After antibody incorporation, an experimental design using Response Surface Methodology (RSM) followed to indicate the concomitant conditions for all of the factors to obtain the maxi-mum colorimetric response, as described by Liyana-Pathirana andShahidi [22]. The four-factors and two level face-centered cubedesign (FCD) consisting of thirty experimental runs was employedincluding six replicates in the center point. The effects of unex-plained variability in the observed response due to experimentalerrors were minimized by randomizing the order of the exper-iments. The design variables were antibody concentration, pH,incubation temperature and incubation time, while response vari-able was particle size, zeta potential and colorimetric response [22].For obtained the pH intended, solution of NaOH (0.1 M) and HCl(0.1 M) was used. Symbol codes, experimental range and levels ofthe independent variables were described in Table 1. Statisticalanalysis of the model was performed to evaluate the analysis ofvariance (ANOVA)