Interactions between mouse IgG2 antibodies are common and mediated by plasma C1q

Abstract

The simultaneous use of multiple antibodies for multicolor flow cytometry is increasingly common and presents the opportunity for antibody interactions. Antibodies of differing isotypes were evaluated in pair-wise combinations for the presence of antibody interactions, using a standard whole blood lysing method or variations thereof. Artifactual interactions were identified and preferentially involved IgG2a (58%) or IgG2b (33%) antibodies and a single IgG1 antibody (3%). The interactions were present in all 10 random peripheral blood samples evaluated and required only whole blood and the two antibodies. Plasma removal prior to antibody incubation eliminated the interaction, as did switching any single IgG2 antibody to an IgG1 clone. Heat-inactivated plasma eliminated the interactions, and the addition of purified C1q to washed cells was capable of recreating the interaction in its entirety, confirming the mediation by complement C1q. Complement C1q mediates significant interactions between mouse IgG2 class antibodies; these interactions are very common and may be prevented by either complete serum removal or use of alternate IgG1 clones. Such interactions represent a significant potential source of artifact in the use of whole blood lysis methods for specimen preparation for multicolor flow cytometry.