Yth53

Rituximab (Genentech, San Francisco, CA) was acquired through a drug exchange program at Duke University Medical Center, Department of Hematology and Oncology. Human CFH mAb 7968 was generated by cloning and expressing antibody genes derived from a single B cell as previously described [12 (link)]. The IgG1subclass-matched negative control antibody 7B2 recognizes gp41, an HIV-1 envelope glycoprotein, and was a gift of Dr. H. Liao of the Duke Human Vaccine Institute. The rat anti-human CD59 mAb YTH53.1 was obtained from Santa Cruz Biotechnology (Dallas, TX). The FITC mouse anti-human CD20 mAb 2H7 was obtained from BD Biosciences (San Jose, CA).

Exosome-bead conjugates containing 10 μg of exosome protein were labeled with 25 μM calcein AM (BD Biosciences, #564061) overnight at 37°C. Exosome-beads were washed three times in wash buffer (PBS with 0.5% BSA) to remove unincorporated dye, and incubated with antibodies. In the human plasma exosome lysis experiment, GT103 and/or anti-CD59 mAb YTH53.1 (Santa Cruz Biotechnology) were used at 50 μg/ml. In the cell line exosome lysis experiments, GT103, a subtype-matched negative control antibody, or a Fab fragment of GT103 [14 (link), 22 (link)] were used at 200 μg/ml. Mixtures were incubated for 1 hr at 37°C and washed twice in wash buffer. Following antibody treatment, exosome-bead conjugates were incubated with 10% normal human serum (NHS), heat-inactivated NHS, or specific complement factor-depleted serum for 1 hr at 37°C and washed twice. (NHS and complement C1q, C4, and Factor B depleted sera were obtained from Complement Technology, Inc., Tyler, TX.) Beads were incubated with buffer containing 5% Triton X-100 for 10 minutes as a positive control for calcein release. Mixtures were analyzed for calcein release by flow cytometry on a FACSCanto flow cytometer (BD Biosciences). A decrease in signal indicates lysis.