| 主权项 |
1. A method for determining antibody sequences in circulation from a subject, wherein the subject has a tumor, an infectious disease, an autoimmune disease, has been immunized or has been exposed to an antigen which is an infectious agent, a tumor antigen, a tumor cell or a self-antigen, comprising:
a) obtaining mature B cells from the subject; b) isolating nucleic acids encoding VH and VL gene repertoires encoded by the mature B cells of the subject, and sequencing the isolated nucleic acids using a high-throughput sequencing method, wherein the high-throughput sequencing method is selected from the group consisting of sequencing-by-synthesis, sequencing-by-ligation, sequencing-by-hybridization, single molecule DNA sequencing, multiplex polony sequencing and nanopore sequencing, and determining the nucleic acid sequence and the corresponding amino acid sequence information of the VH and VL gene repertoires encoded by mature B cells of a subject; c) isolating antibodies from the biological fluid of the subject and preparing CDR3-containing peptide fragments derived from the isolated antibodies, separating the peptide fragments using chromatography, and generating mass spectra of the separated CDR3-containing peptide fragments; d) using the nucleic acid sequence information and the mass spectra to determine the amino acid sequence of VH and VL regions of one or more antibodies in circulation of the subject; e) determining the abundancy level of the amino acid sequences of the antibodies in circulation of the subject, and identifying the antibody sequences that exhibit at least a threshold level of abundancy; and f) expressing the one or more antibodies or antigen-binding fragments identified in step e) comprising one or more of the abundant amino acid sequences. |
