Improvement of a low pH antigen-antibody dissociation procedure for ELISA measurement of circulating anti-Aβ antibodies

BACKGROUND: Prior work from our group found that acid dissociation (pH 2.5 incubation) of serum from APP transgenic mice vaccinated against Aβ increased the apparent anti-Aβ titers, suggesting antibody masking by antigen in the ELISA assay. Subsequently, we found that pH 2.5 incubation of serum from unvaccinated non-transgenic mice showed antibody binding to Aβ1–42, but no increase when other proteins, including shorter Aβ peptides, coated the ELISA plate. To investigate further the effects of low pH incubation on apparent anti-Aβ1–42 signals, we examined normal sera from nonTg unvaccinated mice, nonTg mice vaccinated with Aβ peptide (to produce authentic anti-Aβ antibodies) or a monoclonal antibody against Aβ (6E10) using competitive-inhibition ELISA and Aβ epitope mapping assays. In addition, we examined use of a less stringent low pH procedure at pH 3.5, to ascertain if it had the same effects as the pH 2.5 procedure. RESULTS: We believe there are three distinct effects of pH 2.5 incubation.; A) an artifactual increase in binding to full length Aβ by mouse immunoglobulin which has low affinity for Aβ, B) an inactivation of anti-Aβ antibodies that is time dependent and C) unmasking of high affinity anti-Aβ antibodies when high levels of circulating Aβ is present in APP transgenic mice. All three reactions can interact to produce the final ELISA signal. Incubation of sera from unvaccinated nonTg mice at pH 2.5 enhanced ELISA signals by process A. Conversely, pH 2.5 incubation of sera from vaccinated nonTg mice with caused a time dependent reduction of antibody signal by process B (overcoming the increase caused by A). The artifactual anti-Aβ ELISA signal enhanced by pH 2.5 incubation of normal mouse sera could not be effectively competed by low to moderate concentrations of Aβ, nor bind to shorter Aβ peptides in a manner similar to authentic anti-Aβ antibodies. Incubation of mouse sera at pH 3.5 caused neither an apparent increase in anti-Aβ ELISA signal, nor an inactivation of the ELISA signals resulting from either vaccination or monoclonal antibodies. However, incubation at pH 3.5 was able to completely reverse the reduction in ELISA signal caused by Aβ complexing with antibodies in sera from vaccinated mice or monoclonal anti-Aβ antibodies. CONCLUSION: Incubation at pH 3.5 is sufficient to dissociate Aβ bound to anti-Aβ antibodies without producing artifactual increases in the signal, or inactivating authentic antibody binding. Thus, use of pH 3.5 is a considerable improvement over pH 2.5 incubation for unmasking anti-Aβ antibodies in ELISA assays to measure antibodies in APP transgenic mouse sera.