IgG Avidity Assay Protocol

The IgG avidity SIAs are based on the principle of elution of the antigen-bound antibodies with urea (Fig. 4), under the experimental conditions presented in Table 8. Briefly, from each serum sample, two dilution series were made in PBST, series 1 (1:20, 1:80, 1:320, and 1:1,280) and series 2 (1:80, 1:320, 1:1,280, and 1:5,120). These dilutions were placed into a 96-well plate and incubated with 1.75 × 10³ antigen-coated microspheres/well/analyte for 45 min. Next, series 1 samples were washed three times for 5 min each with 6 M freshly prepared urea (Promega, USA) in PBS, as opposed to series 2, with PBS only. Subsequently, biotinylated protein G (Thermo Scientific, USA) and SA-PE were added, and the MFIs were measured, as for the IgG SIAs (18 (link)).