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

ABSTRACT: 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.

SUBMITTER: Taylor CJ

PROVIDER: S-EPMC5457814 | biostudies-other | 2017 Jun

REPOSITORIES: biostudies-other

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

Taylor Craig J CJ Kosmoliaptsis Vasilis V Martin Jessie J Knighton Graham G Mallon Dermot D Bradley J Andrew JA Peacock Sarah S

Transplantation 20170601 6

<h4>Background</h4>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.<h4>Methods</h4>Sera from 25 highly sensitized ...[more]

PMID: 27306532

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Project description:Monoclonal antibodies directed against the CD20 surface antigen on B cells are widely used in the therapy of B cell malignancies. Upon administration, the antibodies bind to CD20 expressing B cells and induce their depletion via cell- and complement-dependent cytotoxicity or by induction of direct cell killing. The three antibodies currently most often used in the clinic are Rituximab (RTX), Ofatumumab (OFA) and Obinutuzumab (OBI). Even though these antibodies are all of the human IgG1 subclass, they have previously been described to vary considerably in the effector functions involved in therapeutic B cell depletion, especially in regards to complement activation. Whereas OFA is known to strongly induce complement-dependent cytotoxicity, OBI is described to be far less efficient. In contrast, the role of complement in RTX-induced B cell depletion is still under debate. Some of this dissent might come from the use of different in vitro systems for characterization of antibody effector functions. We therefore set out to systematically compare antibody as well as C1q binding and complement-activation by RTX, OFA and OBI on human B cell lines that differ in expression levels of CD20 and complement-regulatory proteins as well as human primary B cells. Applying real-time interaction analysis, we show that the overall strength of C1q binding to live target cells coated with antibodies positively correlated with the degree of bivalent binding for the antibodies to CD20. Kinetic analysis revealed that C1q exhibits two binding modes with distinct affinities and binding stabilities, with exact numbers varying both between antibodies and cell lines. Furthermore, complement-dependent cell killing by RTX and OBI was highly cell-line dependent, whereas the superior complement-dependent cytotoxicity by OFA was independent of the target B cells. All three antibodies were able to initiate deposition of C3b on the B cell surface, although to varying extent. This suggests that complement activation occurs but might not necessarily lead to induction of complement-dependent cytotoxicity. This activation could, however, initiate complement-dependent phagocytosis as an alternative mechanism of therapeutic B cell depletion.

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

Publications

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

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