*2.2. Luminex Assay with Human Premixed Multi-Analyte Kit*

Two human premixed multi-analyte kits from the R&D system were used to assess the levels of 10 different biomarkers. First kit (cat: LXSAHM-09) for analyzing Angiopoietin-1, HGF, MMP-7, Osteopontin, TIMP-1, Galectin, Mesothelin, NRG1-b1 and Syndecan-1 simultaneously and second kit (cat: LXSAHM-01) for analyzing VEGF. In total, we analyzed 118 pleural effusions, of which 42 were from MM patients, 36 from AD patients and 40 benign effusions. Effusions were diluted 5-fold, using the dilution buffer included in the kit. All standards and samples were assayed in duplicate.

Analyte specific antibodies are pre-coated onto magnetic microparticles embedded with fluorophores at set ratios for each unique microparticle region. Two spectrally distinct light emitting diodes (LEDs) illuminate the microparticles. One LED excites the dyes inside each microparticle, to identify the region and the second LED excites the Streptavidin-PE to measure the amount of analyte bound to the microparticles. A minimum of 50 beads per region were counted. The median fluorescence intensities were determined on a Luminex® 100/200™ analyzer.
