Proteomic identification of antibodies
Methods and compositions for identification of candidate antigen-specific variable regions as well as generation of antibodies or antigen-binding fragments that could have desired antigen specificity are provided. For example, in certain aspects, methods for determining amino acid sequences of serum antibody CDR3 and abundancy levels are described. In some aspects, methods for determining nucleic acid sequences of antibody variable region sequences and the frequency thereof in biological samples are provided. Furthermore, the invention provides methods for identification and generation of antibodies or antigen-binding fragments that comprise highly-represented CDR domains.
1. A method of identifying a repertoire of different antibodies specific to an antigen in a biological fluid of a subject comprising:
a) obtaining a biological fluid sample from the subject;
b) isolating multiple single B-cells from the biological fluid of the subject;
c) co-isolating nucleic acids encoding VH and VL genes from the single B-cells, thereby obtaining the natively paired VH and VL coding sequences that are expressed in the same B-cell, and sequencing the co-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 natively paired VL gene repertoire encoded by the single B cells;
d) 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;
e) using the sequence information and the mass spectra to determine the amino acid sequence of the V H and V L of antibodies in multiple single B cells; and
f) expressing one or more antibodies or antigen-binding fragments determined in step e).
2. The method of claim 1 , wherein a repertoire of at least 20 V H chains or antibodies is identified.
3. The method of claim 2 , wherein a repertoire of 20 to 250 V H chains or antibodies is identified.
4. The method of claim 1 , wherein the biological fluid is serum.
5. The method of claim 1 , wherein the biological fluid is intestinal lavage or bronchoalveolar lavage.
6. The method of claim 1 , wherein the nucleic acid sequences are determined from a cDNA library.
7. The method of claim 1 , wherein the nucleic acid sequences are determined from genomic DNA.
8. The method of claim 1 , wherein the subject has or has been exposed to an antigen that is an infectious agent, a tumor antigen, a tumor cell, or a self-antigen.
9. The method of claim 1 , wherein the method further comprises determining the relative abundancy level or relative frequency of the amino acid sequences of the antibodies in the sample.
10. The method of claim 1 , wherein each of the antibodies or antigen-binding fragments so generated comprises similarly abundant amino acid sequences of V H and V L or is part of a cluster of highly homologous amino acid sequence that are similarly abundant.