Characterization of monoclonal and patient-derived antiplatelet factor 4 antibodies in platelet factor 4 and platelet factor 4/polyanion chemiluminescence assays
Abstract
BACKGROUND: Heparin-induced thrombocytopenia, vaccine-induced immune thrombocytopenia and thrombosis (VITT), and VITT-like disorders are caused by antiplatelet factor 4 (PF4)/polyanion and/or anti-PF4 immunoglobulin G antibodies. The rapid anti-PF4/polyanion and anti-PF4 chemiluminescence assays differentiate between typical heparin-induced thrombocytopenia (anti-PF4/heparin) and VITT antibodies (anti-PF4). OBJECTIVES: To characterize monoclonal antibodies (mAbs) and recombinant anti-PF4 antibodies in the 2 chemiluminescence assays specific for anti-PF4/polyanion and anti-PF4 antibodies, respectively, and to characterize anti-PF4/polyanion antibodies in patients whose sera tested positive in both assays. METHODS: For anti-PF4/heparin and anti-PF4 antibodies, the chimeric mAbs 5B9, 1C12, and 1E12 were generated using standard technology. Recombinant antibodies from VITT and VITT-like patients were generated based on the amino acid sequences of affinity-purified patient anti-PF4 antibodies. KKO was humanized by cloning its Fab sequences and expressing them as a chimeric recombinant human immunoglobulin G1 antibody. Anti-PF4 antibodies from patient sera testing positive in both chemiluminescence assays were affinity-purified using PF4/polyanion and PF4 beads. RESULTS: In chemiluminescence assays, 5B9, humanized KKO, 1C12, and 1E12 bound to PF4/polyanion complexes but differed largely in their PF4 reactivity. VITT (-like) recombinant antibodies bound only to PF4. Some patients' sera contained antibodies that showed similar reactivity in both assays, regardless of whether purified with PF4/polyanion or PF4 beads, suggesting cross-reactive antibodies, while sera from other patients contained both anti-PF4/heparin and anti-PF4 antibodies. CONCLUSION: The different reactivities of anti-PF4 mAbs/heparin antibodies need to be considered when planning experiments and interpreting results. Patients may have very different antibody reactivities despite giving the same results in the 2 chemiluminescence assays.
Related
- Novel monoclonal antibodies for an activated protein C cleavage neoepitope of coagulation factor Va
- Editorial: Monoclonal antibodies in treating multiple sclerosis (MS), and related diseases
- Targeting Integrins in Triple Negative Breast Cancer: Therapeutic Potential of Monoclonal Antibodies, PARP Inhibitors, Synthetic Peptides, and Aptamers
- 83 Translational PBPK modeling of PET imaging reveals enhanced predicted tumor exposure of antiCTLA4 singledomain antibody fragments vs. monoclonal antibodies
- Anti-Nectin4 monoclonal antibodies attenuate airway inflammation and epithelial barrier dysfunction in asthma
BibTeX
@article{dabbiru2026characterization,
title = {{Characterization of monoclonal and patient-derived antiplatelet factor 4 antibodies in platelet factor 4 and platelet factor 4/polyanion chemiluminescence assays}},
author = {Venkata A.S. Dabbiru and Marta Broto and Jan Wesche and Jing Jing Wang and Luisa Müller and Patrycja Gebicka and Jérôme Rollin and Yves Gruel and Marta Palicio and Thomas Thiele and Andreas Greinacher and Linda Schönborn},
journal = {Journal of Thrombosis and Haemostasis},
year = {2026},
month = nov,
doi = {10.1016/j.jtha.2026.01.016},
url = {https://hal.science/hal-05849033v1},
note = {CC BY},
}
RIS
TY - JOUR
TI - Characterization of monoclonal and patient-derived antiplatelet factor 4 antibodies in platelet factor 4 and platelet factor 4/polyanion chemiluminescence assays
AU - Venkata A.S. Dabbiru
AU - Marta Broto
AU - Jan Wesche
AU - Jing Jing Wang
AU - Luisa Müller
AU - Patrycja Gebicka
AU - Jérôme Rollin
AU - Yves Gruel
AU - Marta Palicio
AU - Thomas Thiele
AU - Andreas Greinacher
AU - Linda Schönborn
PY - 2026
DA - 2026/11/02
JO - Journal of Thrombosis and Haemostasis
DO - 10.1016/j.jtha.2026.01.016
UR - https://hal.science/hal-05849033v1
AB - BACKGROUND: Heparin-induced thrombocytopenia, vaccine-induced immune thrombocytopenia and thrombosis (VITT), and VITT-like disorders are caused by antiplatelet factor 4 (PF4)/polyanion and/or anti-PF4 immunoglobulin G antibodies. The rapid anti-PF4/polyanion and anti-PF4 chemiluminescence assays differentiate between typical heparin-induced thrombocytopenia (anti-PF4/heparin) and VITT antibodies (anti-PF4). OBJECTIVES: To characterize monoclonal antibodies (mAbs) and recombinant anti-PF4 antibodies in the 2 chemiluminescence assays specific for anti-PF4/polyanion and anti-PF4 antibodies, respectively, and to characterize anti-PF4/polyanion antibodies in patients whose sera tested positive in both assays. METHODS: For anti-PF4/heparin and anti-PF4 antibodies, the chimeric mAbs 5B9, 1C12, and 1E12 were generated using standard technology. Recombinant antibodies from VITT and VITT-like patients were generated based on the amino acid sequences of affinity-purified patient anti-PF4 antibodies. KKO was humanized by cloning its Fab sequences and expressing them as a chimeric recombinant human immunoglobulin G1 antibody. Anti-PF4 antibodies from patient sera testing positive in both chemiluminescence assays were affinity-purified using PF4/polyanion and PF4 beads. RESULTS: In chemiluminescence assays, 5B9, humanized KKO, 1C12, and 1E12 bound to PF4/polyanion complexes but differed largely in their PF4 reactivity. VITT (-like) recombinant antibodies bound only to PF4. Some patients' sera contained antibodies that showed similar reactivity in both assays, regardless of whether purified with PF4/polyanion or PF4 beads, suggesting cross-reactive antibodies, while sera from other patients contained both anti-PF4/heparin and anti-PF4 antibodies. CONCLUSION: The different reactivities of anti-PF4 mAbs/heparin antibodies need to be considered when planning experiments and interpreting results. Patients may have very different antibody reactivities despite giving the same results in the 2 chemiluminescence assays.
SN - 1538-7933
SN - 1538-7836
ER -