Questioning the added value of Luminex single antigen beads to detect C1q binding donor HLA-specific antibodies.

Luminex single antigen bead (SAB) technology enables highly sensitive and rapid characterization of immunoglobulin G (IgG) human leukocyte antigen (HLA)-specific antibodies. It is widely used to determine antibody compatibility for renal transplantation and to aid the diagnosis of antibody-mediated rejection and response to therapy. HLA-specific antibodies may contribute to allograft rejection through a variety of mechanisms, including activation of the classical complement pathway, endothelial cell activation, and recruitment of Fc-dependent effector cells. Of these, complement-mediated cytotoxicity has long been associated with hyperacute rejection and, more recently, antibody-mediated rejection for which the deposition of the complement component C4d on peritubular capillaries is a diagnostic marker. In the standard SAB assay, patient serum is incubated with microbeads coated with purified HLA proteins. Human leukocyte antigenYspecific IgG antibody binding is then detected using a fluorescent anti-IgG detection antibody. Increasing levels of donor HLA-specific antibodies detected by this assay predict inferior long-term graft outcome, but not all patients with IgG donor-specific HLA antibodies (DSA) have a poor graft outcome (1, 2). This observation is consistent with the notion that HLA antibodies with the same specificity may differ in their ability to cause graft injury, and this variability may be related to differences in complement fixing activity. In an effort to improve the clinical utility of the IgGSAB assay, a novel C1q-SAB Luminex assay was developed, which aims to detect only complement fixing HLA-specific antibodies. In this assay, heat-inactivated patient serum is incubated with HLA-coated microbeads, with the addition of purified human C1q. C1q is the first component of the classical pathway of complement activation and binds to the Fc region of complement fixing IgG isotypes, mainly IgG1 and IgG3. Bound C1q is then detected by the addition of fluorescent-conjugated anti-human C1q detection antibody (Fig. 1A, panel a). Several studies have suggested that in patients after renal transplantation with DSA detected by IgG-SAB, the use of the C1q-SAB assay provides improved prediction of those most at risk of graft failure (3Y5). In the most comprehensive analysis to date, Loupy et al. (4) found that for patients where DSA were identified within the first year of renal transplantation, those with C1q-binding DSA showed a much worse graft survival than those with nonYC1q-binding DSA (54% vs. 93% 5-year graft survival). Although the presence of C1q-binding DSA are associated with inferior transplant outcome, it is by no means clear that this is a direct consequence of the ability of detected antibodies to activate complement. First, there are technical limitations to the C1q-SAB assay that have important consequences for interpretation of the test results. C1q is only able to bind when two IgG molecules are spaced 30 to 40 nm apart (6), which occurs readily when IgG crosslinks target HLA glycoproteins that have mobility within the lipid bilayer of a cell membrane. In solid phase binding assays, if HLA target molecules immobilized on the surface of SAB meet this strict spatial requirement for C1q, high levels of complement binding IgG will give a strongly positive readout (Fig. 1A, panel a). However, high levels of DSA IgG isotypes that are complement binding may not be detected in the C1q-SAB assay if the density of HLA protein is low and there are high levels of denatured HLA protein unable to bind antibody that interfere with the spatial position of intact HLA (Fig. 1A, panel b). Complement fixing antibodies of potential clinical importance may also be present, but at levels below the threshold required for effective binding of C1q in the C1q-SAB assay (Fig. 1A, panel c). Second, the C1q-SAB assay may reveal high levels of C1q binding in a serum that apparently has only low levels of IgG detected in the standard IgG-SAB. However, this apparent anomaly can be explained by the presence of blocking factors (particularly C1q itself ) present in undiluted patient serum that interfere with binding of the fluorescent anti-IgG detection antibody (Fig. 1B panel a) (7, 8). Pretreatment of test sera by heat inactivation or dithiothreitol to denature ANALYSIS AND COMMENTARY