Characterization of the 3E8 anti-HK antibody. (A) Western blot shows 3E8 specifically recognizes HK in NHP and cHK in Aβ42-treated human plasma. Purified human HK and cHK were included as controls. In KN-DF human plasma, HK was not detected by 3E8, indicating the specificity of 3E8. The membrane was stripped and reprobed with anti-LMWK antibody. LMWK was detected in normal and Aβ42-treated human plasma but not in KN-DF plasma. 3E8 did not recognize LMWK. (B) The 3E8 and 2B7 anti-HK antibodies pulled down HK, but not FXII, PK, or TF from human plasma. Human plasma was incubated with biotinylated 3E8, 2B7, and 4B12 anti-HK antibodies and control IgG, and streptavidin was added to pull down the antibody-antigen complex. The samples were analyzed by western blot using commercial antibodies against HK, FXII, PK, and TF. The 3E8 and 2B7 HK antibodies immunoprecipitated HK from human plasma, but they did not pull down FXII, PK, or TF. The 4B12 antibody, which recognizes cleaved HK but not intact HK, did not pull down HK, FXII, PK, or TF from human plasma. (C) HK ELISA shows the K_D of 3E8 anti-HK antibody binding to HK is 168 ± 38 pM. The experiments were performed in triplicate and repeated 3 times.