Monoclonal Antibodies to Discriminate the EF Hand Containing Calcium Binding Adaptor Proteins EFhd1 and EFhd2.

Small Ca(2+) binding adaptor proteins of the EF hand family play important roles in neuronal and immune cell Ca(2+) signaling. Swiprosin-1/EFhd2 (EFhd2) and Swiprosin-2/EFhd1 (EFhd1) are conserved and very homologous Ca(2+) binding adaptor proteins of the EF hand family, with possibly redundant functions. In particular, EFhd2 has been proposed to be involved in B cell signaling and neuropathological disorders. Little is known thus far about the expression of EFhd2 on the single cell level in tissue sections or blood cells. Here we describe the generation of four specific anti-EFhd2 monoclonal antibodies. These recognize murine and human EFhd2, but not murine EFhd1, and their binding site maps to a region in the N-terminal part of EFhd2, where EFhd2 and EFhd1 differ most. Moreover, to detect EFhd1 specifically, we also generated anti-EFhd1 polyclonal antibodies, making use of a singular peptide of the N-terminal part of the protein. Using anti-EFhd2 MAb, we reveal two EFhd2 pools in B cells, one at the membrane and one cytoplasmic pool. Staining of human peripheral blood mononuclear cells shows EFhd2 expression in B cells but a ∼5 fold higher expression in monocytes. Taken together, EFhd2 monoclonal antibodies will be valuable to assess the real subcellular localization and expression level of EFhd2 in healthy and diseased primary cells and tissues.