Analysis of sensitivity of the custom anti-human ORF2p monoclonal antibody. (A) Western blot analysis of protein generated from expression plasmids containing wild-type ORF2 endonuclease sequence (ENwt), codon-optimized ORF2 endonuclease sequence (ENco), and codon-optimized ORF2 sequence (ORF2) transiently transfected in 293 cells with our monoclonal antibody; 5 or 10 μg of the whole cell lysate was used for analysis as indicated. Control lane indicates cells transiently transfected with an empty vector. Bacterially purified endonuclease was loaded at 0 (empty, buffer only), 10, 20, and 40 ng. GAPDH is used as a loading control; 15 to 150 kDa on the right indicate positions of molecular markers. Arrows denote bands of expected sizes for each protein. (B) A standard curve was generated using the quantitation of the increasing amounts of the bacterially purified endonuclease shown in A. Signals detected for ORF2co, ENco, and ENwt are plotted and labeled with the respective names of the proteins.