Complement component C3 activation: the leading cause of the prozone phenomenon affecting HLA antibody detection on single-antigen beads.

Luminex-based anti-HLA IgG detection on single-antigen flow beads (SAFB) represents a valuable tool for characterization of allosensitization patterns. Assay interpretation, however, may be impeded by false-low test results caused by the prozone effect. Recent experimental data have related this artifact to direct blockade of IgG detection by complement component C1 as a possible candidate mechanism. To dissect a causative role of C1 complex formation and subsequent steps of classical complement activation, native or modified sera obtained from transplant candidates with HLA class I sensitization were evaluated applying SAFB-based IgG, IgM, C1q, C1r, C1s, or C4 and C3 split product detection, respectively. Evaluating a total of 1,164 single-antigen reactions, serum dilution (1:10) revealed an 11% incidence of the prozone effect as defined by a greater than 100% increase in IgG mean fluorescence intensity. Prozoning was found to be related to the amount of antibody-triggered C1q, C4 or C3 split product deposition, and was eliminated by serum modifications affecting the integrity of the C1 complex (dithiotreitol, ethylenediaminetetraacetic acid, heat inactivation). Remarkably, the same effect was achieved without C1 disintegration, either by serum treatment with methylamine to block C4 and C3 split product binding or by cobra venom factor to trigger C3 consumption. Our results reinforce a central role of C1 as a trigger of prozoning. However, we provide evidence that C1 may exert its effects only indirectly, namely via inducing C3 fragment deposition, which by coating of the bead surface may block the binding of IgG detection reagents.