Technical Limitations of the C1q Single-Antigen Bead Assay to Detect Complement Binding HLA-Specific Antibodies

Abstract

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 high-level 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 = 0.42). ethylene diamine tetraacetic acid and serum dilution improved the correlation (r = 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 (r = 0.40) and highest using diluted sera and SAB with 30% or less denatured HLA (r = 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.

FIGURE 1

Effect of serum treatment on IgG-SAB and C1q-SAB binding. Undiluted sera (panel A), EDTA treated sera (panel B) and 1 in 20 diluted sera (panel C) obtained from 25 highly sensitised patients were tested using Luminex HLA class I IgG-SAB. The results (IgG-SAB MFI, x-axis) were compared with that obtained for undiluted sera tested using C1QScreen (C1q-SAB MFI, y-axis). The results show improved correlation coefficients (r²) between IgG-SAB MFI and C1q-SAB MFI after correction for the prozone effect (EDTA-treated sera) and taking account of high titre IgG HLA specific antibody (diluted sera) compared with the conventional assay performed using untreated sera.

FIGURE 2

Assessment of levels of denatured HLA class I protein on single antigen bead populations. The level of denatured HLA class I protein expressed on SAB was determined by comparing HC-10 MFI value for each SAB population tested using untreated SAB as a percentage of maximum HC-10 MFI value tested using acid treated (denatured) SAB.

FIGURE 3

Effect of denatured HLA class I protein expression on IgG-SAB and C1q-SAB binding. Undiluted sera (panels A and B), EDTA treated sera (panels C and D) and 1 in 20 diluted sera (panels E and F) were tested using Luminex HLA class I IgG-SAB. The results (IgG-SAB MFI, x-axis) were compared to that obtained for undiluted sera tested using C1QScreen (C1q-SAB MFI, y-axis). SAB populations were stratified into 2 groups according to 30% or less denatured HLA protein expression (panels A, C, and E) and greater than 30% denatured protein (panels B, D, and F). The results show that for each serum treatment, the correlation coefficient between IgG-SAB MFI and C1q-SAB MFI was higher for SAB populations that express low levels (≤30%) of denatured HLA compared to high levels (>30%) of denatured HLA.

FIGURE 4

Examples of IgG-SAB and C1q-SAB MFI binding profiles for 2 selected patients. Undiluted sera (panels A and B), EDTA treated sera (panels C and D) and sera diluted 1 in 20 (panels E and F) obtained from 2 selected patients (patient 1 and patient 2) were tested using Luminex HLA class I IgG-SAB. MFI values for IgG-SAB (x-axis) were compared to that obtained for undiluted sera tested using C1QScreen (C1q-SAB, y-axis). For patient 1 untreated serum, the correlation coefficient (r²) between MFI values obtained using IgG-SAB MFI and C1q-SAB was poor, but after correction for the prozone effect (EDTA treated serum) and taking account of high level IgG (diluted serum), the correlation was near perfect. For patient 2, the correlation between IgG-SAB MFI and C1q-SAB MFI using untreated serum was poor, and EDTA treatment and serum dilution gave only a relatively modest improvement.

FIGURE 5

Effect of denatured HLA class I protein expression on IgG-SAB and C1q-SAB binding profiles for patient 2. For patient 2 serum, MFI values obtained using IgG-SAB (x-axis) were compared with those tested using C1QScreen (C1q-SAB, y-axis). The results were stratified according to the level of denatured HLA (≤30% versus >30%) expressed on the different SAB populations. For SAB that expressed 30% or less denatured HLA there was a high correlation between MFI values obtained using IgG-SAB and C1q-SAB assays, particularly when using EDTA treated and diluted serum. In contrast, for SAB that expressed greater than 30% denatured HLA, for undiluted serum the correlation was very poor, and showed only a modest improvement using EDTA-treated and diluted serum.