OA1‐AM23‐ST‐10 | A Novel Protocol for Determining the Absolute Concentration of Anti‐RBC Isotype‐Specific IgG Alloantibodies

• DOI: 10.1111/trf.24_17554
• Publication Date: 2023-10-12T10:32:37Z
• Authors: C. Niebuhr; O. Seldon; J. Medved; A. Arneja; A. Prakash; J. Zimring; C. Luckey
• Keywords: Isotype; Subclass

Current methods of measuring anti-red blood cell (RBC) antibody levels are limited in their ability to provide absolute quantification. This is due inherent differences the affinity for RBC antigens among primary antisera as well IgG isotypes secondary reagents. Given important biological effector function observed between different subclasses, we set out develop a research-based flow cytometry protocol that would quantification subclass-specific bound RBCs. We took advantage recent development four anti-KEL recombinant antibodies carry same Kpb binding CDR regions genetically modified express each 4 major C57Bl/6 constant (IgG1, IgG2b, IgG2c, and IgG3). Thus, these purified monoclonal constructs encode an binds KEL antigen (Kpb) with differs only region. Subclass-specific anti-Kpb monoclonals were used standards dilution series where anti-RBC was measured by cytometry. Our novel cytometry-based allowed us generate standard curves, providing generated C57BL/6J mice transfused KELmed RBCs (that also Kpb). found many commercially available anti-IgG showed significant biases toward certain subclasses over others. Furthermore, some isotype-specific demonstrated cross-reactivity other isotypes, rendering them useless. By using validated antibodies, quantified median at weeks one post-KEL2med transfusion. Week 1 IgG1, IgG3 67.3, 4.6, 0.0, 1022.7 ng/mL, respectively. At four-week time point, concentrations 2892.3, 60.5, 273.0, 1445.2 have developed provides anti-isotype-specific responses mice. data KEL2med system show abundant early response. Interestingly, relative subclass distribution significantly changes time, IgG1 dominating overall response post-transfusion. assay will allow more accurate study dynamics class switching mouse models. our proof principle how similar human measure patient samples.