Measurement of anti-Aβ1-42 antibodies in intravenous immunoglobulin with indirect ELISA: The problem of nonspecific binding

Improvement in cognitive scores in patients with Alzheimer's disease (AD) has been reported in two trials in which intravenous immunoglobulin (IvIg) preparations were administered. IvIg's benefits in AD patients have been suggested to be due to antibodies to amyloid-beta (A beta). Our previous study using indirect enzyme-linked immunosorbent assay (ELISA) indicated that much of IvIg's apparent binding to A beta 1-42 is nonspecific; it is detectable even when ivIg is incubated on specificity controls (bovine serum albumin [BSA] and A beta reverse sequence A beta 42-1) rather than A beta 1-42. The objective of this study was to evaluate procedures that might reduce this nonspecific binding. The IvIg preparation used was Gamunex (Talecris Biotherapeutics). Multiple blocking agents were evaluated, but even the most effective blocker only reduced nonspecific binding by 48%. Dissociating Gamunex's antibody-antigen complexes had no effect on specific binding when A beta 42-1 was used as the specificity control, although it increased this binding when BSA was the specificity control. Decreasing Gamunex's dilution from 1:1,500 to 1:500 resulted in a slight (7.4%) but significant (p = 0.027) increase in specific binding. Using a sandwich ELISA to measure Gamunex's anti-A beta antibodies resulted in even less specific binding to A beta 1-42 than with the indirect ELISA. Despite Gamunex's low percentage of specific binding to A beta 1-42, it inhibited All oligomer formation. We conclude that, when anti-Ail antibodies in IvIg are measured by indirect ELISA, extensive nonspecific binding occurs despite procedures taken to prevent it. This must be subtracted from total binding to accurately measure specific anti-Ail antibody concentrations. (C) 2010 Elsevier B.V. All rights reserved.