Blood group type A antibody repertoire

Mononuclear cells from 400 ml peripheral blood of a blood group type B healthy donor were isolated and equally divide into two parts. One stained with CD20 and CD5 and sorted 2.0 * 106 B cells; the other stained with CD20/CD38/CD27 and sorted 3.5 * 105 memory B cells. B cell receptor sequences were reverse transcribed and amplified with a set of primers. High through-put sequencing was performed with PE300 platform considering the length of BCR variable region. After quality filtering and merging paired reads, we obtained about 1.9 *106 sequences for each sample. Using IgRepertoireConstructor, which constructs antibody repertoire and keeps mass spectra validation in mind, we identified 2.9 * 105 antibody clusters (including both heavy chain and light chain). At the same time, plasma from blood of the same individual was incubated Sepharose conjugated with KLH coupled with blood group A antigen. The bound antibody was identified with HPLC-MS/MS with the above sequenced antibody sequences as a reference database. Sequences with at least two peptides identified in both trypsin and chymotrypsin digestion samples were designated as blood group A antibody sequences, the sequences collection was considered as blood group A antibody repertoire.