Allotypes matter: Evaluation of twelve commercially available antihuman IgG2 antibodies for subclass specificity.

Antibody responses to individual antigens vary widely in their relative contributions of the four human IgG subclasses. Detection of IgG subclasses is usually done using specific monoclonal antibodies. Ideally, such antibodies recognize a subclass irrespective of polymorphic structural variation (also called allotypes), while not recognizing any of the other subclasses. For IgG2 this is especially challenging. There are many monoclonal antibodies commercially available, but whether they meet the abovementioned requirements is unclear. In this study, we examined twelve commercially available monoclonal antibodies against human IgG2 (HP6002/AB4-DH4, HP6014/AC3-AA11/MH162.1, HP6200/HG2-56F, KT138, 985028/MAB9794, 31-7-4, MTG211E, 52G1, EPR4418, 2348B/MAB97941, and RM118) for their ability to selectively bind to human IgG2, and not to the other three subclasses. We used a panel of recombinant monoclonal antibodies comprising different human IgG subclasses and allotypes, and tested binding in ELISA. Of the twelve anti-human IgG2 clones tested, only three were found to be highly specific for IgG2 (>1000-fold). Two further clones demonstrated good selectivity albeit with measurable cross-reactivity to IgG4 of up to ca. 1-2%. Three clones were cross-reactive to certain IgG4 and IgG3 allotypes. These clones may possess G4m(b) specificity. The remaining clones showed only low selectivity for IgG2. In conclusion, specificity determinations of anti-IgG subclass reagents should take into account the genetic/allotypic variation across IgG subclasses. Antibodies with proclaimed IgG2 specificity do not always meet requirements for subclass-specific antibody detection.