Brigham and Women's Hospital Boston, Massachusetts, United States
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Background/Case Studies: The affinity of an antibody for its antigen is an important factor in the regulation of its activity.This feature is often defined by its inverse, the dissociation constant (Kd), which measures the propensity of an antibody to separate from its target antigen. Even high affinity antibody-antigen interactions are subject to factors that regulate rates of association/dissociation, termed antibody equilibrium.While equilibrium is often characterized in vitro, how this translates to equilibrium in vivo has not been thoroughly studied.
Study
Design/Methods: MIMA-29, a monoclonal mouse IgG2a anti-Duffy antibody (binding to epitope Fy3) was fluorescently labeled with Alexa Fluor 647 (AF647 MIMA-29) or AF660 (AF660 MIMA-29).Binding of AF647 or AF660 MIMA-29 was studied on HOD RBCs, which express the transgenic triple fusion protein hen egg lysozyme (HEL), ovalbumin, and human Duffy b antigen.DiO-labeled B6 RBCs and AF660 M29 coated DiI-labeled HOD RBCs were either incubated in an in vitro solution of AF647 MIMA-29 or transfused into FcRγ KO mice which were passively immunized with AF647 MIMA-29.FcyR KO recipients were used, as MIMA-29 is unable to clear RBCs or cause antibody-mediated antigen loss (antigen modulation) in the absence of FcyR. Flow cytometry was used to analyze binding properties of the anti-Duffy monoclonal antibody at multiple timepoints.An unpaired t test was used to analyze the results.IACUC approval was obtained prior to experimentation.
Results/Findings: Despite AF647 and AF660 having similar excitation and emission spectra, these labeled antibodies could be distinguished from one another by flow cytometry.At approximately 28ug/mL these antibodies reached a saturating concentration for staining HOD RBCs.AF660 MIMA-29 coated HOD RBCs, which were either incubated in a saline solution or injected into FcyR KO mice passively immunized with saline, retained a very high AF660 signal and negative AF647 signal from 5 minutes to 6 hours post-exposure.HOD RBCs, incubated with saturating levels of AF647 MIMA-29 in vitro or injected into FcyR KO mice passively immunized with AF647 MIMA-29, maintained a very high AF647 signal and negative AF660 signal from 5 minutes to 6 hours post-exposure.HOD RBCs coated with saturating levels of AF660 MIMA-29 and then incubated with AF647 MIMA-29 in vitro, reached ~5% saturating level of AF647 MIMA-29 at 5 minutes and ~50% saturation between 1-2 hours, while it was >50% saturation in FcyR KO mice passively immunized with AF647 MIMA-29 at the 5-minute timepoint (P< 0.01). Conclusions: RBC antibodies rapidly associate and dissociate from antigens on RBCs.These results do not support a therapeutic approach that uses cleaved antibody fragments (e.g. Fab or F(ab')2) to prevent harmful effects of intact antibodies in transfused recipients.
