Figure 3

Performance of the MMP-9:TIMP-1 proximity ligation assay. A) The dark grey bar represents the specific signal from buffer when both MMP-9 and TIMP-1 antigens are present. The light grey bar represents the unspecific signal from a buffer sample with only MMP-9 as spike-in, while the medium grey bar represent the unspecific signal from a buffer sample with only TIMP-1 as spike-in. For all spike-in antigens the concentration stated on the x-axis applies. Further, the cross-reactivity in chicken plasma was demonstrated to be below buffer level. Values are reported as raw Cp signals. B) Standard curve for the MMP-9:TIMP-1 complex in PBS + 0.1% BSA buffer. The TIMP-1 (5’probe) and MMP-9 (3’ probe) were incubated with increasing amount of the MMP-9 and TIMP-1 antigens. This curve demonstrates the performance of the assay by assessing linear range in buffer settings and thereby the correlation between dose and Cp signal. The linear range is illustrated by the dotted lines and values are reported as raw Cp values. C) Inter-assay variation was determined by measuring the same breast cancer plasma sample on four different days. The four lines represent the sample measured on four different days (run 1, 2, 3 and 4). Values are reported as raw Cp signals. D) Plasma dilution curves of the MMP-9:TIMP-1 complex from four different breast cancer patients. Samples A and C have a high level of TIMP-1 protein, while samples B and D have a low level of TIMP-1 protein. The combined linear range was set between the 1:10 and 1:100 dilutions, illustrated by the dotted lines. Values are reported as raw Cp signal. Error bars represent standard deviation.