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-speci fic 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 sur face high-affinity Fc receptors (Fc epsilon RIs) 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 Fc epsilon RIs on the cell surface. However, the required antigen-specific Ig E can be specifically detected after the addition of the corresponding anti gen. The resulting increase in intracellular calcium concentration ([Ca2+]( i)), monitored by Ca(2+)fluorometry, was found to be an analytical measure for the screening of IgEs. Two kinds of rodent mast cells, cell-lined RBL-2 H3 cells and primary cultured BMMCs, were used as a representative model sy stem of mast cells. A DNP hapten (DNP35-HSA) and ovalbumin (OVA) were chose n for illustrative antigens, and these antigen-specific IgEs (DNP-specific IgE, OVA-specific IgE) in the corresponding rodent sera were target antibod ies. 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 a ntigens were found to be 10 ng/mL and 1 mu g/mL for DNP35-HSA and OVA, resp ectively. It was concluded that by monitoring the increase of [Ca2+](i) in mast cells, we could determine the antigen-specific IgEs, The present immun ological assay based on the Ca2+ signal transduction in mast cells offers n ew possibilities for efficient screening of antigen-specific IgEs and the i mmunogenicity of IgE in sera.