In recent years, the extent of our vulnerability to misinterpretation due to poorly characterized reagents has become an issue of great concern. Antibody reagents have been identified as a major source of error, contributing to the “reproducibility crisis.” In the current report, we define an additional dimension of the crisis; in particular, we define variation of the targets being analyzed. We report that natural variation in the immunoglobulin “constant” region alters the reactivity with commonly used subtype-specific anti-IgG reagents, resulting in cross-reactivity of polyclonal regents with inappropriate targets and blind spots of monoclonal reagents for desired targets. This raises the practical concern that numerous studies characterizing IgG subtypes in human disease may contain errors due to such previously unappreciated defects. These studies also focus attention on the broader concern that genetic variation may affect the performance of any laboratory or research test that uses antibodies for detection.
Heather L. Howie, Meghan Delaney, Xiaohong Wang, Lay See Er, Linda Kapp, Jenna N. Lebedev, James C. Zimring
Human IgG isoallotype genetic variation and reactivities to anti-IgG subclass reagents.
Canonical sequences for each IgG subclass are shown in light blue, with all known intrasubclass variants within the hinge, CH2, and CH3 region shown below each canonical sequence. A summary of the reactivity with either monoclonal or polyclonal anti-IgG subclass–specific antibody is shown to the right, with blind spots (and their corrections) shown in red and cross-reactive antibodies (and their corrections) shown in blue. For clarity, variant amino acids that differ between IgG subclasses but that do not vary within a subclass (i.e., isoallotype) are not shown. For a more comprehensive view of all known amino acid differences between the 29 isoallotypes, please see ref.