Antibody Avidity Assay Protocol

Antibody avidity (functional affinity), defined as the overall binding strength of an antibody to an antigen was measured by competitive inhibition^{45 (link)}. Briefly, 96 well microtiter plates were coated with 5 µg/mL of SF2a LPS incubated for 1 h at 37 °C and blocked with 150 µl of blocking buffer (containing 0.5% BSA and 0.5% Casein) for another 1 h. Double dilutions of SF2a LPS, here as free antigen, in blocking buffer were added to the microtiter plate (initial concentration 5 µg/mL). The last well was covered only with blocking buffer (control well). Sera at O.D. of approx. 1.0 at A₄₀₅ were added to the wells containing free antigen or blocking buffer and plates were incubated ON. After washing plates six times, 100 µl of AP-conjugated anti-human IgG (KPL Inc. USA) was added followed by ON incubation. Wells were washed four times, 100 µl of pNPP one component substrate was added and the plates were incubated in the dark for 15 min at RT. Plates were read at 405 nm until the O.D. of control wells reaches 0.8 to 1.2. The results of relative avidity, avidity index (AI), represent the free antigen concentration that was required to inhibit antibody binding to the coated SF2a LPS wells by 50%. This value could also be expressed as the log of the free antigen concentration that is necessary to cause 50% inhibition (I₅₀).