Measuring Antibody Affinity by ELISA

IgG₁ or λ1 antibody specific for the NP hapten was detected by ELISA using two different coupling ratios of NP–BSA as the coating antigens. In brief, 96-well flat bottom plates (Falcon; Becton Dickinson, Oxnard, CA) were coated with 50 μg/ml NP₅–BSA or NP₂₆– BSA in 0.1 M carbonate buffer (pH 9.0) at 4°C overnight, and blocked with 0.5% BSA in carbonate buffer. Serially diluted sera were then added to each well and incubated at 4°C overnight. On each plate, serially diluted H33Lγ1/λ1, a monoclonal antibody recognizing the NP hapten (K_a = 2.0 × 10⁷ M⁻¹)² was also included as a control. After washing with PBS containing 0.1% Tween 20, HRP-conjugated goat anti–mouse IgG₁ or biotinylated Ls136 was added and incubated at room temperature for 2 h. HRP-conjugated streptavidin was added to detect biotinylated Ls136; HRP activity was visualized using a TMB peroxidase substrate kit (Bio-Rad Laboratories, Hercules, CA) and optical densities were determined at 450 nm. The concentrations of anti-NP IgG₁ or λ1 antibodies were estimated by comparison to standard curves created from the H33Lγ1/λ1 control on each plate. To estimate the affinity of NP-binding antibody in the sera, the ratio of NP₅-binding antibody to NP₂₆-binding antibody was calculated (43 (link)).
The affinity threshold of antibody binding to each NP–BSA conjugate was determined by using several monoclonal antibodies with different affinities for NP. H33Lγ1/λ1 bound equally well to both NP–BSA conjugates, whereas a monoclonal antibody with a K_a = 10⁶ M⁻¹ showed a 20-fold lower binding to NP₅–BSA than to NP₂₆–BSA. A monoclonal antibody with a K_a = 2.3 × 10⁵ M⁻¹ had a 10-fold lower binding to NP₂₆–BSA than one with a K_a = 1.0 × 10⁶ M⁻¹. Thus, antibody with a K_a ⩾2.0 × 10⁷ M⁻¹ can be detected with NP₅–BSA, and one with a K_a ⩾10⁶ M⁻¹ can be detected with NP₂₆–BSA.