Background and aim:
Glycosylation is an essential posttranslational modification used primarily for cellular communication. The formation of complex glycans as secondary gene products is dependent on the activity of various glycosyltransferases (GTs). Mutated GTs, however, can cause significant alterations in the glycan patterns expressed on cancerous cells, making those patterns potent biomarkers. Whereas the detection of these tumor-associated carbohydrate antigens (TACA) require primary tumor tissue, autoantibodies (AAbs) against cancer-specific glycans in human plasma can be detected with a simple and minimal invasive technique.

Methods:
The Glyco-Multiplex assay enables the simultaneous detection of AAbs against (i) various glycans with (ii) minor amounts of plasma from (iii) a large number of patients in (iv) a short time. For this approach, multiple, potentially tumor-associated, glycans are coupled to distinct Luminex® bead regions. The unique color-coding of the beads allows the measurement of an individual’s isotypespecific AAb profile against the selected glycans simultaneously in one single well. The adaption of the coupling protocol for the use of the KingFisherTM Flex enables the upscale of this assay to up to 96 different regions with negligible additional hands-on time.

Results:
A protocol for the coupling of glycans to carboxylated MagPlex® beads was established and optimized. Subsequently, suitable combinations of plasma concentrations and Ig-specific (IgM, IgG and IgA) detection antibodies and assay controls were evaluated to detect α-TACA AAbs in human plasma samples. To get a first impression on α-TACA AAb signals, plasma samples from colon cancer patients, lung cancer patients and healthy donors were measured using the Glyco-Multiplex. In accordance with literature, (auto)antibodies in both healthy and diseased individuals could be detected. Furthermore, the analysis revealed individual isotype (IgM, IgG, IgA)-specific α-TACA-AAbs profiles for the tested samples. MagPlex® beads that were coupled using the protocol for the KingFisherTM Flex yielded results that were comparable to those obtained with manually coupled beads.

Conclusion:
Taken together, we are now able to generate individual autoantibody profiles against glycan structures, using minor amounts of plasma, in a high throughput manner.