Soluble thymic stromal lymphopoietin receptors are absent in murine sera – detection with anti‐mTSLPR monoclonal antibodies

Abstract

Two monoclonal antibodies, termed nnIE11 and nnIG11, were generated against the murine thymic stromal lymphopoietin receptor, mTSLPR, using traditional hybridoma technology. The antibody-producing hybridoma clones were obtained by fusing P3X63-Ag8.653 myeloma cells with splenocytes from Balb/c mice immunized with anti-FLAG M2 affinity-purified FLAG-tagged mTSLPR from pSVL-mTSLPR-FLAG-transfected COS cells and Ni-NTA-purified his-tagged mTSLPR from recombinant FastBacHisB-mdelta1 baculovirus-infected Sf9 cells. Several monoclonal anti-mTSLPR-specific hybridoma clones were obtained and two of these clones are further characterized here. The generated antibodies could in an immunoblotting identify baculovirus-expressed mTSLPR proteins with a molecular weight corresponding to 50 kDa. Both immunoblotting and ELISA with recombinant mouse TSLPR/Fc chimera as antigen, having only the N-terminal domain of mTSLPR present, indicated that the generated monoclonal antibodies identify the C-terminus of mTSLPR. Although sandwich ELISAs performed with a goat anti-mTSLPR antiserum as capture antibody and nnIE11 as indicator antibody were able to detect mTSLPR in the range of 5 ng/ml, no souble mTSLPR could be observed in serum samples from CBA/H, Balb/c and C57Bl/6 mice.