Journal
JOURNAL OF IMMUNOLOGY
Volume 185, Issue 9, Pages 5377-5383Publisher
AMER ASSOC IMMUNOLOGISTS
DOI: 10.4049/jimmunol.1001587
Keywords
-
Categories
Funding
- Marie Curie Research and Training Network (European Commission) [MRTN-CT-2006-036032]
- Research Council of Norway
- National Institutes of Health/National Institute of Allergy and Infectious Diseases [5U19AI062629, 2U19AI057266]
Ask authors/readers for more resources
The gut mucosal surface is efficiently protected by Abs, and this site represents one of the richest compartments of Ab-secreting cells in the body. A simple and effective method to generate Ag-specific human monoclonal Abs (hmAbs) from such cells is lacking. In this paper, we describe a method to generate hmAbs from single Ag-specific IgA- or IgM-secreting cells of the intestinal mucosa. We found that CD138-positive plasma cells from the duodenum expressed surface IgA or IgM. Using eGFP-labeled virus-like particles, we harnessed the surface Ig expression to detect rotavirus-specific plasma cells at low frequency (0.03-0.35%) in 9 of 10 adult subjects. Single cells were isolated by FACS, and as they were viable, further testing of secreted Abs by ELISPOT and ELISA indicated a highly specific selection procedure. Ab genes from single cells of three donors were cloned, sequenced, and expressed as recombinant hmAbs. Of 26 cloned H chain Ab genes, 22 were IgA and 4 were IgM. The genes were highly mutated, and there was an overrepresentation of the VH4 family. Of 10 expressed hmAbs, 8 were rotavirus-reactive (6 with K(d) < 1 x 10(-10)). Importantly, our method allows generation of hmAbs from cells implicated in the protection of mucosal surfaces, and it can potentially be used in passive vaccination efforts and for discovery of epitopes directly relevant to human immunity. The Journal of Immunology, 2010, 185: 5377-5383.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available