Figure 3. Identification of Residues Critical for B4-2/55 Binding.
A: Multiple sequence alignment of NSP4 representing each genogroup. Primary NSP4 sequence corresponding to amino acids 99–119 for SA11 cl. 3 (genogroup A), Wa-attenuated (genogroup B and the immunogen for B4-2/55), OSU (genogroup B), RRV (genogroup C), ECTC (gengroup D) and Ty-1 (genogroup E) are shown. The B4-2/55 epitope (aa100–118) is highlighted in bold type on the SA11 cl. 3 sequence. Conservative changes relative to SA11 NSP4 are shown in bold type, and non-conservative changes are in underlined bold type. Residues that differ between genogroups A–D and genogroup E within the B4-2/55 epitope are indicated by arrows. B: Immunoblot analysis of alanine substitution mutants. Alanine mutants of NSP490–175 were expressed in BL21(DE3) E. coli, purified by Ni-NTA beads, separated by SDS-PAGE, blotted onto nitrocellulose, and stained by Ponceau S to determine equal loading (lower panels). Identical blots were detected with either MAb B4-2/55 or αNSP4-FL. Arrows indicate NSP4 monomers or oligomers in the immunoblot. C: ELISA end-point titer of B4-2/55 against NSP4 alanine mutants. * indicates p < 0.01 by students T-test. D: The SA11 NSP4 sequence is shown highlighting the B4-2/55 epitope (aa100–118) in bold with the critical residues (105, 108, & 111) underlined.