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J Immunol Methods. 1991 Aug 9;141(2):263-75.

A sensitive and efficient induction system for murine IgE. Single cell analysis at the clonal level.

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1
Research Department, CIBA-GEIGY, Ltd., Basel, Switzerland.

Abstract

A culture system is described which permits the analysis of IgE expression by single murine cells within clones of B cells. The system is based on the use of a CB5.1 stroma cell line as a feeder which optimally supports the IL-4-induced switch to IgE of LPS-stimulated B cells in culture. In this system 100 U/ml IL-4 induces the switch to IgE, in 3-5% of B cells and the switch frequency to IgG1 was as high as 2%. Five ng IgE or 12 ng IgG1 were produced per clone containing on average 13-15 PFC. The detection of single IgE secreting B cells was possible due to two newly developed, highly specific rat anti-mouse IgE antibodies used in a sandwich-ELISA. The frequency of IgE-secreting B cells was enhanced 2.5 times when the fibroblastoid CB5.1 cells rather than thymocytes were used as feeder cells. CB5.1 cells supported the differentiation of B cells to IgM-PFC almost as well as rat thymocytes (which have, to date, been used as the standard feeder layer) whereas the amount of secreted IgG1 was about 3 times lower than in thymocyte cultures. Optimal switching to IgE occurred at concentrations of IL-4 which were 10-fold lower than that required for IgG1 expression, a situation quite opposite to that observed in the rat thymocyte-supported culture system. In confirmation of established data the switch of B cells to IgE or IgG1 occurred randomly. The advantages of CB5.1 cells as feeder cells are (1) the use of a homogeneous and defined cell line, (2) their limited release of defined lymphokines (IL-6 and GM-CSF), and (3) the low degradation and consumption of cytokine factors. The combination of the CB5.1 cell line with a highly specific IgE ELISA assay made it possible to analyse the appearance of IgE producing cells within a developing B cell clone.

PMID:
1880431
DOI:
10.1016/0022-1759(91)90153-7
[Indexed for MEDLINE]

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