PB1063 - A sensitive and user-friendly method for quantifying VWF collagen binding (VWF:CB) using a fiber optic - surface plasmon resonance biosensor

Background: Determination of both von Willebrand factor (VWF) levels and functionality is crucial to classify patients with von Willebrand disease (VWD). VWF functionality primarily encompasses the quantification of both collagen (VWF:CB) and platelet binding of VWF (VWF:RCo) and can, if needed, be supplemented with more specialized tests.
Fiber optic – surface plasmon resonance (FO-SPR) biosensors allow fast evaluation of interactions between biomolecules via a simple plasma dip. We recently developed a FO-SPR-based VWF:Ag assay and developing similar assays for VWF:CB and VWF:RCo would further facilitate VWD classification.

Aims: Establishing a VWF:CB assay using FO-SPR biosensors.

Methods: Gold-coated optical fibers were made specific by coupling either collagen fibers type 3 or a synthetic triple-helical-peptide (THP) onto the carboxyl-SAM layer. Next, normal human plasma (NHP) was added to allow binding of VWF to either the collagen fibers or THP. Finally, the end SPR signal was generated by adding biotinylated polyclonal anti-VWF antibodies followed by anti-biotin coated gold nanoparticles. Serial dilutions of NHP were used to establish a dose-response curve. Assay sensitivity was determined using a VWD type 3 sample.

Results: Immobilization of collagen fibers type 3 to the optical fibers was inconsistent and variable in contrast to that of THP. Using the THP, antibody concentrations and buffer conditions in all assay steps were optimized and showed good reproducibility (CV < 20%). A calibration curve was established using NHP and an excellent non-linear fitting (R² = 0.9920) was found. Detection and quantification limit were 0.36% and 2.56%, respectively. Further fine-tuning is running and based on comparison studies with our in-house VWF:CB ELISA.

Conclusion(s): We developed a FO-SPR-based VWF:CB assay using a synthetic THP which showed less variability than with the organic collagen source. In combination with our already developed FO-SPR-based VWF:Ag measurement, these FO-SPR assays could be ideal candidates for a multiplex set-up to determine VWF parameters simultaneously.