Effect of time and temperature on the sensitivity of C3d complement detection with HLA single antigen beads

The C3d Detection assay, consisting of PE-labeled anti-human C3d antibody, Complement Serum and a Positive Control for C3d, is a reagent set used for the qualitative detection of complement C3d bound to antibodies/antigen complex. These reagents used in conjunction with a solid phase assays, such as Immucor’s HLA recombinant single antigen beads, have the ability to detect donor specific HLA antibodies (DSA) and may help identify C3d+DSA+ patients with higher risk of organ rejection (Sicard A et al. J Am Soc Nephrol. 2015 26: 457–67). C3d is one of the final degradation product of C3, it binds to antigen and interacts with CR2 on B-cells stimulating there proliferation. C3 is a key protein in the complement system, since it is the most abundant protein and has the ability to activate itself which greatly magnifies the response and C3d production. The other major end product of C3 are the components of the membrane attack complex (MAC) that form the basis of the complement dependent cytotoxicity (CDC) assay. The objective of this study was to examine the correlation of the C3d/HLA single antigen assay with CDC and at parameters that may increase C3d production thus increasing sensitivity of C3d Detection in serum with low titer HLA antibody. In vitro studies have shown that the formation of the final components of the complement cascade are temperature and time dependent. Our results with the Luminex-based C3d Detection assay confirmed that observation and showed that incubation with Complement Serum at increasing temperatures (from 20 °C to 27 °C) and/or longer times (from 30 to 120 minutes) will result in several fold increase of the C3d specific MFI values of low titer serum. This results in increased sensitivity, easier interpretation of the results and significant increase in the correlation of C3d with the CDC assay. In conclusion, the ability to vary the sensitivity of the C3d assay may increase its utility in analyzing patient samples and potentially complement (no pun intended) the standard CDC assay. D. Dalfo : Employee; Company/Organization; Immucor . N. Jiang : Employee; Company/Organization; Immucor . A. Contreras : Employee; Company/Organization; Immucor . I. Balazs : Employee; Company/Organization; Immucor .