Recombinantly Expressed Tagged SUrface Protein (RETSUP) assay - A new diagnostic system for the detection of antibodies to platelets.

BACKGROUND: Current assays for the detection of (allo-)antibodies to platelet antigens are often laborious and widely based on the presence of well-characterized donor platelets.

OBJECTIVES: To develop an easy-to-perform, sensitive, and specific test for the detection of antibodies against platelet antigens, in particular, glycoprotein (GP) antigens, called 'Recombinantly Expressed Tagged SUrface Protein' (RETSUP) assay, which does not require donor platelets.

METHODS: Twin-Strep-tagged GP complexes were recombinantly expressed in HEK-293 cells after stable transfection. These cell lines were used as antigen sources in the RETSUP assay, combining cell-based and ELISA-based assay procedures. The assay performance was tested with recombinant antibodies, anti-human platelet antigen (HPA) reference plasmas, and anti-HPA patient sera.

RESULTS: HEK-293 cell lines stably expressing either Twin-Strep-labelled GPIa/IIa, GPIIb/IIIa, GPIb/IX, or GPIb/IX/V complexes or GPV, as well as the distinct HPA-1, HPA-3 and HPA-5 epitopes, were successfully generated. Applying the generated GP-expressing cell lines, the developed RETSUP assay proved very sensitive and specific with recombinant antibodies targeting different GPs and human plasma/serum samples. The results of the test were not affected by the GP carrying the Twin-Strep-tag or by using freshly harvested or cryopreserved cells.

CONCLUSION: The RETSUP assay is an easy-to-perform, sensitive, and specific assay for the detection of plasma/serum antibodies to platelet GP, with performance comparable to or better than current state-of-the-art assays in anti-platelet antibody diagnostics. Due to the recombinant nature of the target antigens, it can be easily adapted to detect antibodies in other antibody-mediated diseases.