Establishment of Sandwich ELISA for Quality Control in Rotavirus Vaccine Production

Figures

• 

  Figure 1. The reactivity of the antibody pairs for P[4], P[6], P[8]-VP4*, and Bl21(DE3) E. coli lysate as examined by checkerboard. (A–D) The reactivity of P[4]-VP4*-specific mAb pairs for detecting 100 ng/mL P[4]-VP4*, 100 ng/mL P[6]-VP4*, 100 ng/mL P[8]-VP4*, and the cell lysate of Bl21(DE3); (E–H) the reactivity of P[6]-VP4*-specific mAb pairs for detecting 50 ng/mL P[4]-VP4*, 50 ng/mL P[6]-VP4*, 50 ng/mL P[8]-VP4*, and the cell lysate of Bl21(DE3); (I–L) the reactivity of P[8]-VP4*-specific mAb pairs for detecting 20 ng/mL P[4]-VP4*, 20 ng/mL P[6]-VP4*, 20 ng/mL P[8]-VP4*, and the cell lysate of Bl21(DE3).

• 

  Figure 2. The detection range and specificity of P[4], P[6], and P[8]-VP4*-specific detection systems. The purified VP4* proteins were 2-fold serially diluted from 2 μg/mL to 0.97 ng/mL by 20% NBS, and detected by P[4], P[6], and P[8]-specific VP4* detection system. (A) The reactivity of P[4]-VP4* antibody pair 7E3:5D8-HRP to different genotype VP4* proteins; (B) the reactivity of P[6]-VP4 antibody pair 1E9:3D4-HRP to different genotype VP4 proteins; (C) the reactivity of P[8]-VP4* antibody pair 15D9:5F10-1-HRP to different genotype VP4* proteins. All the experiments were performed in three replicates, and the error bars represent the standard deviation of each group.

• 

  Figure 3. The recovery rate of P[4], P[6], and P[8]-VP4* proteins in E. coli lysate. The purified P[4], P[6], and P[8]-VP4* proteins were added to the supernatant of Bl21(DE3) E. coli lysate to a final concentration of 100, 20, and 4 μg/mL, respectively, and detected by the corresponding genotype-specific antibody pairs. (A) The results detected by P[4]-VP4*-specific mAb pair (7E3: 5D8-HRP); (B) the results detected by the P[6]-VP4*-specific mAb pair (1E9: 3D4-HRP); (C) the results detected by the P[8]-VP4*-specific mAb pair (15D9: 5F10-1-HRP). All the experiments were performed in three replicates, and the error bars represent the standard deviation of each group. The concentration of each protein was calculated from the corresponding standard curve (Supplementary Figure S2). The recovery rate was calculated according to the formula: VP4* calculated/VP4*theoretical*100%.

• 

  Figure 4. The evaluation of the identity of purified VP4* proteins by the genotype-specific mAb pairs. In order to confirm whether the ELISA systems can be used to detect protein contamination, we mixed 10 μg/mL of the other two VP4* proteins into P[4], P[6], and P[8]-VP4* protein, respectively, and then the mixtures were detected by the three genotype-specific mAb pairs. As shown in the (A–C), the three ELISA detection systems can accurately identify the P[4], P[6], and P[8]-VP4* in the mixed samples. All the experiments were performed in two replicates, and the error bars represent the standard deviation of each group.