A screening method for antigen-specific IgE using mast cells based on intracellular calcium signaling.
A simple screening method is presented for the measurement of antigen-specific IgEs in sera in which mast cells are used. This method is based on the intracellular calcium signal in mast cells induced by cross-linking the surface high-affinity Fc receptors (FcepsilonRIs) with IgEs and multivalent antigens. When a serum containing various antigen-specific IgEs is added to the mast cell suspension, various antigen-specific IgEs are captured by FcepsilonRIs on the cell surface. However, the required antigen-specific IgE can be specifically detected after the addition of the corresponding antigen. The resulting increase in intracellular calcium concentration ([Ca2+]i), monitored by Ca2+-fluorometry, was found to be an analytical measure for the screening of IgEs. Two kinds of rodent mast cells, cell-lined RBL2H3 cells and primary cultured BMMCs, were used as a representative model system of mast cells. A DNP hapten (DNP35-HSA) and ovalbumin (OVA) were chosen for illustrative antigens, and these antigen-specific IgEs (DNP-specific IgE, OVA-specific IgE) in the corresponding rodent sera were target antibodies. It was found that [Ca2+]i increased linearly with IgE concentrations ranging from 25 to 5000 ng/mL for DNP-specific IgE and from 5 to 50 ng/mL for OVA-specific IgE. For these dynamic ranges, optimum concentrations of antigens were found to be 10 ng/mL and 1 microg/mL for DNP35-HSA and OVA, respectively. It was concluded that by monitoring the increase of [Ca2+]i in mast cells, we could determine the antigen-specific IgEs. The present immunological assay based on the Ca2+ signal transduction in mast cells offers new possibilities for efficient screening of antigen-specific IgEs and the immunogenicity of IgE in sera.
Aketani
S
S
Department of Chemistry, School of Science, The University of Tokyo, Japan.
Teshima
R
R
Sawada
J
J
Umezawa
Y
Y
D000818:Animals; D000918:Antibody Specificity; D020013:Calcium Signaling; D002460:Cell Line; D006801:Humans; D007073:Immunoglobulin E / Q000032:analysis* / Q000378:metabolism; D008407:Mast Cells / Q000737:chemistry* / Q000378:metabolism; D051379:Mice
D007073:Immunoglobulin E