Neutralization Antibody Assay for CDI

For the neutralization antibody assay, 96-well plates (TPP) were prepared to obtain monolayers of Vero cells using the procedure described in the cytotoxicity assay section. Antibodies used in these assays were obtained from serum samples collected from CDI-recovered patients included in the SERODIFF study. In order to confirm that the recombinant toxins were also susceptible to the neutralizing activity of these antibodies, 75 µL of a 4 µg.mL⁻¹ solution of toxins was mixed with 75 µL of 1/10 to 1/160 dilutions of serum from CDI-recovered patients and incubated at 37 °C for 60 min [27 (link),28 (link)]. The mixtures were then added to monolayers of Vero cells, and the plates were incubated in 5% CO₂ at 37 °C for 18 h. A negative control (cells in DMEM and 0.1% of BSA only) and positive controls (4 µg.mL⁻¹ and 0.8 µg.mL⁻¹ of rTcdA or rTcdB alone) were also added. Cell morphological alterations were observed under phase contrast microscopy (Zeiss, Oberkochen, Germany).