Fig 2: Antibodies against modified NS1-WD peptide recognize all four serotypes of DENV-infected cells and show reduced cross-reactivity to HUVECs. (A) Consensus sequence of mAb 19-5 and 33D2 phagetopes shared with LYRIC and NS1 were analyzed. The amino acids depicted with different color backgrounds are identical between the four serotypes of DENV NS1 and LYRIC. (B) The sequence of the modified NS1-WD peptide was compared to those of the original NS1-WD and LYRIC peptide (C) HUVECs were infected with DENV 2 (moi = 10) for 48 h. Cells were stained with anti-NS1 mAb 33D2, modified NS1-WD immune sera, or KLH immune sera (1:100 dilution) and analyzed by surface or intracellular IIF staining as described in the Methods. The scale bar indicates 20 µm. (D) The binding of purified pAbs from modified NS1-WD peptide-immunized mouse sera to NS1, modified NS1-WD peptide-conjugated BSA, or BSA was detected by ELISA. (E) The binding of different doses of purified pAbs from original NS1-WD, modified NS1-WD peptide immune sera or cmIg to HUVECs was analyzed by flow cytometry. The shift ratio (%) represents the percentage of binding compared with the control group (Anti-mouse IgG Alexa 488 only). All data are presented as the mean ± S.D. from at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ns indicates no significance.