OR14 Next-generation HLA antibody reagents

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Aim A humanized monoclonal antibody (Ab) pan-reactive for HLA Class I is useful for many HLA testing applications, including validation and confirmation of HLA Ab assays and platforms. By engineering the Ab so it is also available in subtype-specific (IgG1-4) and allotype-specific forms, it becomes useful for validating and characterizing Ab assays dependent upon complement-fixation, and validating the specificity of secondary Abs used as detection reagents. Methods The IgG1-4 and allotype-specific forms of this Ab were tested with the detection reagents for our flow cytometric crossmatch (FCXM) assay and single-antigen bead (SAB) assay. The IgG1-4 Abs were also tested in the CDC assay, with and without AHG. The IgG1 Ab was tested at dilutions against cells in the FCXM. The IgG1-4 Abs were tested at multiple dilutions in the SAB and C1q assays, and with and without heat-inactivation and EDTA treatment of sera. Results All lots of AHG used as detection reagents in the FCXM and SAB assays were able to detect the IgG1-4 Abs, and the FCXM reagents detected all 29 isoallotypes. No false-reactivity was seen in the Class II SAB assay. In the CDC assay, IgG1-3 were positive both with and w/out AHG, but IgG4 was only positive when AHG was added. When tested against cells from 15 different individuals, the IgG1 Ab provided consistent reactivity, regardless of the HLA type of the donor cells. When serial dilutions of the IgG1-4 Abs were tested in the SAB and C1q assays, a distinct hierarchy of sensitivity in the C1q assay was apparent, with IgG3 > IgG1 > IgG2. IgG4 was negative in the C1q assay. Conclusions These IgG1-4 monoclonal Abs, directed against a monomorphic Class I epitope, are valuable reagents for validating and characterizing the multiple HLA antibody assays typically employed in a modern HLA laboratory. In Red Cell serology, it has been demonstrated that some IgG isoallotypes are NOT detected by the secondary AHG detection reagents, making these Abs useful to confirm detection with different lots of AHG. These antibodies are also useful as controls in antibody assays that rely on complement-fixation (i.e. CDC and C1q). Finally, when titrated appropriately, these Abs can give consistent low, medium, or strongly-positive reactivity in the SAB and FCXM assays, making them useful as a reagent to standardize and/or normalize results.