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Craig J.Taylor, Vasilis Kosmoliaptsis, Jessie Martin, Graham Knighton, Dermot Mallon, J. Andrew Bradley, and Sarah Peacock

Background. Solid-phase assays to distinguish complement binding from noncomplement binding HLA-specific antibodies have been introduced, but technical limitations may compromise their interpretation. We have examined the extent to which C1q-binding to HLA-class I single-antigen beads (SAB) is influenced by denatured HLA on SAB, antibody titre, and complement interference that causes a misleading low assessment of HLA-specific antibody levels. Methods. Sera from 25 highly sensitized patients were tested using Luminex IgG-SAB and C1q-SAB assays. Sera were tested undiluted, at 1:20 dilution to detect highlevel IgG, and after ethylene diamine tetraacetic acid treatment to obviate complement interference. Conformational HLA and denatured HLA protein levels on SAB were determined using W6/32 and HC-10 monoclonal antibodies, respectively. Denatured HLA was expressed as HC-10 binding to untreated SAB as a percentage of maximal binding to acid-treated SAB. Results. For undiluted sera, Luminex mean fluorescence intensity (MFI) values for IgG-SAB and C1q-SAB correlated poorly (r 2=0.42). ethylene diamine tetraacetic acid and serum dilution improved the correlation (r 2 = 0.57 and 0.77, respectively). Increasing levels of denatured HLA interfered with the detection of C1q binding. Consequently, the correlation between IgG-SAB MFI and C1q-SAB MFI was lowest using undiluted sera and SAB with greater than 30%denatured HLA (r2 = 0.40) and highest using diluted sera and SAB with 30% or less denatured HLA (r2 = 0.86). Conclusions. Antibody level, complement interference, and denatured HLA class I on SAB may all affect the clinical interpretation of the C1q-SAB assay. The C1q-SAB assay represents a substantial additional cost for routine clinical use, and we question its justification given the potential uncertainty about its interpretation.

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