 48 patients with and without ovarian cancer. The ABO blood group glycan antigens were selected as well recognized ligands for sensitivity and specificity assessments. As another ligand we selected P(1), a member of the P blood group system recently identified by PGA as a potential ovarian cancer biomarker. All three glyco-immunoassays reflected the known ABO blood groups with high performance. In contrast, anti-P(1) antibody binding profiles displayed much lower concordance. Whilst anti-P(1) antibody levels between benign controls and ovarian cancer patients were significantly discriminated using PGA (p=0.004), we got only similar results using SA (p=0.03) but not for ELISA. Our findings demonstrate that whilst assays were largely positively correlated, each presents unique characteristic features and should be validated by an independent patient cohort rather than another array technique. The variety between methods presumably reflects the differences in glycan presentation and the antigen/antibody ratio, assay conditions and detection technique. This indicates that the glycan-antibody interaction of interest has to guide the assay selection.

SUBMITTER: Pochechueva T

PROVIDER: S-EPMC3228963 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Comparison of printed glycan array, suspension array and ELISA in the detection of human anti-glycan antibodies.

Pochechueva Tatiana T Jacob Francis F Goldstein Darlene R DR Huflejt Margaret E ME Chinarev Alexander A Caduff Rosemarie R Fink Daniel D Hacker Neville N Bovin Nicolai V NV Heinzelmann-Schwarz Viola V

Glycoconjugate journal 20110923 8-9

Anti-glycan antibodies represent a vast and yet insufficiently investigated subpopulation of naturally occurring and adaptive antibodies in humans. Recently, a variety of glycan-based microarrays emerged, allowing high-throughput profiling of a large repertoire of antibodies. As there are no direct approaches for comparison and evaluation of multi-glycan assays we compared three glycan-based immunoassays, namely printed glycan array (PGA), fluorescent microsphere-based suspension array (SA) and ...[more]

PMID: 21948103

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Project description:Slaughter pigs are monitored for the presence of the zoonotic pathogen Salmonella, using both serology and bacteriology. ELISAs used to investigate pig herds are based on the detection of antibodies against components of the Salmonella cell envelope. Nearly all Salmonella isolates in food-producing animals are serovars of Salmonella enterica subspecies enterica, distributed over various serogroups as determined by the composition of their lipopolysaccharide (LPS). ELISAs for Salmonella serology are usually based on serogroup B and C1 LPS, often combined with serogroup D or E LPS. Although C2 LPS may improve serology, use of C2 LPS in a broad ELISA was never achieved.To enable detection of serum antibodies against Salmonella in pigs, a bead-based suspension array was developed with five LPS variants (B, 2× C1, C2, D1), each conjugated to a different bead set using triazine chemistry. Reactivity of the beads was confirmed with rabbit agglutination sera and with experimental pig sera. With a mixture of bead sets, 175 sera from slaughter pigs were investigated for the presence of antibodies against Salmonella. With a combination of ROC analysis (B and D LPS) and a prevalence estimation based on historic data (C LPS), individual cut-offs were defined for each LPS-conjugated bead set, and assay performance was evaluated. Results of the suspension array (BC1C1C2D) suggest that more pigs are seroconverted than indicated by a commercial BC1D1-ELISA, and that most of these extra seropositive samples give a signal on one of the beads with C LPS. These results show that expansion of a standard panel with more C LPS variants improves antibody detection.A suspension array for Salmonella serology in pigs was developed, that detects more seropositive sera than ELISA, which is achieved by expanding the panel of Salmonella LPS variants, including C2 LPS. The results demonstrate that bead-based suspension arrays allow for testing of pig sera, with the advantage of being able to set cut-offs per antigen. Ultimately, this type of assay can be applied in routine veterinary serology to test for antibodies against multiple Salmonella serovars (or other pathogens) in one single serum sample, using up-to-date antigen panels.

Dataset Information

Comparison of printed glycan array, suspension array and ELISA in the detection of human anti-glycan antibodies.

Publications

Comparison of printed glycan array, suspension array and ELISA in the detection of human anti-glycan antibodies.

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