Fig 2: as-APF binds directly and specifically to the CKAP4 extracellular domain. Non-glycosylated APF control peptide (TVPAAVVVA) was immobilized to the Fc1 channel of a CM5 chip through amine-coupling as the control (immobilized RU = 530); as-APF was immobilized to the Fc2 channel as the ligand (immobilized RU = 347). Purified CKAP4 extracellular domain CKAP4127–602 (0.22 uM) and the extracellular domain with the transmembrane domain CKAP4106–602 (0.17 uM) were used as the analytes for the binding assay by SPR. Adjusted sensorgrams (Fc2-Fc1) are shown above

Fig 3: Detection of as-APF in spiked urine. Control urine was mixed 1:1 with as-APF stock (200 μM) solution. Various dilutions of this mixture in SPR running buffer were prepared and injected over a CM5 chip surface immobilized with CKAP4127–360 by amine-coupling on the Fc2 channel. Fc1 channel was similarly treated but without CKAP4, as the reference. Final concentrations of as-APF were 40 μM (a), 20 μM (b), 5 μM (c), and 1.25 μM (d). Sensorgrams were corrected for background interference from urinary constituents by subtracting the RU traces resulting from unspiked urine injections from the RU traces resulting from the corresponding urine dilutions with spiked as-APF, and re-plotted after subtracting the RU trace from the reference (Fc1) channel

Fig 4: Binding of as-APF to CKAP4525–602 mutant is blocked by epitope specific antibody. SPR sensorgrams of equimolar injections of as-APF over a CM5 chip surface before (a), and after (c) injection of anti-CKAP4 (epitope region: 552–602) antibody (b). CKAP4525–602 was immobilized on Fc2 channel by amine-coupling. Fc1 channel was similarly treated but without CKAP4, as the reference

Fig 5: Characterization of purity and antigenicity of CKAP4 constructs. a SDS-PAGE of normalized amounts of purified CKAP4 constructs followed by silver staining. b–f Western blots using anti-His tag, anti-CKAP4 (G1/296), anti-CKAP4 (epitope region: 216–265), anti-CKAP4 (epitope region: 375–425), and anti-CKAP4 (epitope region: 552–602) primary antibody, respectively. Separate blots were used for each antibody. Molecular weight standards are indicated on left and right