Measurement of Low Avidity, Polyreactive Immunoglobulin G Antibodies with Increased Sensitivity by Using Low Ionic Strength Buffers

Author(s): Andrea Engelmaier, Harald Arno Butterweck, Alfred Weber

Low avidity polyreactive immunoglobulin G (IgG) antibodies have a broad range of affinity with dissociation constants of 10-5 to 10-8. Both their low concentration and avidity require low dilutions for measurement in solid-phase assays, which can cause issues due to the high total IgG levels probably interacting with the solid support. Here, we show that using an assay buffer system with low conductivity obviously increases the sensitivity of direct ELISAs. The antigens amyloid-β (Aβ) 40 and Aβ42 peptide, Aβ40 oligomers, Aβ42 fibrils, DNA, tubulin, and thyroglobulin, diluted in 0.1 M carbonate buffer, pH 9.5, were coated to the wells of polystyrene plates. Human citrated normal plasma or the intravenous IgG preparation GAMMAGARD LIQUID was diluted in phosphate-buffered saline or 20 mM HEPES, pH 7.4. Both buffers contained 10 mg/mL human serum albumin. Serial dilution series were loaded to antigen-coated and blank wells after blocking the plate with the respective dilution buffer. Detection of binding was achieved by an anti-human IgG peroxidase and peroxidase staining. Reduced dilution buffer’s conductivity increased signals by at least 50-fold without affecting the binding selectivity. Within the NaCl concentration range from 0 to 150 mM in 20 mM HEPES buffer, the conductivity correlated well with the signal height (R²=0.98). Competition experiments confirmed the assay’s adequate selectivity. In summary, buffer systems with low conductivity significantly increase the signals of direct ELISA without negatively affecting their selectivity. This simple assay modification increases the validity of results obtained by direct ELISAs for measurement of autoantibodies.

© 2016-2024, Copyrights Fortune Journals. All Rights Reserved