A New Rapid, Specific, and Simple ELISA for von Willebrand Factor Propeptide

Scientific Research Question: The ratio between the von Willebrand factor propeptide (VWFpp) and the mature VWF can be used to diagnose von Willebrand disease (VWD) patients with an increased clearance rate of VWF. However, the implementation of the currently available ELISA-based VWFpp assays is complicated. They are not easy to perform and could not be completed within 24 h. The aim of the present feasibility study was to develop and validate a rapid, specific and simple ELISA that could be easily implemented in routine.

Methodology: Monoclonal antibodies against VWFpp were used for coating of microtiter plate strips, and for horseradish peroxidase (POD) labeling. A sandwich type ELISA was designed with a total assay time of about 90 min based on precoated strips (ready to use). Potential interference by rheumatoid factor and heterophilic antibodies is prevented by specific additives. The fzmb VWFpp assay was calibrated against the SSC/ISTH Secondary Coagulation Standard Lot #4, 0.97 IU/ml vWFpp). Citrate plasma samples obtained from ostensibly healthy controls (VWF > 40%) and from VWD patients were investigated using the fzmb VWFpp assay and results were compared to a reference VWFpp ELISA and lab data.

Findings: Assay performance: For the fzmb VWFpp assay a detection limit of 1.7 mIU/ml and a measurement range from 1.7-120 mIU/ml were found. VWFpp above 120 mIU/ml require sample dilution; the recovery (CV of several dilution steps) was 4.9% at 583 mIU/ml in sample 1 and 9.7% at 2170 mIU/ml in sample 2. The intra-assay precision (CV) was 8.1% at 3.8 mIU/ml and 0.1% at 60.6 mIU/ml. The inter-assay precision (8 days-CV) was 9.8% at 3.8 mIU/ml and 0.6% at 60.6 mIU/ml. Assay comparison: A study of 150 samples from healthy controls and VWD patients revealed a strong correlation between the new assay and the reference research assay from Sanquin (y=0.91x+4.43, Kendalls Tau= 0.75; mean difference -2,9%) with comparable reference intervals of 508 mIU/ml to 2225 mIU/ml and of 487 mIU/ml 2499 mIU/ml for the new and the reference ELISA, respectively.

Conclusions: The newly developed fzmb VWFpp ELISA demonstrated results comparable to the Sanquin ELISA assay. However, the new test is much more suitable for implementation in routine diagnostic medical laboratories. Short assay time, less hands-on steps, ready-to-use microtiter strips, and good performance characteristics make it a useful tool for better categorizing VWD patients, specifically for the workup of increased VWF clearance rate.