Skip to main content
. 2018 May 31;13(5):e0198375. doi: 10.1371/journal.pone.0198375

Fig 1. Surface-immobilization of pCRP induced the formation of an intermediate with dual antigenicity.

Fig 1

(A) a.a.22-45 (recognized by 8C10; orange) is mostly packed inside the subunit in pCRP (PDB ID: 1B09), while a.a.199-206 (recognized by 3H12; green) lies at the subunit interface of the pentamer. (B) The subunit structures extracted from the crystal structure of pCRP (medium blue; PDB ID: 1B09) or de novo modeled by I-TASSER (orange red; C-Score = -0.78) were superposed. For I-TASSER modeling, templates with a homology over 25% were excluded. a.a.199-206 segment lies on the subunit interface of pCRP and packs closely against the subunit body. By contrast, this segment extrudes from the subunit body in the modeled structure, which is why 3H12 can report the pentamer disassembly. (C-F) Indicated concentrations of pCRP in TBS-Ca or TBS-EDTA (pH 7.4) were added into Nunc MaxiSorp microtiter wells and incubated overnight at 4°C or for 1 h at 37°C. The antigenicity expression of the immobilized protein was assayed with mAbs 3H12 (C), 8C10 (D), 8D8 (E) or 1D6 (F). Immobilized pCRP showed dual antigenicity of both pCRP (8D8 and 1D6) and mCRP (3H12 and 8C10). (G) 5 μg/ml pCRP or mCRP was immobilized in bicarbonate coating buffer (pH 9.6), TBS-Ca or TBS-EDTA (pH 7.4) at 4°C overnight. Alternatively, 2 μg/ml pCRP was captured by immobilized polyclonal sheep anti-human CRP antibody for 1 h at 37°C in TBS-Ca (pH 7.4). The antigenicity of the immobilized or bound antigens were determined by 8D8, 1D6 or 3H12. When mCRP was immobilized, only mCRP antigenicity (3H12) could be detected. By contrast, pCRP bound to immobilized pAb showed only pCRP antigenicity (8D8 and 1D6). However, directly immobilized pCRP showed significant 3H12 antigenicity (***p < 0.001). (H) Binding of unlabeled (the left panel) or biotin-labeled pCRP (the right panel) to mCRP (5 μg/ml) immobilized in TBS-Ca (pH 7.4) overnight at 4°C. The binding status was detected by mAbs and HRP-avidin, respectively. There was no binding of pCRP to the immobilized mCRP, excluding the possibility that the dual antigenicity was contributed by mCRP and survived pCRP. Data were obtained from at least three independent experiments and represented as mean ± SEM. For C-F, values underwent a nonlinear curve fit with OriginPro 8 software, during which the category was set as “Growth/Sigmoidal” and the function was set as “Hill1”.