Immunoreactivity of Cygnus E. coli HCP kit antibody to the standards used and to the residual proteins in MK-1454 Prep. Lab. batch API
To find out the composition difference between Cygnus kit standard and the in-house lysate supernatant standard, different concentrations ofE. coli control antigen used as the kit standard and lysate supernatant standard (concentration determined by micro BCA protein quantitation kit) were loaded and resolved on duplicate 4-12% SDS-PAGE gels as described in the Materials and methods. One of the gels was then Coomassie blue stained and the other transferred to PVDF membrane using iBlot and blotted with Cygnus anti-E. coli HCP antibodies provided in the kit. The protein profile differences between the two standards are shown in Figure 4a, and their immunoreactivity to the Cygnus antibody are shown in Figure 4b. Cygnus antibodies show stronger immunoreactivity to the Cygnus kit standard than the in-house standard in the Western blot results (Figure 4b). The Cygnus standard also shows a broader representation of E. coli HCPs ranging from high molecular weight to low molecular weight (10-180 kDa) while the in-house lysate supernatant standard has relatively less amount of low molecular weight (≤40 kDa) portion of proteins (Figure 4a and 4b). A densitometry analysis shows higher number of bands and higher total volume of proteins in E. coli control antigen than in the in-house standard detected both on the Coomassie blue stained gel and the Western blotted membrane. Cygnus antibody is also able to detect certain residual proteins in Prep. Lab batch API, with the immunoreactivity decreases along with the decrease of API loading concentration (Figure 4d). Comparing the bands detected by Cygnus antibody in Prep. Lab batch API samples (Figure 4d) to those detected by Coomassie blue staining at the high API concentration (Figure 5a) indicates that not all proteins detected by Coomassie blue staining is detectable by the Cygnus antibody, further indicating the need of using orthogonal methods for total residual protein quantitation.