BIAS IN MURINE IGG ISOTYPE IMMOBILIZATION IMPLICATIONS FOR IGG GLYCOFORM ANALYSIS ELISA PROCEDURES

This study investigates immobilisation of murine IgG in various ELISA procedures. Monoclonal murine IgG isotypes and polyclonal IgG from ser a were studied. Similar binding curves to plastic were found for all f our individual murine IgG isotypes. Single isotypes displayed differen t affinities for both protein A and protein G, in particular IgG1 was poorly and IgG3 strongly bound to both of these proteins. When mixture s 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 reve aled that IgG3 had the slowest binding rate. Heating of bound IgGs res ulted in significant but isotypically non-selective losses from the pl ates. The data demonstrate that despite obtaining equivalent individua l IgG isotype binding curves, mixtures of IgG isotypes behave very dif ferently, with competition for binding occurring even on plastic. The IgG isotype levels of murine sera were measured for individual mice, a nd the capture efficiency of each Ige isotype by protein A determined at different serum dilutions. Comparisons were made between the observ ed capture levels of IgG isotypes and their known serum levels. At all dilutions tested, greater than expected binding of IgG3, IgG2b and Ig G2a was observed. At a serum dilution of 1/100 the binding of these th ree isotypes was increased 16-, 2.9- and 0.4-fold, respectively. These increases were balanced by a decrease in IgG1 binding which was the m ost prevalent serum IgG isotype. The results described above suggest t hat 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 pr otein A or protein G, and the errors further compounded by direct comp etition between isotypes whatever the capture surface. Induced coalesc ence of IgG3 may explain the latter observations.