## 2 Materials and Methods
### SPRI Analysis
#### 2.3.2 SPRi Data Processing  
The changes in RI were continuously monitored by the DAX software version 1 and converted into response units (RUs) (1 RU represents a RI change of \(1\times 10^{-6}\)). For the ROIs, the changing RUs were plotted over time, creating sensorgrams. The SPR-generated trix files were converted to ibmx files using SprintX software version 2.4.1.2 (IBIS Technologies, Enschede, The Netherlands) and an in-house-generated Matlab script was used in order to allow data analysis. To increase the quality of the experimental results, the local ligand density response (RLL) values were evaluated. Spots with large deviations in ligand density between triplicates were excluded. Subsequently, all sensorgrams were subjected to two signal modification steps, namely zeroing and local referencing. Zeroing was performed to correct for spot-to-spot variation and was achieved by setting the SPR signals to zero at the  
Figure 1: Schematic experimental set-up for SPRi. (**A**) Gold-plated sensor chip with immobilized antibodies on a prism. Upon illumination with P-polarized light from different angles, SPR causes a dip in the intensity of the reflected light at the incident angle or the so-called SPR angle, which is recorded by a CCD camera. Refractive index changes are caused by the binding of biomarkers with the antibodies. (**B**) The procedure starts with the injection of PBS to create a baseline (1), followed by flowing the body fluids over the sensor to bind the antigens with the specific antibodies on the surface (2, association phase). To remove unbound antigens, PBS is flowed again over the sensor (3, dissociation phase). Subsequently, a regeneration buffer is added to remove all bound antigens (4), to allow a new sample to bind to the antibodies.  
start of the initial baseline. Local referencing was performed to correct for background noise and was realized by subtracting the SPRi signals of reference spots from the signals of the ROIs. After the corrections, the mean SPRi signal between 60 and 70 s of the baseline was subtracted from the mean SPRi signal between 120 and 130 s of the dissociation phase to calculate the biomarker quantities. The first minute of the baseline and the second minute of the dissociation phase were excluded to guarantee the removal of signals induced by bulk shift changes, which are the result of RI differences between the buffers. When the calculated response resulted in a negative value, the value was set to 0. A negative response could be the result of a higher signal of the reference spot compared to the antibody spot, or because the baseline response was higher than the dissociation response. Since the antibodies were printed in triplicate, the mean response signal and standard deviation were calculated to determine the mean response signal per biomarker. To ensure data quality and reliable results, samples with negative response signals were excluded when the triplicate responses of a single biomarker were all three negative and/or when all the body fluid-specific markers provided negative responses. Negative signals imply that the samples were too diluted or that the signals fall within the range of instrumental noise. The biomarkers were considered present in the body fluids when the positive responses signals were >0.