IgG antibodies in patients with COVID-19 or VITT against anti-SARS-CoV-2 spike protein and PF4. (A) Individual OD results of sera tested by ELISA. Error bars are medians with interquartile ranges (indicated by red lines). Graph shows sera of patients with COVID-19 (n = 20), patients with VITT (n = 24), and PF4-affinity-purified (blue stars) IgG or PF4-heparin affinity-purified (^) IgG from VITT sera (n = 14). The 14 sera used for affinity purification of anti-PF4 IgG are indicated by green filled circles. All sera and the respective affinity-purified anti-PF4 IgG fractions were tested against SARS-CoV-2 S1 domain, RBD-SD1 domain, spike full-length ectodomain, PF4, and PF4-heparin complexes. Sera of patients with COVID-19 reacted with the spike protein and its S1 and RBD domains but not with PF4 or PF4-heparin complexes. VITT sera reacted with spike protein epitopes and PF4, but reactions were strongest with PF4-heparin complexes, whereas the affinity-purified anti-PF4 antibody fraction reacted with PF4 and PF4-heparin complexes but not with the spike protein or its S1 and RBD-SD1 domains. All negative controls (n = 15) gave negative results (supplemental Figure 3). The positive controls in the experiment with affinity-purified antibody for binding of antibodies to the S1 domain, the RBD domain, and the spike protein were positive (data not shown). (B) As a control to show that the affinity-purified anti-PF4 antibodies could still activate platelets, we incubated 75 µL washed platelets in Tyrodes buffer with PF4 (10 µg/mL) and added 10 µL of the affinity-purified antibodies, which reacted in the same manner as the original serum. Results of 14 affinity-purified antibody fractions are shown. Twelve showed strong platelet activation in the presence of PF4. Two antibody fractions still reacted positively by PF4-heparin ELISA but no longer activated platelets, most likely as a result of an antibody yield that was too low after affinity purification.