Figure 4.

Development of a sandwich immunoassay for the detection of heterodimeric PirAB^Vp complex. (A, B) Detection of PirA and PirB in protein lysates obtained from several Vibrio spp. Total proteins were extracted from culture pellets of six Vibrio strains and coated on a 96-well plate for ELISA. Anti-PirA 1A8 and anti-PirB 3A5 IgGs were used to detect (A) PirA, and (B) PirB, separately in the lysates. Data represent the mean ± SD of duplicate tests (A, B). *P < 0.05, **P < 0.01, and ***P < 0.001. (C) Detection of heterodimeric PirAB^Vp by a sandwich immunoassay. 1A8 and biotinylated 3A5 were used as a capture and a detector, respectively (left), and detectability of the sandwich immunoassay was assessed using PirA, PirB, and PirAB^Vp (right). Values represent the mean ± SD for a duplicate. *P < 0.05, **P < 0.01. (D) Sensitivity of the immunoassay in detecting PirAB. PirA and PirB were mixed together in 1:1 molar ratio, serially diluted, and detected by the immunoassay. Linear regression analysis was performed. (E) Detection of PirAB^Vp naturally produced by Vibrio spp. Protein lysates extracted from culture pellets of 19-021-D1 (left) and CH49 (right) were subject to the immunoassay.