Bias in murine IgG isotype immobilisation. Implications for IgG glycoform analysis ELISA procedures.

Journal Article (Academic article)

This study investigates immobilisation of murine IgG in various ELISA procedures. Monoclonal murine IgG isotypes and polyclonal IgG from sera were studied. Similar binding curves to plastic were found for all four individual murine IgG isotypes. Single isotypes displayed different affinities for both protein A and protein G, in particular IgG1 was poorly and IgG3 strongly bound to both of these proteins. When mixtures of the isotypes were bound to either plastic, protein A or protein G, competition was observed in which IgG3 was dominant. Paradoxically, studies on the binding rates of single isotypes direct to plastic revealed that IgG3 had the slowest binding rate. Heating of bound IgGs resulted in significant but isotypically non-selective losses from the plates. The data demonstrate that despite obtaining equivalent individual IgG isotype binding curves, mixtures of IgG isotypes behave very differently, with competition for binding occurring even on plastic. The IgG isotype levels of murine sera were measured for individual mice, and the capture efficiency of each IgG isotype by protein A determined at different serum dilutions. Comparisons were made between the observed capture levels of IgG isotypes and their known serum levels. At all dilutions tested, greater than expected binding of IgG3, IgG2b and IgG2a was observed. At a serum dilution of 1/100 the binding of these three isotypes was increased 16-, 2.9- and 0.4-fold, respectively. These increases were balanced by a decrease in IgG1 binding which was the most prevalent serum IgG isotype. The results described above suggest that capture techniques are biased and unlikely to provide a coating of IgG isotypes that accurately reflects that of the serum. This bias is derived from the specificity of the individual isotypes for either protein A or protein G, and the errors further compounded by direct competition between isotypes whatever the capture surface. Induced coalescence of IgG3 may explain the latter observations.

Duke Authors

Cited Authors

  • Jones, RH; Rademacher, TW; Williams, PJ

Published Date

  • October 1996

Published In

Volume / Issue

  • 197 / 1-2

Start / End Page

  • 109 - 120

International Standard Serial Number (ISSN)

  • 0022-1759

Conference Location

  • netherlands