Following the incorporation of LFA control lines, we initially conducted a matrix effect study in diluted human sera to identify an appropriate sample dilution that could be used to effectively compare the traditional and AmpliFold formats of the assay. Immunoassays typically observe matrix effects in complex physiological fluids due to competing interactions between various components of the assay and the sample. In this study, the assays were run as previously, but with the volume fraction of human serum to running buffer adjusted to cover a range of human serum fractions from 0 (pure running buffer) to 50% v/v. These samples were prepared such that the concentration of HER2 added was