Fig. 4

Residues of 295–300 were key binding sites for MAb 2D11. a To map the epitope for MAb 2D11, six variable regions were selected by an alignment of four time-ordered strains of GII.17 and sets of changes were introduced into these regions. Production of chimeric P proteins expressed by CFPS system was similar among the six mutants (evaluated by MAb 2A11) and residues 293–300 were found to lose binding affinity with MAb 2D11. b Continous three residues’ mutants were generated to narrow the binding site of MAb 2D11. Q293 was less relevant to MAb 2D11 binding. c Double alanine substitution mutants were introduced into 294-300aa. D300 existed across the four time-ordered strains of GII.17, but GII.17 clade A and GII.17 clade C could not restore the binding activity with MAb 2D11. Comparing these combined changing residues, loss of 298aa resulted in a decrease in binding of the antibody, while residues of 295, 299, and 300 might play a subordinate role in the binding site. The dotted line marked the negative control detection on panel B. d Binding of wild type P proteins of the GII.17 clade D variant, as well as reverse mutants to MAb 2A11 and 2D11. Residues of epitope A in GII.17 clade A and GII.17 clade C were replaced by those in GII.17 clade D. All reverse mutants restored the MAb 2D11 binding capability, while changes of these residues in GII.17 clade A resulted in the loss of binding capability with MAb 2A11. The statistical differences were shown by star symbols (*P < 0.05, ** P < 0.01, *** P < 0.001)