FIG. 4.

(A) Blocking ELISA in the absence of detergent with native virions and MAbs A1, A2, A4, B2, and B4 as described previously (27). A predetermined fixed dilution of the respective MAb was incubated with decreasing concentrations of native TBEV. The mixture was then added to microtiter plates that had been coated with purified virus at a concentration of 0.5 μg/ml, a procedure that leads to the exposure of the FP loop, allowing its reaction with FP-specific MAbs (27). Antibody that was not blocked by the antigen in solution bound to the solid-phase antigen and was detected using a peroxidase-labeled rabbit anti-mouse immunoglobulin G (27). Results are expressed as percentages of the absorbance value obtained with each MAb in the absence of a blocking antigen. The data are representative of results of at least two independent experiments. (B) Four-layer ELISA with TBEV and MAb A1 to analyze the transient exposure of the A1 binding site upon acidification. Native TBEV in phosphate-buffered saline (pH 7.4; protein concentration, 0.5 μg/ml) was captured by polyclonal anti-TBEV immunoglobulin G for 1 h at 37°C as described previously (27). A1 epitope exposure was tested with the following combinations of pH and biotin-labeled MAb: column A, A1 was added in phosphate-buffered saline (pH 7.4); column B, A1 was added in MES buffer (pH 5.5; 50 mM MES, 100 mM NaCl); column C, the captured virus was exposed to MES buffer (pH 5.5) for 10 min followed by MAb A1 in the same buffer. After incubation for 1 h at 37°C, bound antibodies were detected by using streptavidin-peroxidase (Sigma-Aldrich). The data are the averages from five independent experiments performed in duplicate, and the error bars represent the standard errors of the means.