Fig 2: 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 3: 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

Fig 4: Detection of as-APF binding to CKAP4 mutants by LC-MS. Various CKAP4 mutant-immobilized His-tag affinity beads were used to interact with as-APF, and the captured as-APF was analyzed by MS as described in “Materials and Methods”. a The protein staining of SDS-PAGE to show the efficiency of experiment; b The quantitation of as-APF bound to various CKAP4 mutants based on the as-APF standard curve (not shown) also performed with the same LC-MS conditions

Fig 5: 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