Identifying human B cells expressing anti-allergen antibodies
In one aspect, methods of generating human monoclonal antibodies that specifically binds to an allergen are provided. In some embodiments, the monoclonal antibodies are generated from sequences identified from isolated single B cells from a human subject who is allergic to the allergen.
1. A method of identifying a human monoclonal antibody that specifically binds to an allergen, the method comprising:
isolating single B cells from a tissue or body fluid sample from a subject known to have an allergy to an allergen, wherein the isolating step comprises selecting single B cells that express cell surface IgE;
generating cDNA from RNA of individual single B cells;
sequencing the cDNA to obtain sequence data of heavy chain transcripts from the individual single B cells;
analyzing the sequence data to select a candidate single B cell that expresses an immunoglobulin heavy chain that comprises an IgE constant region;
identifying, from the candidate single B cell, (i) a heavy chain variable region sequence, and (ii) a light chain variable region sequence that are co-expressed with the immunoglobulin heavy chain in the candidate single B cell;
expressing antibodies comprising the heavy and light chain variable region sequences; and
determining that said antibodies bind said allergen, thereby identifying a human monoclonal antibody comprising an IgE constant region that specifically binds to the allergen.
2. The method of claim 1 , wherein said isolating step comprises sorting cells by fluorescent activated cell sorting (FACS).
3. The method of claim 1 , wherein said isolating step comprises contacting cells from the sample with an anti-human CD19 antibody and an anti-human IgE antibody and selecting for CD19+ IgE-expressing B cells.
4. The method of claim 1 , wherein said isolating step comprises contacting cells from the sample with an anti-human CD19 antibody, an anti-human IgE antibody, an anti-human IgM antibody, and an anti-human IgG antibody and selecting for CD19+IgM−IgG−IgE− expressing B cells.
5. The method of claim 1 , wherein said isolated B cells express a B cell marker and are negative for non-IgE isotypes.
6. The method of claim 1 , wherein said isolating step comprises contacting cells from the sample with an anti-human CD19 antibody, an anti-human IgM antibody, an anti-human IgG antibody, an anti-human IgA antibody, and an anti-human IgD antibody and selecting for CD19+IgM−IgG−IgA−IgD− B cells.
7. The method of claim 1 , wherein the method further comprises substituting the constant region of an antibody identified in said sequencing step with a wild-type IgG4 constant region or a modified IgG4 constant region sequence.
8. The method of claim 1 , wherein the sample is selected from the group consisting of blood, sputum, saliva, and tissue biopsy.
9. The method of claim 1 , wherein the allergen is a food allergen, a plant allergen, a fungal allergen, an animal allergen, a drug allergen, a cosmetic allergen, or a latex allergen.
10. The method of claim 9 , wherein the allergen is a food allergen selected from the group consisting of a milk allergen, an egg allergen, a nut allergen, a fish allergen, a shellfish allergen, a soy allergen, a legume allergen, a seed allergen, and a wheat allergen.