Procedure for constructing the 35-plex panel. Through using 4500 serum samples from cancer patients, five markers were discovered and identified through two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), six markers through surface-enhanced laser desorption/ionisation time-of-flight (SELDI-TOF) and 24 markers through conventional sandwich ELISA method. After optimisation in capturing and detecting the antibody pair for a target analyte, the capturing antibody was conjugated with beads, and the simplex kit was validated for its dynamic range, recovery rate, parallelism with standard curve, interference, matrix effect, and lower and upper limits of detection. Twenty-three simplex kits could be grouped together according to dilution factor and absence of cross reactivity. The remaining 12 were left and used as a simplex kit. BC = breast cancer; SC = stomach cancer; LC = lung cancer; CC = colon cancer; HCC = hepatocellular carcinoma.