Extended Data Fig. 1: Enzyme selection strategy and evaluation against group A and B RBCs.
Overview of the discovery and selection of exoglycosidases for conversion of: a B antigen, b ExtB antigen, c A and A type 3 antigens, d H type 3 antigen and e Gal-A antigen. Initial screening against chromogenic substrates guided initial selection of enzymes, which were further assayed against unconjugated saccharides using thin layer chromatography. Normalized rates (Vo/E) of best performing enzymes were determined using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), then these enzymes were evaluated against target antigens on RBCs using flow cytometry. The blood drop symbol denotes top-performing enzymes, which were selected for crossmatch reactivity (compatibility) analyses. f Crossmatch reactivity analyses were performed by enzymatic conversion of RBCs from A1, A2 and B donors. Single-enzyme treatments targeting A or B antigens as well as sequential and one-pot treatments with enzyme cocktails targeting A or B antigens and their respective extended forms were performed. Blood group A or B RBCs of secretor/non-secretor backgrounds were converted with either a single enzyme or enzyme cocktails and converted RBCs from each treatment were crossmatched against 100 group O plasmas, resulting in 1522 crossmatch tests, besides lower numbers of crossmatch assays with plasmas from other ABO groups. Created with BioRender.com.
