Investigating the relationship between class I HLA-specific immunoglobulin-G subclasses, Pan-IgG single antigen bead assays and complement mediated interference in sera from renal transplant recipients.

Introduction: Antibody mediated rejection is the leading cause of kidney transplant failure. Not all antibodies are harmful and some may be protective. Immunoglulin Gs, of which there are four subtypes, are detected by single antigen bead testing. The aims of this study were to characterise the IgG subclass profiles for class I HLA-specific antibodies in an uncensored post-transplant population and to determine the underlying relationship between reactivity patterns and MFI cut-offs with the pan-IgG assay.

Methods: Patients were recruited to the study who were transplanted in our centre between 2009 and 2014. Prospectively stored post-transplant serum initially underwent a Labscreen Mixed assay and those positive for class I HLA-specific antibody underwent standard SAB testing, EDTA, 1 in 10 dilution and IgG subclass modifications using the Luminex platform. A total of 4947 bead reactions from 51 patients were analysed.

Results: A 1 in 10 dilution was used as a comparator pan-IgG assay for summed subclass and individual subclass linear regression analyses. Using a dilution to standard assay ratio we characterised all reactions for prozone potential i.e. how likely there is to be inhibition related to complement complex formation. We stratified samples into degrees of association and were able to determine suggested MFI thresholds of Log 5.35 for the dilution assay and Log 5.05 for the summed subclass assay when considering a Log MFI of 6.9 (1000) in the standard assay. Using individual subclass dominant reactions (>70%) we were able to determine linear relationships between the 1 in 10 dilution pan-IgG assay and the individual subclass assays (excluding prozone potential reactions for IgG1/3) enabling us to suggest Log MFI thresholds of 5.03, 3.58, 4.3 and 4.05 respectively for IgG1-4.

Discussion: We recommend a 1 in 10 dilution as the optimum pan-IgG comparator assay for a subclass analysis. We advocate the utilisation of the summed subclass assay to determine overall relationships and potential subclass failures. Following others, we recommend serum pre-treatment of the subclass assays to mitigate prozone. We suggest cut-offs for each IgG subclass which should be used with caution given the many inhibitory influences which may include competitive inhibition for bead binding, IgM and IgA interference and under-representation of specific subclasses on the bead panel.