65-P

• Published in: Human immunology 2013-11, Vol.74, p.97-97
• Main Authors: Timofeeva, Olga A, Li, Dong, San Juan, Vinna P, Thai, An T, Sochacki, Richard R, Guan, Jing, Awwad, Mariam K, Rosen-Bronson, Sandra
• Format: Article
• Language: English
• Subjects: Allergy and Immunology
• ISSN: 0198-8859
• DOI: 10.1016/j.humimm.2013.08.140

Aim Preliminary clinical data suggest that the C1q assay is a promising noninvasive technique for prediction of antibody mediated rejection (AMR). However, conflicting reports exist suggesting that assay performance and sensitivity vary between laboratories. Our aim was to evaluate a modified protocol for the commercially available C1qScreen™ assay to achieve increased sensitivity. We hypothesized that sensitivity of the assay could be increased by preincubation of serum with LABScreen beads prior to incubation with C1q. The rationale for this modification was that individual heads on a C1q molecule bind to the Fc region with weak affinity. Antibody aggregation on an antigenic surface facilitates binding of several heads significantly enhancing C1q affinity to low-MFI antibody. Methods The standard and modified C1q assays were performed in parallel. The difference between protocols was that in the standard C1qScreen™ protocol, heat-inactivated serum was spiked with human C1q and incubated with LABScreen beads for 20 min at RT. In the modified protocol, heat-inactivated serum was first incubated with LABScreen beads for 20 minutes followed by a 20 minute incubation with C1q. The rest of the assay was performed as described in the standard protocol. Results Of 30 samples tested, 17 samples that were positive for class I and 19 samples that were positive for class II C1q-binding antibody by both protocols. Some specificities that were weak or negative for C1q-binding using the standard protocol were strongly positive using the modified protocol. Also, of 13 ClassI and 11 ClassII samples that were negative for C1q-binding antibody by the standard protocol, 5 and 2 samples, respectively, were positive by the modified protocol. Conclusions Preincubation of serum with LABScreen beads prior to addition of human C1q facilitates increased sensitivity for detection of C1q-binding antibody. Further investigation will reveal whether increase in sensitivity offers better prediction of AMR.