Capture-stabilize approach for membrane protein SPR assays

Chu, Ruiyin; Reczek, David; Brondyk, William

We further extended the application of the capture-stabilize approach to virus-like particles and demonstrated its utilities analyzing antibodies against CD52. Alemtuzumab (Campath-1H), specific to human CD52, is currently approved as a treatment for B-cell chronic lymphocytic leukaemia. Although Alemtuzumab is capable of binding to naked synthetic CD52 peptide, other anti-CD52 mAb clones require glycan as part of their binding target and the synthetic peptide method with these mAbs is not a viable option. The virus-like particles were captured by Alemtuzumab and further stabilized by limited cross-linking and this stabilized surface worked well in binding kinetics assays for comparing all types of antibodies, including the capturing antibody. VLPs produced by recombinant expression systems in Saccharomyces cerevisiae have been reported at 30-50 nM in size 28 . We also determined that the CD52 VLPs produced in HEK293FT cells were similar in size (30-50 nM, data not shown). It is presumed that only a portion of the CD52 molecules displayed on VLP surface is bound by the capturing antibody; those unoccupied CD52 molecules are therefore available for binding by the analyte antibodies. Similarly, we expect that this approach can be applied to other types of membrane targets, such as multi-transmembrane domain protein ion channels.