(A) Human RBCs with a phenotype of K⁺k⁺ (blue histograms) or K^–k⁺ (red histograms; negative control) were stained with PUMA1 (specific for K⁺k⁺ cells) of the indicated human IgG subclass (labeled columns) and with the human IgG-specific reagents (labeled rows). While only canonical isoallotypes are shown, all 29 isoallotypes were tested in a similar manner, and data are presented in Figure 1. (B) Titration of the Sanquin anti-IgG4 polyclonal reagent with staining of canonical IgG4-01, as well as cross-reactive isoallotypes IgG3-03 and IgG3-13. (C) Cross-reactivity of IgG3-03 and IgG3-13 with polyclonal anti-IgG4 reagents is eliminated by mutating glutamic acid at amino acid position 419 in the CH3 region to glutamine. Binding of the pan-IgG detection reagent Ortho-AHG is shown for each variant to indicate that loss of staining is not due to loss of antibody expression. (D) Adsorption of cross-reactive antibodies using IgG3-03 and IgG3-13 backbones fused to a variable domain that binds an epitope (HLA) not expressed on RBCs results in elimination of cross-reactive detection while maintaining binding to canonical IgG4-01. Data shown are MFIs representative of 3 replicate experiments; stain indices are shown (upper right corner) for each histogram as an indicator of staining intensity above background (see Methods). S.Bio, Southern Biotech; BindSite, The Binding Site Group; Ortho, Ortho Clinical Diagnostics (see Methods for antibody details)