These DLS results, when considered with other data reported in the study support the conclusions drawn in this section. HE-4 interactions with serine proteases were confirmed with surface plasmon resonance and kinetic constants were calculated. HE-4 was immobilized on Amthamine dihydrobromide research grade CM5 chip using EDC/NHS chemistry as described in methods. Different proteases were flowed over the chip in varying concentrations, ranging from 75�C300 nM. Concentration of proteases above this range was shown to reduce the signal, possibly due to ����Hook effect����. Among all the proteases tested, the highest affinity and association constant was found to be for proteinase K followed by AR-C 118925XX chymotrysin, PSA and then trypsin. Trypsin had the lowest association and dissociation constants among all the proteases tested. Converse study was also performed with SPR, where all serine proteases were on the chip and HE-4 was flowed. No significant changes were observed in binding affinity of the proteases and HE- 4. Unfortunately, we could not determine the kinetic constants for papain and pepsin as we faced an unexpected problem of negative sensograms. Although, negative sensograms are not uncommon they are usually ascribed to the differences in buffer composition pre and post injection. In the present study, there were no differences in the buffer compositions therefore we sought to further investigate the reason of signal dropping below baseline. For this, papain was incubated with increased concentration of HE-4 at pH 8.5 for 1 hr, and as a control HE-4 and papain was incubated separately for 1 hr at room temperature. Later mixtures were resolved on 14% SDS-PAGE under reducing conditions. HE-4 and papain, when incubated alone, showed band at their own molecular weight, but when they are incubated together two new bands appeared in the mixture below HE-4 band as seen in fig. 7A. In SDS-PAGE, we observed two bands that are probably the cleavage product of HE-4 by papain because with increasing concentrations of HE-4, the band at approximately 10 kDa increase in intensity while the original band of HE-4 does not increase in intensity which would be explainable by HE-4 cleavage by papain. This was confirmed with western blot which showed a low molecular weight HE-4 band lower than full length HE-4. This explains the negative sensogram of papain suggesting that after initial interaction with HE-4, papain cleaves and releases HE-4 from the chip bringing sensogram below baseline. In case of pepsin, we were surprised by the results as seen in fig. 7B when pepsin is incubated with HE-4 at pH 5.0 for 1 hr it undergoes self-cleavage, as evident by the band present at approximately 20 kDa and with increasing concentration of HE-4, the decrease in intensity of pepsin band. This band at 20 kDa was definitely of pepsin as western blot using HE-4 antibodies revealed that there was no band of HE-4 at that position.