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
Reactivity of anti-IgG1 monoclonal and polyclonal reagents with canonical IgG isoallotypes.
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). Data shown are MFIs representative of 3 replicate experiments; stain indices are shown (upper right corner) for each histogram as indicator of staining intensity above background (see Methods). While only canonical isoallotypes are shown, all 29 isoallotypes were tested in a similar manner, and data are presented in