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. Author manuscript; available in PMC: 2015 Dec 1.
Published in final edited form as: Transfusion. 2014 May 21;54(12):3208–3215. doi: 10.1111/trf.12717

Figure 2. Detergent solubilization of recombinant CTL2 disrupts the epitope recognized by Type 2 antibodies.

Figure 2

A) HEK293 cells expressing full-length CTL2 (R154) were recognized by both Type 1 (1, 5) and Type 2 (2–4, 6, 13) HNA-3a antibodies in flow cytometry; values on the ordinate indicate median fluorescence intensity (MFI) relative to the signal obtained with cells expressing CTL2 Q154 (HPA-3a-negative). B) HEK293 cells expressing CTL2 were sensitized with the HNA-3a antibody panel and lysed with detergent. CTL2 was then captured with anti-myc monoclonal 9E10 and CTL2-bound IgG was detected with ELISA. Only Type 1 antibodies (1, 5) were detected with CTL2 (R154) (black bars). No antibodies recognized CTL2 (Q154) (gray bars). C) Same as Figure 3B except that cell bound antibody was marked with alkaline phosphatase (AP)-labeled goat anti-human IgG (H + L) before lysis. Released IgG was then captured with bovine anti-human IgG (Fc specific) and AP was measured by ELISA. All seven HNA-3a antibodies were detected (black bars); no antibodies were detected when cells expressing CTL2 (Q154) were used (gray bars). Studies were done in triplicate. Error bars represent ± 1.0 SD. *** = p<0.001, ** = p<0.01, * = p<0.05 relative to reaction with the Q154 construct. HD=Healthy donor serum.