Recombinantly Expressed Tagged SUrface Protein (RETSUP) assay: a new diagnostic system for the detection of antibodies to platelets

Susanne Weber; Jasmin Birgit Zuzana Arnold; Ulrich Jörg Sachs; Peter Bruno Luppa

doi:10.1016/j.jtha.2023.12.030

Recombinantly Expressed Tagged SUrface Protein (RETSUP) assay: a new diagnostic system for the detection of antibodies to platelets

J Thromb Haemost. 2024 Apr;22(4):1187-1201. doi: 10.1016/j.jtha.2023.12.030. Epub 2024 Jan 4.

Authors

Susanne Weber¹, Jasmin Birgit Zuzana Arnold², Ulrich Jörg Sachs³, Peter Bruno Luppa²

Affiliations

¹ Institute of Clinical Chemistry and Pathobiochemistry, Klinikum rechts der Isar of the Technische Universität München, Munich, Germany. Electronic address: su.weber@tum.de.
² Institute of Clinical Chemistry and Pathobiochemistry, Klinikum rechts der Isar of the Technische Universität München, Munich, Germany.
³ Institute for Clinical Immunology, Transfusion Medicine, and Haemostasis, Justus Liebig University, Giessen, Germany; Department of Thrombosis and Haemostasis, Giessen University Hospital, Giessen, Germany.

PMID: 38184205
DOI: 10.1016/j.jtha.2023.12.030

Abstract

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 human embryonic kidney 293 cells after stable transfection. These cell lines were used as antigen sources in the RETSUP assay, combining cell-based and enzyme-linked immunosorbent assay-based assay procedures. The assay performance was tested with recombinant antibodies, anti-human platelet antigen (HPA) reference plasmas, and anti-HPA patient sera.

Results: Human embryonic kidney 293 cell lines stably expressing either Twin-Strep-labeled 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 those of current state-of-the-art assays in antiplatelet antibody diagnostics. Owing to the recombinant nature of the target antigens, it can be easily adapted to detect antibodies in other antibody-mediated diseases.

Keywords: antibody detection; in vitro testing; membrane glycoproteins; neonatal alloimmune thrombocytopenia; platelets.

MeSH terms

Antigens, Human Platelet* / genetics
Blood Platelets
Enzyme-Linked Immunosorbent Assay / methods
Humans
Isoantibodies
Membrane Proteins*
Platelet Glycoprotein GPIIb-IIIa Complex

Substances

Membrane Proteins
Isoantibodies
Platelet Glycoprotein GPIIb-IIIa Complex
Antigens, Human Platelet