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



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.



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).



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.
1 Histocompatibility and Immunogenetics Laboratory, Cambridge University Hospitals NHS Foundation Trust, Cambridge, United Kingdom. 2 Department of Surgery, University of Cambridge, Cambridge, United Kingdom.
Craig J
CJ
Taylor
Vasilis
V
Kosmoliaptsis
Jessie
J
Martin
Graham
G
Knighton
Dermot
D
Mallon
J Andrew
JA
Bradley
Sarah
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Peacock
D000328:Adult; D000918:Antibody Specificity; D003167:Complement Activation; D015922:Complement C1q / Q000276:immunology*; D005260:Female; D006084:Graft Rejection / Q000097:blood / Q000276:immunology; D006085:Graft Survival; D006680:HLA Antigens / Q000737:chemistry / Q000276:immunology*; D006648:Histocompatibility; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood / Q000276:immunology*; D007518:Isoantibodies / Q000097:blood / Q000276:immunology*; D008297:Male; D008875:Middle Aged; D011237:Predictive Value of Tests; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D015203:Reproducibility of Results; D055815:Young Adult
D006680:HLA Antigens; D007074:Immunoglobulin G; D007518:Isoantibodies; D015922:Complement C1q