Development of an Enzyme-Linked Immunosorbent Assay for Detection of the Native Conformation of Enterovirus A71

Figures

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  FIG 1. Expression and purification of 16-2-2D scFv. (a) Schematic of the expression cassette used for the production of scFv in P. pastoris. (b and c) Western blotting (b) and Coomassie blue staining (c) to determine the presence of scFv in IMAC elution fractions. Samples were denatured in Laemmli buffer, separated using SDS-PAGE, and visualized by Coomassie blue staining or anti-His Western blotting. Representative data are shown (n = 3).

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  FIG 2. Determining the dynamic range of 16-2-2D scFv. Sandwich ELISA was performed using EVA71 cell culture supernatant and 16-2-2D scFv to identify a suitable working concentration for scFv. (a) Virus concentrations between 5 × 106 and 1.25 × 106 TCID50/mL and scFv concentrations between 40 and 2.5 μg/mL were used to identify the linear range of the assay. Graphed data are mean ± standard error of the mean (SEM) (n = 3, each in duplicate), and the dotted lines indicate the 95% CI. (b) To determine the suitable working concentration, a linear regression was performed using four successive 2-fold dilutions of EVA71. The linear regression is indicated by the solid line, the dotted lines indicate the 95% CI, and graphed data are mean ± SEM (n = 3, each in duplicate).

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  FIG 3. Antigenic specificity of scFv1 and mAb979. (a) Gradient-purified viral samples were assessed for the presence of VP0 and VP2 by Western blotting with MAb 979 (n = 3). (b to d) The titers of fractions were determined by TCID50 assay (n = 3) (b), and sandwich ELISAs were used to determine their reactivity with MAb 979 (c) and scFv (d) (n = 3, each in duplicate), with a representative Western blot. Graphed data are mean ± SEM.

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  FIG 4. Antigenic conversion of virus. Gradient-purified and concentrated virus samples were heated to a range of temperatures to induce antigenic conversion. (a to c) Graphed infectious viral titers (a), 16-2-2D reactivity (b), and MAb 979 reactivity (c) over a range of incubation temperatures are shown. Data are mean ± SEM (n = 3), with ELISAs conducted in duplicate. (d) The relationship between infectious viral titer and 16-2-2D scFv reactivity is graphed. (e) Negative-stain TEM shows particles with or without heating to 55°C. Scale bar = 100 nm.